archie lovatt, phd thesis 1994
TRANSCRIPT
Molecular Analysis of a P-N-Acetyl-Hexosaminidase Gene From Porphyromonas gingivalis W83
Archie Lovatt
Thesis presented for the degree of Doctor of Philosophy
University of Leicester
1994
UMI Number: U539289
All rights reserved
INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
DiSËürtâtion Publishing
UMI U539289Published by ProQuest LLC 2015. Copyright in the Dissertation held by the Author.
Microform Edition © ProQuest LLC.All rights reserved. This work is protected against
unauthorized copying under Title 17, United States Code.
ProQuestProQuest LLC
789 East Eisenhower Parkway P.O. Box 1346
Ann Arbor, Ml 48106-1346
Contents
Chapter 1 Introduction
1.1 Porphyromonas gingivaUs
1.1.1 General Biology1.1.2 Role in Periodontal Disease
1.2 Bacterial Adhesion and Colonisation1.2.1 Dental Plaque1.2.2 Bacterial Colonisation Factors and their Interactions
9
910
1.3 The Degradation of Host Components and ExtracellularMatrix Molecules 14
1.3.1 Proteolytic Degradation 151.3.1.1 The Role of Proteolytic Enzymes from P. gingivalis 151.3.1.2 The Activation of Host Proteases by P. gingivalis 17
1.3.2 The Breakdown of Glycosaminoglycans by Exoglycosidases 18
1.4 P-N-Acetyl-Hexosaniinidases and
P-N-Acetyl-Glucosaminidases 221.4.1 Specificity 221.4.2 Possible Roles in Glycoconjugate Degradation 24
1.4.2.1 The Modification of Host Extracellular MatrixGlycosaminoglycans 24
1.4.2.2 The Degradation of Glycoproteins and Glycolipids 26
1.4.2.3 The Hydrolysis of Asparagine-Linked Oligosaccharides on
Human IgG 291.4.2.4 Autolysins that Degrade Peptidoglycan 31
1.4.2.5 P-N-Acetyl-Hexosaminidases in Glycobiology 33
1.5 The Aims of This Th^is 36
Chapter 2 Materials and Methods 37
2.1 Bacterial Strains and Plasmids 372.1.1 Growth Condition and Media 37
2.2 Transformation of Bacterial Cells 392.2.1 Production of Competent Cells 39
2.2.1.1 Calcium Chloride Method 392.2.1.2 Electrotransformation Method 402.2.2 Transformation with Plasmid DNA 402.2.2.1 Calcium Chloride Method 402.2.2.2 Electrotransformation Method 412.2.3 Transformation with Bacteriophage DNA 41
2.3 Procedures for DNA Extraction 42
2.3.1 Extraction of Chromosomal DNA 42
2.3.2 Small Scale Extraction of Plasmid DNA 432.3.3 Large Scale Extraction of Plasmid DNA 432.3.4 Extraction of M13mpl8/19 Template DNA 44
2.3.5 Phenol Extraction and Ethanol Precipitation 45
2.4 Techniques used in Routine DNA Manipulation 45
2.5 DNA Hybridisation Proœdures 462.5.1 Transfer of DNA to Nylon Filters 462.5.2 Preparation of Filters for Colony Hybridisation 47
2.5.3 Production of RadiolabelW Probe 482.5.4 Hybridisation of DNA Immobilised on Filters with the Probe 48
2.6 DNA Sequencing 49
2.7 Polymerase Chain Reaction Proœdures 51
2.8 Radioactive Labelling of Proteins 522.8.1 Minicell Analysis 522.8.2 DNA Directed Transcription-Translation System 53
2.9 Biochemical Assay of P-N-Acetyl-GIucosaminidase
and P-N-Acetyl-Galactmiaminidase Activity 54
2.10 Computer Analysis 55
2.10.1 Database Searching and Multiple Sequence Alignment 55
2.9.2 The Hydropathy Profile 56
Chapter 3 The Cloning and Expression of the nah Regionfrom P. gingivaUs W83 in E. coli 57
3.1 Introduction 57
3.2 Results 58
3.2.1 Detection of P-N-Acetyl-Hexosaminidase Activity inf . gmghufw W83 58
3.2.2 Isolation and Restriction Endonuclease Analysis of the
nah Region from P. gingivalis W83 593.2.2.1 Construction of a P. gingivalis W83 Expression Library
in E. coli 59
3.2.2 2 Screening of the P. gingivalis W83 Gene Library
for Exo-P-N-Acetyl-Glucosaminidase Activity 61
3.2.2.3 Restriction Endonuclease Analysis of the nah Region 633.2.2.4 Southern Blot Analysis of Chromosomal DNA 653.2.3 Localisation and Expression of the Gene on pALl that
Encodes for P-N-Acetyl-Hexosaminidase Activity 67
3.2.3.1 Localisation on pALl 67
3.2.3 2 IPTG-Induced Expression of P-N-Acetyl-Hexosaminidase 69
3.2.3.3 Expression of Proteins in a Cell-Free and Minicell System 70
3.3 Discussion 72
IV
Chapter 4 Molecular Characterisation of the nah Regionby DNA Sequence Analysis 75
4.1 Introduction 75
4.2
4 .2.1
4 .2 .1.1
4 .2 . 1.2
4 .2 .1.3
4 .2 . 1.4
4 .2 . 1.5
4.3
Results 76
DNA Sequence and Computer Analysis of the nah Region from P. gingivalis W83 76
The DNA Sequencing Strategy 76The DNA Sequence of the nahA Gene and the Predicted
Amino-Acid Sequence of the P-N-Acetyl-Hexosaminidase
Protein (NahA) 76Homology Between NahA and Other Enzymes 87The NahA Protein of P. gingivalis is Homologous to the
Central Domain of CrylA(c) 6-endotoxin from Bacillus thuringiensis 97
Analysis of orfl 98
Discussion 106
Chapter 5 Further Studies on the P-N-Acetyl-Hexosaminidase From P. gingivalis W83 114
5.1 Introduction 114
5.2 Results and Discussion 115
5 .2.1 Construction of Plasmid pMUT for the Generation of a
nahA Isogenic Mutant of P. gingivalis 1155.2.2 Strategy for the Generation of a Glutathione
S-transferase-NahA Fusion Protein 120
Chapter 6 Discussion 124
References 129
List of Abbreviations
Amp AmpicillinCc ClindamycinEDTA Ethylenediaminetetraacetic acidEm ErythromycinECM Extracellular matrixFuc FucoseGAG GlycosaminoglycanGal GalactoseGalNAc N-Acetyl-galactosamineGlcA Glucuronic acidGlcNAc N-Acetyl-glucosamineIdoA Iduronic acidIPTG Isopropyl-P-D-galactopyranosideKan KanamycinMan MannoseMES 2-[N-Morpholino]ethanesulfonic acidPEG Polyethylene glycolSA Sialic acidSm StreptomycinTEMED N, N, N ', N'-Tetramethylethylenediamine
Acknowledgements
A special thanks to Dr I. S. Roberts for his continuous supervision,
guidance, help and encouragement throughout all of this work. I would also like to thank past and present members of the Department of Microbiology and
Immunology, University of Leicester for their help and expertise. I thank Dr C.
Pazzani, A. Wallace and G. Rigg for their endless advice, assistance and
friendship. I thank Dr J. Milner, J. Maley, R. Pearce, Dr T. McGenity, R.
Willmouth and A. K. Barton and for their help, friendship and interest. Thanks
also to Dr J. Eastgate, S. Gordon, D. Simpson, Dr M. Camara, A. Smith, R.
Feely, A. Owtrowski, Dr C. R. Drake, N. Taylor, Dr C. Petite, Dr S. O’
Brien, Dr. R. Morse, Dr M. Coleman, B. J. Roberts, Dr J. Hill, A. Sheperd,
Dr T. Mitchell, P. Esumeh and J. R. Canvin for helping me in a number of
situations. For those who gave friendship, advice or inspiration, I extend my
thanks. I thank the Medical Research Council for my stipend and funding my
attendance at conferences. My extra special thanks goes to my parents for
everything they have done and continue to do.
vm
Abstract
Molecular Analysis of a P-N-Acetyl-Hexosaminidase Gene From Porphyronwnas gingivaUs W83
Archie Lovatt
The black-pigmented Gram-negative anaerobe Porphyromonas gingivalis has been implicated in human pericxlontal diseases and expresses a number of exoglycosidase activities that may be capable of degrading the oligosaccharides from host glycoconjugates. The first stage in characterising the role of the p-N- Acetyl-hexosaminidase activity from this microorganism was the cloning of the nahA gene from P. gingivalis W83 which encodes for a protein with this enzyme activity. The nahA gene was cloned in E.coli by constructing a plasmid expression library of 5fl«3A-generated P. gingivalis W83 chromosomal DNA fragments. Expression of p-N-Acetyl-hexosaminidase activity was detectW by cleavage of the fluorogenic substrates 4-melhylumbelliferyl-N-Acetyl-P-D- glucosaminide and 4-methylumbelliferyl-N-Acetyl-P-D-galactosaminide. Southern blot analysis suggested that the nahA gene was present as a single copy in all P. gingivalis strains tested. In contrast, sequences homologous to the nahA gene were not detectable in the closely related species P. endodontalis and P. asaccharolytica. The nahA gene was 2331 base pairs long and encoded a protein of 111 amino acids with a predicted molecular weight of 87 kDa. A characteristic signal peptide for an acylated lipoprotein was present at the amino terminus suggesting that the mature p-N-Acetyl-hexosaminidase may be a lipoprotein attached to the outer membrane of P. gingivalis. Protein homology studies suggested that NahA and p -N-Acetyl-hexosaminidases from eukaryotes and prokaryotes contain homologous active site domains with similar catalytic arginine residues. DNA sequence analysis 5’ to the nahA gene identified another open reading frame, and a potential hairpin structure which may be involved in regulating gene expression. Lastly, a suicide plasmid that may allow the site specific inactivation of the nahA gene on the chromosome of P. gingivalis was constructed. The results presented in this thesis may contribute to future studies including the generation of an isogenic mutant of P. gingivalis lacking p-N-Acetyl-hexosaminidase activity.
CHAPTER 1
Introduction
1.1 Porphyromonas gingivaUs
1.1.1 General Biology
Black-pigmented Gram-negative anaerobes were originally describe as
Bacterium melaninogenicum, producing dark brown-black pigmented colonies
when grown for 6-10 days on blood agar plates (Oliver and Wherry, 1921).
This species was shown to be very heterogenous, comprising of both
saccharolytic and asaccharolytic strains (Sawyer et al, 1962). These strains
were assigned to a single species, Bacteroides melanimgenicus, which
containW the three subspecies, melaninogenicus (saccharolytic), intermedius
(moderately saccharolytic) and asaccharolyticus (asaccharolytic). (Holdeman
and Moore, 1974). The saccharolytic (fermentative) species now belong to the
genus Prevotella and the asaccharolytic (non fermentative) species have been
placed into the genus Porphyromonas, which includes Porphyromonas
endodontalis, Porphyromonas asaccharolytica and Porphyromonas gingivalis
(Finegold et al., 1993; Kulekci et al., 1993; van Steenbergen et al., 1993).
When P. gingivalis is grown under chemostat conditions, the cells utilise
arginine, cysteine, histidine, serine and tryptophan as their carbon and energy
source, whereas sugars inhibit growth rate (Mayrand and Holt, 1988). P.
gingivalis utilises a polyaspartate/glutamate heteropolymer at a slower rate than
aspartate homopolymer (Shah and Gharbia, 1993). When grown on peptides die
yield of cells is greater than that grown on amino acids (Shah and Gharbia,
1993). Moreover, when grown in an equimolar mixture of polyaspartate and
free aspartate, the uptake of free aspartate appears to be suppressed until there
is utilisation of the homopolymer. Such observations imply that peptides are the
favoured substrates for growth of P. gingivalis and amino acids are utilised to
lesser extent (Shah and Gharbia, 1993).
P. gingivalis requires vitamin K and the iron compound, haemin, for growth
(Gibbons and MacDonald, 1960; Mayrand and Holt, 1988). It has been
proposed that haemin and vitamin K function as electron carriers in the electron
transport system of P. gingivalis (Gibbons and MacDonald, 1960; Mayrand and
Holt, 1988). The black pigment produced by P. gingivalis has been described
as a ‘haemoglobin derivative’ and has been shown to be protohaemin with
traces of protoporphyrin (Duerden, 1975; Shah et al., 1979). The concentration
of haemin available for growth appears to have an effect on the physiology and
virulence of P. gingivalis (McKee et al., 1986). For example, P. gingivalis
cells grown with no haemin in their media are avirulent when injected into
mice, whereas those grown in haemin excess cause 100% mortality in mice
(MC Kee et al., 1986). Furthermore, those grown in haemin excess appear to
have more fimbriae than those grown under haemin limitation (M^ Kee et al.,
1986). A 26 IcDa protein is predominant on the cell surface of P. gingivaUs
when grown under haemin limitation, but is not expressed under haemin
excess. This protein, termed Omp26, appears to be exported to the outer
membrane for haemin binding and imported across the outer membrane for
haemin transport (Bramanti and Holt, 1992a & b).
Isolates of P. gingivalis from in vivo infections are often associated with a
mixed bacterial population (Mayrand and Holt, 1988). The presence of vitamin
K-related compounds, such as naphthoquinone, produced by associated bacteria
may enhance the growth of P. gingivalis (MacDonald et al., 1963). Succinate
has been shown to replace vitamin K or haemin as a growth factor for P.
gingivalis (Mayrand and Holt, 1988). When produced by Treponema denticola
via fermentation of amino acids, succinate may act as a growth factor for P.
gingivalis in vivo (Grenier, 1992a). Moreover, succinate may be incorporated
into the lipids and phospholipids of the cell envelope and is suggested to play an
important role in bacterial nutritional interaction during periodontal disease
(Lev et al., 1971; Grenier, 1992a).
1.1.2 Ro!e in Periodontal Disease
Periodontal disease is a collective term for a variety of conditions
characterised by the inflammation and degeneration of the gingivae, connective
tissue, periodontal ligament, cementum and the alveolar bone that supports
teeth. While gingivitis refers to inflammation that is confined to the gingival
tissues, periodontitis is considered to be an advancement of gingivitis into the
bone that surrounds the teeth. The initial symptoms of gingivitis are
enlargement and inflammation of the soft tissue with bleeding of the gums. The
inflammatory reaction results in swelling of the tissue and the formation of
periodontal pockets between the teeth and their supporting tissue (Hirsch and
Clarke, 1989; Tortora and Anagnostakos, 1990). At the advanced stage,
extensive degradation of the host extracellular matrix (ECM) takes place,
together with loss of alveolar bone and tooth support (Uitto, 1991). A
hypothetical representation of chronic adult periodontal disease is shown in
Figure 1.1.
Figure 1.1 A Hypothetical Representation of Chronic Adult Periodontal Disease
Enamel
Ine pathological periodontal pocket
GE
EPITHELIAL CELLS COLLAGEN ANCHOR
CELLULAR MATRIX
GE=GmGIVAL EPITHELIUM Cl^CONNEClTVE TISSUE AB=ALVEOLAR BONE PDL=PEmODONrAL LIGAMENT CE=CEMENTUM
BASEMENT MEMBRANE
Figure 1.1 Hypothetical representation of chronic adult periodontitis. The structure of the periodontium is shown. The small boxed region of the periodontal pocket is shown in more detail within the large box. Dental plaque bacteria colonise the periodontal pocket and their toxins/enzymes/extracellular vesicles may gain access from the pocket to the subepithelium. All host cells are surrounded by a complex extracellular matrix. During disease, bacteria may directly degrade host extracellular matrix and immune components and indirectly trigger host-mediated tissue degradative pathways (Constructed from information present in Saglie et al., 1988; Holt and Bramanti, 1991; Uitto, 1991; Lamont et al., 1992; Birkedal-Hansen, 1993; Couchman and Woods, 1993; Duncan et al., 1993; Sandros et al., 1993).
The pathogenesis of periodontal diseases is thought to be a complex series of
events that involves both host factors and dental plaque. Although periodontal
diseases are multifactorial in nature, studies indicate that the inflammation and
destruction of tissues is initiated and maintained by the bacteria of dental plaque
(Lindhe et al., 1975; Christersson et al., 1991; Corbet and Davies, 1993;
Offenbacher et al., 1993; Ready and Jeffcoat, 1993). Dental plaque is a highly
organised mixture of bacterial species and the co-aggregation or cell-to-cell
recognition of genetically distinct partner-types is essential for its formation
(Kolenbrander, 1988; Kolenbrander and London, 1993). The estimated
composition of dental plaque is over 300 bacterial species, but only certain
types of Gram-negative anaerobes are associated with the various forms of
periodontal diseases (Christersson et al., 1991; Holt and Bramanti, 1991). For
example, Actinobacillus actinomycetemcomitans is associated with localised
juvenile periodontitis, Prevotella intermedius ! Treponema denticola with acute
necrotizing ulcerative gingivitis and Capnocytophaga spp. with juvenile
diabetes advanced periodontitis (Holt and Bramanti, 1991). Porphyromonas
gingivalis has received considerable attention in past years and is thought to
play an important role in the formation of lesions during chronic adult
periodontal disease (Slots and Genco, 1984; Holt and Bramanti, 1991).
P. gingivalis is found in adult periodontitis lesions at high frequency and
increased numbers. However, in plaque from healthy patients P. gingivalis is
either not detected or shows significantly lower frequency and numbers
(Christersson et al., 1991; Dahlen, 1993). Therefore, it appears that an
ecological niche of P. gingivalis is the diseased periodontal pocket that occurs
between the tooth and its supporting tissue. Patients suffering from periodontitis
exhibit high levels of antibodies that are specific for P. gingivalis (Ebersole et
al., 1985; De Nardin et al., 1991; Lopatin and Blackburn, 1992; Kinane et al.,
1993), and eradication of this organism from the subgingival microflora
correlates with resolution of the disease (Loesche et al., 1981; van Dyke et al.,
1988). Several reports demonstrate that P. gingivalis can invade gingival and
oral epithelial cells in vitro (Lamont et al., 1992; Duncan et al., 1993; Sandros
et al., 1993). Such invasion and the detection of P. gingivalis within gingival
tissue (Saglie et al., 1988), may suggest that this organism penetrates the
mucosal epithelial barrier. Although the invasive mechanisms are not fully
understood, it is clear that P. gingivalis expresses an arsenal of potential
virulence factors (Holt and Bramanti, 1991).
Virulence factors that may aid colonisation, tissue destruction and
impairment of host defence mechanisms have been proposed for P.gingivalis.
These include extracellular hydrolytic enzymes such as trypsin-like protease,
collagenase, neuraminidase, p-N-Acetyl-hexosaminidase and glycosulphatase,
bacterial surface components such as lipopolysaccharide (LPS), polysaccharide
capsule, fimbriae and haemagglutinins (Slots and Genco, 1984; Mmhas and
Greenman, 1989; Holt and Bramanti, 1991; Socransky and Haffajee, 1991;
Meghji et al., 1993; Slomiany et al., 1993; Sundqvist, 1993; van WinkeUioff et
al., 1993). hi addition, P. gingivalis releases outer-membrane vesicles (OMV)
into the extracellular environment when grown in vitro and may also secrete
these factors in vivo (Grenier and Mayrand, 1987; Mayrand and Holt, 1988).
The OMV structures possess haemagglutinating, haemolytic, proteolytic and
exoglycosidase activity, mediate bacterial co-aggregation and may activate
alveolar bone resorption (Grenier and Mayrand, 1987; Smalley and Birss,
1987; Minhas and Greenman, 1989; Bourgeau and Mayrand, 1990; Kay et al.,
1990a; Mihara et al., 1993). The small size of OMV may allow them to cross
epithelial barriers that are impermeable to bacterial cells, hi this way, they
could serve as vehicles for toxins and enzymes that extend the ability of the
bacterial cell to obtain nutrients (Grenier and Mayrand, 1987; Mayrand and
Holt, 1988). Moreover, since OMV are highly proteolytic, they have been
7
implicated in the degradation and penetration of epithelial tissue by P.
gingivalis (Grenier and Mayrand, 1987). These potential virulence factors may
also compete for antibodies and inhibit specific antibacterial immune defence
mechanisms (Mayrand and Holt, 1988).
Many studies have assessed the pathogenicity of P. gingivalis using
experimental animal model systems (Holt and Bramanti, 1991; Sundqvist,
1993). The cynomolgus monkey model, Macaca fascicularis has a similar
periodontal morphology to that of humans, with gingivitis developing
spontaneously in the presence of calculus and plaque (Komman et al., 1981;
Holt et al., 1988; Birek et al., 1989; Nemeth et al., 1993). The implantation of
P. gingivalis into the periodontal microbiota of this monlcey results in high
levels of antibodies to this microorganism plus rapid and significant alveolar
bone loss (Holt et al., 1988). Immunisation of the cynomolgus monlcey with
Idlled P. gingivalis protects against such bone loss (Dahlen, 1993).
Periodontal destruction can also be induced in gnotobiotic rats by mono
infection with P. gingivalis. Periodontal destruction is estimated by measuring
horizontal and vertical bone changes in the animal’s periodontium (Klausen et
al., 1991). Immunisation of the gnotobiotic rat with Idlled P. gingivalis cells or
highly purified fimbriae before gingival challenge with this microorganism
results in a reduction of periodontal bone loss (Klausen et al., 1991; Evans et
al., 1992). Recently, infection with a nonfimbriated mutant of P. gingivalis 381
showed that this strain was unable to induce the extent of periodontal bone loss
that was observed with the wild type strain (Malek et al., 1994)
Subcutaneous injection of mice with invasive strains of P. gingivalis
produces spreading lesions that frequently result in death (Mayrand and Holt,
1988). Invasive strains of P. gingivalis spread to distant sites and produce
abdominal abscesses, whereas non-invasive strains produce localised lesions at
the challenged site (Mayrand and Holt, 1988; Genco et al., 1991; Naito et al.,
1993). Although the mouse system does not mimic the human pathological
periodontal pocket, this model may be useful when studying the pathogenic
mechanisms of P. gingivalis. For example, Genco et al., 1991 have described
the development of the mouse subcutaneous chamber model. Bacteria within
the chamber can be studied throughout the course of infection and the chamber
contents can be used to examine specific host factors that are produced in
response to P. gingivalis.
hi summary, P. gingivalis is implicated in human adult periodontitis and
studies indicate that alveolar bone loss or experimental periodontitis in animal
models can be induced by P. gingivalis. P. gingivalis is suggested to be an
opportunistic periodontopathogen that survives and multiplies within the
periodontal pocket, resisting host defence mechanisms and damaging host tissue
(Slots and Genco, 1984; Holt and Bramanti, 1991; Loos et al., 1992). The
pathogenicity of P. gingivalis is more than likely multifactorial, requiring
several virulence factors that may play an important role in the pathogenesis of
adult periodontal disease. As a result, the determination and analyses of factors
that may influence bacterial colonisation, the impairment of host defences and
the destruction of host tissue is essential to understanding the pathogenesis of
human periodontal disease.
1.2 Bacterial Adhesion and Colonisation
1.2.1 Dental Plaque
Dental plaque development on tooth surfaces begins with the precipitation of
a salivary pellicle (Christersson et al., 1991). The salivary pellicle is a thin coat
that covers the freshly cleaned tooth surface and consists of glycoproteins,
mucim and salivary enzymes (Mayhall, 1970; Kolebrander and London, 1993).
Bacterial colonisation of the salivary pellicle takes place rapidly and the first
microorganisms that attach to the tooth surface are mainly Streptococcus
species and Gram-positive rods. It is thought that facultative bacteria proliferate
first and create an environment suitable for the growth of anaerobes (Hirsch
and Clarke, 1989). As plaque matures, its composition becomes more complex
and the early colonising population diversifies to include Actinomyces,
Capnocytophaga, Haemophilus, Prevotella and Fusobacterium species
(Kolenbrander and London, 1993). Late colonisers which include A.
actinomycetemcomitans, P. gingivalis and T. denticola are linked to early
colonisers, such as A. israelii, C. gingivalis, H. parainfluenzae, Pr. loeochei
and S. oralis. The linkage that bridges the attachment of late colonisers to early
colonisers is thought to be Fusobacterium nucleatum (Kolenbrander and
London, 1993).
P. gingivalis attaches to a variety of bacteria from dental plaque, including
A. viscosus, A. naeslundii, A. israelii, S. sanguis, S. mitis, T. deiuicola and F.
nucleatum (Kolenbrander, 1990; Grenier, 1992b; Kolenbrander and London,
1993). The current idea is that P. gingivalis and T. denticola adhere to F.
nucleatum and to each other within bacterial plaque (Grenier, 1992b;
Kolenbrander and London, 1993). Moreover, the occurence of T. denticola in
diseased periodontal sites requires a detectable level of P. gingivalis (Simonson
10
et al., 1992), and the co-aggregation and nutritional interaction between these
two microorganisms is thought to be important for the initiation and
progression of certain forms of periodontal disease (Grenier, 1992a & b; Nilius
et al, 1993). P. gingivalis may also provide growth factors for F. nucleatum,
which could explain why these two organisms frequently coexist in
periodontally diseased sites (Rogers et al., 1992).
1.2.2 Bacterial Colonisation Factors and their Interactions
P. gingivalis adheres to a variety of host components, including fibronectin-
collagen complexes (Naito and Gibbons, 1988), lactoferrin (Kalfas et al.,
1991), fibrinogen (Lantz et al., 1991), epithelial cells (Isogai et al., 1988) and
erythrocytes (Hoover et al., 1992b). Surface structures of P. gingivalis
proposed to be involved in adherence in vivo are fimbriae (Yosmimura et al.,
1984; Sharma et al., 1993), proteases (Grenier, 1992c; Hoover et al., 1992b;
Stinson et al., 1993) and haemagglutinins (Desluariers and Moutoun, 1992;
Dusek et al., 1993). Haemagglutinins of P. gingivalis have been reported, to
range in size (Chandad and Mouton, 1990; Dusek et al., 1993), may have
proteolytic activity (Grenier, 1992c; Hoover et al., 1992b), and form
complexes with proteases (Pilce et al., 1994). The protease-haemagglutinin
complexes of P. gingivalis may be involved in the adhesion and subsequent
hemolysis of host erythrocytes, thereby facilitating the acquisition of haemin in
vivo (Dusek et al., 1993; Pike et al., 1994). Besides agglutinating erythrocytes,
haemagglutinins may be involved in the aggregation of Actinomyces spp. that is
mediated by the OMV of P. gingivalis (Bourgeau and Mayrand, 1990).
Early studies indicated that fimbriae were associated with the
haemagglutination activity, but they are now believed not to be involved in this
process (Yoshimura et al., 1984 & 1985; Watanabe et al., 1992; Hamada et
11
al., 1994; Malek et al., 1994). Fimbriae may play an important role as
adtiesins in vivo and bave been shown to be a major target for antibody
responses in patients with advanced periodontal disease (Yoshimura et al.,
1987). Fimbriae from P. gingivalis have been purified (Sojar et al., 1991), the
fimA gene has been characterised (Dickinson et al., 1988), and the structural
subunit fimbrülin has an apparent molecular weight of 43 IcDa (Washington et
al., 1993). The fimbriae of P. gingivalis are thought to be involved in the
adhesion to salivary pellicle, epithelial cells, collagen, periodontal ligament and
gingival fibroblasts (Watanabe et al., 1992; Naito et al., 1993). Naito et al.,
1993 suggest that the fimbriae of non-invasive strains (ATCC 33277, 381 and
Su63) have a higher relative hydrophobicity and stronger collagen binding
capacity than than the fimbriae firom invasive strains (ATCC 53977, ATCC
49417, 16-1 and W83). Further, a comparison of non-invasive and invasive
strains suggest that non-invasive strains have relatively more cell surface
hydrophobicity than invasive strains (Watanabe et al., 1992). These
observations have led to the hypothesis that non-invasive strains have fimbriae
which strongly attach to collagen in lesions, but because of their high cell
surface hydrophobicity can be readily phagocytosed. However, invasive strains
may bind weakly to collagen, and because of their high cell surface
hydrophilicity remain in lesions by avoiding phagocytosis (Naito et al., 1993).
Recently, Hamada et al., 1994 and Malek et al., 1994 have reported no change
in the relative cell surface hydrophobicity of fimA mutants of P. gingivalis
33277 and 381.
A number of investigators have described the construction and
characterisation of fimA mutants of P. gingivalis (Hamada et al., 1994; Malek
et al., 1994). Hamada et al., 1994 showed that inactivation of the fimA gene m
P. gingivalis 33277 causes no alteration in haemagglutinating activity, however
decreases the adherence of this strain to human gingival fibroblasts. The
12
adhesion of wM-type P. gingivalis to gingival fibroblasts causes changes in the
normal architechure of the fibroblast, with the appearance of long microvilli
surrounding large bacterial clumps. No such changes are observed with the
fimA mutant, which could imply that fimbriae trigger a sequence of events in
the fibroblast that facilitate bacterial contact (Hamada et al., 1994). hi a similar
report, Malek et al., 1994 have shown that Q.fimA mutant of P. gingivalis 381
has no change in haemagglutination, however is less able to bind saliva-coated
hydroxy apatite. Moreover, in the gnotobiotic rat model, this fimA mutant was
unable to induce the extent of periodontal bone loss that was observed with
wild-type strain (Malek et al., 1994). The failure of ihofimA mutant to cause
significant periodontal damage in gnotobiotic rats may be due to the inability of
the mutant to adhere to saliva-coated oral surfaces in the animal (Malek et al.,
1994). Alternatively, fimbriae may play a role in other reactions that are
important in periodontal disease. For example, they have been shown to
stimulate the release of interleuldn-1 (IL-1) from mouse monocytes (Hanazawa
et al., 1991). IL-1 stimulates osteoclastic bone resorption (Roodman, 1991),
and antibodies directed towards the fimbriae may inhibit their ability to
stimulate IL-1 production (Evans et al., 1992). This could explain why
immunisation of the gnotobiotic rat with highly purified fimbriae elicits an
immune response that interferes with bone loss induced by P. gingivalis (Evans
et al., 1992) (Section 1.1.2).
Many cell-to-cell adhesive interactions and bacterial co-aggregations can be
inhibited by the addition of simple sugars, suggesting that many adhesins are
lectin-like proteins. (Kolenbrander, 1988; Kolenbrander, 1989; Holt and
Bramanti, 1991; Kolebrander and London, 1993). The F. nucleatum-P.
gingivalis co-aggregation has been characterised and represents a typical
carbohydrate-lectin interaction. This interaction is inhibited by lactose and
appears to be mediated by a carbohydrate receptor on P. gingivalis that
13
interacts with a 42 IcDa outer-membrane protein on F. nucleatum (Kinder and
Holt, 1993). The specific co-aggregation between F. gingivalis and T. denticola
is inhibited by D-galactosamine and arginine and is thought to be bimodal, that
is, both microorganisms contain specific adhesins that recognise complementary
receptors on the other partner cell (Grenier, 1992b; Kolenbrander and London,
1993). Further, the co-aggregation between A. israelii and C. gingivalis is
inhibited by SA, GalNAc and GlcNAc (Kagermeier et al., 1984). A GlcNAc
residue is thought to be a receptor for the attachment of T. denticola to
epithelial cells, fibroblasts and erythrocytes (Weinberg and Holt, 1988;
Grenier, 1991; Milcx and Keulers, 1992; Keulers et al., 1993). Interactions
between a GalNAc residue and a bacterial cell surface lectin appear to be
involved in the adhesion of Prevotella loeschei to both prokaryotic and
eukaryotic cells (London and Allen, 1989), and the co-aggregation between
Streptococcus sanguis 34 and Actinomyces viscosus T14V appears to be
dependent on a lectm-GalNAc association (Mclntre, 1985). The aggregation of
the plaque bacterium Eikenella corrodens with salivary glycoprotein is thought
to involve a bacterial cell surface adhesin that interacts with a complementary
GalNAc sugar receptor (Ebisu et al., 1992). Moreover, salivary glycoprotein is
thought to play an important role in the accumulation of dental plaque but its
aggregation with P. gingivalis does not involve GalNAc (Ebisu et al., 1992).
Several studies suggest that the binding of P. gingivalis to certain oral
bacteria and host components involves non-lectin type adhesin(s) (Olcuda et al.,
1986; Nagata et al., 1990; Bourgeau and Mayrand, 1990; Kalfas et al., 1991).
Unlike bacterial lectins, these adhesin(s) are not inhibited by sugars but are
inhibited by L-lysine or L-arginine (Olcuda et al., 1986; Bourgeau and
Mayrand, 1990; Nagata et al., 1990). Studies indicate that L-arginine can
inhibit the aggregation of Actinomyces spp. that is mediated by the OMV of P.
gingivalis, and the specific co-aggregation between S. mitis and P. gingivalis
14
(Nagata et al., 1990; Bourgeau and Mayrand, 1990). L-arginine can also
inhibit the trypsin-like protease and haemagglutination activity from P.
gingivalis (NisMkata et al., 1989).
1.3 The Degradation of Host Immune Components and Extracellular Matrix Molecules
The extracellular matrix (ECM) is made up of collagens and glycoconjugates
such as proteoglycans, glycosaminoglycans and glycoproteins. Collagens are
thought to be the main constituents of the connective tissue matrix, whüe
proteoglycans and the glycosaminoglycan hyaluronic acid are supposedly
present in the intercellular material of the epithelium. The subepithelial
basement membranes are specialised extracellular matrices and contain
collagen, chondroitin sulphate and glycoproteins such as lanainin and
fibronectin. (Uitto, 1991; Couchman and Woods, 1993). In periodontal
diseases, several factors may interfere with ECM interaction. Bacterial
enzymes and toxins may directly act on epithelial cells, resulting in degradation
of host cell surface and adhesion molecules. The host cells may react with
increased proliferation, production of inflammatory mediators and extracellular
hydrolytic enzymes, resulting in local degradation of the subepithelial basement
membrane (Uitto, 1991).
15
1.3.1 Proteolytic Degradation
1.3.1.1 Ilu;31ole(%fIhroteolyde Ibazymes jBnanijP.apmypwYRKc
Proteolytic enzymes are produced by a number of microbial pathogens and
have been implicated as pathogenicity determinants (Stephen and Peitrowsld,
1986; Mirelman, 1988; Hase and Finlcelstein, 1993). It has been suggested that
the secretion of proteases by P. gingivalis may have important roles in the
degradation of host immune system and ECM components, and the generation
of oligopeptides or amino-acids for bacterial growth (Schenken, 1986; Shah and
Gharbia, 1989; Holt and Bramanti, 1991; Madden et al., 1992; Kato et al.,
1992). A secreted protease has been shown to lyse erythrocytes (Shah and
Gharbia, 1989), and P. gingivalis ean degrade IgAl, IgA2, IgG and the C3
component of complement (Kdian, 1981; Schenlcen, 1986). The inhibition of
the proteolytic degradation of IgG and C3 by P. gingivalis enhances the
phagocytosis of P. gingivalis suggesting that protease(s) contribute to
phagocytosis resistance (Cutler et al., 1993). Scott et al., 1993 have purified a
70 IcDa membrane bound thiol-protease from P. gingivalis that is able to render
fibrinogen non-clottable and suggest this enzyme is the one of the most potent
fibrases described to date. It is proposed that the fibrinolytic activity of P.
gingivalis may serve by degrading the fibrinous matrix within periodontal
lesions, allowing the baeteria to enter underlying connective tissue (Holt and
Bramanti, 1991; Lantz et al., 1991).
Trypsin-lilce activity is suggested to cause morphological changes in gingival
fibroblasts and polymorphonuclear leukocytes (PMN) (Morioka et al., 1993;
Sundqvist, 1993). It is also thought that trypsin-lilce activity of P. gingivalis
decreases the expression of complement (CRl) and IgG (FcyRII and FcyRIII)
reeeptors on PMNs. The decrease of CRl may impair the attachment of C3b-
16
opsonised bacteria to PMNs. The decreased IgG receptor expression on PMNs
may result in reduced antibody-dependent cell cytotoxicity and phagocytosis of
IgG-coated bacteria (Tai et al., 1993). Sinee the PMN is thought to be
important in the maintenance of health in periodontal tissues, the impairment of
this immune eell by the trypsin-like protease activity from P. gingivalis may
allow the proliferation of this microorganism and other plaque bacteria within
periodontal tissue (Lambster and Novak, 1992).
The trypsin-lilce protease activity of P. gingivalis appears to be related to
virulence. Studies show that trypsin-like activity of the invasive strain W50 is
more than 3-fold higher than that of the avirulent mutant W50/BE1. Further,
the virulence of P. gingivalis W50 in the mouse lesion model is reduced under
haemin limitation, which is associated with a 3-fold reduction in trypsin-lilce
activity (Sundqvist, 1993). Recently, a gene from P. gingivalis that encodes
trypsin-lilce protease activity iprtT) has been isolated, characterised and the
deduced amino-aeid sequence eorrresponds to a 54 IcDa protein (Otogoto and
Kuramitsu, 1993). Two proteins (Mj. 120,000 and 150,000) that degrade
fibrinogen have been isolated from P. gingivalis and these enzymes appear to
have trypsin-lilce specificity (Lantz et al., 1991).
Collagenolytic activity is a characteristic feature of virulent P. gingivalis
strains and has been implicated in the degradation of collagen within underlying
connective tissue (Holt and Bramanti, 1991). A gene encoding collagenase
activity has been isolated from P. gingivalis and encodes a polypeptide with a
predicted molecular weight of 35 IcDa. The active enzyme, PrtC, appears to
behave as a dimer following gel filtration chromatography. The purified protein
degrades fibrillar type I collagen but not the synthetic collagenase substrate 4-
phenylazobenzyloxycarbonyl-Pro-Leu-D-Arg (Kato et al., 1992). Sojar et al.,
1993 have purified a collagenase that migrates as a 50 IcDa major band and a 60
17
kDa minor band on SDS-PAGE. This enzyme hydrolysed type I collagen from
rat skin, rat plasma kininogen and transferrin. The authors also suggest that this
collagenase has a specificity for the Pro-X-Gly sequence found in several
proteins, including collagen (Sojar et al., 1993). Recently Bedi and Williams,
1994 demonstrated that a 55 kDa trypsin-like protease from P. gingivalis is
capable of degrading type I, III and IV collagen. This enzyme was also
reported to hydrolyse the C3 component of complement, fibrinogen,
fibronectin, al-antitrypsin, a2-macroglobulin, apotransferrin and human serum
albumin. These activities may suggest that this protein is involved in the
degradation of serum proteins, subepithelial basement membrane and
underlying connective tissue, which may be a potent virulence mechanism of P.
gingivalis (Bedi and Williams, 1994).
1.3.1.2 The Acüvaüom of Host Proteases by P. giMgiWis
Host matrix metalloprotemases (MMP) are a family of nine or more highly
homologous endopeptidases that cleave many components from ECM, and play
a role in the metabolic degradation of the ECM in health and disease (Birkedal-
Hansen et al., 1993). MMPs are thought to be stored in the ECM and may be
released during periodontal tissue damage (Uitto, 1991; Birkedal-Hansen et al.,
1993). The expression of MMPs can be stimulated in a variety of cultured cells
derived from human periodontal tissues, including PMNs, fibroblasts,
macrophages and kératinocytes (Birkedal-Hansen et al., 1993). MMP activity
may be stimulated either directly by microbial products or indirectly by
inflammatory mediators or cytokines generated in response to oral
microorganisms (Birkedal-Hansen, 1993).
18
The fimbriae of P. gingivalis can induce the expression of IL-1 and TNFa in
mouse macrophages (Hanazawa et al., 1991; Murakami et al., 1993). Also, the
LFS of P. gingivalis can induce IL-1 production in human gingival fibroblasts
(Sisney-Durrant and Hopps, 1991). In response to IL-1 and TNFa, the host’s
fibroblasts, kératinocytes, macrophages, PMNs and endothelial cells may in
turn upregulate the synthesis and release of MMPs that degrade host tissue
(Birkedal-Hansen, 1993). Proteases of P. gingivalis may be capable of
converting mucosal kératinocytes and fibroblasts to destructive cellular
phenotypes by inducing expression of, or mediating activation of MMPs
(Birkedal-Hansen et al., 1984; Uitto et al., 1989; Birkedal-Hansen et al, 1993).
Therefore, the proteases, fimbriae and LPS of P. gingivalis may contribute to
periodontal tissue damage by activating host MMPs.
1.3.2 The Breakdown of Glycosaminoglycans by Exoglycosidases
Recent advances in glycoconjugate research have highlighted the significanee
of oligosaccharide structures in mammalian cellular interactions, protein
conformation, host-parasite recognition and antibody function (Rademacher et
al., 1988; Karlson, 1989; Feizi, 1991 & 1993; Hakomori, 1993; Oppdenaldcer
et al., 1993). The oligosaccharides linlced to glycolipids and glycoproteins are
generally branched struetures, where two to four monosaccharides can be
attached to a single monosaccharide within the chain (International Union of
Biochemistry (lUB) recommendations, 1976; Kornfeld and Komfeld, 1980).
This property distinguishes them from other glycoconjugates such as
peptidoglycans, proteoglycans and glycosaminoglycans (GAG), where
polypeptides are linked to linear carbohydrate chains composed of variable
numbers of specific repeating disaccharide units (lUB recommendations, 1980).
19
Glycosaminoglycans (GAG) are present in mammalian connective tissue
(Roden, 1980; Rahemtulla, 1992), cell surfaces and the extracellular matrix
(Couchman and Woods, 1993; Yanagishita, 1993). GAGs are integral parts of
the proteoglycans (Alberts et a l , 1980; Roden, 1980; Couchman and Woods,
1993) and interact with a wide range of biolological molecules, including
fibronectins (Ruoslahti, 1988; Hynes, 1990), protease inhibitors (Bourin and
Lindahl, 1993; Yanagishita, 1993), blood coagulation factors (Bourin and
Lindahl, 1993), cytokines and growth factors (Wright et a l , 1992; Tanaka et
al, 1993). It is suggested that the GAGs are widely distribute in periodontal
tissue (Table 1.1) (Rahemtulla, 1992).
Table 1.1 Glycosaminoglycans within Periodontal Tissue
PERIODONTAL TISSUE
GLYCœiAMINOGLYCAN(GAG)
GingivalFibroblasts
Gin^valEpithelium
PeriodontalLigament
AlveolarBone
Hyaluronic acid 4- 4- 4- 4-
Chondroitin sulphate 4- 4- 4- 4-
Dermatan sulphate 4- 4- ■+- -
Keratan sulphate - - - 4-
Table 1.1 The distribution of glycosaminoglycans in periodontal tissue.
GAGs are thought to be involved in the overall integrity of gingival tissue,
where as protein-linked aggregates they interact with collagen and other cell
adhesive molecules to form a sieve-like, three-dimensional network (Bartold et
al. 1982; Uitto, 1991; Couchman and Woods, 1993). Hyaluronic acid may
represent between 30 and 40% of the total GAG in gingival tissue, with the
remainder being dermatan and chondroitin sulphate (Embery et a l , 1979). The
composition of GAGs in periodontal tissue is thought to alter during
20
periodontitis and may fluctuate with disease severity (Kirldiam et al., 1992;
Rahemtulla, 1992; Shibutani et al., 1993). It has been reported that the ratio of
glucuronic acid to hexosamine is the same in normal and diseased human
gingivae, with the polysaccharide components of GAGs being significantly
reduced in the diseased tissue (Rahemtulla, 1992). It has been suggested that
direct enzyme attack on GAGs by bacterial plaque, or indirect attack by
lysosomal enzymes as part of an inflammatory response may lead to an
imbalance in the structure of periodontal tissue (Tipler and Embery, 1985). It is
possible that such disruption of GAGs is due to brealcdown of GAGs by
exoglycosidases, which in turn could have pronounced effects on the functional
integrity of periodontal tissue. Interestingly, the exoglycosidase P-N-Acetyl-
hexosaminidase participates in the step-wise degradation of the
glycosaminoglycans hyaluronic acid (Bach and Geiger, 1978; Roden, 1980;
Kresse and Glossl, 1987), keratan sulphate (Yutaka et al., 1982; Kresse and
Glossl, 1987), chondroitin sulphate and dermatan sulphate (Kresse and Glossl,
1987) (Figure 1.2).
21
Figure 1.2 Structure and Sequential Hydrolysis of Glycosaminoglycan Glycosidic Linkages by Exoglycosidases
coo
O alN A cY o, OalNAcOlcA
(%B AA B
(i) Structure and sequential degradation of hyaluronic acid.A= P-glucuronidase B= P-N-Acetyl-hexosaminidase
ooo
GalNAcGalNAc^GlcA
DB
O-SOj pooQ y—
GalNAc
OhOH
a tD
O t-O -S O ; O t-O -a O ) ÇH20H qt-o-8 0 .
00CHj
B D
(ii) Structure and sequential degradation of dermatan sulphate.A= p-glucuronidase B and F = N-Acetyl-galactosamine-4- sulphate sulphataseC and H=P-N-Acetyl-hexosaminidase D=iduronate-2-sulphate sulphatase E =a-L-iduronidase
(iii) Structure and sequential degradation of chondroitin-4/6-sulphate. A and D= P-glucuronidase B=N-Acetyl-galactosamine-6-sulphate sulphataseC and F = P-N-Acetyl-hexosaminidase E=N-Acetyl-galactosamine-4-sulphate sulphatase
(iv) Structure and sequential degradation of keratan sulphate.A= N-Acetyl-galactosamine-6-sulphate sulphataseB and E=P-galactosidase C and F = N-Acetyl-glucosamine-6- sulphate sulphataseD and G= P-N-Acetyl-hexosaminidase
Figure 1.2 The sequential degradation of glycosaminoglycans by lysosomal exoglycosidases. The exoglycosidases recognise monosaccharides at the outer non-reducing end of the oligosaccharide and release them in turn (Modified from Kresse and Glossl, 1987)
22
1.4 p-N-Acetyl-Hexosaminidases and P-N-Acetyl-Glncosaminidases
These enzyme activities have been reported in organisms from microbes to
higher animals (Gibson and Fullmer, 1969; Neufield, 1989; Esaiassen et al.,
1991; Conoi et at., 1992; Cesari gf oZ., 1992). P-N-Acetyl-hexosaminidases can
hydrolyse terminal non-reducing p-linked NAcGlc and NAcGal, while exo-P-
N-Acetyl-glucosaminidases are described as enzymes that hydrolyse terminal
non-reducing NAcGlc (Cabezas, 1989). Since P-N-Acetyl-hexosaminidases (EC
3.2.1.52) can also be termed as exo-P-N-Acetyl-glucosaminidases (EC
3.2.1.30) under enzyme nomenclature, both names have been used for the same
exoglycosidase activity (Cabezas, 1989). Exo-P-N-Acetyl-glucosaminidases are
also occasionally termed chitobiases (formerly EC 3.2.1.29 now EC 3.2.1.30)
due to their ability to release terminal non-reducing GlcNAc residues from the
homopolymer, chitin (lUB recomendations, 1978; Cabib, 1987; Flach et at.,
1992).
1.4.1 Specificity
Exoglycosidases usually show a very high degree of specificity for a
particular monosaccharide, which is controlled by at least two factors. Firstly,
the glycon specificity is directed towards the terminal sugar moiety, its
anomeric configuration (a/p) and its sterioisomericity (D/L) (Kobata, 1979;
Dwek et al, 1993). Secondly, the aglycon (the rest of the oligosaccharide)
influences the hydrolysis of a monosaccharide and in certain cases affects the
specificity of the enzyme (Kobata, 1979). For example, the P-N-Acetyl-
hexosaminidase isolated from Streptococcus pneumoniae culture filtrate
(Glasgow et al., 1977), can cleave terminal GlcNAc/GalNAcp 1 -2Man linkages
23
from N-linlced oligosaccharides only if the mannose is not substituted at C-6
(Furulcawa and Kobata, 1993) (Figure 1.3). Its reported activity against O-
linked oligosaccharides suggests that terminal GlcNAcpl-3Gal and GlcNAcpi-
6Gal can be hydrolysed (Yamashita et al., 1981). The P-N-Acetyl-
hexosaminidase from Jack Bean has a broad specificity (Li and Li, 1970), and
can cleave terminal GlcNAc/GalNAcP 1-2, 3, 4 or 6Man linkages (Welply,
1989; Dwek et a l , 1993). One study suggests that Jack Bean P-N-Acetyl-
hexosaminidase may not be able to hydrolyse the GlcNAcP l-4GlcNAc
oligosaccharides derived from chitin (Iwamoto et al., 1993).
The p-N-Acetyl-hexosaminidases from human lysosomes are extensively
characterised since defects result in lysosomal storage of their substrates and
neurodegenerative disease (Cantz and Kresse, 1974; von Figura and Hasilk,
1986; Neufield, 1989). The p-N-Acetyl-hexosaminidases from human
lysosomes are formed by dimérisation of two different polypeptide chains, a
and p. In normal human tissues, at least two different isoenzyme forms of P-N-
Acetyl-hexosaminidases occur, namely A (aP) and B (PP). The A form has a
broad substrate specificity and can remove terminal non-reducing p-linlced
NAcGal or NAcGlc from glycoconjugates that occur in human cells. The B
isoenzyme has a similar specificity with the key exception that it does not
hydrolyse the glycolipid GM% ganglioside or the synthetic substrate MU-
G1cNAc-6-S04 (Price and Dance, 1972; Kytzia and Sandhoff, 1985; Neufield,
1989).
P -N-Acetyl-hexosaminidases and exo-P -N-Acetyl-glucosannnidases can
hydrolyse terminal NAcGlc monosaccharides, while endo-P-N-Acetyl-
glucosaminidases usually cleave internal NAcGlc linkages within
oligosaccharides (Figure 1.3) (Kobata, 1979; Cabezas, 1989; Furulcawa and
Kobata, 1993). The exoglycosidases usually differ from the endoglycosidases in
terms of substrate specificity (Kobata, 1979; Valisena et al., 1982; Morinaga et
al., 1983; Greber et al, 1989; Sugai et al., 1989; Furulcawa and Kobata, 1993),
and amino-acid sequence similarity (Henrissat and Bairoch, 1993). Many endo-
P-N-Acetyl-glucosaminidases that have been described do not contain exo-P-N-
Acetyl-glucosaminidase activity (Valisena et al., 1982; Morinaga et al., 1983;
Greber et al, 1989; Sugai et al., 1989; Furulcawa and Kobata, 1993), however,
a Neisseria gonorrhoeae enzyme that contains both exo- and endo-P -N-Acetyl-
glucosaminidase activity has been reported (Gubish et al., 1982).
1.4.2 Possible Roles in Glycocopjngate Degradation
1.4.2 .1 The ModiHcation of Host Extracellular MatrixGlycosaminoglycans
The invasive parasites Trichinella spiralis and Entamoeba histolytica contain
high levels of P-N-Acetyl-hexosaminidase (Lundblad et al., 1981; Rhoads,
1985). These microorganisms penetrate the intestinal mucosa, invade and
damage other host tissue (Ravidin, 1986; Prescot et al., 1990). Mice infected
with T. spiralis are reported to contain 40-50% less muscle-parasite when
immunised with the P-N-Acetyl-hexosaminidase from T. spiralis, suggesting
this enzyme is important for pathogenicity (Rhoads, 1988). The p-N-Acetyl-
hexosaminidase and several other hydrolases from E. histolytica are suggested
to be involved in the pathogenesis of invasive amoebiasis (Ravidin, 1986;
Mirelman, 1988). Lundblad et al., 1981 purified the secreted P-N-Acetyl-
hexosaminidase from E. histolytica strains and postulated that this enzyme has a
role in erythrocyte brealcdown or intestinal epithelial damage. In degradation
experiments. Worries et al., 1983 showed that the p-N-Acetyl-hexosaminidase
25
from E. histolytica could degrade oligosaccharides from hyaluronic acid, which
may suggest that this enzyme participates in the degradation of host submucosa
ECM.
P. gingivalis is reported to degrade the GAG chondroitin sulphate (Holt and
Bramanti, 1991), and express an extracellular N-Acetyl-galactosamine-4-
sulphatase capable of releasing sulphate from gingival GAG (Slomiany et al.,
1993). P. gingivalis has several exoglycosidase activities that have the potential
to hydrolyse a number of host oligosaccharides. These enzyme activities
include P-galactosidase, neuraminidase and p-N-Acetyl-hexosaminidase
(Laughon et al., 1982; van WinlceUioff et al., 1985; Nash, 1987; Syed et al.,
1988; Minhas and Greenman, 1989; Moncla et al., 1990; A. Wallace, personal
communication, 1993).
The increased levels of P-N-Acetyl-hexosaminidase activity in gingival
crevicular fluid from periodontal disease patients and the serum of subjects with
cancer may implicate this enzyme activity in tissue damage (Dobrossy et al.,
1980; Tucker et al., 1980; Chatterjee et al., 1982; Beighton et a l , 1992). In
cancer, it is accepted that the joint action of host hydrolytic enzymes degrade
the ECM and contribute to tissue breakdown, whereas in periodontal disease,
tissue damage may be due to both bacterial and host enzymes (Nicolson, 1982;
Uitto, 1991; Lambster and Novak, 1992). Studies in cancer research have
demonstrated that highly metastatic murine leukemic and human ovarian
carcinoma cells release substantial amounts of glycosidases, with P-N-Acetyl-
hexosaminidase showing high secretory activity (Bosman and Bernacki, 1970;
Niedbata et al., 1987). Moreover, both tumour cell- and P-N-Acetyl-
hexosaminidase-mediated ECM degradation can be inhibited with sugar analogs
loiown to be competitive inhibitors of P-N-Acetyl-hexosaminidase (Niedbata et
a l, 1987; Woynarowska et al., 1989 & 1992). Although the site of action for p
26
-N-Acetyl-hexosaminidase in ECM is unclear, it can be postulated that this
enzyme may act on macromolecules such as the glycosaminoglycans. Also, the
treatment of basement membrane material with P-N-Acetyl-hexosaminidase
significantly increases its sensitivity to trypsin degradation (Jensen and Lendet,
1986). Therefore, it could be implied that p-N-Acetyl-hexosaminidase may
unmask new proteolytic sites of action allowing more efficient brealcdown of
the ECM (Woynarowska et al., 1989). If, in a similar manner, the P-N-Acetyl-
hexosaminidase and trypsin-like protease of P. gingivalis can degrade oral
basement membrane, this could farther implicate these enzyme activities as
virulence factors of P. gingivalis. With this in mind, it is tempting to speculate
about the increased level of p -N-Acetyl-hexosaniinidase activity in gingival
crevicular fluid during periodontal disease. For example, this activity could be
due to the action of bacterial exoglycosidases and/or host lysosomal enzymes as
part of an inflammatory response. Moreover, this enzyme activity from P.
gingivalis may play a role in ECM degradation during chronic adult periodontal
disease.
1.4.2.2 The Degradaüom of (glycoproteins and Glycolipids
The oligosaccharides of host cell surface glycosphingolipids and
glycoproteins have roles as molecular attachment sites for a number of
pathogenic bacteria (Karlson, 1989). Moreover, the action of glycosidases on
cell surface structures may influence bacterial colonisation or attachment
(Hoskins, 1991). For example, the attachment of T. denticola to laminin and
fibrinogen is significantly decreased when the bacteria are treated with a
mixture of glycosidases (Haapasalo et al., 1991). Treatment of epithelial cells
with neuraminidase increases the binding of P. gingivalis but decreases the
attachment of Streptococci to these host cells (Socransky and Haffajee, 1991).
Therefore, it could be suggested that the extracellular neuraminidase activity of
27
P. gingivalis may eliminate the binding of Streptococci to SA residues on
epithelial cells, thereby facilitating the attachment of P. gingivalis to these
cells. Since a number of bacterial adhesins recognise complementary
GlcNAc/GalNAc residues (Section 1.2.2), a decrease of GalNAc/GlcNAc
lectin-receptors on the surface of host cells, bacteria and salivary glycoprotein
would directly hinder the adhesive ability of certain oral bacteria in the
periodontal microbiota. If the extracellular P-N-Acetyl-hexosaniinidase from P.
gingivalis can deplete GalNAc/GlcNAc lectin-receptors this enzyme may
modulate bacterial adhesion.
p-N-Acetyl-hexosaminidase activity is also assumed to play a role in the
sequential degradation of digestive tract mucin oligosaccharides (Prizont, 1982;
Hoskins, 1991; Boureau et al., 1993). Mucins are glycoproteins that form a gel
covering the mucosal surface, which protects the delicate epithelial cells against
the extracellular environment and selects substances for binding and uptake by
these epithelia (Süberberg, 1989; Strous and Dekker, 1992). Mucins contain
oligosaccharide structures with large quantities of GalNAc, GlcNAc, Gal, Fuc,
and SA, and carbohydrate may be roughly 50 % of the dry weight (Strous and
Dekker, 1992). Since non-covalent interactions between carbohydrate moitiés
on mucins are thought to be important in the formation mucin-clusters and gel
like properties (Strous and Deldcer, 1992), the degradation of oligosaccharide
structures on mucins may modify the effectiveness of epithelial hmction and
protection (Hoskins, 1991). Moreover, Eposito et al., 1983 has shown that in
vivo treatment of rat mucosa with a mixture of the exoglycolytic enzymes P-N-
Acetyl-hexosaminidase, neuraminidase, P-galactosidase, sulphatase, a-
mannosidase, P-glucosidase and P-glucuronidase causes the mucosa-to-serosa
permeability to increase dramatically.
28
It is known that certain faecal bacteria can degrade mucin oligosaccharides
(Salyers et al., 1977; Robertson and Stanley, 1982; Hoskins et al., 1985;
Ruseler van Embden et al., 1989). The bacterial degradation of
oligosaccharides on mucin glycoproteins and intestinal glycosphingolipids
appears to require extracellular neuraminidase, a-glycosidases, p-galactosidase
and p-N-Acetyl-hexosaminidase activities (Hoskins and Boulding, 1981;
Hoskins et al., 1985; Larson et al., 1988; Falk et al., 1990; Hoskins, 1991). hi
Bacteriodes fragilis, the production of exoglycosidases with the potential to
degrade mucin is inversely related to growth rate when mucin is provided as
the sole carbon and nitrogen source (MacFarlane and Gibson, 1991). Mucin has
been shown to induce the production of the both extracellular and cell-bound
neuraminidase, a-glucosidase, p-galactosidase and p-N-Acetyl-hexosaminidase
activities of feacal bacteria (MacFarlane et al., 1989). Moreover, when
subgingival plaque bacteria are grown in BM medium (medium that supports
the growth of black-pigmented anaerobes see section 2.1.1), with added mucin,
these glycosidase activities and P-glucuronidase increase significantly (Beighton
et a/., 1988). Since mucin increases the production of exoglycosidases in the
bacteria of subgingival plaque, it is possible that these enzymes may degrade
the oligosaccharides of mucin structures within the gingival epithelium and
salivary pellicle (Dabelsteen et al., 1991). Additionally, in host cell plasma
membranes, the oligosaccharides that are linlced to glycoproteins and
glycosphingolipids are thought to be important for host cell-to-cell adhesion and
interaction (Hakomori, 1993). Consequently, the combined action of
glycosidases degrading these oligosaccharides could have important effects on
host cell-to-cell interactions.
The release of carbohydrate molecules might also enhance the growth of
certain bacterial species. This hypothesis is based on the observation that a lack
of glycosidic enzymes in Clostridium difficile may be correlated with its
29
nutritional supression and inability to compete for sugars in the normal colonic
microflora. Moreover, free sugars added to a normal colonic microflora in
continuous culture enhance the growth of C. difficile (Wilson and Perini,
1988). Further, a faecal strain of Ruminococcus gnavus which has a-
galactosidase activity but lacks P-N-Acetyl-hexosaminidase activity cannot
degrade the backbone of blood group B oligosaccharides on salivary
glycoprotein. When this strain is grown in symbiotic association with a strain
that produces P-N-Acetyl-hexosamdnidase, extensive degradation occurs and
bacterial growth is enhanced (Hosldns et al., 1985; Hoskins, 1991). If
comparable circumstances exist in the periodontal microflora, then it can be
postulated that the action of p-N-Acetyl-hexosamdnidase on salivary
glycoprotein and cell surface glycoconjugates may provide growth factors that
nutritionally enhance certain saccharolytic members of bacterial plaque.
1.4.2.3 Hydrolysis of Asparagine-Linked Oligosaccharides onHnman IgG
The two CH2 domains in the Fc region of human IgG are thought to be held
apart by two asparagine-linlced oligosaccharides of the general structure in
Figure 1.3. Some IgG molecules contain a l-3 arms and a l-6 arms that lack the
terminal SA-Gal and terminate in GlcNAc. The a l-3 arms from each sugar are
thought to provide a bridge between the two CH2 domains, whereas the a l-6
arms are thought to be directed towards the surface of the CH2 domains
(Rademacher, 1988; Roitt, 1991). The al-3 and a l-6 arms can be completely
removed by the sequential enzymatic action of neuraminidase, P-galactosidase
and P-N-Acetyl-hexosaminidase (Figure 1.3) (Koide et al., 1977).
30
Figure 1.3 The Structure and Enzymatic Degradation of the Desialylated Oligosaccharides of Human IgG
S. pneumoniae Jack Bean or S. pneumoniaefi-galactosidase /J-N-Acetyl-hexosaminidase
X X±Gaip 1—4GlcNAcP 1—2M anal +F ucal
Jack Bean \ \P-N-Acetyl-hexosaminidase^ 5 5
±GlcNAcP 1—4ManP 1—4GlcNAcP 1— 4G 1cN A c-asp
3 X/ S. pneumoniae
±Gaip 1—4GIcNAcP 1—2M anal endo-p-N-Acetyl-glucosandnidase
% %S. pneumoniae Jack Bean or S. pneumoniaep-galactosidase P-N-Acetyl-hexosandnidase
monosaccharide sequence ►
Figure 1.3 The action of P-N-Acetyl-hexosaminidase, P- galactosidase and endo-P-N-Acetyl-glucosaminidase on desialylated (neuraminidase-treated) asparagine-linked sugar chains of human IgG. The exoglycosidases cleave each glycosidic linkage down the monosaccharide sequence begining at the outer non-reducing end. The basic structure required for endoglycosidic hydrolysis by the endo-P-N-Acetyl-glucosaminidase is shown in bold. The endo-P-N- Acetyl-glucosaminidase hydrolyses the internal glycosidic linkage most efficiently when the al-3-linked mannose residue is not substituted at C-2 (Modified from Kiode et at., 1977; Mizuochi et at., 1982; Kobata era/., 1989; Furukawa and Kobata, 1993).
If the neuraminidase, p-galactosidase and P-N-Acetyl-hexosaminidase
activities from P. gingivalis can sequentially remove the a l-3 and a l-6 arms of
each oligosaccharide in vivo, this may aid modification of IgG interactions.
This idea is based on the importance of these oligosaccharides in antibody-
monocyte interaction and the molecular stability of IgG (Leatherbarrow et al.,
31
1985; Rademacher, 1988; Oppdenaldcer et al., 1993). Moreover, the treatment
of intact IgG with neuraminidase and P-galactosidase has no effect on rosette
formation or antibody-dependent cell-mediated cytotoxicity. However,
additional manipulation with p-N-Acetyl-hexosaminidase and endo-P-N-Acetyl-
glucosaminidase from S. pneumoniae decreased these immunological reactions
(Koide et al., 1977).
1.4.2.4 Autolysins that Degrade Pepddoglycan
Bacterial peptidoglycan hydrolases that can degrade their own cell walls are
refered to as autolysins (Ghuysen et al., 1966). Autolysins can be classified as
N-Acetyl-muramidases, endopeptidases, transglycosidases, N-Acetyl-muramyl-
L-alanine-amidases and P-N-Acetyl-glucosaminidases (Ghuysen et al., 1966).
Autolysins may be required for several important cellular functions, including
cell wall growth, turnover and splitting of the septum for cell separation (Holtje
and Tuomanen, 1991). Some autolysins may be important for bacterial
pathogenicity and may cause release of highly inflammatory cell wall
components or may lyse a portion of the cell that results in the liberation of
toxins (Berry et al., 1989; Holtje and Tuomanen, 1991; Wuenscher et al.,
1993)
Endo-P-N-Acetyl-glucosaminidase mutants of Bacillus subtilis 168 appear to
form long chains of unseparated cells. Therefore, this endoglycosidase activity
has been suggested to participate in cell growth, division and shape formation
(Fein and Rogers, 1976). However, an endo-P-N-Acetyl-glucosaminidase gene
from B. subtilis AC327 was disrupted using site specific mutagenesis and the
mutant strain appeared normal in growth and morphology (Rashid et al., 1993).
It was proposed that the endo-p-N-Acetyl-glucosaminidase together with
32
amidase may be important for B. subtilis motility rather than growth and
morphology (Rashid et al., 1993). Endo-P-N-Acetyl-glucosaminidases may
play a significant role in bacterial pathogenicity (Berry et al., 1989; Valisena et
al., 1991). For example, an endo-P-N-Acetyl-glucosaminidase from
Staphylococcus aureus inhibits the response of human lymphocytes to mitogens
and interferes with the production of antibodies in mice (Valisena et al., 1991).
This enzyme is also suggested to share common epitopes with microbial and
mammalian exoglycosidic P-N-Acetyl-hexosaminidases (Guardati et al., 1993).
Bacterial exo- and endo-P-N-Acetyl-glucosaminidases are reported to
hydrolyse peptidoglycan and they may play a role as endogenous prokaryotic
hydrolases that modify peptidoglycan during cell growth (Kawagishi et al.,
1980; Gubish e/fl/., 1982; Chapman and Perldns, 1983). However, in bacterial
peptidoglycan modification, exo-P-N-Acetyl-glucosaminidase activity has an
uncertain physiological role and some authors are sceptical of its dhect
involvement in peptidoglycan turnover or metabolism (Doyle and Koch, 1987).
This may be due to the observation that exo-P-N-Acetyl-glucosaminidase
mutants of Bacillus subtilis B and Escherichia coli K12 appear normal in
growth, division and morphology (Ortiz, 1974; Yem and Wu, 1976). Also, it
has been reported that B. subtilis cells do not reincorporate pre-radiolabelled
cell wall GlcNAc into their 'new' peptidoglycan during growth, which could
suggest a more indirect role for exo-P-N-Acetyl-glucosanimidase enzymes in
wall metabolism or turnover (Doyle and Koch, 1987). Further studies are
needed to examine the role of these exoenzymes in bacterial cell wall
metabolism.
33
1.4.3 p-N-Acetyl-Hexosaminidases in Glycobiology
The deglycosylation enzymes are powerful tools in the analyses of
oligosaccharide structure and function (Maley et al., 1989; Welply, 1989;
Dwek et a l , 1993; Furukawa and Kobata, 1993). A variety of specific
glycosidases are generally used and enzymatic deglycosylation is carried out by
sequential exoglycosidase digestion or by endoglycosidases (Nageswara and
Bahl, 1987; Welply, 1989; Dwek et a l , 1993; Furukawa and Kobata, 1993).
The detennination of oligosaccharide structures linked to the glycoconjugates
shown in Table 1.2 involved the use of P-N-Acetyl-hexosaminidase cleavage. P
-N-Acetyl-hexosaminidase cleavage has also contributed to the analysis of the
oligosaccharides linked to fibrinogen (Townsend et al., 1982), intracellular
glycoproteins (Hanover et al., 1987; Holt et al., 1987), human chorionic
gonadotropin (Goverman et al., 1983), plus the epidermal growth factor
(Cummings et al., 1985) and acetyl-choline receptors (Herron and Schimerlick,
1983). The wide use of P-N-Acetyl-hexosaminidases in mixed glycosidase
digestion procedures that analyse oligosaccharide structure and function
demonstrates that these enzymes are valuable tools for glycobiologists.
Therefore, it is lUcely that commercially available and novel P-N-Acetyl-
hexosaminidases will play an integral role in the future of carbohydrate
research. Together, P-N-Acetyl-hexosaminidases and other glycosidases may
be pivotal in the identification and commercialisation of new developments in
gly cobiology.
34
Table 1.2 Some Selected Glycoproteins and Glycolipids
Glycoconjugate Function Role of CHO ReferencesImmunoglobulin G Host defence Fc interaction
Stability Protease resistance
Mizuochi et al., 1982 Taniguichi et al., 1985
Rademacher et al., 1986 Rademacher et al., 1988
aj-acid glycoprotein
Immunomodulator ? Jeanloz, 1972 Yoshima e ta l., 1981 Bouten e ta l., 1992
Interleukin-6 T/B cell activation ? van Snick, 19W Parekh et al., 1992
p67 Eukaryotetranslation
Regulation? Datta eta l., 1989
ABH cell surface antigens
Tissue markers and
Cellular adhesion
Cell-cell interactions Ito et a t., 1989a&b Dabelsteen et al., 1991
Hakomori, 1993
a 2 -HS-glycoprotein Bone metabolism? ? Colclasure e ta l., 1988 Watzlawick eta l., 1992
Fetuin Developmentand
Lipogenesis
? Takasaki & Kobata, 1986 Cayatte et al., 1990
Table 1.2 Selected examples of glycoconjugates where P-N-Acetyl- hexosaminidase cleavage has been applied to determine oligosaccharide structure or function. The oligosaccharide functions are shown.
In summary, new advances in glycobiology have demonstrated that
oligosaccharides play a key role in interactions between pathogens and host
cells. Since oligosaccharides are also involved in many host cell fimctions and
molecular interactions, then extracellular glycosidic enzymes liberated by
pathogenic bacteria may be important in the pathogenesis of disease. Although
microbial p-N-Acetyl-hexosaminidases have been shown to hydrolyse a wide
range of carbohydrate structures, there is little information about the exact
role(s) of these enzymes from pathogenic microorganisms. However, the
35
growing interest in microbial P-N-Acetyl-glucosamdnidases and P-N-Acetyl-
hexosaminidases is reflected by the cloning of the genes from Vibrio harveyi
(Jannatipour et al., 1987; Soto-Gil and Zyskind, 1989), V. vulnificus (Wortman
et al., 1986; Somerville and Colwell, 1993), V. fumissii (Bassler et a l , 1991),
V. parahaemolyticus (Zhu et a l , 1992), Serratia liquefaciens (Joshi et a l ,
1988), S. marcescens (Kless et a l , 1989; Tews et a l , 1992), P. gingivalis
(Lovatt and Roberts, 1991), Dictyostelium discoideum (Graham et a l , 1988)
and Candida albicans (Cannon et a l , 1994). P-N-Acetyl-glucosaniinidases and
P-N-Acetyl-hexosanhnidases might function as virulence factors that degrade
host oligosaccharides, and play a role in the hydrolysis of peptidoglycan, acting
as autolysins. Although numerous studies have characterised and exploited
enzymes within this group of exoglycosidases, there is sthl much to loiow about
their regulation and physiological significance in the degradation of
glycoconjugates.
Lastly, the occurence of densely packed mixtures of diverse bacterial species
in dental plaque suggests that bacterial interactions play an important role in
species survival. The in vivo formation of bacterial aggregates may provide a
network for the growth and retention of selected oral bacterial species. Some
interspecies relationships may be favourable, in that one species produces
growth factors for, or facilitates the attachment of, another. Other relationships
may be antagonistic due to the competition for nutrients, or the production of
substances that inhibit the growth or attachment of a second species. For
successful colonisation of the periodontal pocket, the adhesion to host
components may be required, and the fimbriae, trypsin-lilce proteases and
haemagglutinins of P. gingivalis may serve in the attachment process. The
production of proteases and glycosidases by P. gingivalis may modulate
bacterial adhesion and damage host immune components. In addition, proteases
and glycosidases may degrade bacterial/host cell surfaces and ECM molecules
36
in an effort to gain ecological advantage, perhaps by providing nutrients and/or
assisting in microbial attachment and spreading. The glycosidases produced by
P. gingivalis and the bacteria of subgingival plaque may release the
carbohydrate from human IgG, protective mucin glycoconjugates and basement
membrane glycosaminoglycans. In response to ECM damage by bacteria,
hydrolytic enzymes and LPS, the host may amplify tissue destruction by
releasing or expressing matrix-metalloproteinases that degrade the subepithelial
basement membrane. Although there is no evidence that P-N-Acetyl-
hexosaminidase activity contributes to the degradation of host ECM molecules
during periodontal disease, considerations may offer future research directions
that wül contribute to the understanding of molecular mechanisms in tissue
damage and periodontopathogenesis.
1.5 The Aims of This Thesis
Firstly, the aim of this thesis is to speculate on the function of the P-N-
Acetyl-hexosaminidase from P. gingivalis and to adopt a genetic manipulation
approach for defining the role of this enzyme. Secondly, to use this strategy to
isolate and produce structural information on the gene that encodes for P-N-
Acetyl-hexosaminidase activity. To use this information to construct a site-
directed gene replacement strategy for the generation of a isogenic mutant of P.
gingivalis lacldng P-N-Acetyl-hexosaminidase activity, which can be compared
with the wild type P.gingivalis in appropriate model systems. The last aim is
for this thesis is to provide guidance and inspiration for future work and new
investigators.
37
CHAPTER 2
Materials and Methods
2 .1 B acterial Strains and Plasm ids
The bacterial strains and plasmids that were used in this study are listed in
Table 2.1 and Table 2.2 respectively.
2.1.1 Growth Conditions and Media
Porphyromonas gingivalis, Porphyromonas asaccharolytica and
Porphyromonas endondontalis strains were grown anaerobically at 37°C in
Bacteroides Medium (BM) (10 g/litre ' trypticase peptone; lOg/litre ' proteose
peptone; 5g/litre'^ yeast extract; 5g/litre ' glucose; 5g/litre ‘ NaCl; 0.7g/litre ‘
cysteine HCl; Ig/litre^ NaHCOg; 5pg/ml ' hemin; lOpg/ml ' menadione) with
the addition of 1.5% w/v agar (BBL) as required or on 7% v/v horse-blood
agar plates (Oxoid). E. coli srains were grown in Luria broth (L-broth) (25
g/litre ' peptone; 12.5g/litre ‘ NaCl; 25g/litre ‘ yeast extract) at 37°C with the
addition of 1.5% w/v agar as required. B-agar (10 g/litre ' peptone; 8g/litre '
NaCl, 1.5% w/v agar) was used where stated. For detecting P-N-Acetyl-
hexosaminidase activity, the flourogenic substrates 4-methylumbelliferyl-N-
Acetyl-p-D-glucosanunide (MUAG) and 4-methylumbelliferyl-N-Acetyl-P-D-
galactosaminide (MUAGal) were added to media at a concentration of lOOp
g/ml. Antibiotics at concentrations of lOOpg/ml ampicillin, 25pg/ml
tetracycline and 25pg/ml kanamycin were used when required. Antibiotics and
substrates were obtained from Sigma Chemical Comp. Ltd.. Bacterial cells
38
were routiiüey harvested by centrifiigation in a Sorval centrifiige (3300g at 4°C
for 10 minutes (mins)) or in a bench top minifuge (13400g at room temperature
for 5 mins).
Table 2.1 Bacterial Strains
Bacterial strain Relevant characteristic Source *E.COÜ
SURE™ recB r e d sbcCZOl uvrC umuC::Tn5 (konO lacA (hsdRMS) endAl gyrA96 thi relAl supE44 F'lproAB'^ lacB lacZAMlS TnlO (tef)]
Stratagene® Cloning Systems
JMlOl supE thil A(lac-proAB) F'[froD36 proAB'^ lacP
Yanisch-Perron et al., 1985
DS410 minA minB ora xyl m l azi thi
Dougan and Sherratt, 1977
P. gingivaüsW83 Clinical specimen H. Shah"
WpH35 Clinical specimen MRC Dental Unit, London
23A3 Clinical spœimen MRC Dental Unit, London
ATCC 33277 Type strain ATCC^
P. endodontalisATCC 35406 Type strain ATCC
P. asacharolyticaATCC 8503 Type strain ATCC
Table 2.1 Bacterial strains. * Addresses; ^, Eastman DentalHospital, London. , American Type Culture Collection, Rockville, Maryland, USA
39
Table 2.2 Bacterial Plasmids / Vectors
Plasmid/vector Relayent characteristic Construction or sourcepTTQlS pUC based expression
vector (Amp' lacN)Stark et at., 1987
M13mpl8/19 M13 cloning/sequencing vector
Yanisch -Perron gfo/., 1985
pNJR6 Colonic Bacteroides suicide vector derived from the shuttle cosmid pNJRl and does not contain the pB8-51 region that enables replication in Bacteroides hosts (Kan' Cc' Em' Sm')
Shoemaker et al., 1989
PGEX-2T Vector that directs the synthesis of foreign polypeptides as fusions with glutathione S-transferase (Amp')
Smith and Johnson, 1988
Table 2.2 Bacterial plasmids / vectors.
2.2 Transformation of Bacterial Cells
2.2.1 Production of Competent CeUs
2.2.1.1 Calcium Chloride Method
lOOpl of an E. coli overnight culture grown at 37°C in 10ml L-broth were
diluted 1:100 with 10ml of L-broth and grown to mid exponential phase
(ODgoty» 0.4). The cells were harvested (3300g at 4°C for 10 mins), washed in
10ml of lOmM NaCl, pelleted (3300g at 4°C for 5 mins) and resuspendW in
4ml ice-cold CaCfy (lOOmM). The cells were placed on ice for 30 mins and
collected by gentle centrifugation (1800g) at 4°C for 5 mins. The cell pellet was
resuspended in 1ml ice-cold CaCl] (lOOmM) and used immediately in
transformation,
2.2.1.2 Electrotransfbrmadon Method
lOOpl of an overnight culture grown at 37°C in 10ml L-broth were diluted
1:100 with 10ml of L-broth and grown to mid exponential phase (ODggo^O.S).
The cells were chilled on ice for 15 mins and harvested (3300g at 4°C for 10
mins). The cell pellet was washed 4 times in 10ml of nanopure water and once
in 10% v/v glycerol with centrifugation as before between the washes. The cell
pellet was then resuspended in SOpl of 10 % v/v glycerol and used immediately
in transformation.
2.2.2 Transformation with Plasmid DNA
2.2.2.1 Calcium Chloride Method
Competent cells (lOOpl) were mixed with 5-20pl of DNA (in water) and
placed on ice for 1 hour (hr). The cells were heat shocked at 42°C for 3 mins.
Immediately after heat-shocldng, SOOpl of L-broth were added and the cells
incubated for 1 hr at 37°C. The transformed cells were plated onto L-agar
plates (lOOpl per plate), which contained the appropriate antibiotic(s).
41
2.2.2.2 Electrotransfbrmation Method
Competent cells (40pl) were mixed with l-2pl of DNA (in water) and
transferred to an ice-cold BIO-RAD 0.2cm gene puiser cuvette. The cell
suspension was pulsed (2.4kVcm-i, 25pF, 2000) on a BIO-RAD Gene-Pulser
apparatus. Immediately after pulsing, 1ml of ice cold SOC recovery medium
(20 g/litre'^ tryptone; 5g/litre'^ yeast extract; lOmM NaCl; 2.5mM KCl; lOmM
MgSO^; 20mM glucose) was added and the cells were incubated for Ihr at 37°
C with shaldng. The transformed cells were plated onto agar plates (lOOpl per
plate), which contained the appropriate antibiotic(s).
3 TTraauüRariaadioïkivith IkacterioidbaypeiDOSYl
Competent cells of E. coli JMlOl (lOOpl; see Section 2.2.1.1) were mixed
with DNA, incubated on ice for 1 hr and heat-shocked at 42°C for 3 mins. The
transformed cells were mixed with lOOpl of JMlOl (ODggg^O.S) and then 3ml
of molten B-agar (held at 45°C and containing 20pl lOOmM IPTG, SOpl 2%
w/v X-gal in dimethylformamide) were added. The suspension was immediately
mixed and poured onto a B-agar plate, rocked to disperse and once set
incubated at 37°C overnight.
42
2.3 Procedures for DNA Extraction
DNA extraction protocols used the following solutions:
solution I : 50mM glucose
25mM Tris-HCl pH 8.0
lOmM EDTA
5mg/ml lysozyme
solution II 0.2M NaOH
35mM sodium dodecyl sulfate (SDS)
solution III : 5M acetate (11.5ml glacial acetic acid)
3M potassium ions (60ml 5M potassium acetate)
nanopure water (28.5ml)
2.3.1 Extraction o f Chromosomal DNA
The method used to extract chromosomal DNA was based on that described
by Saito and Muira, 1963. Bacterial cells from 10ml stationary phase cultures
were washed in lOmM NaCl and resuspended in 5ml solution I for 30 mins on
ice. SDS was added to a final concentration of 35mM and EDTA to 50mM.
The preparation was left at room temperature until the solution was clear
(typically 20 mins). The protein was removed by repeated phenol:chloroform
(1:1 w/v) extraction followed by one chloroform:isoamyl alcohol (24:1 v/v)
extraction (see Section 2.3.5). Chromosomal DNA was finally retrieved by
gently pouring 2 volumes of ethanol (chilled at -20°C) down the side of a tube
containing the clear aqueous phase. DNA precipitated at the interface and was
43
spooled out using the rounded end of a pasteur, resuspended in sterile nanopure
water and stored at -20°C.
2.3.2 Small Scale Extraction of Plasmid DNA
Small scale preparation of plasmid DNA used 1.5ml of an overnight culture.
Cells were suspended in lOOpl of a freshly made solution I for 30 mins on ice.
Solution II (200|Lil) was added and the tube was gently mixed and placed on ice
for 5 mins. HOpl of solution III were added to the clear mixture and the tube
was gently mixed and left on ice for 5 mins. The supernatant was recovered
after centrifugation (13400g for 10 mins) avoiding the white pellet. Protein was
removed by one phenol:chloroform (1:1 w/v) extraction followed by one
chloroform: isoamyl alcohol (24:1 v/v) extraction. The DNA was precipitated
by adding two volumes of ethanol (see Section 2.3.5)
2.3.3 Large Scale Extraction of Plasmid DNA
Overnight cultures (400ml) were used for large scale preparation of plasmid
DNA (Birboim and Doly, 1979). The cells were collected in large pots by
centrifugation (3300g at 4°C for 10 mins), resuspended in 10ml of a freshly
made solution I and left on ice for 30 mins. Fresh solution II (20ml) was added,
gently mixed and the whole left on ice for another 10 mins. Solution III (7.5ml)
was added, gently mixed and the whole left on ice for 10 mins. Cell debris was
removed from the plasmid preparation by centrifugation at 4°C for 20 mins at
35000g. Isopropyl alcohol (0.6 volumes) was added to the supernatant, mixed
and left to stand at room temperature for a minimum of 15 mins. DNA was
collected by centrifugation at 4000g for 30 mins at 20°C. The DNA pellet was
air dried for 15 mins and resuspended in sterile nanopure water to a final
44
volume of 17ml. Caesium chloride was added to a final concentration of
Img/ml and ethidium bromide to SOpg/ml. Chromosomal and plasmid DNA
were separated by centrifugation at 40000 rpm using a Sorval TV850 rotor in a
Sorval OTD 60 centrifuge for 20 hrs at 20°C. DNA was visualised under UV
light and the lower band of plasmid DNA extracted. Ethidium bromide was
removed by equilibration with caesium chloride-saturated isopropanol. Caesium
chloride was removed by exhaustive dialysis against distilled water at room
temperature. Plasmid DNA was stored dissolved in sterile distilled water at -20
°C.
2.3.4 Extraction of M13mpl8/19 Template DNA
The recombinant bacteriophage M13mpl8/19 were transformed into JMlOl
(Section 2.2.3) and white plaques were picked into 5ml L-broth containing 100
pi of an overnight culture and incubated at 37°C for 5 hrs with vigorours
aeration. Replicative form DNA and template DNA were obtained from two
1.5ml aliquots of a bacterial culture. The replicative form DNA was extracted
as described for small scale extraction of plasmid DNA (see Section 2.3.2),
whereas the template DNA was isolated from the supernatant. The supernatant
(1.2ml) was mixed with 300pl of a solution containing 2.5M NaCl and 20%
w/v PEG 6000. The mixed solution was left at room temperature for 30 mins.
The phage pellet was recovered by two sequential centriftigations (the second to
remove traces of PEG 6000), then resuspended in 120pl I.IM sodium acetate
pH 7.0, and extracted with an equal volume of phenol:chloroform (1:1 w/v)
followed by one chloroform: isoamyl alcohol (24:1 v/v) extraction (see Section
2.3.5). The template DNA was precipitated with ethanol (see Section 2.3.5).
The template DNA was collected by centrifugation at 13400g for lOmins, dried
in vacuo and resupended in 20pl nanopure water. 2pl of template DNA were
45
visualised by agarose gel electrophoreseis (see Section 2.4) and 4-7pl were
typically used in a sequencing reaction (see Section 2.6)
2.3.5 Phenol Extraction and Ethanol Precipitation
Phenol extraction was performed using one volume of phenol:chloroform
(1:1 w/v) containing 0.1% w/v hyroxyquinoline and equilibrated with lOOmM
Tris-HCl pH 8.0. Chloroform extraction was performed using one volume of a
mixture of chloroform:isoamyl alcohol (24:1 v/v). The aqueous phase was
separated in a Sorval centrifuge (3300g at 20°C for 20 mins) or in a bench top
minifuge for 5 mins at 13400g, then collected avoiding the inter-phase. Ethanol
precipitation was performed with sodium acetate to a final concentration of
300mM and 2 volumes of ethanol at -20°C for minimum of 30 mins. The DNA
was collected by centrifugation either in a bench top niinifuge for 5 mins at
13400g or in a Sorval centrifuge for 30 mins at 3500g.
2.4 Techniques Used in Routine DNA Manipulation
Restriction endonucleases and DNA modifying enzymes were purchased
from Pharmacia Biochemicals Inc. or Life Technologies Ltd (GIBCO/BRL) and
used according to the manufacturers recomendations. Restriction endonuclease
cleavage of DNA was performed typically in 20pl reactions with one unit of
enzyme per pg of DNA at 37°C. T4 DNA ligase was used at 14°C overnight in
T4 ligase buffer (50mM Tris-HCl pH 7.5; lOmM MgClz; ImM ATP). DNA
fragments were separated by agarose gel electrophoresis using 0.7-1.2% w/v
Seakem agarose in TAE buffer (40mM Tris-acetate; ImM EDTA) with 0.5p
g/ml ethidium bromide and visualised using a longwave UV transilluminator.
DNA samples were mixed with the appropriate volume of 6x gel-loading buffer
46
(0.25% w/v bromophenol blue; 0,25% w/v xylene cyanol; 15% w/v Ficoll)
prior to loading. The DNA size markers used was 1 kb ladder (BRL/GIBCO).
For subcloning, the DNA fragment was excised from the gel and the DNA
recovered by Sephaglass-Band-Prep-Kit (Pharmacia) according to the
manufacturers recommendations.
For routine dephosphorylation of plasmid vector, lOpg DNA was incubated
in 100pi calf-intestinal-phosphatase (CIP) buffer (50mM Tris-HCl pH 9.0;
lOmM MgCl2 ; ImM ZnCl2 ; lOmM spermidine) containing ten units of calf-
intestinal alkaline phosphatase and incubated at 37°C for 30 mins. Another ten
units of calf-intestinal phosphatase was added to the reaction and the whole
incubated once more at 37°C for 30 mins. Nanopure water was added to a final
volume of 300pl. The DNA was extracted twice with an equal volume of
phenol: chloroform (1:1 w/v), then once with an equal volume of
chloroform:isoamyl alcohol (24:1 v/v) (section 2.3.5). The phosphatased DNA
was precipitated with ethanol and sodium acetate and collected by
centrifugation at 13400g for 5 mins. The DNA was washed in fresh 70% v/v
ethanol, centrifuged as before, and resuspended in a final volume of 50pl
2.5 DNA Hybridisation Procedures
2.5.1 Transfer of DNA to Nylon Filters
DNA was transferred to filters as described by Southern, 1975. DNA
samples were separated by agarose gel electrophoresis as described above and
the gel photographed along side a linear rule. The DNA was de-purinated by
soaking the gel in 0.25M HCl for 7 mins. The gel was rinsed briefly in distilled
water and placed in denaturing solution (0.5M NaOH; 1.5M NaCl) for 30 mins
47
with occasional shaking. The gel was again rinsed in distilled water and placed
in neutralising solution (0.5M Tris-HCl pH 7.5; 3M NaCl) for another 30 mins
with occasional shaking as before. The gel was rinsed again and placed on six-
sheets of pre-wet (20x SSC) Whatman paper (3mm) without trapping any air
bubbles (20x SSC is 3M NaCl; 0.3M trisodium citrate). A pre-wet (3x SSC)
sheet of nylon membrane (Hybond-N, Amersham International pic) was placed
on the gel with a pre-wet sheet (3x SSC) Whatman paper on top, again taking
care to avoid bubbles. Four sheets of dry Whatman paper were placed above
this with a stack of paper towels. Finally, a glass plate and a 500g weight were
placed on top. The lower sheets of Whatman paper were regularly soaked with
20x SSC and the paper towels changed. The apparatus was left overnight for
the DNA to transfer and then dismantled. The nylon filter was air dried,
wrapped in Saran wrap and exposed to UV light from a long wave
transilluminator for 5 mins to fix the DNA to the filter. Filters were stored at
room temperature in the dark until required for DNA hybridisation (see Section
2.5.4).
2.5.2 Preparation of Filters for Colony Hybridisation
Bacteria were grown overnight at 37°C on an L-agar plate containing the
appropriate antibiotics. A nylon filter (Hybond-N) was placed on top of the
bacterial colonies and left for 10 mins. Whatman paper (3mm) was placed m a
shallow tray and soaked in denaturing solution. The nylon filter was removed
from the L-agar plate and placed on the soaked Whatman paper (colony side
up) for 5 mins. The filter was transfer to Whatman paper, this time soaked in
neutralising solution for a further 5 mins and air dried. For details on the
composition of the solutions see Section 2.5.1. DNA was fixed to the filter by
exposing to longwave UV light from a transilluminator for 5 mins. Cell debris
48
was removed from the filters by gentle scrubbing in 5x SSC using polymer
wool and filters left to air dry in preparation for DNA hybridisation.
2.5.3 Production of a Radiolabelled Probe
Plasmid DNA was cleaved with the appropriate restriction endonucleases and
the fragments separated by agarose gel electrophoresis on a 1% w/v low
melting point agarose gel (BRL). The required DNA fragments were excised
from the gel and added to sterile nanopure water (1.5ml water per gram of
agarose). The sample was placed in a boiling water bath for 7 mins then stored
at -20°C. Prior to use the sample was boiled for an additional 3 mins.
Approximately lOng of DNA was radiolabelled using random hexanucleotide
primers exactly as described by Feinberg and Vogelstein, 1983. Nucleotides
and hexanucleotides were obtained from Pharmacia and [a-32p]dCTP from
Amersham International pic.
2.5.4 Hybridisation of DNA Dnmobilised on Filters with the Probe
Southern blot or colony hybridisation filters were shaken at 65°C m 100ml
of pre-hybridisation solution (see below) for 2 hr. This solution was discarded
and replaced by 20ml hybridisation solution (see below) containing the
radiolabelled probe DNA which had been boiled for 5 mins before adding. The
filter was shaken overnight at 65°C. Hybridisation solution (3x SSC; 2x
Denhardts; 200|Lig/ml salmon sperm DNA; 0.1% w/v SDS; 6% w/v PEG
6000). Prehybridisation solution is the same except with 5x Denhardts (lOOx
Denhardts is 2% w/v Bovine serum albumin V; 20% w/v Ficoll 400; 2% w/v
polyvinylpyrollidone). Solutions were stored at -20°C without salmon sperm
49
DNA. Salmon sperm DNA was sheared by forcing it through a narrow gauge
syringe needle and denatured by boiling prior to use.
After the hybridisation period the filters were washed twice by shaldng in
250ml 2x SSC 0.1 % w/v SDS at 65°C for 15 mins and twice in 0.5x SSC 0.1 %
w/v SDS (0.5x SSC 0.1% w/v SDS is a high stringency wash condition that
allows for approximately 75% DNA homology) (Drake, 1991). SDS
concentration (0.1% w/v) and temperature (65°C) were not varied. The filters
were then air dried completely. The filters were wrapped in Saran wrap for
autoradiography and placed in a cassette carrying intensifying screens. Kodak
X-Omat AR film was exposed to the filters at -70°C. Films were developed in
an Agfa-Geveart automatic processing machine.
2 .6 D N A Sequem clng
Nucleotide sequence was determined by the chain termination method
described by Sanger et al., 1977, in which DNA synthesis from
deoxynucleotide triphosphates is terminated by the addition of
dideoxynucleotide triphosphates. The M13 cloning vectors, M13mpl8 and
M13mpl9 were used to generate single stranded DNA templates (Section
2.3.4). Sequence reactions were performed using the Sequenase Version 2.0 Idt
produced by United States Biochemical Corporation, U.S.A.. The protocol
recommended by the manufacturers was followed using the universal (-40)
primer or oligonucleotide primers synthesised for this purpose. DNA fragments
were radiolabelled by incorporating [a-35S]dATP in the extension reactions.
The radiolabelled fragments were separated by gradient gel electrophoresis
(Biggin et al., 1983). Preparation of the gels used the following solutions.
50
Gel Solution 1 Gel Solution 2
7ml 5x TBE acrylamide/urea mix 40ml O.Sx TBE acrylamide/urea mix
45pi 10% w/v ammonium persulphate 180pl 10% w/v ammonium persulphate
2.5pl TEMED 7.5pl TEMED
0.5x TBE acrylamide/urea mix 5x TBE acrylamide/urea mix
430g urea 430g urea
50ml lOx TBE 150ml lOx TBE
150ml 40 % acrylamide 150ml 40 % acrylamide
per litre 50g sucrose
50mg bromophenol blue
per litre
Electrophoresis grade ammonium persulphate was purchased from BIO
RAD, TEMED from Sigma Chemical Company Ltd. and SEQUEGEL 40%
acrylamide from BDH. lOx TBE is 0.089M Tris-borate, 0.089M boric acid,
0.002M EDTA. To prepare the gel, gel plates (20cm x 50cm) were taped
together separated by 0.4mm spacers. 10ml gel solution 2 followed by 14ml gel
solution 1 were drawn up into a 25ml pipette. Air bubbles were introduced to
form a rough gradient. The liquid was run between gel plates and the cavity
filled with the remaining of gel solution 2. The comb was positioned and the
plates clamped along each side. Gels were routinely freshly made. A vertical
electrophoresis system was used. Running buffer in the top tank was 0.5x TBE
and the lower Ix TBE in accordance with the gradient itself. The gel was
clamped in position with aluminium sheets of a similar dimension as the gel
plates on either side for even heat distribution. The gel was pre-run for 30 mins
at a constant power of 40W and the wells rinsed with running buffer prior to
loading. Electrophoresis was performed at constant power of 40W for 3, 7 and
51
9 hrs. After electrophoresis the gel plates were prised apart and the gel was
soaked in fixing solution (10% v/v methanol and 10% v/v acetic acid) for 15
mius and then rinsed with distilled water. The gel was transferred to a pre-wet
filter paper, covered with Saran wrap and dried under vacuum at 80°C.
Autoradiography used Dupont Cronex film and took place at room temperature.
2.7 Polymerase Chaim Reaction Procedures
PCR amplification reactions contained Ix Taq buffer (lOmM Tris-HCl pH
8.8; 1.5mM MgCfy; 50mM KCl; 0.001% w/v gelatin), lOng template DNA,
primers at 2.5pM, dNTPs at 40|LiM and 2.5 units of Taq polymerase (Sigma
Chemical Company Ltd.) in a total volume of lOOpl. Ultrapure dNTPs were
purchases from Pharmacia Biochemicals Inc.. Reaction mixtures were UV
irradiated on a transilluminator for 15 mins prior to the addition of template
and Taq polymerase. Reaction mixtures were vortexed collected by
centrifugation and overlain with lOOpl sterile mineral oü prior to amplification.
DNA amplification was performed in a Perkin-Elmer Cetus thermal cycler. 30
cycles of the following conditions were performed:
Denaturing step 95°C 1 min
Annealing step 55°C 1 min
Extension step 72°C 3 mins
Following amplifications the PCR product was analysed by agarose gel
electophoresis and the DNA band excised and purified. Direct cloning of the
PCR product was by a modified method of Holton and Graham, 1991. Plasmid
vector DNA (0.5pg aliquots) (for direct cloning of the PCR product) was
cleaved with Sma\ restriction endonuclease to generate linear blunt end vector.
52
incubated at 70°C for 2 hrs in Ix Taq buffer with 10 pM ddTTP and 5 units of
Taq polymerase. The T-tailed vector was extracted once with
phenol:chloroform (1:1 w/v) and once with chloroform:isoamyl alcohol (24:1
v/v), resupended in nanopure water, pooled and stored at -20°C.
2.8 Radioactive Labelling of Proteins
2.8.1 Minicell Analysis
Minicells were isolated using the procedure described by Hallewell and
Sherratt, 1976. Minicell strains {E. coli DS410) were grown to stationary phase
in 400ml Brain Heart Infusion (if necessary the appropriate antibiotic was
added to the medium). The cells were separated from culture by centrifugation
at 600g for 5 mins. The supernatants were centrifuged at 8500g for 15 mins
and the pellets retained. The pellets were resuspended in 3ml of Ix M9 salts
and niinicells were further purified by two successive sedimentations through
20ml linear gradients of 5-20% sucrose (w/v) in Ix M9 salts at 4650g for 20
mins (4°C). Purified minicells were collected by centrifugation at 9500g for 10
mins and resuspended in Ix M9 salts to a final OD6qo=2.0. Minicells were
either immediately used for protein labelling or aliquoted (lOOpl) in 30% v/v
sterile glycerol and stored at -20°C (for a period of time not exceeding 3
months).
lOx M9 salts per litre: 60g Na2HP0 4 (337mM)
30g KH2PO4 (220mM)
5g NaCl ( 85mM)
lOg NH4CI (187mM)
53
Proteins were labelled for 45 mins at 37°C in Ix M9 nainimal medium
containing 35§_niethionine (lOOpCi/ml). After a 15 mins chase with cold-
methionine-supplemented broth minicells were lysed by boiling m loading
buffer (0.08M Tris-HCl pH 6.8; O.IM dithiothreitol; 2% w/v SDS; 10% v/v
glycerol; O.lmg/ml bromophenol blue). 20pl (from a final volume of 25pi)
were analysed on a SDS polyacrylamide gel. The upper stacldng gel contained
4.5% acrylamide in 0.125M Tris-HCl pH 6.8 and 0.1% SDS. The lower
running gel contained 15% acrylamide in 0.037M Tris-HCl pH 8.8 and 0.1%
SDS. The running buffer contained 0.02M Tris-HCl, 0.2M glycine, 0.1% SDS
and 2.4mg/l sodium thioglycollate. The gel was run at a constant current of
25mA. After running the gel was soaked in fixing solution (10% v/v acetic
acid; 25 % v/v isopropanol) for 30 mins and treated with Amplify (Amersham)
according to the manufacturers recommendations. The gel was dried under
vacuum at 80°C and autoradiographed at room temperature.
2.8.2 DNA Directed Transcription-Translation System
DNA directed transcription-translation was carried out and used according to
the manufacturers recommendations. The DNA directed transcription-
translation kit was purchased from Amersham International pic. DNA was
prepared as described in Section 2.3.3. DNA (2-5pg) was transcribed and
translated in vitro with an E. coli S30 extract at 37°C for 60 mins. Proteins
were labelled in vitro with L-[35S]methionine (lOOpCi/ml) and the reactions
terminated by placing on ice. The samples were diluted 1:1 with loading buffer
(0.08M Tris-HCl pH 6.8; O.IM dithiothreitol; 2% w/v SDS; 10% v/v glycerol;
O.lmg/ml bromophenol blue) and heated to 100°C for 5 mins prior to loading.
lOpl of sample was loaded onto a SDS polyacrylamide gel and analysed as
described in Section 2.8.1.
54
2.9 Biochemical Assay of p-N-Acetyl-Glacosaminidase and p -N-Acetyl-Galactosaminidase Activity
Bacterial cultures (100ml) supplemented with the appropriate antibiotics
were incubated at 37°C until mid exponential phase (OD6Qo=0.5), at which
point isopropyl-p-D-thiogalactopyranoside (IPTG) was added to a final
concentration of lOmM when necessary. The cultures were then grown to
00^00=1.0, harvested by centrifugation (3300g at 4°C for 10 mins) and
resuspended in 3ml O.IM MES buffer pH 6.5. The samples were kept on ice
and sonicated with a Braun Labsonic 200 sonicator using a medium probe. The
samples were sonicated for 15 seconds (secs) with 30 secs cooling intervals,
repeatedly, until clearing was visible. Quantitative P-N-Acetyl-glucosaminidase
(EC 3.2.1.30) and N-Acetyl-galactosaminidase (EC 3.2.1.53) biochemical
assays performed in a final volume of 1.0ml. 500pl of O.OIM p-nitrophenyl-N-
Acetyl-P -D-glucosaminide or p-nitrophenyl-N-Acetyl-P-D-galactosaminide,
300-480pl of O.IM MES buffer pH 6.5 and 20-200pl of cellular sonicate were
mixed on ice then incubated at 37°C for 1 hr. The 1ml reaction was terminated
by adding 3mls of 0.2M Borate pH 9.8 and the absorbance measured at 420nm
(measures the amount of p-nitrophenol liberated from the substrate). One
enzyme unit was defined as the amount of enzyme which produced Immol of p-
nitrophenol in 1 min. The amount of protein in the assay reaction was estimated
using a BIO-RAD protein assay Idt with lysozyme as a standard. Enzyme
activity was expressed as units per milligram of protein.
55
2.10 Computer Analysis
The DNA sequence produced by this thesis was analysed using the
Wisconsin (Dereveux et al., 1984), Lipman-Pearson (Lipman and Pearson,
1985) and Clustal-V (Higgins et al., 1992) molecular biology programs on the
Vax VMS cluster, IRIX or DARESBURY. The nucleotide sequence produced
by this thesis has been given the Genbank accession number X78979.
2.10.1 Database Searching and Multiple Sequence Alignment
Protein sequences can be aligned with respect to their amino acid homology
and/or their amino acid identity. The homologies between proteins sequences
produced by this thesis and the SWISSPROT/National Biomedical Research
Foundation (NBRF) protein data bases were identified using the computer
program FastA. The FastA alogrithm uses the method of Lipman and Pearson
to search for similarities between one sequence (the query) and any group of
sequences already available in databases (Lipman and Pearson, 1985). FastA
displays the best similarities aligning horizontally the query sequence with the
identified homologous sequences. Double vertical lines identify identical amino
acids, whereas horizontal lines (gaps) within the protein sequence are
automatically inserted to maximise the alignment. FastA can also be used to
search for similarities between nucleotide sequences.
Protein homology searches using FastA was used to identify the NahA and
ORFl protein homologues within the SWISSPROT/NBRF databanlc. Proteins
with the top scores were identified and further analysed by the Clustal-V
computer program. Clustal-V quicldy and automatically produces multiple
alignments of proteins that are almost impossible to improve by eye, thereby
identifying common amino acid sequences present in these proteins.
56
2.10.2 The Hydropathy Profile
The hydropathy profile of predicted amino acid sequences was analysed on
the DNA strider software package (Kyte and Doolittle, 1982). This computer
program evaluates the hydrophilicity and hydrophobicity of a protein along its
amino acid sequence. The hydropathy profile of a protein is plotted on the basis
of a hydropathy scale, which takes into account the hydrophilic and
hydrophobic properties of each of the 20 amino acid side chains. The
hydropathy scale is based on experimental observations derived from the
literature. The program uses a movement-segment approach that continuously
determines the hydropathy within a segment of predetermined length (window)
as it advances through the sequence. The consecutive scores are plotted from
the amino to the carboxy terminus. The upper part of the hydropathy profile
indicates protein hydrophobicity and the lower part indicates protein
hyrophilicity.
57
CHAPTER 3
The Cloning and Expression of the waA Region from P. giMgzra/fs W83 in E. coZz
3.1 Introduction
The use of genetic manipulation to construct a DNA library of a pathogen is
an initial approach for isolating and characterising putative virulence factors of
pathogenic bacteria. Gene(s) that encode for the putative virulence factor can be
isolated, mutated and then reintroduced onto the chromosome of the original
pathogenic host strain. The generated isogenic mutants that differ in the
expression of a single factor can then be assayed for virulence and compared to
the wüd-type strain (Finlay, 1992).
The expression of the potential virulence factor under investigation is often
associated with pathogenic strains of a species or pathogenic members of a
genus (Falkow, 1988). The genus Porphyromonas contains at least three
species and exo-P -N-Acetyl-glucosannnidase activity is a feature of the most
virulent species, P. gingivalis (van Winlcelhoff et al., 1985; Mayrand and Holt,
1988; van Steenbergen et al., 1993). The cloning of a DNA fragment from P.
gingivalis W83 that encodes for p-N-Acetyl-hexosanimidase activity in E. coli
is the first step to determine if this enzyme activity functions as a virulence
factor. The cloned DNA fragment is termed the nah region.
58
Results
3.2.1 Detection of P-N-Acetyl-Hexosaminidase Activity in P. W83
P-N-Acetyl-hexosaminidase activity can be detected with a range of
chromogenic and fluorogenic synthetic substrates (Lewy and Concilie, 1966;
Cenci et al., 1992; Kouichi and Yamaji, 1992). The flourogenic substrates 4-
methylumbelliferyl-N-Acetyl-P-D-glucosaminide (MUAG) and 4-
methylumbelliferyl-N-Acetyl-p-D-galactosaminide (MUAGal) can detect exo-p-
N-Acetyl-glucosaminidase activity and exo-P-N-Acetyl-galactosaminidase
activity, respectively. Moreover, hydrolysis of both of these substrates suggests
the presence of P-N-Acetyl-hexosaminidase activity. To confirm the presence
of P -N-Acetyl-hexosaminidase activity in P. gingivalis W83, single colonies of
this strain were grown with MUAG or MUAGal incorporated into the agar
growth medium. Under long wavelength ultra-violet light, single colonies of P.
gingivalis W83 contained peripherial zones of fluorescence if either MUAG or
MUAGal was incorporated into the agar growth medium at a concentration of
lOOpg/ml. This observation demonstrates that P. gingivalis W83 expresses P-
N-Acetyl-hexosaminidase activity.
59
3.2.2 Isolation and Restriction Endonuclease Analysis of the naA Region from f . gingivalfs W83
3.2.2.1 Construction of a f . gmgiva&s W83 Expression Libraryin E. coK
Laboratory strains of E. coli do not express P-N-Acetyl-hexosaminidase
activity since they presumably lack the structural gene. This phenotype was
exploited to isolate and express gene(s) from P. gingivalis that encode p-N-
Acetyl-hexosaminidase activity in E. coli. To increase the expression of the
cloned gene(s) in E. coli, it was decided to use the plasmid expression vector
pTTQlS (Stark, 1987). This vector contains a tac promoter which can be
induced by IPTG to increase the transcription of the cloned gene(s), as well as
an added lac repressor gene to obtain tight transcriptional regulation of the
cloned insert (Figure 3.1a). Therefore, this vector is useful when expression of
the cloned gene may be detrimental to cell growth (Stark, 1987). Also, to limit
DNA rearrangement events in the genomic library the recombination-deficient
E. coli SURE™ was chosen as a transformation host strain.
High molecular weight chromosomal DNA from P. gingivalis W83 was
partially digested with the resriction endonuclease SauSA. This generated
fragments of 1-4 Idlobases (kb) which were analysed by agarose electrophoresis
(Figure 3.1b). The plasmid vector pTTQ18 was cleaved with BamUl and
treated with calf-intestinal-phosphatase to prevent self-ligation. Such treatment
removes the 5' phosphates from cohesive termini and prevents self-ligation,
thereby maximising the number of recombinant plasmids generated during
construction of the genomic DNA library. The phosphatased vector was self
ligated and visualised by agarose gel electrophoresis. Linear vector was
predominantly visible, suggesting that 5’ phosphates had been removed (Figure
3.1c).
60
Figure 3.1 The Cloning Vector and P.gingivalis W83 Genomic DNA
A
hla
pTTQlS
lacf
EcoRlS a dKpnlSmal
BamHl<=‘XbalP stlSphl
H in dm
1 2 3 1 2 3 4
21.6
0.5
421
B
Figure 3.1 (A) A diagramatic representation of the plasmid expressionvector pTTQlS. Polycloning-linker restriction enzyme sites are shown. The horizontal arrow highlights the BamHl restriction site that was used in the construction of the DNA library. The vertical arrow indicates the position of the tac promoter. The P-lactamase (bla) and lac repressor f/acP) genes are shown. (B) Size selected P gingivalis W83 chromosomal DNA. Lane 1; kilobase ladder (KBL). Lane 2; Sau3A treated W83 Chromosomal DNA ~ 1-4 kb in length. Lane 3; KBL. Numbers on the left are sizes in kilobases. (C) BamH 1 -phosphatased pTTQ18. Lane 1; KBL. Lane 2; BomH 1 -phosphatased pTTQ18. Lane 3; Self-ligated BamH 1 -phosphatased pTTQ18. The lower band in lane 3 may represent a small amount of uncut vector. Lane 4; KBL. Numbers on the left are sizes in kilobases. KBL molecular weight markers (kilobases): 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.6, 1, 0.5
61
The Sauih chromosomal DNA fragments, which contain 5’ phosphates,
were ligated into the phosphatased BaniEl restriction site immediately
downstream of the pTTQlS tac promoter (Figure 3.1a) (Stark, 1987).
Approximately O.Spg vector DNA was self-ligated or ligated with
approximately 0.5|iig of SauSA chromosomal DNA fragments, in a final
volume of lOpl. 2\xl of each ligation was used to electrotransform E. coli
SURE™ and ampicillin resistant colonies selected. Calculated yields in
transformations were 70 transformants/pg vector DNA for phosphatased-self-
ligated pTTQlS, and 1 x lO'̂ transformants/|Lig vector DNA for the
chromosomal DNA to phosphatased-pTTQ 18 ligation. The total number of
transformants generated in the DNA library was approximately 5000. To
estimate the number of recombinants in the 5000 transformants, 400 ampicillin-
resistant colonies were isolated and streaked on L-agar+ IPTG (10mM)4-X-gal
(0.004%) to test for blue or white colour. Of these, 98% were white (Lac ),
suggesting that approximately 98% of the transformants contained a cloned
DNA insert
3.2.2.2 Screening of the f . gingzy/w Gene Library For Exo-P -N-Acetyl-Glncosaminidase Activity
The P. gingivalis W83 gene library was screened on agar growth medium
containing lOOiug/ml MUAG and lOmM IPTG. In this way, E. coli colonies
expressing exo-P-N-Acetyl-glucosaminidase activity could be isolated and then
tested for exo-P -N-Acetyl-galactosaminidase activity. After screening
approximately 3500 recombinants a single fluorescent E. coli colony that
expressed both exo-p-N-Acetyl-glucosaminidase and exo-P-N-Acetyl-
galactosaminidase activity was identified. Figure 3.2 illustrates the detection of
this recombinant. Recombinant plasmid DNA was isolated from this fluorescent
62
colony and was used to retransform the host strain E. coli SURE™. All the
transformants expressed P-N-Acetyl-hexosaminidase activity. The isolated
recombinant plasmid was termed pALl and was used for further studies.
Figure 3 .2 Detection o f Exo-P-N-Acetyl-Glucosaminidase Gene Expression in E. coli SURE™(pALl)
MUAG
Exo-P-N-Acetyl-ghicosaminidase
+
4-Methylumbelliferone
GlcNAc
Figure 3.2. Detection of exo-P-N-Acetyl-glucosaminidase gene expression in E. coli SURE™ (pALl). Hydrolysis of the MUAG substrate by exo-P-N-Acetyl-glucosaminidase activity is shown. The fluorescent product 4-methylumbelliferone was detected by long-wavelength ultraviolet light.
63
3 2.2.3 Restriction Endonuclease Analysis of the naA Region
Plasmid pALl was cleaved with different restriction endonucleases. The
restriction endonucleases were used singly or in combinations when necessary.
Figure 3.3 shows the DNA fragments generated when plasmid pALl is cleaved
singly with different restriction enzymes and analysed by agarose gel
electrophoresis. The fragment sizes generated by the restriction enzymes used
are present in Table 3.1. It was noted that cleavage with BaniHl yehded DNA
fragments of approximately 4 kb and 4.5 kb. Since the size of pTTQ18 is close
to 4.5 kb, this suggested that pALl contained a cloned BamRl fragment of
approximately 4 kb (Figure 3.3 Lanes 2 and 3). The restriction enzyme profile
of pALl and the restriction map of pTTQ18 was used to generate a partial
physical map of pA L l. The generated partial physical map of the cloned nah
region present on pALl is shown in Figure 3.5.
64
Figure 3 .3 The Restriction Enzyme Profile o f pALl
1 2 3 4 5 6 7 8 9 10 11
4
2
1
Figure 3.3 The restriction enzyme profile of the recombinant plasmid pALl. Lanes (1) and (11); KBL. (2) BamHl. (3) BamHl pTTQlS control. (4) EcoRl. (5) Kpnl. (6) Xbal. (7) HincW. (8) Bjrl. The 0.7 kb DNA fragment is not shown in this lane. (9) Sphl. (10) Hindlll. Numbers on the right indicate sizes in kilobases. KBL molecular weight markers (kilobases): 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.6, 1
Table 3.1 Predicted DNA Fragments After Cleavage o f pALl with the Given Restriction Endonuclease
BamHl EcoRl Kpnl Xbal HincH Pstl Sphl JTindin
4.5 6.2 7.7 5.8 2.8 4.7 5.9 5.24.0 2.3 0.8 2.7 2.5 2.2 2.6 3.3
1.9 0.91.3 0.7
Table 3.1 Fragment sizes in kilobases after restriction enzyme cleavage of pALl.
65
3.2.2 4 Southern Blot Analysis of Chromosomal DNA
To confirm that the cloned nah region was present on the chromosome of P.
gingivalis W83, genomic DNA from this strain was cleaved with Pst\, Kpnl
and ffindlll and analysed using southern blot analysis with the cloned nah
region as a probe. Further, the Pstl restriction enzyme profile of chromosomal
sequences homologous to the cloned nah region was analysed in three other P.
gingivalis strains and in P. asaccharolytica 8503 and P. endodontalis 35406.
This would determine whether these other Porphyromonas strains contained
sequences that were homologous to nah region. The blot was probed with the 4
kb BamHl insert of pALl that contained the nah region and washed under high
stringency conditions (0.5x SSC 0.1% w/v SDS). As expected from the
restriction map of pALl, the probe hybridised to four Pstl fragments, two
Kpnl fragments and two Hindlll fragments on P. gingivalis W83 chromosomal
DNA (Figure 3.4 Lanes 1,2 and 3 respectively). Apart from the presence of an
additional Pstl fragment of approximately 0.3 kb in the chromosomal DNA of
strain 33277 (Lane 8), the Pffl-digested chromosomal DNA from three other
P. gingivalis strains gave similar patterns of hybridisation as that of P.
gingivalis W83 (Lanes 1 , 6 , 7 and 8). Sequences homolgous to the nah region
were not detected in the closely related species P. asaccharolytica and P.
endodontalis (Lanes 4 and 5 respectively). This observation suggests that these
Porphyromonas spp. do not contain a copy of the nah region.
66
Figure 3.4 Southern Blot Analysis of Porphyromonas Genomic DNA
1 2 3 4 5 6 7 8
• 8 . 0
2 .8
2 . 0
■1.3
■ 0 . 6
Figure 3.4 DNA-DNA hybridisation of Porphyromonas genomic DNA using the nah region as a probe. Lanes 1-3; restriction enzyme digested P. gingivalis W83 genomic DNA. Lane 1; Pstl. Lane 2; Kpnl. Lane 3; Hmdlll. Lanes 4-8; Pstl digested Porphyromonas genomic DNA. Lane 4; P. asaccharolytica ATCC 8503. Lane 5; P. endodontalis ATCC 35406. Lane 6; P. gingivalis WpH35. Lane 7; P. gingivalis 23A3. Lane 8; P. gingivalis ATCC 33277. Numbers on the right side indicate estimated molecular weight in kilobases.
67
3.2.3 Localisation and Expression of the Gene on pALl that Encodes for P-N-Acetyl-Hexosaminidase Auüdvity
3.2.3.1 Localisadoh on pALl
Localisation of the cloned gene on pALl that encodes for p-N-Acetyl-
hexosaminidase activity was determined by constructing various subclones and
deletions of pALl. pALl contains a number of restriction sites within the
multiple cloning site of pTTQlS (Stark, 1987), and these restriction sites
proved useful for the construction of subclones and deletions of pALl (Figure
3.5). DNA fragments from the nah region were either deleted from pALl or
isolated and resubcloned into pTTQ18. The subcloning of the purified 2.7 kb
Xbal fragment from pALl into the Xbal restriction site on pTTQ18 resulted in
the plasmid pAL2. Similarly, pAL3 and pAL4 were constructed by subcloning
the purified 2.6 kb Sphl and 2.3 kb EcdRl fragments respectively into the
corresponding restriction sites on pTTQ18. The deletion of the 2.3 kb EcdRl
fragment on pALl resulted in plasmid pAL5 (Figure 3.5). All subclones and
deletion derivatives of pALl shown in Figure 3.5 contain cloned DNA
fragments that have a similar orrientation to that of pA Ll.
It was noted that when E. coli SURE™ carried pAL2 or pAL3 these strains
were fluorescent on MUAG and MUAGal growth medium. In contrast, when
E. coli carried the EcoRl subclone (pAL4) or the EcoRl deletion (pAL5) no
fluorescence was detected with either substrate. These observations suggest that
the DNA encoding for P-N-Acetyl-hexosaminidase activity has been localised
to the 2.6 kb Sphl fragment on pAL3.
68
Figure 3 .5 Physical Map o f the Cloned DNA on pALl and its Derivatives.
PLASMID
pTTQIS
£ S Kp Sm B Xb Ps Sp H
pALI
E S Kp Sm Xb Ps Sp H
I I I IB P H/Kp Xb Sp E Ps
1 IPs B
pAL2
Xb
IXb
pAL3
Sp" T
Sp
pAL4
pAL5
1 kb
Figure 3.5 Physical map of the cloned DNA on pALl and its derivatives. The polycloning-1 inker site on pTTQlS and pALl is shown by boxes, with vertical arrows showing position. The thick black horizontal lines show P. gingivalis DNA. The thin black horizontal lines refer to pTTQlS DNA. The hatched horizontal arrow shows the position of the tac promoter on the various plasmids. Restriction sites: E; EcoRl. S; Sacl. Kp; Kpnl. Sm; Smal. B; BamHl. Xb; Xbal. Ps; Pstl. Sp; Sphl. H; Hindlll
69
3.2.3.2 IPTG-Induced Expression of P-N-Aceiyl-Hexosaminidase Activity
To determine the level of P-N-Acetyl-hexosaminidase activity, both P-N-
Acetyl-glucosaminidase (NAGase) and p-N-Acetyl-galactosaininidase
(NAGalase) activities were quantified for E. coli SURE™ carrying pALl and
its derivatives. As compared to P. gingivalis W83, the expression of P-N-
Acetyl-hexosaminidase activity in E. coli SURE™(pALl) is at low levels even
if the tac promoter is induced (Table 3.2). However, induction of the tac
promoter when E.coli SURE™ is carrying pAL2 or pAL3 causes P-N-Acetyl-
hexosaminidase activity to be significantly increased in these E. coli strains.
This strongly suggests that when E. coli SURE™ carries pAL2 or pAL3 the
expression of P-N-Acetyl-hexosaminidase activity is under the control of the
tac promoter on the cloning vector. Such increased expression with pAL2 and
pAL3 may be due to the elimination of transcriptional termination sequences
that are present on pALl in the upstream region of the gene that encodes for P-
N-Acetyl-hexosaminidase activity. Since the carriage of pAL4, pAL5 or
pTTQ18 by E. coli SURE™ results in a specific activity that is less than 1.0, it
is assumed that these plasmids do not encode for P-N-Acetyl-hexosaminidase
activity.
70
Table 3.2 p-N-Acetyl-Hexosaminidase Activities
NAGase Activity “ NAGalase Activity “Strain +IPTG -IPTG +IPTG -IPTG
E.CO& SURE™ (pTTQlS) 0.20 ± (0.03) 0.10 ±(0.01) 0.10 ±(0.05) 0.20 ± (0.03)
E.m/f SURE™ (pALl) 1.5 ±(0.5) 1.4 ±(0.1) 1.2 ±(0.1) 1.3 ±(0.1)
E.coZ:SURE™(pAL2) 23.3 ± (8.0) 3.3 ±(1.2) 18.8 ± (8.5) 2.2 ±(0.1)
SURE™ (pAL3) 32.5 ± (16.0) 3.7 ±(1.1) 12.1 ±(4.1) 2.1 ±(1.6)
E.co/i SURE™ (pAIA) 0.40 ±(0.10) 0.30 ±(0.10) 0.40 ± (0.20) 0.50 ± (0.20)
E.CO& SURE™ (pAL5) 0.30 ±(0.20) 0.20 ±(0.10) 0.30 ±(0.10) 0.30 ±(0.10)
P. gingivalis W83 ND^ 22.5 ±(7.5) ND* 21.5 ±(3.5)
Table 3.2 P-N-Acetyl-glucosaminidase (NAGase) and P-N-Acetyl- galactosaminidase (NAGalase) specific activities. Activity is expressed as units/mg protein. “ The mean ± (the standard deviation) of at least two independant observations. * ND; not determined
3.2.3.3 Expression of Proteins in a Cell-Free and Minicell System
Recombinant plasmids can be transfered to mutant strains of E. coli that
segregate into minicells. Minicells are small anucleate cells and contain only
plasmid DNA (Macrina, 1984). Minicells carrying pTTQlB encode for at least
two major polypeptides, namely the LaclQ repressor (38.1 kD monomer) and
the P-lactamase (28.9 kD processed) proteins (Figure 3.6a Lane 1) (Stark,
1987). Initial experiments with DS410 minicells carrying pALl, pAL2 or
pAL3 failed to radiolabel the cloned gene(s) product(s) even when minicells
were induced with IPTG (data only shown for pAL2) (Figure 3.6a Lane 2).
An in vitro DNA directed transcription-translation system is a cell-free kit
derived from E. coli S30 extract. A DNA-directed transcription-translation kit
71
was used to radiolabel the proteins encoded by the recombinant plasmid pALl
and the cloning vector pTTQlS (Figure 3.6b). With this system, it was possible
to identify two non-vector polypeptides o f approximately 69 kD and 90 kD
(Figure 3.6b Lane 2).
Figure 3 .6 Expression o f nah Region Proteins with an E. coli in vitro Transcription-Translation and Minicell System
100
69
46
30
100
69
46
30
B
Figure 3.6 Autoradiographs of L-[35S]methionine-labelled polypeptides analysed by SDS PAGE. (A) IPTG induced minicells carrying pTTQlS and pAL2. Lanel; pTTQlS. Lane 2; pAL2. (B) DNA directed transcription-translation of plasmids pTTQlS and pALl. Lane 1; pTTQlS. Lane 2; pALl. Numbers on the right side indicate molecular weight markers in kilodaltons. The large horizontal arrow highlights the 90 kD polypeptide. The small horizontal arrow shows the position of the 69 kD polypeptide.
72
3.3 Discussion
A single E. coli recombinant from a P. gingivalis W83 gene library has been
isolated and shown to express P-N-Acetyl-hexosaminidase activity. The isolated
recombinant plasmid pALl contains a P. gingivalis W83 chromosomal DNA
insert of approximately 4 kb in length, which is termed the nah region.
Subclone and deletion analyses of the nah region, suggest that the smallest
DNA fragments that encode for P-N-Acetyl-hexosaminidase activity are the
2.7kb Xbal insert on pAL2 and the 2.6 kb Sphl insert on pAL3, and that the
DNA encoding for this enzyme activity spans the EcoRl restriction site in the
nah region. Since it was not possible to separate the exo-P-N-Acetyl-
glucosaminidase and exo-P -N-Acetyl-galactosaminidase activities by
subcloning, one gene may be responsible for the p-N-Acetyl-hexosaminidase
activity.
The nah region probe hybridises to chromosomal DNA fragments of P.
gingivalis W83 as expected from restriction mapping pALl. Hybridisation to
DNA fragments of approximately 0.7, 0.9, and 2.2 kb in the Pstl digested
chromosomal DNA of P. gingivalis strains is in agreement with the sizes of the
Pstl fragments that are predicted from restriction mapping pALl. The only
Pstl restriction enzyme site polymorphism between the different strains of P.
gingivalis at this region of the chromosome is the presence of an additional
fragment of approximately 0.3 kb in the chromosomal DNA of stram 33277.
Taken together, these hybridisation patterns could suggest that the nah region is
present as a single copy on the chromosome of P. gingivalis W83, WpH35,
23A3 and 33277. Further, sequences homologous to the nah region are not
detected in the closely related species P. asaccharolytica and P. endodontalis.
The expression of exo-p-N-Acetyl-glucosaminidase activity is one of the
73
discriminatory tests used to distinguish between P. gingivalis and other
members of the genus Porphyromonas (Laughon et al., 1982; van Winlceüioff
et al., 1985). The southern blot results and the lack of exo-p-N-Acetyl-
glucosarninidase activity in P. asaccharolytica and P. endodontalis indicates
that these strains that do not contain a copy of the nah region. Since the nah
region appears to be present in P. gingivalis and absent from other
Porphyromonas spp., the cloned DNA fragment is potentially useful as a DNA
probe for species identification. DNA probes have already been used for the
detection of P. gingivalis in subgingival plaque samples from periodontal
disease (Yasui et al., 1993), however many other oral microrganisms will be
required to evaluate the effectiveness of the nah region as a species-specific
probe for the detection of P. gingivalis in clinical samples.
When E. coli SURE '̂' ̂ carries pAL2 or pAL3 induction of the tac promoter
increases the expression of P-N-Acetyl-hexosaminidase activity. If the P-N-
Acetyl-hexosaminidase activity seen in E. coli SURE™ (pALl) is a result of
transcription from the tac promoter, IPTG-inducible enzyme activity would be
expected. However, IPTG-inducible enzyme activity was not detected in this
strain. Moreover, E. coli SURE™ (pALl) has P-N-Acetyl-hexosaminidase
activity that is independent of IPTG addition, which suggests that the E. coli
transcriptional apparatus is recognising a promoter or promoter-lilce sequence
5' to the P-N-Acetyl-hexosaminidase gene. Several other reports also indicate
that E. coli can recognise promoter or promoter-like sequences on DNA
fragments from P. gingivalis (Bourgeau et al., 1992; Park and Me Bride, 1992;
Joe et al., 1993).
74
Applying pALl and pTTQlS to an in vitro transcription-translation system
identified two non-vector encoded proteins of approximately 69 kDa and 90
IcDa that may be encoded by the nah region. The reason for the failure to
obtain expression in E. coli minicells is not clear. One possibility is that
expression of nah region protein(s) may be detrimental to E. coli, since the
presence of pA Ll, pAL2 or pAL3 seemed to reduce the yeüd of minicells and
alter the colonial morphology of E. coli strains. Further, it may be possible that
the in vitro system incorporates radioactive label into proteins more efficently
than in vivo methods (Pratt et al., 1981), or allows the high level expression of
proteins that are repressed in vivo (Collins, 1979).
Lastly, the cloning and expression of the nah region in E. coli is an initial
approach for testing Koch's postulates in the molecular form. Since, the aim is
to inactivate the expression of P-N-Acetyl-hexosaminidase activity in P.
gingivalis by allelic replacement, we must understand the genetic organisation
of the nah region. The molecular characterisation of the nah region is the
subject of the next chapter.
75
CHAPTER 4
Molecular Characterisation of the waA Region by DNA Sequence Analysis
4 .1 In tro d u ctio n
DNA sequence analysis can be used to define the boundaries of genes that
are important for virulence and may yield clues about gene regulation and
function. The DNA sequence can be used to determine the structure of the gene
and predict the primary amino acid sequence of the virulence factor. Discrete
areas of amino-acid sequence homology can be then be identified and may
provide information about the functions of various domains (Macrina, 1984;
Macrina et al., 1990; Finlay, 1992). Further, the search for functional motifs
in the predicted amino-acid sequence of proteins and for sites of covalent
modification may predict the cellular location of the final gene product and aid
in the study of a purified protein (Aitken, 1990).
76
4.2 Results
4.2.1 DNA Sequence and Computer Analysis of the nah Region from P. gingivaUs W83
4.2.1.1 The DNA Sequencing Strategy
The DNA sequences that code for p-N-Acetyl-hexosaminidase activity are
suggested to span the EcoRl site within the nah region (Chapter 3). As a result,
initial analysis resolved the nucleotide sequence on both sides of this EcoRl
site. This analysis eventually led to the DNA sequence of the 2.9 kb Kpnl-
BamHl fragment from pA Ll. The DNA sequence of the entire nah region was
then completed by determining the nucleotide sequence of the 1.3 kb EamHl-
Xbal from pALl. The nucleotide sequence of the nah region was resolved at
least once for each DNA strand. The DNA sequencing strategy adopted and a
sequencing gel used in this study are shown in Figure 4.1 and 4.2 respectively.
4.2.1.2 The DNA Sequence of the nahA Gene and the Predicted Amino-Acid Sequence of the P-N-Acetyl-Hexosaminidase Protein (NahA)
Computer analyses of the DNA sequence of the Kpni-BanMl fragment from
pALl identified a single open reading frame (prj) of 2334 base pairs (bp) Üiat
encoded a protein of 111 amino-acids with a predicted molecular mass of 87
kDa. This orf is termed the nahA gene and the predicted polypeptide encoded
by this orf is referred to as the NahA protein. The position of the nahA gene
and the predicted amino acid sequence of the NahA protein is shown within the
complete DNA sequence of the nah region (Figure 4.3).
77
Figure 4.1 The DNA Sequencing Stategy used in this Study
M - T I I I I IB P Kp Xb Sp I e I Ps Ps
Ikb
Figure 4.1 The DNA sequencing strategy. The restriction map of the nah region is shown. Vertical lines indicate restriction sites. The spanning EcoRl site is boxed. Arrow length indicates DNA fragments that were subcloned from the 4 kb EomHl insert on pALl into appropriately cleaved M13mpl8 and M13mpl9. Arrow direction shows from 5’ to 3’. The shaded boxes refer to the part of the DNA strand that was sequenced from 5’ to 3’. The box with diagonal lines at the bottom of the figure indicates the sequence that is shown in Figure 4.2 (sequencing gel). Abbreviations: B; BomHl. P; Pstl. Kp; Kpnl. Xb; Xbal. Sp; Sphl. E; EcoRl.
78
Figure 4.2 A DNA Sequencing Gel used in this Study
A T C G
P. gingivalis DNA
M13 mp18 Polylinker
3' ■ G
A C G T
L_ C 5'
Asfl
Figure 4.2 Autoradiograph of a DNA sequencing gel used in this study. The DNA sequence is read from bottom to top. The bottom of the gel contains part of the M13mpl8 polyclonal linker DNA sequence. As the sequence continues it is interupted by the Pstl site that is proximal to the EcoRl in the nah region. The DNA sequence that follows this Pstl site would eventually lead into the EcoRl site in the nah region. Abbreviations: A; Adenosine. T; Thymidine. C; Cytidine. G; Guanosine
79
Figure 4.3 The DNA Sequence of the 3974 bp BamRl Fragment from pALl and the Position of the nahA Gene.
1
6 1
121
1 8 1
2 4 1
3 0 1
3 6 1
4 2 1
4 8 1
5 4 1
6 0 1
6 6 1
7 2 1
7 8 1
8 4 1
9 0 1
9 6 1
1 0 2 1
1 0 8 1
1 1 4 1
1 2 0 1
1 2 6 1
1 3 2 1
1 3 8 11
1 4 4 11 7
1 5 0 13 7
5a/nH1GGATCCGTGAAGAGTATGGAGGATTCTACCTTGCCGGTACCCAAACATGTGGGACAACAC
TCTTCGGTATGGATGACCATCGCCGGACGAACAGGCTAGCGCGTAATCTGCATAACGCCG
A A TTTGCTCAATGGCAAGATATTGTGTCGCGCTCTGTCAGCCGACATCAATTTGACCATG
TGTTCGTACAACTGCTGACGGTGCTGGGCTTCGCTCATATCGATGAAGTCCACTACGATG
Pst^ATACCACCCATATCGGAAGCGCAATTGTCTGGCCAGTTCTTCGGCTGCAGCCATATTGAC
ATCGACGGCCGTTGCTTCCTGCTCGGTACAGCCACGCGAGCGGTTACCGCTATTAACATC
CTCCACGTGCAAGGCCTCGGTCTGCTCAATAATCAGATAAGCACCACTCTTATACGTAAC
AGTACGTCCGAAGAGAGCTTTAATCTGCTTCGTAATGGCAAAATGGTCGAAAATGGGTAG
TTCGCCCTGATAATACTGAACGATTTCCTCCCGTCCCGGAGCTATCAACTCGACATAGTC
GCTCAAACTGTCCCGAAAAGCCTTGTCATTGACAATAATACTCTGGTAAGAGGGATTGAA
GTTGTCCCTCAAGAGTCCCAGAGTACGGCTGGCTTCCTCATAAACAATGGACGGAGCCTT
Hinm\GGCACGCAAAAGCTTCTTGATGTTGTCATCCCAACGACGTAGTAGGCTCTGCAACTCTTT
GTCCAATTCGGATGCCCGTTTACCTTCAGCAGAAGTGCGTATGATAACGTCAAAATTCTT
GGGCTTGATGCTGATAATCAACTGCGCAAGCGTGCTCGCTCTTCGGCAGAACGAATCTTC
TGAGACACCGACACTTTGTCTGCAAAGGGTAACCAAAACCAATGAACGGCCGGCAAAAGA
AAGCTCGGCAGTCAGGCGTGGGCCTTTGGTAGAGATCGATCTTCGTCCGAAGTTCCCCGA
ACTGACAGGTAAAGTGGCAGAGAAAGCCCTCGTGGCAGCGGATATTACCGTCAATAAGAA
Kpn^CATGGTACCGTTCGATTCTCGCTCTGCATTCCAGACATCGGGCTTCCGCGTGGGTACTCC
GGCCATCACCACTCGTGGCGTAAAAGAAGATAAGATGGGCTATATCGTGGAGTTGATAGA
CCGTGTGCTCTCCGCACCGGAGGACGAAGCCGTAATAGCATCGGTTCGTACCGAAGTCAA> > > > > > > >
CCG G A TG A TG GCCG A TTA TCC TCTCTTTGCTTGGTAAGAAATCCCAAGAAGTCTA T C C C C
> > > > > > > < < < < < < < < < < < < < < XbayCTATCCTTCTCTTAGGATAGGGGATAGTTCTTTACTGATATTCTAGAAAGCACGCAAGGT
TGTGTG A TG CCG G TG GTG CCCG G AC A C CCTCA CTTA CG TGC TTTTTTG TTTG A TCA CTA A
Sph\TTCATA CTTGACAATGAAACGACTGACTTTCGGAGCATGCATTTGCTGCCTCCTGTCTCT
L ̂ r-r-c-TT-rTTATGGCCTGCTCACAGAAAGCAAAGCAGGTGCAAATCCCCGAATACGACAAGGGTATAAA H m a 1 C S Q K A K Q V Q I P E Y D K G I N
'^P o te n tia l signal peptidase II cleavage siteCATCATTCCCTTGCCGATGCAGCTGACCGAATCGGACGACAGCTTTGAGGTCGATGATAA
I I P L P M Q L T E S D D S F E V D D K
6 0
120
1 8 0
2 4 0
3 0 0
3 6 0
4 2 0
4 8 0
5 4 0
6 0 0
6 6 0
7 2 0
7 8 0
8 4 0
9 0 0
9 6 0
1 0 2 0
1 0 8 0
1 1 4 0
1 2 0 0
1 2 6 0
1 3 2 0
1 3 8 0
1 4 4 01 6
1 5 0 03 6
1 5 6 05 6
80
1 5 6 1 GACCACTATCTGCGTATCTGCCGAAGAGCTAAAGCCTATCGCTAAACTTCTTGCCGACAA 1 6 2 05 7 T T I C V S A E E L K P I A K L L A D K 7 6
1 6 2 1 GCTAAGAGCATCAGCCGACCTCTCTCTCCAGATAGAGATAGGCGAGGAGCCTTCGGGGAA 1 6 8 07 7 L R A S A D L S L Q I E I G E E P S G N 9 6
Potential Shine Dalgamo Region %1 6 8 1 TG CTA TTTACA TCG G TG TCG A TA CG G CTC TTC CTC TTA A AG AAGAQQGTTA TA TG C TC C G 1 7 4 0
9 7 A I Y I G V D T A L P L K E E G Y M L R 1 1 6
1 7 4 11 1 7
1 8 0 11 3 7
ATCCGATAAGCGTGGTGTCAGTATCATCGGCAAATCTGCCCATGGTGCTTTCTACGGTAT S D K R a V S I I G K S A H G A F Y G M
% Potential nucleotide binding motifQCAGACTTTGCTCCAGCTCCTTCCTGCCGAAGTGGAATCTTCGAATGAGGTACTGCTCCC
Q T L L Q L L P A E V E S S N E V L L P
1 8 0 01 3 6
1 8 6 01 5 6
1 8 6 1 CATGACGGTGCCCGGCGTCGAGATCAAGGACGAACCGGCATTCGGCTATCGTGGCTTTAT 1 9 2 01 5 7 M T V P G V E I K D E P A F G Y R G F M 1 7 6
1 9 2 1 GCTGGATGTATGCCGTCATTTCCTTTCGQTGGAGGACATCAAGAAGCATATCGACATCAT 1 9 8 01 7 7 L D V C R H F L S V E D I K K H I D I M 1 9 6
1 9 8 1 GGCCATGTTCAAGATCAATCGTTTCCATTGGCACCTGACAGAGGATCAGGCATGGCGTAT 2 0 4 01 9 7 A M F K I N R F H W H L T E D Q A W R I 2 1 6
2 0 4 1 CGAAATCAAGAAATACCCACGACTGACCGAAGTGGGGTCTACAAGGACGGAAGGGGACGG 2 1 0 02 1 7 E I K K Y P R L T E V G S T R T E G D G 2 3 6
2 1 0 1 TACGCAGTACTCCGGTTTCTACACGCAGGAGCAAGTACGGGATATTGTACAATACGCATC 2 1 6 02 3 7 T Q Y S G F Y T Q E Q V R D I V Q Y A S 2 5 6
2 1 6 12 5 7
2 2 2 12 7 7
G GATCATTTCATTACGGTGATTCCCATGATCGAAATGCCCGGACATGCCATGGCTGCCCT 2 2 2 0 D H F I T V I P M I E M P G H A M A A L 2 7 6
EcoR^CGCTGCTTATCCGCAGTTCCGTTGCTTCCCACGCGAATTCAAGCCACGGATTATCTGGGG 2 2 8 0
A A Y P Q F R C F P R E F K P R I I W G 2 9 6
2 2 8 1 AGTGGAGCAGGATGTTTATTGTGCCGGTAAGGACAGCGTCTTCCGTTTTATCTCTGATGT 2 3 4 02 9 7 V E Q D V Y C A G K D S V F R F I S D V 3 1 6
2 3 4 1 TATCGACGAGGTAGCACCCCTTTTCCCCGGCACATACTTCCATATCGGAGGGGACGAATG 2 4 0 03 1 7 I D E V A P L F P G T Y F H I G G D E C 3 3 6
2 4 0 1 CCCTAAAGATCGATGGAAGGCTTGTTCGCTTTQTCAGAAGCGTATGCGTGACAATGGGTT3 3 7 P K D R W K A C S L C Q K R M R D N G L
/»Sf12 4 6 1 GAAAGACGAACACGAGCTGCAGAGTTATTTCATCAAACAAGCTGAAAAGGTCTTACAAAA
3 5 7 K D E H E L Q S Y F I K Q A E K V L Q K
2 4 6 03 5 6
2 5 2 03 7 6
2 5 2 1 GCACGGCAAGAGACTGATCGGTTGGGATGAAATCCTCGAAGGCGGGCTTGCACCTTCTGC 2 5 8 03 7 7 H G K R L I G W D E I L E G G L A P S A 3 9 6
2 5 8 1 CACCGTTATGAGCTGGCGTGGAGAGGATGGTGGCATCGCAGCGGCTAATATGAATCACGA 2 6 4 03 9 7 T V M S W R G E D G G I A A A N M N H D 4 1 6
2 6 4 1 TGTGATCATGACTCCGGGTAGCGGAGGTCTCTACTTGGATCATTATCAGGGAGATCCGAC 2 7 0 04 1 7 V I M T P G S G G L Y L D H Y Q G D P T 4 3 6
2 7 0 1 CGTCGAGCCTGTTGCCATCGGAGGTTATGCTCCATTGGAOCAAGTGTATGCTTACAATCC 2 7 6 04 3 7 V E P V A I G G Y A P L E Q V Y A Y N P 4 5 6
Nae^2 7 6 1 TTTGCCGAAAGAATTGCCGGCCGATAAGCATCGCTACGTGCTCGGAGCACAGGCCAATCT 2 8 2 0
4 5 7 L P K E L P A D K H R Y V L G A Q A N L 4 7 6
81
2 8 2 1 GTGGGCAGAATACCTCTATACTTCCGAACGATACGACTATCAGGCCTATCCAAGGCTACT 2 8 8 04 7 7 W A E Y L Y T S E R Y D Y Q A Y P R L L 4 9 6
2 8 8 14 9 8
GGCTGTGGCAGAGCTTACCTGGACACCGTTGGCCAAGAAAGATTTTGCCGATTTCTGTCGA V A E L T W T P L A K K D F A D F C R
2 9 4 05 1 6
2 9 4 1 CCG TTTGGATAATGCCTGCGTTCGTCTGGACATGCATGGTATCAATTACCACATTCCGCT 3 0 0 05 1 7 R L D N A C V R L D M H G I N Y H I P L 5 3 6
3 0 0 1 GCCCGAACAACCGGGTGGCTCTTCCGACTTTATAGCCTTTACGGACAAGGCTAAGCTGAC 3 0 6 05 3 7 P E Q P G G S S D F I A F T D K A K L T 5 5 6
3 0 6 1 CTTCACGACATCGCGTCCGATGAAAATGGTCTATACGCTGGACGAAACCGAACCATCCCT 3 1 2 05 5 7 F T T S R P M K M V Y T L D E T E P S L 5 7 6
3 1 2 1 CACATCGACTCCTTACACGGTCCCTCTTGAATTTGCACAAACGGGCCTTCTGAAGATTCG 3 1 8 05 7 7 T S T P Y T V P L E F A Q T G L L K I R 5 9 6
3 1 8 1 TACCGTCACGGCCGGTGGGAAGATGAGTCCCGTACGCCGCATTCGTGTGGAGAAACAACC 3 2 4 05 9 7 T V T A G G K M S P V R R I R V E K Q P 6 1 6
3 2 4 1 CTTCAATATGTCAATGGAAGTACCGGCACCGAAACCCGGACTGACCATTCGTACGGCTTA 3 3 0 06 1 7 F N M S M E V P A P K P G L T I R T A Y 6 3 6
PsH3 3 0 1 C GGTGACTTATATGATGTGCCTGATCreCAOCAGGTAGCCTCATGGGAAGTAGGGACCGT 3 3 6 0
6 3 7 G D L Y D V P D L Q Q V A S W E V G T V 6 5 6
3 3 6 16 5 7
TAGCTCTTTGGAGGAAATCATGCACGGGAAAGAGAAGATAACTTCTCCTGAAGTACTGGAS S L E E I M H G K E K I T S P E V L E
3 4 2 06 7 6
3 4 2 16 7 7
GCGCAGAGTTGTAGAGGCTACCGGTTATGTGCTTATTCCGGAGGATGGGGTATATGAGTT R R V V E A T G Y V L I P E D G V Y E F
3 4 8 06 9 6
3 4 8 1 CTCTACGGAAAACAACGAGTTTTGGATTGATAATGTGAAGCTGATCGACAATGTGGGCGA 3 5 4 0
6 9 7 S T E N N E F W I D N V K L I D N V G E 7 1 6
3 5 4 1 AGTAAAGAAATTCTCCCGTCGCAATAGCAGTCGTGCCCTTCAGAAAGGCTACCATCCGAT 3 6 0 07 1 7 V K K F S R R N S S R A L Q K G Y H P I 7 3 6
3 6 0 1 CAAGACGATATGGGTCGGAGCCATACAAGGTGCCTGGCCTACTTATTGGAACTACAGCAG 3 6 6 07 3 7 K T I W V G A I Q G A W P T Y W N Y S R 7 5 6
3 6 6 1 GGTAATGATACGGCTCAAGGGAGAAGAAAAGTTCAAGCCGATCTCGTCCGATATGCTCTT 3 7 2 07 5 7 V M I R L K G E E K F K P I S S D M L F 7 7 6
3 7 2 1 TCAATAAAGTCTCATCACTTTATGGTTAAAGACGGCAACCTTTCCGCAGAAAAAAACGCA
7 7 7 Q *
3 7 8 0
3 7 8 1 AGGCCTAAATTAACCCCAACTACCAGAAGAGGGTGTGTCAAAATTCAGTTTAAGCAGGGG 3 8 4 0> > > > > > > > > > < < < < < < < < < <
3 8 4 1 GCCGGGATCATCGTTTGTATAGAAAAACCGCTATCAAATCAATAGTGTCCTAAATGTTTG 3 9 0 0
3 9 0 1 CTTAGAGGGGGGAGTTTTTAGTATGAGAGCTGAGTCGCTCCAATACAAGACTTTTGGGAT 3 9 6 0
BamHA3 9 6 1 TTGAGGGGCGATCC 3 9 7 4
Figure 4.3 DNA sequence of the nah region. The nucleotide sequence and the predicted amino acid sequence are numbered. Restriction enzyme sites are shown in bold. Hairpin structures are denoted by > > < < and potential -10 and -35 regions are double underlined. The predicted N-terminal signal peptide of NahA is shaded and shows the potential signal petidase II cleavage site. The nucleotide binding motif of NahA is singly underlined. A potential internal Shine-Dalgarno region at position 1724 of the nucleotide sequence is shown. The arrow at position 1086 refers to the first ATG of the orfl gene.
82
Many bacterial toxins and virulence factors are secreted proteins that have
hydrophobic signal peptides located at the N-terminus (von Heijne and
Abrahmsen, 1989; Finlay, 1992). Signal peptides help to translocate the protein
across the cytoplasmic membrane and they have similar structural features, that
is, a positively charged amino terminal segment (> 1 K or R), followed by a
long hydrophobic region (rich in A and L, devoid of F, K, D, E, K, H and R)
and a processing site (von Heijne and Abrahmsen, 1989; Fugsley, 1993). The
processing site of bacterial lipoproteins is usually a L-X-(AG)>1<C sequence,
were the cysteine residue must be modified to glycerylcysteine to allow
cleavage by a cytoplasmic membrane signal peptidase II (the downward arrow
between AG and C shows the cleavage site) (Fugsley, 1993). The most
probable site of signal peptidase cleavage within a given amino acid sequence
can be predicted by the computer program SIGSEQ (von Heijne, 1986). Using
this computer program, the predicted amino acid sequence and the hydrophathy
profile of the NahA protein (Figure 4.4), it was possible to identify a potential
lipoprotein signal peptide and signal peptidase II cleavage site at the N-terminus
of the of the NahA protein. From the predicted amino acid sequence of the
NahA protein the site of action of the signal peptidase II would be the bond
between the alanine at position 18 and the cysteiue at postion 19, with the
mature form of NahA being acylated at this cysteine residue (Figure 4.3).
A large number of diverse enzymes with a requirement for adenosine
triphosphate (ATP) or guanosine triphosphate (GTF) contain a highly conserved
glycine-rich motif involved in binding of the nucleotide (Aitken, 1990). The
Walker A motif (G-X-S-G-X-G-G-K-T/S) and the Walker B motif R-(l-3)-G-
(3)-L-0-0-0-0-(O-2)-D, where 0 indicates any hydrophobic amino acid and
numbers (0-3) indicate the spacing between the conserved residues, are
characteristically present on proteins with ATPase, Idnase or ATP synthetase
activities (Walker et al., 1982; Fugsley, 1991). It is thought that the Walker A
83
motif forms a loop, with the first glycine in contact with the ribose and the last
glycine lying adjacent to the pyrophosphate (Aitken, 1990). Walker motifs are
also found on membrane proteins involved in the transport of various solutes
across membranes (Pugsley, 1991). Using the computer program MOTIFS, it
was possible to identify a sequence homologous to the Walker A motif in the
predicted amino acid sequence of the NahA protein. This sequence, 121-
GVSIIGKS-128 of the NahA protein shares homology to the Walker A motif at
the residues shown in bold above. The position of this motif is shown in the
predicted amino acid sequence of NahA (Figure 4.3), and m the hydropathy
profile of the NahA protein (Figure 4.4). It may be possible that this region of
the NahA protein can bind ATP/GTP, however the possible role of this
hypothetical binding site is not clear.
Figure 4.4 The Hydropathy Profile of the NahA Protein
2
1
0
- 1 -
100 20 0 3 0 0 40 0 500 6 0 0 7 00111111111-[ m 111111111111111111 I I I 11 n 11 M 111111111 n 111111111111111111111111
l l l l l l l l l l l l l l l l l l l l l l l l i l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l i l l
10
-1
100 200 3 0 0 400 500 6 0 0 70 0
Figure 4.4 The hydropathy profile of the predicted amino acid sequence of the NahA protein. The small downward arrow indicates the hydrophobic N-terminal signal peptide. The large upward arrow shows the position of the potential nucleotide binding motif. The hydropathy profile was plotted with a window 20.
Promoters that are recognised by the E. coli holoenzyme have the
conserved (5’)TATaaT(3’) sequence at the -10 region, followed by an
interconsensus spacing of 16-18 bp and a conserved (5’)TTGAca(3’) sequence
at the -35 region (the most highly conserved sequence bases are shown in
uppercase) (Hawley and McClure, 1983; Cowing et al., 1985). At this level of
degeneracy, promoters would occur approximately every 200 bp in a random
sequence. Therefore, only on the basis of the -35 and -10 consensus sequence,
it can sometimes be difficult to predict the position of an genuine promoter
(Dykes et al., 1975; Pazzani, 1992). No DNA sequences highly homologous to
the E. coli -10 and -35 consensus sequence were detected 5’ to the nahA gene.
Nevertheless, approximately 130 bp 5’ to the nahA gene is a region that
contains 4 bases (TTGgtA) homologous to the -35 consensus sequence and 3
bases (TATccc) homologous to the -10 consensus sequence, with an
interconsensus spacing of 16 bp (Figure 4.3). It is possible that this region of
pALl may act as a recognition sequence for E. coli Ea^® holoenzyme and allow
transcription of the nahA gene to be independent of the upstream tac promoter.
Computer analysis identified a potential hairpin structure both 5’ and 3’ to
the nahA gene (Figure 4.3). Figure 4.5 shows the proposed structure of the
hairpin that is 5 ’ to the nahA gene. Position 1223-1325 of the nucleotide
sequence has an estimated AG value of -31 kcal/mol as determined by the
computer program FoldRNA. This hairpin structure may act as a
transcriptional terminator in E. coli SURE™(pALl) and could partly explain
why expression of P-N-Acetyl-hexosarninidase activity in this strain could not
be increased by IPTG. Indeed, in pAL2 and pAL3 this hairpin structure is
absent and P-N-Acetyl-hexosaminidase activity could be increased by the
addition of IPTG (Figure 3.5 and Table 3.1). The 5’ hairpin structure also
contains the implicated -10 region and because of the position of the -10 region,
it could be imagined that the binding of E. coli RNA polymerase may be
85
repressed (Figure 4.5). This suggestion and the lack of homology with the -10
and -35 consensus sequence of Eo-’° holoenzyme may explain why E. coli
(pALl) has poor expression of P-N-Acetyl-hexosaminidase activity as
compared to P. gingivalis (Chapter 3). The second potential hairpin loop
structure 3’ to the nahA gene (Figure 4.3 nucleotide position 3841-3900) has a
calculated AG value of -8.6 kcal/mol and may act as a rho-dependant
transcriptional termination sequence (Rosenberg and Court, 1979; Brendel and
Trifonov, 1984). In addition, no other was detected 3’ to the nahA gene,
which may indicate that the nahA gene is a single transcriptional unit.
The initiation reaction of protein biosynthesis plays a major role in gene
expression (Gold and Stormo, 1987). Initiation of translation in prokaryotes is
commonly directed by an AUG start codon («90%of cases), in combination
with a Shine-Dalgarno (SD) sequence (AGGAGG) that possesses homology to
the 3’ end of 16S RNA incorporated into the 30S ribosomal subunit (McCarthy
and Gualerzi, 1990). The SD sequence is not essential for translation, but its
interplay may determine the efficiency of the translational initiation region
(McCarthy and Gualerzi, 1990). No obvious SD sequence was identified 5’ to
the putative AUG start codon of the nahA gene, however at position 1724 in the
nucleotide sequence a AGAGG sequence was found 5’ to an internal AUG
codon (Figure 4.3). If translation begins at this AUG codon, this internal orf
has a predicted molecular weight of 73 kDa, which might correspond with the
69 kDa polypeptide seen in the in vitro transcription-translation system.
86
Figure 4.5 The Proposed Hairpin Structure that is 5’ to the nahA Gene
c uu u
T-AC-GC-GT-AA-TT-A AG=-31 kcal/mol
CC-GC-GC-G
-10 region C-GT-A A-T T-A C-G
(5’)TCTTTGCÎTGGrAAGAAATCCCAAGAAGT TTCTTTACTGATATTCTAGAAAGCACGCAAGGTTGTO’l =» -35 region Xba 1
1223 1325
Figure 4.5 Structure of the implicated stem-loop between nucleotide 1223 and 1325 in the nah region. The -35 and -10 sequence that may be recognised be E. coli RNA polymerase are shown in bold and italic. The Xbal site 5’ to the nahA gene is underlined. The arrow indicates the downstream sequences that lead to the first AUG of the nahA gene. The region 1223-1325 has a AG value of -31 kcal/mol as determined by the computer program FoldRNA in the GCG program Irix.
87
4.2.1.3 Homology Between NahA and Other Enzymes
Protein data base (SWISSPROT/NBRF) searches revealed homology
between the predicted amino acid sequence of the NahA protein and a number
of P-N-Acetyl-hexosaminidase enzymes, including human lysosomal P-N-
Acetyl-hexosaminidase (Table 4.1 and Figures 4.6a-g). For nomenclature of the
proteins see Table 4.1. In human P-N-Acetyl-hexosaminidase, arginine residues
at position 178 of the a-subunit (HexA) and at postion 211 of the P-subunit
(HexB) have been shown to be essential amino acids within the catalytic site of
each subunit (Figure 4.6a and 4.6b respectively) (Kytzia and Sandhoff, 1985;
Brown and Mahuran, 1991).
A region of homology between the NahA protein and the other P-N-Acetyl-
hexosaminidase enzymes was most pronounced when all proteins were aligned
with each other, hi this region of the protein alignment there was the catalytic
domains of the a and p-subunits of human P-N-Acetyl-hexosaminidase and
twelve identical amino acids in all eight proteins. Moreover, the catalytic
arginine residues were present at a similar position in all proteins (Figure 4.7
position 23). This alignment would suggest the involvement of arginines in the
catalytic activity of these other enzymes, including the P-N-Acetyl-
hexosaminidase of P. gingivalis W83.
88
Table 4.1 Similarities of the P. gingivalis W83 p-N-Acetyl-Hexosaminidase (NahA) with Other Proteins
Organism Protein (amino adds)
Idmtity(*)
Homology(%)
Overlap(aminoadds)
Shown in figure
Man HexA (529) 25.1 67.3 426 4.6aMan HexB (556) 25.5 72.3 325 4.6bMouse HexA' (528) 25.7 67.0 373 4.6cMouse HexB’ (536) 26.5 67.3 325 4.6dSlime Mould NagA (532) 26.3 69.3 312 4.6eVibrio harveyi Chb (883) 29.3 70.6 208 4.6fVibrio vulnificus Hex (848) 30.8 73.3 208 4.6g
Table 4.1 Similarities of NahA with other proteins. Identity values are calculated by the amount of identical amino acids in a given overlap. The homology values are expressed taking into account conservative amino acid changes. The number of amino acids in each protein is shown in brackets beside the protein name. Protein name: HexA; a-subunit of human lysosomal P-N-Acetyl-hexosaminidase (Korneluk et at., 1986). HexA’; a-subunit of mouse lysosomal P-N-Acetyl-hexosaminidase (Beccari et al., 1992). HexB; P-subunit of human lysosomal P-N-Acetyl- hexosaminidase (O’Dowd et al., 1985). HexB’; P-subunit of mouse lysosomal P-N-Acetyl-hexosaminidase (Bapat at al., 1987). NagA; P-N- Acetyl-hexosaminidase of Dictyostelium discoideum (slime mould) (Graham et al., 1988). Chb; chitobiase outermebrane lipoprotein of Vibrio harveyi (Jannatipour et al., 1987; Soto-Gil et al., 1989). Hex; P-N- Acetyl-hexosaminidase of Vibrio vulnificus (Somerville and Colwell, 1993).
89
Figure 4.6a Protein Homology Between NahA and the a-subunit of HumanLysosomal p-N-Acetyl-Hexosaminidase
NahA
HexA
90 100 110 120 130 140ADLSLQIEIGEEPSGNAIYIGVDTALPLKESGYMLRSDKRGVSIIGKSAHGAFYGMQTLL
TGKRHTLEKNVLW SW TPGCNQLPTLESVENYTLTINDDQCLLLSETVW GALRGLETFS 90 100 110 120 130 140
NahA
HexA
QLLPAEVESSNEVLLPMTVPGVEIKDEPAFGYRGFMLDVCRHFLSVEDIKKHIDIMAMFK
QLVWKSAEGT-150
: : : I I : I I I : I I : : I I = : I I : I : : = : I : : I : I I I- FFINKTEIEDFPRFPHRGLLLDTSRHYLPLSSILDTLDVM AYNK
NahA
HexA
NahA
HexA
NahA
HexA
NahA
HexA
NahA
HexA
NahA
HexA
210 22 0 230 240 25 0 260INRFHWHLTEDQAWRIEIKKYPRLTEVGSTRTEGDGTQYSGFYTQEQVRDIVQYASDHFI: I I I I I I : : I : : : : | : : | | | | : : : : : | | | : : : : : | | : : |
LNVFHWHLVDDPSFPYES FTFPEU4RKGSYNPVTH------------1YTAQDVKEVIEYARLRGI2 00 210 220 230 240 250
27 0 280 290 3 00 310TVIPM IEM PGHAM AALAAYPQF--RCPPREFKPRIIW GVEQDVYCAGKDSVFRFISDVID
I : : : : | | | : : : : : | : : | : : : | : : : | : : : : : : : | : | : :RVLAEFDTPGHTLSW GPGIPGLLTPCYS-GSEPSGTFGPVN P8LNNTYEPM STFFL
2 60 270 280 290 300
3 3 0 34 0 350 360 370EVAPLFPGTYFHIGGDECPKDRWKACSLCQKRMRDNGLKDE-HELQSYFIKQAEKVLQKH
I I : : : I I : I : I : I I I I : I I : = I : I I : : I : = = : : I : I : : I : : : : —EVSSVFPDFYLHLGGDEVDFTCWKSNPEIQDBWRKKGFGEDFKQLESFYIQTLLDIVSSY
310 320 330 340 350 360
380 390 4 00 410 420 430GKRLIGWDEILEG--GLAPSATVMSWRGEDGGIAAANMNHDVIMTPGSGGLYLDHYQGDP
GKGYWWQEVFDNKVKIQPDTIIQVWRED- 370 380 390
- 1PVNYMKELELVTKAG- 4 00 410
-FRALLSAPW420
4 4 0 45 0 4 60 47 0 48 0 49 0TVEPVAIGGYAPLEQVYAYNPLPKELPADKHRYVLGAQAKLWAEYLYTSERYDYQAYPRL
Y L N R ISY G -- PDWKDFYWEPLAFEGTPEQKALVIGGEACMWGEYV-DNTNLVPRLWPRA 4 3 0 44 0 45 0 460 470
500 510 520 530 540 550LAVAELTWTPLAKKDFADFCRRLDNACVRLDMHGINYHIPLPEQPGGSSDFIAFTDKAKL
GAVAERLWSNKLTSDLTFAYERLSHFRCELLRRGVQAQPLNVGFCEQEFEQT 48 0 4 90 500 510 520
Figure 4.6a Protein homology between NahA and the a-subunit of human lysosomal P-N-Acetyl-hexosaminidase (HexA). Vertical lines align identical amino acids. Double dots align homologous amino acids. Horizontal lines are gaps that are automatically inserted to maximise the alignment. Numbers refer to amino acid position. The catalytic arginine residue of HexA is underlined at position 178.
90
Figure 4.6b Protein Homology Between NahA and the p-subunit of HumanLysosomal P-N-Acelyl-Hexosaminidase
100 110 120 130N ah A SADLSLQIEIGEEPSGNAIYIGVDTALPLKESGÏM LRSDiCRGVSIIGKSAHGAFYGMQTL
: I : I I : : : : : : ; : : | | : | | :H exB AEFQAKTQVQQLLVSITLQSECDAEPNISSDESYTLLVKEPVAVLKANRVWGAUIGLETF
120 130 140 150 160 170
N ah A
HexB
ISO 160 170 180 190LQLLPAEVESSNEVLLPMTVPGVEIKDEPAFGYRGmLDVCRHFLSVEDIKKHIDIMAMF
SQLV-------------Y Q D SY G T FTIN EST IID SPR FSH R G ILID T S|H Y LPV K IILK TLD A M A FN180 190 200 210 220
N ah A
HexB
2 10 22 0 230 240 250KINRFHWHLTEDQAWRIEIKKYPRLTEVGSTRTEGDGTQYSGFYTQEQVRDIVQYASDHF
KFINVLHWHIVDDQSFPYQSITFPEIÆNKGSYS - 2 40 25 0 260
- LSHVYTPNDVRMVIEYARLRG 270 280
NahA
HexB
NahA
HexB
2 70 280 290 300 310ITVIPM IEM PGHAM AALAAYPQF--RCFPREFKPRIIW GVEQDVYCAGKDSVFRFISDVI
IRVLPEFDTPGHTLSWGKGQKDLLTPCYSRQNKLDSFGPINPTIi NTTYSFLTTFF2 90 300 310 320 330
320 330 340 350 360 370DEVAPLFPGTYFHIGGDECPKDRWKACSLCQKRMRDNGLKDE-HELQSYFIKQAEKVLQK
: I : : : | | : : : | : | | | I : I = = : I = I I : : I : — : : | | | : : : : : :KEISEVFPDQFIHLGGDEVEFKCWESNPKIQDBMRQKGFGTDFKKLESFYIQKVLDIIAT 340 350 36 0 370 380 390
3 80 390 40 0 410 4 20 430N ah A HGKRLIGWDEILE--GGLAPSATVMSWRGEDGGIAAANMNHDVIMTPGSGGLYIJ3HYQGD
: I I I : I : : : : | | | : : : | | : : : : : : : : : : : : | : | | |HexB INKGSIVWQEVFDDKAKLAPGTIVEVWKDSAYPEELSRVTASGFPVILSAPWYLDLISYG
40 0 4 1 0 420 43 0 44 0 450
Figure 4.6b Protein homology between NahA and the P-subunit of human lysosomal P-N-Acetyl-hexosaminidase (HexB). Vertical lines align identical amino acids. Double dots align homologous amino acids. Horizontal lines are gaps that are automatically inserted to maximise the alignment. Numbers refer to amino acid position. The catalytic arginine residue of HexB at position 211 is double underlined.
91
Figure 4.6c Protein Homology Between NahA and the a-subunit of MouseLysosomal P-N-Acetyl-Hexosaniinidase
90 100 110 120 130 140NahA ADLSLQIEIGEEPSGNAIYIGVDTALPLKEEGYMLRSDKRGVSIIGKSAHGAFYGMQTLL
I : I I : : : : : : : | | : | | :HexA’ SNKQQTLGKNILWSWTAECNEFPNLESVENYTLTINDDQCLLASETWGAI^GLETFS
90 100 110 120 130 140
NahA
HexA’
150 160 170 180 190 200QLLPAEVESSNEVLLPMTVPGVEIKDEPAFGYRGFMLDVCRHFLSVEDIKKHIDIMAMFK
QLVWKSAEGT-150
-FFINKTKIKDFPRFPHRGVLLDTSRHYLPLSSILDTLDVMAYNK 160 170 180 190
NahA
HexA'
210 220 2 30 240 250 260INRFHWHLTEDQAWRIEIKKYPRLTEVGSTRTEGDGTQYSGFYTQEQVRDIVQYASDHFI
FNVFHWHLVDDSSFPYESFTFPELTRKGSFNPVTH- 200 210 220 230
- IYTAQDVKEVIEYARLRGI 24 0 250
270 280 290 300 310 320NahA TVIPMIEMPGHAMAALAAYPQFRCFPREFKPRIIWGVEQDVYCAGKDSVFRFISDVIDEV
I : : : : | | | : : : : : | : : : : : : : : | : | : : : | : | : | : : : | :HexA’ RVLAEFDTPGHTLSWGPGAPGL--LTPCYSGSHLSGTFGPVN-PSLNSTYDFMSTLFLEI
260 270 280 290 300
NahA
HexA’
330 340 350 360 370 380APLFPGTYFHIGGDECPKDRWKACSLCQKRMRDNGLKDEHELQSYFIKQAEKVLQKHGKR
SSVFPDFYLHLGGDEVDFTCWKSNPNIQAFMKKKGFTDFKQLESFYIQTLLDIVSDYDKG 320 330 340 350 360
NahA
HexA’
390 4 00 41 0 42 0 430LIGWDEILEGGLA--PSATVMSWRGEDGGIAAAMMNHDVIMTPGSGGLYLDHYQGDPTVE
YWWQEVFDNKVKVRPDT11QVWREEMPVEYMLEM - QDITRAGFRALLS APWY - 380 390 400 410 420
45 0 46 0 470 480 490N ah A PVAIGGYAPLEQVYAYNPLPKELPADKHRYVLGAQANLWAEYLYTSERYDYQAYPRLLAV
: I I : : : : I : | | : : : : : : : | : | : : | : | : | | :H e x A ’ RVKYG-- PDWKDMYKVEPLAFHGTPEQKALVIGGEACMWGEYVDSTNLVPRLWPRAGAVA
43 0 440 45 0 460 47 0 480
figure 4.6c Protein homology between NahA and the a-subunit of mouse lysosomal P-N-Acetyl-hexosaminidase (HexA’). Vertical lines align identical amino acids. Double dots align homologous amino acids. Horizontal lines are gaps that are automatically inserted to maximise the alignment. Numbers refer to amino acid position.
92
Figure 4.6d Protein Homology Between NahA and the P-subunit of MouseLysosomal p-N-Acetyl-Hexosaminidase
90 100 110 120 130N ah A SADLSLQIEIQEEPSGNAIYIGVDTALPLKEEGYMLRSDKRQVSIIGKSAHGAFYGMQTI,
: I : I I : : : : : : : | : | | : ( : : | : H e x B ’ ARFRAEAQLQKLLVSITLESECESFPSLSSDETYSLLVQEPVAVLKANSVWGALRGLETF
100 110 120 130 140 150
NahA
HexB’
LQLLPAEVESSNEVLLPMTVPGVEIKDEPATOYRGFMLDVCRHFLSVEDIKKHIDIMAMF
SQLV- --Y Q D SFGTFTINESSIA DSPR FPH RGILIDTSRHLLPVK TIFKTLDA M A FN 160 170 180 190 200
2 1 0 220 230 240 250NahA KINRFHWHLTEDQAWRIEIKKYPRLTEVGSTRTEGDGTQYSGFYTQEQVRDIVQYASDHF
I : I : I I I : : : I I : : : : : : : | | : : | | : | | | : : : | | : : : | | : :HexB’ KFNVLHWHIVDDQSFPYQSTTFPELSNKGSYS------------- LSHVYTPNDVRMVLEYARLRG
220 23 0 24 0 25 0 260
NahA
HexB’
270 280 290 300 310ITVIPM IEM PGHAM AALAAYPQF--RCFPREFKPRIIW QVEQDVYCAGKDSVFRFISDVI
IRVIPGFDTPGHTQSWGKGQKNLLTPCYNQKTKTQVFGPVDPTV- 270 280 290 300
-NTTY AFFNTFF310
320 330 340 350 360 370N ah A DEVAPLFPQTYFHIGGDECPKDRWKACSLCQKRMRDNGLKDE-HELQSYFIKQAEKVLQK
: I ; : : : I I : : : | : | | | | : | : : | | : : | : : : : | : ( : : | | : : : : :HexB’ KEISSVFPDQFIHLGGDBVEFQCW ASNPNIQGFM KRKGFGSDFRRLESFYIKKILEIISS
320 330 340 350 360 370
NahA
HexB’
380 390 400 410 420 430HGKRLIGWDEILEG--GLAPSATVMSWRGEDGGIAAANMNHDVIMTPGSGGLYLDHYQGD
LKKNSIVWQEVFDDKVELQPGTWEVWKSEHYSYELKQVTGSGFPAILSAPWYLDLISYG 380 390 40 0 41 0 42 0 430
Figure 4.6d Protein homology between NahA and the P-subunit of mouse lysosomal P-N-Acetyl-hexosaminidase (HexB’). Vertical lines align identical amino acids. Double dots align homologous amino acids. Horizontal lines are gaps that are automatically inserted to maximise the alignment. Numbers refer to amino acid position.
93
Figure 4.6e Protein Homology Between NahA and the Lysosomal P-N-Acetyl-Hexosaminidase from Dictyostelium discoideum
70 80 90 100 110 120N ah A KPIAiCLLADKLRASADIiSLQIEIGEEPSGNAIYIGVDTALPLKEEGYM LRSDKRGVSIIG
: I I : : I : | | | : : :N ag A SV SM DRY TNLFFPFSNESEPSSNESFLLSVTIYSDDETLQLGIDESYSLSIEQGSYQLKA
60 70 80 90 100 110
NahA
NagA
130 140 150 160 170 180KSAHGAFYGMQTLLQLLPAEVESSNEVLLPMTVPGVEIKDEPAFGYRGFMLDVCRHFLSV
TNIYGAMRGLETFKQLI - 120 130
- VYNSLENSYSIVCVSISDSPRYPWRGFMVDSARHYIPK 140 150 160 170
190 200 210 220 230 240N ah A EDIKKHIDIMAMFKINRFHWHLTEDQAWRIEIKKYPRLTEVGSTRTEGDGTQYSGFYTQE
: I : I I : : : | : | : | | | : : : : | : : : | : : | | | | : : : : : : : : : : :N ag A NMILHMIDSLGFSKFNTLHWHMVDAVAFPVESTTYPDLTKGAFSPSAT----------------- FSHD
180 190 200 210 220
250 260 270 280 290 300N ah A QVRDIVQYASDHFITVIPMIEMPGHAMAAIAAYPQFRCFPREFKPRIIWGVEQDVYCAGK
: : : : : I : I I : : : | | | | : : : | | | | | : | | : : : ; : : : : : : | : : :N ag A DIQEW AYAKTYGIRVIPEFDIPGHAAAW GIGYPELVATCPDYAANVN-NIPLDI- - -S N
230 240 250 260 270
310 320 330 340 350 360N ah A DSVFRFISDVIDEVAPLFPGTYFHIGGDECPKDRWKACSLCQKRMRDNGLKDEHELQSYF
: : I I I : : : : I : I I I I : : | | | : | | | | : : | : : : : : | : | : : : : : : : | | |N ag A PATFTFIQNLFTEIAPLFIDNYFHTGGDELVTGCWLEDPAIANWMTKMGFSTTDAFQ-YF
290 300 310 320 330
NahA
NagA
370 380 390 400 41 0 420IKQAEKVLQKHGKRLIGWDEILEGG--tAPSATVMSWRGEDGGIAAANMNHDVIMTPGSG
ENNLDVTMKSINRTKITWNDPIDYGVQLNPETLVQVWSSGSDLQGIVNSGYKALVSFAWY 350 360 370 380 390
Figure 4.6e Protein homology between NahA and the subunit of slime mould dimeric lysosomal P-N-Acetyl-hexosaminidase (NagA). Vertical lines align identical amino acids. Double dots align homologous amino acids. Horizontal lines are gaps that are automatically inserted to maximise the alignment. Numbers refer to amino acid position.
94
Figure 4.6f Protein Homology Between NahA and the Chitobiase of Vibrio harveyi
NahA
Chb
10 20 30 40 50MKRLTFQACICCLLSmACSQKAKQVQIPEYDKQINIIPLPMQLTESDDSFEV
ASFVTGLEGNNLKRTPDDNNVFANAVSRFEKNEDLATQDVSTTLLPTPMHVEAGKGKVDI 180 190 20 0 210 220
NahA
Chb
60 70 80 90 100 110DDKTTICVSAEELKPIAKLLADKLRASADLSLQIEIGEEPSGNAIYIGVDTALPLKESGY
-GIALPKDAFDA-TQFAAIQDRAEV-VGVDVR- GDLPVSITWPADFTGELAKSGAY 240 250 260 270 280
NahA
Chb
120 130 140 150 160 170MLRSDKRGVSIIGKSAHGAFY(34QTLLQLLPAEVESSNEVLLPMTVPQVEIKDEPAFGYR
EMSIKGDGIVIKAFDQAGAFYAVQSIFGLVDSQNADS- 290 300 310 320
-LPQLSIKDAPRFDYR3 30
180 190 200 210 220 230NahA GFMU3VCRHFLSVEDIKKHIDIMAMFKINRFHWHI,TEDQAWRIEIKKYPRLTEVGSTRTE
I | : | l | : | I : : | =1 II =| : | : : | I l h UUI UI I I l l l hUChb GVMVDVARNFHSKDAILATLDQMAAYKMNKLHLHLTDDEGWRLEIPGLPELTEVQANRCF
340 350 360 370 380 390
figure 4.6f Protein homology between NahA and outer membrane chitobiase lipoprotein of Vibrio harveyi (Chb). Vertical lines align identical amino acids. Double dots align homologous amino acids. Horizontal lines are gaps that are automatically inserted to maximise the alignment. Numbers refer to amino acid position.
95
Figure 4.6g Protein Homology Between NahA and theP-N-Acetyl-Hexosaminidase Vibrio vulnificus
NahA
Hex
4 0 5 0 6 0 70 8 0 90
KGINIIPLPM QLTESDDSFEVDDKTTICVSAEELKPIAîCLIiADKLRASADLSLQIEIGEE
KVKDLGADAVSAHILPTPLETSVHEGSLNIAQGINIVSDALPADQVEALNFRFETIÆVWT 2 1 0 2 2 0 2 3 0 2 4 0
1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0NahA PSGNAIYIGVDTALPLKEEGYMLRSDKRGVSIIGKSAHGAFYffl4QTLLQLLPAEA7ESSNE
: I : : : : : : : : : | : : : | | : : : | | | : | | | | | : | : | | : : : : :Hex GTGVPVNVTIKADSSKKSGSYTLDVTSSGIRIVGVDKAGAFYGVQSLAGLVTVGKD-------
2 5 0 2 6 0 2 7 0 2 8 0 2 9 0 3 0 0
1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 210NahA VLLPMTVPGVEIKDEPAFGYRGmiiDVCRHFLSVEDIKKHIDIMAMFKINRFHWHLTEDQ
Hex TINQVSIKDEPRLDYRGMHMDVSRNFHSKELVFRFLDQMAAYKMNKFHFHLADDE3 1 0 3 2 0 3 3 0 3 4 0 3 5 0
2 2 0 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0
NahA AW RIEIKKYPRLTEVGSTRTEGDGTQYSGFYTQEQVRDIVQYASDHFITVIPMIEMPGHA
: I I : I I : I II = I I = IHex GWRLEINGLPELTQVGAHRCHDVEQNKC34MPQLGSGAEIiPNNGSGYYTREDYKEILAYAS
3 6 0 3 7 0 3 8 0 3 9 0 4 0 0 4 1 0
Figure 4.6g Protein homology between NahA and the P-N-Acetyl- hexosaminidase of Vibrio vulnificus (Hex). Vertical lines align identical amino acids. Double dots align homologous amino acids. Horizontal lines are gaps that are automatically inserted to maximise the alignment. Numbers refer to amino acid position.
96
Figure 4.7 The Potential Active Sites of P-N-Acetyl-Hexosaminidases
Position
HexA 156 HexB 189 HexA' 156 HexB’ 168 NagA 144 Chb Hex
321304
NahA 159
INKTEIEDFPRFPHROLLLDTSRHYLPLSSILDTLDVMAYNKIiNVFHWHLVDDPSFPYES 215INESTIIDSPRFSHRQILIDTSRHYLPVKIILKTLDAMAFNKFNVLHWHIVDDQSFPYQS 248INKTKIKDFPRFPHROVLLDTSRHYLPIÆSILDTLDVMAYNKFNVFHWHLVDDSSFPYES 215INESSIADSPRFPHRQILIDTSRHLLPVKTIFKTLDAMAFNKFWVLHWHIVDDQSFPYQS 227IVCVSISDSPRYPWROFMVDSARHYIPKNMILHMIDSLGFSKFNTLHWHMVDAVAFPVES 203LPQLSIKDAPRFDYROVMVDVARNFHSKDAILATLDQMAAYKMNKLHLHLTDDEGWRLEI 380INQVSINDEPRLDYROMHMDVSRNFHSKELVFRFLDQMAAYKMNKFHFHLADDEGWRLEI 363VPGVEIKDEPAFGYROFMLDVCRHFLSVEDIKKHIDIMAMFKINRFHWHLTEDQAWRIEI 218
# # # # # - # # - - -% putative catalytic arginine residues
Figure 4.7 Alignment of the active sites of the a(HexA) and P(HexB) subunits of human P-N-Acetyl-hexosaminidases with other enzymes. The numbers on the top refer to the amino acids within the active site starting at position 1. The numbers at each end denote the amino acid postions within each protein. Identical amino acids are shown by bold and #. Conservative amino acid changes are shown by ~ . The arrow shows the potential catalytic arginine residues. For the nomenclature of each enzyme see Table 4.1 and the text.
97
4.2.1.4 The NahA Protem of f . gzMgfva&g is Homologous to the Central Domain of CryIA(c) S-endotoxin firom BurifZus tAnnngzenas
During sporulation, B. thuringiensis produces a variety of crystal 5-
endotoxins with different target specificities against insects in the orders
Lepidoptera, Diptera and Coleoptera. The CrylA group of 5-endotoxins
consists of three members, CrylA(a), CrylA(b) and CrylA(c), that are all active
&gmmt Lepidoptera but with different species specificity. The active toxins bind
to specific receptors on the brush border of the intestinal epithelium and create
leakage pores, leading to insect death by starvation (Hofte and Whiteley, 1989).
The 5-endotoxins appear to have three distinct structural domains. The
hydrophobic N-terminal domain (up to residue 283) is thought to insert into the
epithelial cell membrane and result in pores. Amino acid sequences within the
central domain (residues around 286-499) are suggested to be important for the
different specificity and receptor binding of 5-endotoxins (Aronson, 1993). The
C-terminal domain (residues about 500-640) may be important for conformation
and crystal formation (Aronson, 1993; Beitlot et al., 1993).
CrylA(a), CryIA(b) and CrylA(c) share extensive amino acid homology
except for stretches between residues 280-550. This region (mostly within the
central domain) is termed the hypervariable region and is thought to be
important for the different species specificity of the CrylA toxins. (Hofte and
Whiteley, 1989; Aronson, 1993). Activity for Bombyx mori (sük moth) can be
conferred on the CrylA(c) toxin by insertion of an oligonucleotide encoding
amino acids 332-450 from the cryIA(a) gene. In a complementary experiment,
an oligonucleotide encoding residues 335-615 from the cryIA(c) gene inserted
into the crylAia) gene confers activity for Heliothis virescens (tobacco
budworm) larvae. Further, regions 347-349, 370-373 and 428-450 in the
central domain of CrylA toxins are suggested to be important for protein
structure stabilisation (Ahnond and Dean, 1993).
Protein data base searches (see section 2.10.1) revealed the the amino acids
at position 487-714 of the predicted amino acid sequence of the NahA protein
are 17.1% identical and 62% homologous, over 228 amino acids, with part of
the central domain of CrylA(c) 5-endotoxin from Bacillus thuringiensis
subspecies Icurstald HD73 (Figure 4.8) (Andang et al., 1985; Hofte and
Whiteley, 1989). This may suggest that the amino acids at position 487-714 of
the NahA protein have similar structural interactions to a domain of 5-
endotoxin which is involved in binding to receptor(s) on eukaryote epithelial
cell membranes.
4.2.1.5 Analysis of
DNA sequence and computer analysis of the 1.3 kb BamHl-Xbal fragment
from pALl identified a single o ff that appears to be transcribed from the
opposite strand and in the opposite direction to that of nahA. This ‘hypothetical
gene’ is termed the orfl gene and the predicted polypeptide encoded by this
gene is referred to as the ORFl protein. The orfl gene is 927 bp and may
encode for a 309 amino acid protein with a predicted molecular weight of 35
IcDa. Figure 4.9 shows the nucleotide sequence of the 1308 bp BamHl-Xbal
fragment (for clarity shown Xbal to BamUl) and the position of the orfl gene.
See also Figure 4.3 for the location of the orfl gene within the nah region.
Computer analysis identified potential hairpin structures 5’and 3’ to the orfl
gene. The previously described hairpin structure that is 5’ nahA gene
(nucleotide position 1223-1325) is 5’ to the orfl gene (Figures 4.3, 4.5 and
4.9). A hairpin structure 3’ to the orfl gene (from nucleotide position 60-180)
99
has a calculated AG value of -30.7 kcal/mol and might act as a rho-dependant
transcriptional termination sequence (Rosenberg and Court, 1979; Brendel and
Trifonov, 1984) (Figure 4.9).
Figure 4.8 Alignment of the NahA Protein and the Central Domain of CrylA(c) 0-endotoxin of Bacillus thuringiensis subspecies kurstaki HD73
NahA
CrylAc
4 6 0 4 7 0 4 8 0 4 9 0 5 0 0 5 1 0LPKELP/yiKHRYVLGAQANLWAEYLYTSERYDYQAYPRLLAVAELTWTPIAKKDFADFCR
GPDSRDW VRYNQFRRELTLTVLDIVALFPNYDSRRYP-IRTVSQLTREIYTNPVLENF- 2 3 0 2 4 0 2 5 0 2 6 0 2 7 0
NahA
CrylAc
5 2 0 5 3 0 5 4 0 5 5 0 5 6 0 5 7 0RLDNACVRLDMHGINYHIPLPEQPGGSSDFIAFTDKAKLTFTTSRPMKMVYTLDETEPSL
-DGSFRGSAQGIERSIRSPHLMDILNSITIYTDAHRGYYYW SGHQIMASPVGFSGPEF 2 8 0 2 9 0 3 0 0 3 1 0 3 2 0 3 3 0
5 8 0 5 9 0 6 0 0 6 1 0 6 2 0 6 3 0NahA TSTPYTVPLEFAQTGLLKIRTVTAGGKMSPVRRIRVEKQPFNMSMEVPAPKPGLTIRTAY
CrylAc T F P L Y G -- -TMGNAAPQQRIVAQLGQGVYRTLSSTLYRRPFNIGINNQQLSVLDGTEFAY 3 4 0 3 5 0 3 6 0 3 7 0 3 8 0 3 9 0
NahA
CrylAc
6 4 0 6 5 0 6 6 0 6 7 0 6 8 0 6 9 0GDLYDVPDLQQVASWEVGTVSSLEEIMHGKEKITSPEVLERRWEATGYVLIPEDGVYEF
G------TSSNLPSAVYRKSGTVDSLDEIPPQNNNVPPRQGFSHRLSHVSM FRSGFSNSSVSI4 0 0 4 1 0 4 2 0 4 3 0 4 4 0
NahA
CrylAc
7 0 0 7 1 0 7 2 0 7 3 0 7 4 0 7 5 0STENNEFWIDNVKLIDNVGEVKKFSRRNSSRALQKGYHPIKTIWVGAIQGAWPTYWNYSR
IRAPMFSW IHRSAEFNNIIASDSITQIPAVKGNFLFNGSVISGPGFTGGDLVRLNSSGNN 4 5 0 4 6 0 4 7 0 4 8 0 4 9 0 5 0 0
Figure 4.8 Protein homology between the NahA protein and the CryIA(c) 5-endotoxin of Bacillus thuringiensis subspecies kurstaki HD73. Vertical lines align identical amino acids. Double dots align homologous amino acids. Horizontal lines are gaps that are automatically inserted to maximise the alignment. Numbers refer to amino acid position.
1 0 0
Figure 4.9 The DNA Sequence of the 1308 bp Xbal-fiamHlFragment From pALl and the Position of the orfl Gene
Xba"\ » » » » » » » < < < < < < < < < < < < < < <
1 3 0 8 TCTAeAATATCAGTAAAGAACTATCCCCTATCCTAAGAGAAGGATAGGGGGATAGACTTC 1 2 4 8
1 2 4 7 TTGGGATTTCTTACCAAGCAAAGAGAGGATAATCGGCCATCATCCGGTTGACTTCGGTAC 1 1 8 8
1 1 8 7 GAACCGATGCTATTACGGCTTCGTCCTCCGGTGCGGAGAGCACACGGTCTATCAACTCCA 1 1 2 8
1 1 2 71
1 0 6 78
CGATATAGCCCATCTTATCTTCTTTTACGCCACGAGTGGTGATGGCCGGAGTACCCACGCM A G V P T R
Kpn"\GGAAGCCCGATGTCTGGAATGCAGAGCGAGAATCGAACeSTACCATGTTCTTATTGACGG
K P D V W N A E R E S N G T M F L L T V
1 0 6 87
1 0 0 827
1 0 0 728
TAATATCCGCTGCCACGAGGGCTTTCTCTGCCACTTTACCTGTCAGTTCGGGGAACTTCGI S A A T R A F S A T L P V S S G N F G
9 4 847
9 4 748
GACGAAGATCGATCTCTACCAAAGGCCCACGCCTGACTGCCGAGCTTTCTTTTGCCGGCCR R S I S T K G P R L T A E L S F A G R
8 8 867
8 8 768
GTTCATTGGTTTTGGTTACCCTTTGCAGACAAAGTGTCGGTGTCTCAGAAGATTCGTTCTS L V L V T L C R Q S V G V S E D S F C
8 2 887
8 2 788
GCCGAAGAGCGAGCACGCTTGCGCAGTTGATTATCAGCATCAAGCCCAAGAATTTTGACGR R A S T L A Q L I I S I K P K N F D V
7 6 8107
7 6 7108
7 0 7128
TTATCATACGCACTTCTGCTGAAGGTAAACGGGCATCCGAATTGGACAAAGAGTTGCAGA I I R T S A E G K R A S E L D K E L Q S
HintMGCCTACTACGTCGTTGGGATGACAACATCAAGAAGCITTTGCGTGCCAAGGCTCCGTCCA
L L R R W D D N I K K L L R A K A P S I
7 0 8127
6 4 8147
6 4 7148
TTGTTTATGAGGAAGCCAGCCGTACTCTGGGACTCTTGAGGGACAACTTCAATCCCTCTTV Y E E A S R T L G L L R D N F N P S Y
5 8 8167
5 8 7168
ACCAGAGTATTATTGTCAATGACAAGGCTTTTCGGGACAGTTTGAGCGACTATGTCGAQTQ S I I V K D K A F R D S L S D Y V E L
5 2 8187
5 2 7 TGATAGCTCCGGGACGGGAGGAAATCGTTCAGTATTATCAGGGCGAACTACCCATTTTCG 4 6 8189 l A P G R E E I V Q Y Y Q G E L P I F D 207
4 6 7 ACCATTTTGCCATTACGAAGCAGATTAAAGCTCTCTTCGGACGTACTGTTACGTATAAGA 4 0 82 08 H F A I T K Q I K A L F G R T V T Y K S 227
4 0 7228
347248
GTGQTGCTTATCTGATTATTGAGCAGACCGAGGCCTTGCACGTGGAGGATGTTAATAGCG G A Y L I l E Q T E A L H V E D V N S G
Pst̂GTAACCGCTCGCGTGGCTGTACCGAGCAGGAAGCAACGGCCGTCGATGTCAATATGGCTG
N R S R G C T E Q E A T A V D V N M A A
3 4 8247
2 8 8267
2 8 7268
CAOCCGAAGAACTGGCCAGACAATTGCGCTTCCGATATGGGTGGTATCATCGTAGTGGACA E E L A R Q L R F R Y G W Y H R S G L
2 2 8287
2 2 7288
TTCATCGATATGAGCGAAGCCCAGCACCGTCAGCAGTTGTACGAACACATGGTCAAATTGH R Y E R S P A P S A V V R T H G Q I D
168307
101
1 6 7 ATGTCGGCTGACAGAGCGCGACACAATATCTTGCCATTGAGCAAATTCGGCGTTATGCAG 1 0 83 0 8 V G * 3 0 9
> > > > > > > > > > > > < < < < < < < < < < < < < < < < <
1 0 7 ATTACGCGCTAGCCTGTTCGTCCGGCGATGGTCATCCATACCGAAGAGTGTTGTCCCACA 4 8M V I H T E S C C P T
Sa/nHI4 7 TGTTTGGGTACCGGCAAGGTAGAATCCTCCATACTCTTCACGGATCC 1
C L G T G K V E S S I L F T D
Figure 4.9 The DNA sequence of the 1308 bp BamHl-Xbal fragment from pALl and the position of the orfl gene. The nucleotide sequence and predicted amino acid sequence are numbered. Restriction enzyme sites are shown in bold. Hairpin structures are shown by < < < > > > . An uncharactersised orf that extends beyond the first nucleotide of the nah region is underlined.
Protein homology searches (see section 2.10.1) revealed that the predicted
amino acid sequence of the ORFl protein is 30.5% identical and 72.9%
homologous, over 236 amino acids, with the Ams/Rne (altered mRNA
stabilty/ribonuclease E) protein of E.coli (Figure 4.10) (Casaregola et al.,
1992). Further, the predicted amino acid sequence of the ORFl protein was
found to be 30.2% identical and 73.7% homologous over 228 amino acids,
with the ccfA (previously called orfF) gene product from the mre (rrmrem
pathway cluster e) locus of E. colt (Figure 4.11) (Wachi et al., 1991; Okada et
al., 1994). The mre locus plays a role in cell division and is involved in the
formation of rod shape E. coli cells (Wachi et al., 1989). It is suggested that
the mre genes form an operon with cafA being the final downstream gene
(Wachi et al., 1991). The upstream mreBCD genes are required for cell rod
shape and the MreB protein is implicated in protein phosphorylation regulatory
mechanisms (Wachi et al., 1989; Okada et al., 1992; Okada et al., 1994).
Although mutations in the cafA gene do not cause any change in cell shape,
overproduction of the 55 kDa CafA protein affects the morphology of E. coli,
particularly in the formation of singular intracellular structures called
102
cytoplasmic axial filaments (Wachi et al., 1989 &1991; Okada et al., 1992;
Okada 1994).
The ams/rne gene is suggested to encode for a transmembrane ribonuclease
(RNAase) E with multiple functional domains (Casaregola et al., 1992). The C-
terminus of Ams/Rne has been implicated in the catalytic activity and the N-
terminus has two potential nucleotide binding motifs (Taraseviciene et al.,
1991; Casaregola et al., 1992). It is suggested that the first N-terminal
nucleotide binding motif (position 39-59) has homology to the Walker A and B
motifs (Me Dowall et al., 1993), whereas the second potential binding site
(position 172-176) has homology to only the Walker A motif (Casaregola et al.,
1992; Me Dowall et al., 1993). The ams-1 and rne-3071 mutations in the
ams/rne gene cause single amino acid substitiutions (G->S in ams-1, L->F m
rne-3071) next to the first N-termiual nucleotide binding motif of the Ams/Rne
protein (Figure 4.12), which results in decreased mRNA degradation in E. coli
(Me Dowall et al., 1993; Melefors et al., 1993). Interestingly, the first
nucleotide binding motif (position 39-59) of the Ams/Rne protein has extensive
sequence homology to the N-terminus of the cafA gene product (also position
39-59) (Figure 4.12) (Me Dowall et al., 1993). This region does not appear to
be present on the ORFl proteiu, since ORFl did not align with CafA or
Ams/Rne at position 39-59 (Figure 4.10, 4.11 and 4.12). However, by
comparing the Ams/Rne, CafA and ORFl proteins in another region, it was
possible to identify common amino acid sequences in all three proteins. These
homologous sequences are part of the second potential nucleotide binding motif
of the Ams/Rne protein (position 172-176 in Ams/Rne, 175-179 in CafA and
113-115 in ORFl) and a region of the Ams/Rne protein that is suggested to be
involved in membrane spanning (position 113-131 in Ams/Rne, 116-134 in
CafA and 55-73 in ORFl) (Figure 4.12) (Casaregola et al., 1992).
103
Figure 4.10 Alignment of the ORFl Protein and the arm/me Gene Product of E. coli
ORFl
Ams/Rne
20 30 40 50 60 70ERESNGTM FLLTVISAATRAFSATLPVSSGNFGRRSISTKGPRLTAELSFAGRSLVLVIL
lAREYFPANYSAHGRPNIKDVIREGQEVIVQIDKEERGNKGAALTTFISLAGSYLVLMPN
80 90 1 0 0 110 1 2 0 130ORFl CRQSVGVSEDSFCRRASTLAQLIISIK-PKNFDVIIRTSAEGKRASELDKELQSLLRRW D
: : : | : | : : | : : | : : | : : : : : | | | : | | : | : : | : : | | | :Ams/Rne NPRAGGISRRIEGDDRTELKEJUASLELPEGMGLIVRTAGVGKSAEALQWDLSFRLKHWE
140 150 160 170 180 190
ORFl
Ams/Rne
140 150 160 170 180 190DNIKKLLRAKAPSIVYEEASRTLGLLRDNFNPSYQSIIVND- KAFRDSLSDYVELIAPGR
AIKKAAESRPAPFLIHQESNVIVRAFRDYLRQDIGEILIDNPKVLELARQHIAALGRPDF
ORFl
Ams/Rne
EEIVQYYQGELPIFDHFAITKQIKALFGRTVTYKSGAYLIIEQTEALHVEDVNSGNRSRG
SSK IK LY TG EIPLFSH Y Q IESQ IESA FQ R EV R LPSG G SIV ID STEA LTA ID IN SA R A TR G
260 270 280 290 300ORFl CTEQEATAVDVNMAAAEELARQLRFRYGWYHRSGLHRYERSPAPSAWRTHGQIDVG
: : I : I I : : I : : I I : I : I I I I I : IAms/Rne -GDIEETAFNTNLEAADEIARQLRLRDLGGLIVIDFIDMTPVRHQRAVENRLREAVRQDR
320 330 340 350 3 60 370
Figure 4.10 Protein homology between ORFl and the altered mRNA stabilty/ribonuclease E (Ams/Rne) protein of E. coli. Vertical lines align identical amino acids. Double dots align homologous amino acids. Horizontal lines are gaps that are automatically inserted to maximise the alignment. Numbers refer to amino acid position.
104
Figure 4.11 Alignment of the ORFl protein and the cafA Gene Productfrom the mre locus of E. coli
ORFl
CafA
20 30 40 50 60 70SNGTM FLLTVISAATRAFSATLPVSSGNFGRRSISTKGPRLTAELSFAGRSLVLVTLCRQ
CVAGEEQKQFTVRDISELVRQGQDIiMVQWKDPLGTKGARLTTDITLPSRYLVFMPGASH
80 90 1 0 0 110 120 130ORFl SVGVSEDSFCRRAST-LAQLIISIKPKNFDVIIRTSAEGKRASELDKELQSLLRRW DDNI
I I I I : : : : : : | : : : : : : : | | | | : | | | : : | | : : | | | : : :C afA - VGVSQRIESESERERLKKWAEYCDEQGGFIIRTAAEGVGEAELASDAAYLKRVWTKVM
ORFl KKLLRAKAPSIVYEEASRTLGLLRDNFNPSYQSIIVNDKAFRDSLSDYVELIAPGREEIV
CafA ERKKRPQTRYQLYGELALAQRVLRDFADAELDRIRVDSRLTYEALLEFTSEYIPEMTSKL
ORFl
CafA
QYYQGELPIFDHFAITKQIKALFGRTVTYKSGAYLIIEQTEALHVEDVNSGNRSRGCTEQ
EHYTGRQPIFDIiFDVENEIQRALERKVELKSGGYLIIDQTEAMTTVDINTGA-FVGHRNL260 270 280 290 300 310
ORFl
CafA
EATAVDVNMAAAEELARQLRFRYGWYHRSGLHRYERSPAPSAWRTHGQIDVG
DDTIFNTNIEATQAIARQLRLRNLGGIIIIDFIDM NW EDHRRRVLHSLEQALSKDRVKTS 3 20 3 30 340 350 360 370
Figure 4.11 Protein homology between ORFl and the CafA protein of E. coli. Vertical lines align identical amino acids. Double dots align homologous amino acids. Horizontal lines are gaps that are automatically inserted to maximise the alignment. Numbers refer to amino acid position.
105
Figure 4.12 Alignment of the ORFl, Ams/Rne and CafA Proteins
Potential nucleotide-binding motif
A m s
C a f A
0 R F 1
A m s
C a f A
0 R F 1
6 0
6 0
3 4
M KRM LIN A TQ QEELRV A LVD G Q R LY D LD IE S PGHEOKK A N IY K G K IT R IE P S L E A A F V D
M T A E L L V N V T P S E T R V A Y ID G G IL O E IH IE R E A R R G IV GNIYKGRVSRVLPGM OAAFVD
M A G --V PT R K PD V W N A E R E SN G T M F L L T V ISA A T R --------------------------------------------------------
r ams-1
-F rne-3071Y G A E R H G FL P L K E IA R E Y F PA N Y SA H G R P N IK D V LR E G Q E V IV Q ID K E E R G N K
IG L D K A A FL H A SD IM PH TE C V A G EE Q K Q FTV R D ISE LV R Q G Q D L M V Q W K D PLG TK
-------------A F S A T L P V S S G N F ---------------------------------------------------------------------G R R S IS T K
— I f — — — f f
5 9
5 9
3 3
1121 1 5
5 4
A m s
C a f A
0 R F 1
Possible m em brane spanning segm ent of AmsI
113 oAA&ïTrzaïtAesrïiWLjep1 1 6 G A R L T T D M L P a a Y L V m P
5 5 G P R I,T A S î-3 P A ig R S Î.V tV X
tt'- ffff— —————
N N PR A G G ISR R IE G D D R T E L K E A L A SL E L PE G M G L 1 6 6
G A S H V G -V S Q R IE S E S E R E R L K K W A E Y C D E Q G G F 1 6 8
LC R Q SV G V SED S F C R - R A S T L A Q L IIS IK P K N F D V 1 0 7
—if — —
r Potential nucleotide binding motifA m s 1 6 7 IV R T AGVQKSAEA L Q W D L SFR L K H W E A IK K A A E SR PA PFL IH Q E SN V IV R A FR D Y L R Q 2 2 4
C a f A 1 6 9 IIRTAAEGVGEAELASDAAYLKRVW TKVM ERKKRPQTRYQLYGELALAQRVLRDFADA 2 2 6
0 R F 1 1 0 8 IIR T S A E G K R A S E L D K E L Q S L L R R W D D N IK K L L R A K A P S IV Y E E A S R T L G L L R D N F N P 1 6 5
- # - # #
A m s 2 2 5 D IG E IL ID N P K V L E L A R Q H IA A L G R P D F S S K IK L Y T G E IP L F S H Y Q IE S Q IE S A F Q R E 2 8 2
C a f A 2 2 7 E L D R IR V D S - R L T Y E A L L E F T S E Y IP E M T S K L E H Y T G R Q P IF D L F D V E N E IQ R A L E R K 2 8 3
0 R F 1 1 6 6 S Y Q S IIV N D - K A F R D S L S D Y V E L IA P G R E E IV Q Y Y Q G E L P IF D H F A IT K Q IK A L F G R T 2 2 2
if —— — — — # — if ff ff—ff — — —ff — ff
A m s 2 8 3 V R L P S G G S IV ID S T E A L T A ID IN S A R A T R G G D I E E - T A F N T N L E A A D E IA R Q L R L 3 3 6
C a f A 2 8 4 V E L K S G G Y L IID Q T E A M T T V D IN T G A F V G H R N L D D -T IF N T N IE A T Q A IA R Q L R L 3 3 7
0 R F 1 2 2 3 V TY K SG A Y LIIEQ TEA LH V ED V N SG N R SR G C TEQ EA TA V D V N M A A A EELA RQ LRF 2 7 7
# ##— ###— # - #— -#####-
Figure 4.12 Alignment of the ORFl, Ams/Rne and CafA proteins. The numbers at each end denote the amino acid positions within each protein. # denotes identical amino acids. ~ indicates conservative amino acid changes. For the nomenclature of each protein see the text. The ams-1 and me-3071 mutations are displayed. The region of CafA and ORFl that is homologous to the potential membrane spanning segment of Ams is shaded. The potential nucleotide binding sites are double underlined.
106
4.3 Discussion
The nahA gene isolated from P. gingivalis W83 appears to encode for a P-N-
Acetyl-hexosaminidase enzyme (NahA) with an apparent molecular weight of
87 IcDa in the unprocessed form. Therefore, it is possible that the polypeptide
visualised at approximately 90 kDa using the in vitro transcription-translation
system is the NahA protein (Figure 3.6). The 69 kDa protein visualised in the
in vitro transcription-translation system may be either a proteolytic cleavage
fragment of NahA or due to translation begining at an internal AUG codon
within the nahA gene. Otherwise, it might be logical to consider the 69 IcDa
protein as a dimer of the 35 kDa ORFl protein, which is resistant to the
dénaturation conditions used in SDS-PAGE analysis.
When E. coli SURE™ carries pAL2 or pAL3, IPTG induction of the tac
promoter increases the expression of P-N-Acetyl-hexosaminidase activity
(Table 3.1). Plasmid pAL2 contains the entire nahA gene as determined by
nucleotide sequencing, but pAL3 should not contain the first 21 bp of the nahA
gene (Figure 3.5 and Figure 4.3). If the first AUG of the nahA gene is a
genuine start codon, then it can be suggested that the sequences 5’ to the nahA
gene are involved in the initiation of translation (McCarthy and Gualaerzi,
1990). hi theory, plasmid pAL3 does not contain these 5’ sequences, however
this plasmid encodes for P-N-Acetyl-hexosaminidase activity. Since analysis
did not reveal a translational fusion within the polylinlcer of pAL3, this suggests
that the vector is not providing the ribosome-binding-site and the AUG intiation
codon. One possiblity is that the E. coli translational apparatus is recognising
an AUG codon that is internal to the nahA gene, thereby allowing expression of
an active truncated polypeptide from pAL3, which may be the polypeptide with
a predicted molecular weight of 73 kDa, which may in turn be the visualised 69
KDa protein.
107
The predicted amino acid sequence following the first AUG suggests that the
NahA protein is a lipoprotein acylated at the N-terminal cysteine. In Gram-
negative bacteria, it is suggested that the acylation at the N-terminal cytseine
allows the protein to remain associated with the inner or outer membrane by
hydrophobic interactions (Hayashi and Wu, 1990; Penn, 1992; Pugsley, 1993).
Pugsley, 1993 suggests that in Gram-negative bacteria all secretory lipoproteins
are modified in the same way as outer membrane or Braun lipoprotein (Lpp) of
E. coli. It has been shown with the Nip lipoprotein of E. coli, an inner
membrane lipoprotein of unknown function, and the pullanase enzyme of
Klebsiella oxytoca, that an apartic acid (Asp) residue at position +2 ( i.e.,
immediately after the acylated cysteine residue) acts as a lipoprotein sorting
signal. The presence of Asp at -1-2 directs these lipoproteins to the inner rather
than the outer membrane. Since Lpp contains a serine (Ser) residue at position
+2, this lipoprotein is anchored in the outer membrane of E. coli (Pugsley,
1993). Assuming that P. gingivalis has a similar lipoprotein sorting signal to
that of E. coli, the presence of a Ser residue at position -1-2 of the NahA
protein may suggest the P-N-Acetyl-hexosaminidase is directed to the outer
membrane of P. gingivalis. The observation that P-N-Acetyl-hexosaminidase
activity is associated with OMV from P. gingivalis would support the
suggestion that the mature form of NahA is present in the outer membrane of
P. gingivalis (Minhas and Greenman, 1989; A. Wallace personal
communication). Interestingly, it has been reported that the outer membranes
from P. gingivalis W83 and W50 contain a major 90 kDa protein (Holt and
Bramanti, 1991).
Pathogenic bacteria frequently express surface proteins with affinity for
mammalian components. Some of these bacterial proteins are highly specific
for a mammalian receptor, whereas some are multifunctional and express
binding activities towards a number of target receptors. The interactions can be
108
based on protein-protein or on bacterial protein-carbohydrate interaction
(Westerlund and Korhonen, 1993), Knowles et a/., 1984 have reported that N-
Acetyl-galactosamine protects cultured Lepidopteran cells against B.
thuringiensis subspecies kurstaki HDl preparations, suggesting that this sugar is
part of a 5-endotoxin binding site. Interestingly, this strain of B. thuringiensis
subspecies lairstald expresses the CrylA(c) 5-endotoxin, but not the CrylA(a) or
CryIA(b) proteins (Hofte and Whiteley, 1989). The homology between the
CrylA(c) 5-endotoxin and the NahA protein might reflect a similar protein
structure involved in the binding of GalNAc residues. If the region 487-714 in
the NahA protein of P. gingivalis W83 is involved in GalNAc-bindmg, this
domain may facilitate bacterial/enzyme attachment to sugar residues on
eukaryote membranes for their subsequent hydrolysis.
Current work has focused on the homologies between the p-N-Acetyl-
hexosaminidases from different species to assess the potential evolutionary
relationship to the human enzymes (Graham et al., 1988; Soto-Gü and
Zysldnd, 1989; Somerville and Colwell, 1993; Cannon et al., 1994). These
studies and the interest in the active sites of human P-N-Acetyl-hexosaminidase
arises from defects in the human enzyme loiown as the group of
neurodegenerative disorders, G^z gangliosidoses (Kytzia and Sandhoff, 1985;
Brown and Mahuran, 1991; Bassler et al., 1991). The P-subunit of the human
enzyme catalyses the release of GlcNAc and GalNAc, whereas the a-subunit
cleaves terminal GlcNAc-b-SO^ residues (Kytzia and Sandhoff, 1985). A
conservative Arginine-21 l->Lysine-211 amino acid substitution m the P-
subunit (HexB) causes no change in the substrate binding constant (K,„),
however results in a 400-fold decrease in the maximal rate {V,„af) of p-N-
Acetyl-hexosamhiidase activity. It has been suggested that arginine (Arg)
residues in the active sites of many enzymes serve as positively charged
109
recognition residues for negatively charged substrates (Riordan et al., 1977;
Berger and Evans, 1990). The Arg-211 residue of HexB does not appear to be
involved in substrate binding, since there is no change in the K .̂ value of the
mutant HexB enzyme. However, the detection of a different pH optimum in the
mutant HexB enzyme, suggested that Arg-211 of HexB is involved in a charge
transfer process required for substrate hydrolysis (Brown and Mahuran, 1991).
By extrapolation, the Arg-178 of the a-subunit (HexA) is homologous to the
Arg-211 hi the P-subunit. Therefore, the Arg-178 residue of HexA is thought
to be part of the catalytic site within the a-subunit (Brown and Mahuran,
1991). Interestingly, when aligning the predicted amino acid sequences of all P-
N-Acetyl-hexosaminidase enzymes in the SWISSPROT/NBRF database, there
are arginines at a similar position in all proteins (Figure 4.7). A similar
observation was reported when comparing the secreted P-N-Acetyl-
hexosaminidase of Candida albicans with other p -N-Acetyl-hexosaniinidases
(Cannon et al., 1994), which may suggest a common catalytic mechanism in P-
N-Acetyl-hexosaminidase enzymes. Site specific mutagenesis of Arg-182 within
the NahA protein of P. gingivalis may determine that this residue is similar in
function to Arg-211 of the HexB protein. It may also be of interest to determine
if NahA is a dimer, and whether it contains a specifity that is similar to the a-
subunit and/or to the P-subunit of human P-N-Acetyl-hexosaminidase.
Hairpin structures are a common feature of RNA molecules and their
formation in DNA molecules has been postulated in regions with palindromic
sequences, which have been implicated in gene regulation (Menguad et al.,
1989; Bouvet and Belasco, 1992; Segal and Ron, 1993). hi the upstream region
of a number of genes that encode for heat shock proteins there is an inverted
repeat sequence that may form a hairpin loop structure. Transcription initiates
from the upstream end of this inverted repeat and it has been suggested that the
110
hairpin loop causes pausing of the RNA polymerase, which may then be
abolished after heat shock (Li and Wong, 1992; Narberhaus and Bahl, 1992;
Segal and Ron, 1993), This heat-shock regulatory hairpin appears to be
conserved in phylogenetically distinct bacteria, however does not show
sequence similarity to the hairpin structure 5’ to the nahA gene (Narberhaus
and Bahl, 1992; Segal and Ron, 1993). This could suggest that the hairpin
structure 5’ to the nahA gene is distinct from the heat-shock regulatory hairpin
found in Gram-positive and Gram-negative bacteria.
Exo-P-N-Acetyl-glucosaminidase and trypsin-lüce protease activity in P.
gingivalis may be regulated by growth rate. For example, at high levels of
growth rate the cell-bound enzyme activities are higher than those in OMV,
however at low levels of growth rate the enzyme activities in OMV are higher
than those that are cell-bound (Minhas and Greenman, 1989). If the hairpin
structure 5’ to the nahA gene is involved in the regulation of nahA gene
expression in P. gingivalis, it is possible that this structure may act as a
repressor or activator protein binding site, and play a role in gene expression
during growth rate. However, nucleotide sequences that are similar to the 5’
hairpin of nahA were not detected in the 5’ region of a gene that encodes for
trypsin-lüce protease (prtT) (Otogoto and Kuramitsu, 1993). This may suggest
that the 5’ hairpin is not a common repressor/activator site binding site present
in the upstream region of the nahA andprtT genes.
The rate of mRNA decay is an important determinant of the regulation of
gene expression (Belasco, 1993; Gamper and Haas, 1993; Klug, 1993;
Melefors et al., 1993), and 5’ hairpins may play a role in the rate of mRNA
decay. For example, several mRNAs which have long half-lives in E. coli
contain a hairpin structure no more than a few nucleotides from the 5’ end of
the mRNA (Bechhofer, 1993). In fact, a 5’-terminal hairpin in the E. coli ompA
I l l
5’ untranslated region prolongs the in vivo lifetime of most heterologous
messages to which it is fused. Further, the decay of RNA I, a small
untranslated RNA encoded by plasmid pBR322, is intiated by RNAase E at an
internal site. Decay of RNA I is slowed by the addition of a terminal hairpin
structure at the 5’ end of the RNA. Moreover, rapid RNAase E-initiated decay
of RNA I is restored by the addition of several unpaired nucleotides at the 5’
end of the RNA (Bouvet and Balasco, 1992). If transcription of the nahA gene
in P. gingivalis starts 5’ to the hairpin and this structure becomes part of the 5’
end of the mRNA, it is possible that the hairpin may play a role in controlling
the rate of nahA mRNA decay. However, because the hairpin lies close to the
5’ region of the orfl gene, it is possible the hairpin may regulate the expression
of this gene. Alternatively, it may even regulate the expression of both genes.
Moreover, deternaining the transcriptional start site for the nahA and orfl gene
should reveal more information on the role of this hairpin structure.
Processing and degradation of RNA in E. coli and other bacteria involves the
action of a highly specific endoribonuclease RNAase E (Belasco, 1993; Gamper
and Haas, 1993; Klug, 1993; Melefors et a l , 1993). RNAase E activity
appears to be decreased when changing cells from fast to slow growth or from
aerobiosis to anaerobiosis (Georgellis et al., 1993). Mutations in the E. coli
ams/rne gene lead to a deficiency in Ams/RNAase E activity and an increase in
mRNA stability (Mudd and Higgins, 1993). The nucleotide sequence of the
ams/rne gene predicts a polypeptide of 114 kDa (Casaregola et al., 1991),
however functional studies suggest that RNAase E activity may reside in
smaller proteolytic fragments from the intact protein. In one study, proteolytic
fragments of 70 kDa contained RNAase E activity (Mudd and Higgins, 1993),
whereas in another it was suggested that RNAase E activity resides in a 17 IcDa
polypeptide (Sohlberg et al., 1993). It has also been implied that the
Ams/RNAase E protein functions as a multiprotein complex rather than an
112
isolated RNAase (Casaregola at al., 1991; Me Dowall et al., 1993; Mudd and
Higgins, 1993). Interestingly, RNAase E is suggested to functionally interact
with the E. coli heat shock chaperonin GroEL (Sohlberg et al., 1993). In
general, GroEL interacts with polypeptides and is involved in protein folding,
assembly and export, which requires hydrolysis of ATP (Kusukawa et al.,
1989; Kumamoto, 1991; Craig et al., 1993), therefore it has been speculated
that RNA processing is regulated by the interactions between GroEL and
RNAase E (Sohlberg et al., 1993). It has been suggested that both the ams-1
and me-3071 mutations affect mRNA decay and processing by disrupting
structural interactions within a macromolecular complex, rather than by altering
the active site of a ribonuclease (McDowall et al., 1993)
Bacterial cell division involves nucleoid segregation and invagination of the
surface layers during septum formation, and is thought to require a
macromolecular complex (Nanninga et al., 1991; Lutlcenhaus, 1993). Bacterial
contractile proteins that appear to be myosin and actin-lilce are thought to play a
role in the segregation process (Holland et al., 1990; Lutlcenhaus, 1993). The
Ams/Rne protein shows immunological cross-reaction to both yeast and chicken
muscle myosin heavy chain (Holland et al., 1990; Casaregola et al., 1992), and
the CafA protein has been implicated in the formation of contractile
cytoskeletal structures or cytoplasmic axial filaments that participate in cell
division or chromosomal segregation (Okada et al., 1994). Overproduction of
CafA causes the formation of a long axial structure running through chained
cells, with the structure consisting of bundles of filaments assembled in a long
hexagonal pillar several micrometers long and and 0.1-0.2 micrometers in
diameter (Okada et al., 1994). hi additon, overproduction of the CafA protein
causes formation of anucleate cells and minicells, suggesting that the CafA
protein may enhance cell division beyond the normal rate or inhibit partition of
chromosomes after replication, or both (Okada et al., 1994). The homology
113
between CafA and ORFl may reflect a similar structural/functional domain,
therefore, it may be of interest to determine if overproduction of the ORFl
protein in E. coli causes cell morphology changes similar to that of CafA
and/or increased RNAse E-lüce activity. Since RNAase E (in E. coli) and p-N-
Acetyl-hexosaminidase (in P. gingivalis) activity appear to be regulated by
growth rate (Minhas and Greenman, 1989; Georgellis et al., 1993), this factor
may be worth considering in any further experiments that study the regulation
of gene expression in the nah region.
Finally, the determination of the nucleotide sequence of the nah region has
shed light on its genetic organisation and the probable location of the NahA
protein in P. gingivalis. Although, the nahA and orfl genes appear to be
independent single transcriptional units, we cannot rule out the possibility that
these genes are the first in two independent polycistronic mRNAs, or that their
expression is somehow functionally related. Nonetheless, we are now one step
closer to generating isogenic derivatives of P. gingivalis that are absent in P-N-
Acetyl-hexosaminidase activity and then assaying such mutants in appropriate
model systems. The strategy for site specific inactivation of the nahA gene in
P. gingivalis will be shown in the next chapter.
114
CHAPTER 5
Further Studies on the P-N-Acetyl-Hexosaminidase From P, gingivalis
5.1 Introduction
The development of E. coli-P. gingivalis gene transfer systems (Dyer et al.,
1992; Hoover et al., 1992a; Maley et al., 1992) has now made it possible to
use recombinational allelic replacement in P. gingivalis (Hamada et al., 1994;
Joe et al., 1994; Malek et al., 1994; Nakayama, 1994). Recombinational allelic
replacement is a precise method that allows specific gene inactivation and the
generation of isogenic mutants (Foster, 1992). Site-directed insertion, deletion,
substitution and point mutations can be constructed in vitro using E. coli host
vector systems and then moved into the original host strain to inactivate the
wild-type allele. The generated isogenic mutants are excellent tools for defining
the relevance of the virulence factor and characterising the role of specific
enzymes (Macrina et al., 1990; Finlay, 1992; Foster, 1992).
Cloned genes or gene fragments that are joined with other specific genes to
form gene fusions can facilitate purification of recombinant proteins from crude
extracts. From the purified fiision proteins immunogenic peptides can be
produced to raise specific antibodies against the recombinant protein, which in
turn can be used for immunisation studies (Nilsson et al., 1992). Specific
antibodies can also be used to label the protein on the bacterial cell surface and
confirm the disappearence of the virulence factor in the generated isogenic
mutant (Hamada et al., 1994). This chapter will describe an approach for the
115
construction of a nahA isogenic mutant of P. gingivalis and the generation of a
NahA-Glutathione S-transferase fusion protein.
RemMaandD&æwæmn
5 .2 .1 C onstruction o f P lasm id pM UT for the G eneration o f a nuAA Isogenic M utant o f P .
Plasmid pNJR6 is a derivative of the colonic E.coli-Bactewides shuttle
plasmid pNJRS (Shoemaker et al., 1989). Both pNJR5 and pNJR6 contain an
erythromycin (Em) resistance marker which confers resistance on P. gingivalis,
but is not active in E. coli (Figure 5.1). pNJR5 and pNJR6 also appear to
contain DNA elements that allow mobilisation of the plasmid from E. coli to P.
gingivalis via transfer factors expressed from the helper plasmid R751 (Maley
et al., 1992; Joe et al., 1994). pNJR5 and pNJRô also carry an E. coli origin
of replication and a kanamycin (Km) resistance gene for maintenance and
selection in E. coli. While pNJR5 contains the pB8-51 region that enables
replication in Bacteroides hosts, pNJR6 does not contain this region and is not
expected to replicate in P. gingivalis (Shoemaker et al., 1989). Since the
Bacteroides origin of replication (pB8-51) is absent in pNJR6, this plasmid is
an expected suicide vector for P. gingivalis (Shoemalcer et al., 1989; Maley et
al., 1992; Joe et al., 1994).
An approach that may allow homologous recombination and insertion of the
suicide plasmid pNJR6 into chromosomal nahA gene was talcen. Plasmid
pNJR6 was digested with Stul and EcdRl and a 500 bp Nael-EcoBA fragment
from the centre portion of the nahA gene on pALl was isolated. The 500 bp
Nael-EcdKl fragment was ligated into Stul-EcdBA cleaved pNJR6 to generate
plasmid pMUT (Figure 5.1 and 5.2). E. coli JMlOl colonies carrying plasmid
116
pMUT were selected for by their kanamycin resistance and ability to hybridise
to the radiolabeled nahA gene probe under high stringency wash conditions.
The autoradiograph in Figure 5.3 shows two transformants that hybridised to
the nah region specific probe. This observation may suggest that plasmid
pMUT has been isolated in E. coli JMIOI. Only a small amount of DNA could
be isolated from E. coli JMlOl (pNJR6) and E. coli JMlOl (pMUT), therefore
it was difficult to show the presence of pMUT by restriction enzyme digestion
and agarose gel electophoresis.
The next stage in this study may be to transform E. coli JMlOl (pMUT)
with R751 and mobilise pMUT from E. coli JMlOl (R751, pMUT) into P.
gingivalis W83 (Maley et al., 1992; Joe et al., 1994). By isolating the
erythromycin resistant and p -N-Acetyl-hexosaminidase negative P. gingivalis
colonies, further analysis may show that pMUT has integrated into the
chromosome of P. gingivalis and disrupted the nahA gene (Figure 5.4). Indeed,
these studies are now in progress (I. S. Roberts, personnal communication).
117
Figure 5.1 Construction of Plasmid pMUT for Disruption of the chromosomal nahA gene in P. gingivalis
pB8-51 Em
pNJR5 17.4 kb
Km
B Kp St E cos site
E N
pALl 8.5 kbtac
B
N ae\, EcoRl
E N
(0.5 kb)
Ligation
b E
Km Em
pMUT 13.6 kb
Km Em
pNJR6 13.1 kb
Stu\, EcoRl
St
Km Em
pNJR6 13.1 kb
Figure 5.1 Construction of plasmid pMUT. The restriction enzyme sites are shown on each plasmid. The hatched region of pALl denotes the 4 kb BarnUX fragment that contains the nahA gene. Abbreviations: B; BamWl. Kp; Kpnl. E; EcoRl. C; Clal. St; Stul. N; Nael. b\ represents a blunt end ligation between the Stul end of pNJR6 and the Nael end of the 0.5 kb fragment. Km; Kanamycin resitance gene. Em; Erythromycin resitance gene. Arrows beside the plasmids indicate direction of transcription. The large downward arrow on the pNJR6 cos site shows the site of restriction enzyme cleavage.
118
Figure 5.2 The Suicide Plasmid pNJR6 and the 500 bp Nael-EcoPA Fragment From the nahA Gene
32
1 2 3 4
i l i
Figure 5.2 An agarose gel showing pNJR6 and an internal fragment of the nahA gene. Lane 1; KBL. Lane 2; pNJR6 cleaved with Stul and EcoRl. Lane 3; the 500 bp Add-EcoRl fragment from pALl. Lane 4; KBL. Numbers on die left are in kilobases. KBL molecular weight markers (kilobases): 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 1.6, 0.5.
Figure 5.3 Detection of pMUT by Colony Hybridisation using the nah Region as a radiolabelled probe
Figure 5.3 Autoradiograph of E. coli JMlOl (pNJR6) and two suspected E. coli JMlOl (pMUT) colonies. Hybridisation was carried out using the radiolabelled EomHl fragment from pALl as a probe. Position 1 and 2; an E. coli JMlOl (pNJR6) colony. Position 3 and 4; an E. coli JMlOl (pMUT) colony.
119
Figure 5 .4 Strategy for the Disruption o f the Chromosomal nahA Gene in P. gingivalis
TKp
Em'fl I pMUT
U I TXb Sp E N
nahA
The nah region on the P. gingivalis chromosome
TTKp Xb Sp E *
Single cross-over and integration of pMUT into the chromosomal nahA gene
- ^ T rE m ' E N
Disrupted nahA gene on the chromosome of P. gingivalis
Figure 5.4 Inactivation of the chromosomal nahA gene by site specific insertion of plasmid pMUT. Homologous recombination between pMUT and the chromosomal nahA gene may lead to disruption of the nahA gene. The hatched arrow shows the position of the nahA gene on the chromosome of P. gingivalis. The thick black horizontal line refers to the nah region. The thin black horizontal line shows shows the DNA that flanks the nah region on the chromosome. The small black arrow represents direction of transcription. Abbreviations: B; Bam lil. E; EcoRl. Kp; Kpnl. Xb; Xbal. Sp; Sphl. N; Nael. b; denotes a blunt end ligation between the Stul end of pNJR6 and and Nael end of the 0.5 kb fragment. Em'; erythromycin resistance gene.
120
. 2 .2 Strategy for the Generation of a Glutathione S-Transferase-NahA Fusion Protein
The glutathione S-transferase (GST) gene fusion system is designed for high
level expression of genes or gene fragments as fusion proteins. The protein of
interest is fused to the C-terminus of GST from Schistosoma japonicum. This is
achieved by inserting the gene or gene fragment into a pGEX vector in such a
way as to fuse its reading frame with the C-terminus of GST. Upstream of the
gene fusion is the tac promoter for high level expression of fusion proteins.
Fusion proteins can be purified from bacterial lysates by affinity
chromatography using immobilised glutathione, and the GST part of the fusion
protein can then be removed by cleavage with thrombin. The pGEX-2T vector
is shown in Figure 5.5a (Smith and Johnson, 1988).
Plasmid pGEX-2T was cleaved with Smal to generate linear blunt-end
vector. A deoxythymidine (T) was added to the 3’ ends of linear pGEX-2T
using Taq polymerase and dideoxythymidine triphosphate (ddTTP) (Figure
5.5a) (Holton and Graham, 1990). A 2.3 kb nahA gene fragment with 3’ end
deoxyadenosine (A) residues was generated using PCR with pALl as a
template (Figure 5.5b). Figure 5.6 shows the purified 2.3 kb PCR product and
the Smal digested ddT-tailed pGEX-2T. hi theory, ligation of the ddA-taüed
PCR product into the ddT-tailed Smal site of pGEX-2T should allow the
generation of an in-frame GST-NahA fusion protein (Figure 5.5b). Therefore,
the purified ddA-taüed PCR product was ligated into the ddT-taüed Smal site
of pGEX-2T and E. coli SURE™ was transformed with the ligation mixture. E.
coli SURE™ carrying recombinant plasmids were detected by their abüity to
hybridise to the nah region radiolabelled probe at high stringency wash
conditions (Figure 5.7). Although at this stage these positive colonies are not
well characterised, further analysis may show the expression of a GST-NahA
121
fusion protein. A GST-NahA fusion protein could then be purified, cleaved
with thrombin and NahA polypeptides used to generate antibodies that are
specific for the NahA protein. These antibodies could be used to label cell
extracts of P. gingivalis and may show the exact cellular location of the P-N-
Acetyl-hexosaminidase protein in P. gingivalis. Further, antibodies that are
specific for the NahA protein could be used to show the disappearance of the P-
N-Acetyl-hexosaminidase protein in a nahA isogenic mutant of P. gingivalis.
122
Figure 5 .5 Strategy for the Construction o f a GST-NahA Fusion Protein
Lac I’
bla
pGEX-2T 4.9 kbtac
GST
L V P R SG P G I H R N5 ' CTG GTT CCG CGT GGA TCC CCG QGA ATT CAT CGT GAC TGA CTG ACG 3" 3 ' GAC CAA GGC GCA CCT AGO QQC COT TAA GTA GCA CTG ACT GAC TGC 5 '
Smal digestion and ddX-tailing
L V P R G S P5 ' CTG GTT CCG CGT GGA TCC CCT 3 '3 ' GAC CAA GGC GCA CCT AGO GO 5 '
5 ' OGOAATTCATCGTGACTGACTGACG 3 ' 3 ' TÇCCTTAAGTAGCACTGACTGACTGC 5 '
B
1 8 A
n a h A —>
K Q V Q
Primer 23 ' OTTCOOCTAOAOCAOGCTATACOAOAAAOTTATTF K P I S S D M L F Q *
5'7 7 73'5 '
5 ' GCCTGCTCACAGAAAGCAAAGCAGGTGCAAATCCCCGAA. . . AAGTTCAAGCCGATCTCGTCCGATATGCTCTTTCAATAA 3 ' CGGACGAGTGTCTTTCGTTTCGTCCACGTTTAGGGGCTT. . .TTCAAGTTCGGCTAGAGCAGGCTATACGAGAAAGTTATT
5 ' TCACAGAAAOCAAAOCAOOTOCAAATCCCCO 3 'Primer 1
PCR
2 0 S Q K A K Q V Q I P E K F K P I S S D M L F Q * 7 7 75 ' TCACAGAAAGCAAAGCAGGTGCAAATCCCCGAA___AAGTTCAAGCCGATCTCGTCCGATATGCTCTTTCAATAAA 3 '3 ' AAGTGTCTTTCGTTTCGTCCACGTTTAGGGGCTT___TTCAAGTTCGGCTAGAGCAGGCTATACGAGAAAGTTATT 5 '
2.3 kb PCR product containing 3’ end deoxyadenosine (A)
Fused GST-NahA
Ligation of PCR product to Smal digested, ddT-tailed pGEX-2T
Thrombin-0- I
L V P R G S P S Q K A K Q V Q I P E D M L F Q *5 ' CTG GTT CCG CGT GGA TCC CCT TCACAGAAAGCAAAGCAGGTGCAAATCCCCGAA. ■ ■ GATATGCTCTTTCAATAAAGQGAATTCAT 3 ' 3 ' GAC CAA GGC GCA CCT AGO GGA AGTGTCTTTCGTTTCGTCCACGTTTAGGGGCTT. . . CTATACGAGAAAGTTATTTCCCTTAAGTA 5 '
Figure 5.5 Strategy for the construction of a GST-NahA fusion protein. (A) pGEX-2T showing the C-terminus of GST and the thrombin cleavage site. The Sma\ site is underlined. (B) Amplification of the nahA gene on pALl and ligation of the PCR product into pGEX-2T. The expected GST-NahA fusion protein is shown at the bottom of the figure. The horizontal hatched arrow indicates the direction of transcription. The thrombin cleavage site is shown. The single letter amino acid code is shown above the DNA sequence.
123
Figure 5 .6 The pGEX-2T Vector and the Purified nahA Gene Fragment
1 2 3
43
Figure 5.6 Agarose gel showing the ddT-tailed Smal digested pGEX-2T and the purified 2.3 kb PCR product. Lane 1; KBL. Lane 2; Smal digested, dTT-tailed pGEX-2T. Lane 3; The purified 2.3 kb nahA gene fragment generated by PCR. The top bands may indicate an overspill of pGEX-2T. Lane 4; KBL. Numbers on the left are sizes in kilobases. KBL molecular weight markers (kilobases): 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.6, 0.5.
Figure 5 .7 Detection o f the Cloned PCR Product by Colony Hybridisation
1 2 3
VFigure 5.7 Detection of the PCR product ligated into pGEX-2T by colony hybridisation. Position 1, 2, 4, 5 and 6; colonies that hybridise to the nah region probe. Position 3; E. coli SURE™ (pGEX-2T).
124
CHAPTER 6
Discussion
The purpose of this final chapter is to summarise and provide a constructive
discussion for use in any further experiments. The results in this thesis show
the first demonstration that the exo-P-N-Acetyl-glucosaniinidase and P-N-
Acetyl-galactosaminidase activities of P. gingivalis are due to a single enzyme
encoded by the nahA gene. Since the nahA gene may be present as a single
copy on the chromosome, the inactivation of the wild-type allele of P.
gingivalis may be more practically achieved. The isolation of a nahA gene
mutant could allow in vivo and in vitro studies to determine whether the NahA
protein is involved in the degradation of host tissue. It may be of interest to
study the degradation of ECM material by wild type P. gingivalis and nahA
minus strains in the presence and absence of P-N-Acetyl-hexosaminidase
inhibitors (Jensen et al., 1986; Woynarowska et al., 1989 & 1992).
Degradation experiments combined with lectin labelling of GlcNAc/GalNAc
residues on the surface of host cells and IgG may show the release of these
sugars by the p-N-Acetyl-hexosaminidase enzyme (Ito et al., 1989a & b).
Moreover, if disruption of the nahA gene in P. gingivalis leads to reduced
virulence in the gnotobiotic rat or mouse lesion model, the P-N-Acetyl-
hexosaminidase enzyme may play a role in pathogenicity (Falkow, 1988;
Finlay, 1992; Foster, 1992).
Bacteria produce enzymes that are capable of degrading their own
peptidoglycan. These autolysins are found in the cell wall of the organism and
their function may to be to modify existing peptidoglycan (PG), permitting
extension of the wall during cell growth and separation of the cells during
125
division (Nanninga et al., 1991). Clearly, the activity of these enzymes must be
carefully regulated or these enzymes would degrade the bulk PG causing
autolysis (Hammond et al., 1984; Nanninga et al., 1991). During PG
biosynthesis, it could be imagined that a periplasmic p-N-Acetyl-
hexosaminidase could cleave the NAcGlc from the Cgg-isoprenoid lipid-
muramic acid-N-Acetyl-glucosamine pentapeptide during translocation across
the cytoplasmic membrane (Hammond et al., 1984). If this was the case, an
active periplasmic p -N-Acetyl-hexosaniinidase enzyme may inhibit PG
biosynthesis and perhaps regulate PG elongation during growth of the cell.
Therefore, the possibility that the NahA protein may be involved in envelope
growth, cell division or PG recycling cannot be excluded. Moreover, it could
be speculated that any extracellular P-N-Acetyl-hexpsaminidase produced by P.
gingivalis may hydrolyse the PG of certain Gram-positive bacteria in the
periodontal microflora and act as a bacteriocin. Indeed, a commercially
available P-N-Acetyl-hexosaminidase enzyme has been shown to kill the Gram-
positive bacteria Streptococcus agalactiae. Staphylococcus aureus and
Actinomyces pyogenes in vitro, however did not reduce the numbers of the
Gram-negative bacteria E. coli and Enterobacter aerogenes (Hussain et al.,
1992). Therefore, it may be of interest to purify an active form of the NahA
protein and determine whether this enzyme is bactericidal for certain oral
bacteria.
The potential processing site of the NahA protein is similar to a signal
peptidase II cleavage sequence suggesting that the P-N-Acetyl-hexosaminidase
enzyme may be a lipoprotein (Thomas et al., 1992; Penn, 1992; Lundemose et
al., 1993; Thomas and Sellwood, 1993). In Treponema pallidum, it is
speculated that acylation may allow certain lipoproteins to be anchored to the
periplasmic side of the outer membrane and remain antigenically cryptic.
Acylation may also modulate the immunogenicity of a lipoprotein and could be
126
relevant to the interaction of a pathogen with the immune response (Penn,
1992). By combining or palmitic acid labelling studies with globomycin
inhibition, it is possible to show that lipoproteins are lipid modified then
processed during translocation across the cytoplasmic membrane (Soto-Gil and
Zysldnd, 1989; Thomas et al., 1992; Lundemose et al., 1993; Thomas and
Sellwood, 1993). Soto-Gil and Zysldnd, 1989 used ^^S-methionine to label the
Chb lipoprotein from V. harveyi in E. coli minicells and showed that
globomycin inhibits signal peptidase II cleavage of Chb. However, during this
study, the NahA protein could not be expressed in E. coli minicells, therefore it
was not possible to use globomycin inhibition in E. coli minicells. Likewise,
attempts to demonstrate that NahA is a processed lipoprotein by labelling E.
coli SURE™ (pAL2) with '̂‘C-pahnitic acid in the presence or absence of
globomycin also proved unsuccessful. A different approach may needed to
show that the p-N-Acetyl-hexosaminidase enzyme is a lipoprotein. Perhaps
labelling the NahA protein in P. gingivalis or obtaining a higher level of
expression in E. coli wül allow experiments to show lipid modification in the
P-N-Acetyl-hexosaminidase enzyme. It may also be of interest to insert the
TvphoA transposon downstream of and in-firame with the potential signal
peptide of the NahA protein. The secreted alkaline phosphatase activity
encoded by a NahA-TnpAoA fusion may be external to the cytoplasm which in
turn would suggest that the signal peptide of NahA is genuine. Furthermore,
although p-N-Acetyl-hexosaminidase activity is detectable m the OMV from P.
gingivalis, this is not clear enough evidence to indicate that the NahA protein is
responsible for the enzyme activity in OMV. Specific antibodies against the
NahA protein are needed for labelling the OMV, cytoplasmic membrane and
outer membrane of P. gingivalis. Such experiments should define the exact
cellular location of the NahA protein in P. gingivalis.
127
The homology between the NahA protein and the subunits of human P-N-
Acetyl-hexosaminidase appears to stretch over a large amino acid overlap. For
example, the NahA protein is homologous over 426 and 325 amino acids, with
the HexA and HexB subunits respectively, whereas with the Chb protein, NahA
is homologous over only 208 amino acids (Figure 4.6a and 4.6Q. The reason
for this larger stretch of homology between NahA and HexA/B is not clear,
however it may reflect that the NahA protein is more similar in structure to
HexA/B than the Chb protein (perhaps related to dimérisation) (Soto-Gil and
Zysldnd, 1989). If NahA and HexA/B have a similar structure in vivo, then it
could be speculated that they may contain common antigenic epitopes. If this is
the case, the P-N-Acetyl-hexosaminidase of P. gingivalis may be poorly
recognised by the immune system, since the HexA/B proteins are self for the
host. Moreover, any immune response generated against the NahA protein may
cause self P-N-Acetyl-hexosammidase-recognition or an autoimmune reaction
in the host periodontal tissue. Clearly, these suggestions are extremely
speculative, so many other studies will be needed to examine these hypotheses.
Finally, the cloned nah region encodes for at least one protein which is a P-
N-Acetyl-hexosaminidase enzyme. At this stage it is difficult to speculate a role
for the ORFl protein, however it may play a role in cell division and/or serve a
function similar to that of the CafA/Ams/Rne proteius of E. coli. High level
expression of ORFl in E. coli may show increased RNAse E-like activity, or
when combined with microscopy reveal the presence of cytoplasmic axial
filaments. In addition, examination of the genes that flanlc the nah region may
be necessary for a clearer insight into the structure, regulation and function of
this locus. Characterising the nahA and orfl transcripts by northern blotting
should help define whether these genes are mono- or polycistronic. The
generation of a nahA mutation in P. gingivalis that does not affect any other
gene(s) is especially needed to accurately elucidate the role of the P-N-Acetyl-
128
hexosaminidase. Assuming that the ORFl protein is a functional entity,
inactivation of the o tfl gene in P. gingivalis may be useful for further studies
that WÜ1 determine the role of this protein. It wül be necessary to analyse the
transcriptional start sites of the nahA and orfl genes to allow a clearer insight
into the 5’ hairpin structure and its possible role in the regulation of gene
expression. Future studies on the regulation of gene expression may define
parameters important for gene control mechanisms in P. gingivalis. Meanwhüe,
the regulation, the role and the interactions of the NahA and ORFl proteins
from P. gingivalis remain open for speculation.
129
REFERENCES
Adang M. J., Staver, M. J., Rocheleau T. A., Leighton J., Barker R.F.
and R. A. Thompson. 1985. Characterised full length and truncated plasmid
clones of the crystal protein of Bacillus thuringiensis subsp. lairstaki HD-73
and their toxicity to Manduca sexta. Gene 36: 289-300
Aitken, A. 1990. Identification of Protein Consensus sequences: Active Site
Motifs, Phosphorylation and Other Post Translational Modifications. Ellis
Horrwood Series in Biochemistry and Biotechnology. Ellis Horrwood Ltd.,
England
Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. and J. D. Watson.
1980. The Molecular Biology of the Cell. Garland Publication Inc.
Almond, B. D. and D. H. Dean. 1993. Suppression of protein structure
destabilizing mutations in Bacillus thuringiensis ô-endotoxins by second site
mutations. Biochemistry 32: 1040-1046
Aronson, A. I. 1993. The two faces of Bacillus thuringiensis: insecticidal
proteins and post exponential survival. Molecular Microbiology 7: 489-496
Bach, G. and B. Geiger. 1978. Human placental P-N-Acetyl-hexosaminidase
isoenzymes: Activity towards native hyaluronic acid. Archives o f Biochemistry
and Biophysics 189:37-43
Bapat, B., Etbier, M., Neote, K., Mabnran, D. and R. Gravel. 1987.
Cloning and sequence analysis of the P-subunit of mouse P-hexosaminidase.
237: 191-195
130
Bartold; P. M., Wiebkin, O. W. and J. C. Tbonard. 1982. Proteoglycans in
the human gingiva: Molecular size distribution in epithelium and in connective
tissue. Archives in Oral Biology 21% 1-7
Bassler, B. L., Yn, C., Lee, Y. C. and S. Roseman. 1991. Chitin utilization
by marine bacteria: Degradation and catabolism of chitin oligosaccharides by
Vibrio Jurnissii. Journal o f Biological Chemistry 266: 24276-24286
Beccaii, T., Hoade, J., OrlaccMo, A., and J. L. Stirling. 1992. Cloning and
sequence analysis of the cDNA encoding the a-subunit of mouse P-N-Acetyl-
hexosaminidase and comparison with the human enzyme. The Biochemical
Journal 285: 593-596
Becbbofer, D. 1993. 5’ mRNA stabilizers. p31-52. In Control of messenger
RNA stability. Belasco, J. and G. Brawerman (eds.). Academic Press Inc., U.
K.
Bedi, G. S. and T. Williams. 1994. Purification and characterisation of a
collagen degrading protease from Porphyromonas gingivalis. Journal o f
Biological Chemistry 269: 599-606
Beigbton, D. Radford, J . R. and M. N. Naylor. 1992. Glycosidase activities
in gingival crevicular fluid in subjects with adult periodontitis and gingivitis.
Archives o f Oral Biology 37: 343-348
131
Beighton, D., Smith, K., Glenister, D. A., Salamon, K. and C. W. Keevil.
1988. Increased degradative enzyme production by dental plaque bacteria in
mucin limited continuous culture. Microbial Ecology in Health and Disease 1:
85-94
Beidot, H. P., Schemthaner, J. P., Milne, R. E., Clairmont, F. R., Bhella,
R. S. and H. Kaplan. 1993. Evidence that the CrylA crystal protein from
Bacillus thuringiensis is associated with DNA. Journal o f Biological Chemistry
268: 8240-8245
Belasco, J. G. 1993. mRNA degradation in prokaryote cells: An overview. p3-
12. In Control of messenger RNA stability. Belasco, J. and G. Brawerman
(eds.). Academic Press Inc., U. K.
Berger, S. A. and P. R. Evans. 1990. Active site mutants altering the
cooperativity of E. coli phosphofructoldnase. Nature 343: 575-576
Berry, A. M., Lock, R. A., Hansman, D. and J. C. Paton. 1989.
Contribution of autolysin to virulence of Streptococcus pneumoniae. Infection
and Immunity 57: 2324-2330
Biggin, M. D., Gibson, T. J. and G. F. Hong. 1983. Buffer gradient gels and
35s label as an aid to rapid DNA sequence determination. Proceedings o f the
National Academy o f Sciences o f the U. S. A. 80: 3963-3965
Birek, P., Me Cnlloch, C. A. G. and C. M. Overall. 1989. Measurements of
probing velocity with an automated periodontal probe and the relationship with
experimental periodontitis in the cynomolgus monkey (Macaca fascicularis).
Archives in Oral Biology 34: 793-803
132
Birkedal-Hansen, H. 1993. Role of cytoldnes and inflammatory mediators in
tissue destruction. Journal o f Periodontal Research 28: 500-510
Birkedal-Hansen, H., Moore, W. G. 1., Bodden, M. K., Windsor, L. J.,
Birkedal-Hansen, B., DeCarb, A. and J. A. Engler. 1993. Matrix
metalloproteinases: A review. Critical Reviews in Oral Biology and Medicine
4: 197-250
Birkedal-Hansen, H., Wells, R. R., Lin, H-Y., Canfield, P. W. and P. E.
Taylor. 1984. Activation of keratinocyte-mediated collagen (type I) breakdown
by suspected periodontopathogen: Evidence of a novel mechanism of
connective tissue breakdown. Journal o f Periodontal Research 19: 645-650
Birboim, H. C. and J. Doly. 1979. A rapid alkaline extraction procedure for
screening recombinant plasmid DNA. Nucleic Acids Research Is 1513-1523
Bosman, H. B. and R. J. Bemacki. 1970. Glycosidase activity. Experimental
Cell Research 61: 379-386
Bonrean, H., Deere, D., Carlier, J. P., Guichet, C. and P. Bourlioux.
1993. Identification of a Clostridium cocleatum strain involved in an anti-
Clostridium difficile barrier effect and determination of its mucin degrading
enzymes. Research in Microbiology 144: 405-410
Bourgeau, G., Lapointe, H., Peloquin, P. and D. Mayrand. 1992. Cloning,
expression and sequencing of a protease gene {trp) from Porphyromonas
gingivalis W83 in Escherichia coli. Infection and Immunity 60: 3186-3192
133
Bourgeau, G, and D. Mayrand. 1990. Aggregation of Actinomyces strains by
extracellular vesicles produced by Bacteroides gingivalis. Canadian Journal o f
Microbiology 36: 362-365
Bourin, M. and U. Lindahl. 1993. Glycosaminoglycans and the regulation of
blood coagulation. The Biochemical Journal 289: 313-330
Bouten, A., Dehoux, M., Deschenes, M. Rouzeau, J. D., Bories, P. W. and
G. Durand. 1992. aj-acid glycoprotein potentiates lipopolysaccharide induced
secretion of interleuldn-lp, interleuldn-6 and tumour necrosis factor-a by
human monocytes and alveolar peritoneal macrophages. European Journal o f
Immunology 22: 2687-2695
Bouvet; P. and J. G Belasco. 1992. Control of RNase E-mediated RNA
degradation by 5'-terminal base pairing in E. coli. Nature 360: 488-491
Bramanti, T. E. and S. C. Holt. 1992a. Effect of porphyrins and host iron
transport proteins on outer membrane protein expression in Porphyromonas
gingivalis', identification of a novel 26 IcDa haemin repressible surface protein.
Microbial Pathogenensis 13: 61-73
Bramanti, T. E. and S. C. Holt. 1992b. Localisation of a Porphyromonas
gingivalis 26-Küodalton heat-modifiable, haemin-regulated surface protein
which translocates across the outer membrane. Journal o f Bacteriology 174;
5827-5839
Brendel; V. and E. N. Trifonov. 1984. A computer alogrithm for testing
potential prokaryotic terminators. Nucleic Acids Research 12: 4411-4427
134
Cabezas, J. A. 1989. Some comments on the type references of official
nomenclature (lUB) for P-N-Acetyl-glucosaniinidase, P-N-Acetyl-
hexosaminidase and p -N-Acetyl-galactosaminidase. Biochemical Journal 261:
1059-1061
Cabib, E. 1987. The synthesis and degradation of chitin. Advances in
Enzymology 59: 59-101
Cannon, R. D., Kyoko, N., Jenkinson, H. F. and M. G. Shepherd. 1993.
Molecular cloning and expression of the Candida albicans P-N-Acetyl-
glucosaminidase {HEX!) gene. Journal o f Bacteriology 176: 2640-2647
Cantz, M. and H. Buresse. 1974. Sandhoffs Disease: Defective
glycosaminoglycan catabolism m cultured fibroblasts and its correction by P-N-
Acetyl-hexosaminidase. European Journal o f Biochemistry 47: 581-590
Casaregola, S., Jacq, A., Laondj, D., McGnrk, G., Margarson, S.,
Tempete, M., Norris, V. and I. B. Holland. 1992. Cloning and analysis of
the entire Escherichia coli ams gene: ams is identical to hmpl and encodes a
114 IcDa protein that migrates as a 180 IcDa Protein. Journal o f Molecular
Biology 228: 30-40
Cayaite, A. J., Knmbla, L. and M. T. R. Snbbiah. 1990. Marked
acceleration of exogenous fatty-acid incorporation into cellular triglycerides by
fetuin. Journal o f Biological Chemistry 265: 5883-5888
Cenci, G., Beccari, T., Caldini, G., Bellachioma, G. and A. Orlacchio.
1992. P-N-Acetyl-hexosaminidases from Serratia marcescens. Microbios 71:
135-144.
135
CesaH, !. M., Bouty, I. , Bout, D. DeNoya, B. A. and J. Hoebeke. 1992.
Parasite enzymes as a tool to investigate immune responses. Memorias do
Institutio Oswldo Cruz 87: 55-65
Chandad, F. and C. Mouton. 1990. Molecular size variation of
haemagglutinating adhesin HA-Ag2, a common antigen of Bacteroides
gingivalis. Canadian Journal o f Microbiology 36: 690-696
Chapman, S. J . and H. R. Perkins. 1983. Peptidoglycan-degrading enzymes
in ether-treated cells of Neisseria gonorrhoeae. Journal o f General
Microbiology 129; 877-883
Chatteqee, S. K., Chowdhnry, K., Battachaya, M. and J. J. Barlow.
1982. P-N-Acetyl-hexosaminidase activities and isoenzymes in normal human
ovary and ovarian adenocarcinoma. Cancer 49; 128-135
Christersson, L. A., Zambon, J. J. and R. J. Genco. 1991. Dental bacterial
plaques: Nature and role in periodontal disease. Journal o f Clinical
Periodontology 18: 441-446
Colclasnre, G. C., Loyd, W. S., Lamkin, M., Gonnerman, W., Troxler,
R. F., Offher, G. D., Bnrgi, W., Schmid, K. and R. B. Nimberg. 1988.
Human serum a 2 HS-glycoprotein modulates in vitro bone resorption. Journal
o f Clinical Endocrinology and Metabolism 66: 187-192
Collins, J. 1979. Cell-free synthesis of proteins coding for mobilisation
functions of ColEl and transposon functions of Tn3. Gene 6: 29-42
136
Conzelman, E. and K. Sandhoff. 1987. Glycolipid and glycoprotein
degradation. Advance in Enzymology 60: 89-216
Corbet, E. F. and W. I. R. Davies. 1993. The role of supragingival plaque in
the control of progressive periodontal disease: A review. Journal o f Clinical
Periodontology 20: 307-343
Conchman, J . R. and A. Woods. 1993. Structure and biology of pericellular
proteoglycans. p33-82. In Cell Surface and Extracellular Glycoconjugates.
Structure and Function. Academic Press, Inc.
Cowing, D. W., Bardwell, J. C. A., Graig, E. A., Woolfbrd, C., Hendrix,
R. W. and C. A. Gross. 1985. Consensus sequence for Escherichia coli heat
shock gene promoters. Proceedings o f the National Academy o f Sciences o f the
U. S. 82: 2679-2683
Craig, E. A., GambiB, D. and R. J. Nelson. 1993. Heat shock proteins:
Molecular chaperones of protein biogenesis. Microbiological Reviews 57: 402-
414
Cummings, R. D., Soderqnist, A. M. and G. Carpenter. 1985. The
oligosaccharide moitiés of the epidermal growth factor receptor in ALBl cells:
Presence of complex-type-N-linlced chains that contain terminal N-Acetyl-
galactosamine residues. Journal o f Biological Chemistry 260: 11944-11952
Cutler, C. W., Arnold, R. R. and H. A. Schenkein. 1993. Inhibition of C3
and IgG proteolysis enhances phagocytosis of Porphyromonas gingivalis.
Journal o f Immunology 151: 7016-7029
137
CuÜer, C. W.; Arnold, R. R. and H. A. Schenkein. 1993. Inhibition of C3
and IgG proteolysis enhances phagocytosis of Porphyromonas gingivalis.
Journal o f Immunology 151: 7016-7029
Dabelsieen, E., Mandel, U. and H. Clausen. 1991. Cell surface
carbohydrates are markers of differentiation in human oral epithelium. Critical
Reviews in Oral Biology and Medicine 2% 493-507
Dahlen, G. G. 1993. Black-pigmented Gram-negative anaerobes in
periodontitis. FEMS Immunology and Medical Microbiology 6: 181-192
Daita, B., Ray, M. K., Chakrabard, D., Wylie, D. E. and N. K. Gupta.
1989. Glycosylation of eukaryoytic peptide chain initiation factor 2 (eIF-2)-
associated 67 kD polypeptide (p^^) and its possible role in inhibition of eIF-2
kinase catalysed phosphorylation of the eIF-2 a-subunit. Journal o f Biological
Chemistry 264: 20620-20624
Datd, A., Emiliani, C., Capocchi, G. and A. Orlacchio. 1991. P N Acetyl
hexosaminidase in human cerebrospinal fluid and serum of patients with
multiple sclerosis. Clinica ChimicaActalW i 73-80
De Nardin, A. M., Scjar, H. T., Gross, S. G., Christersson, L. A. and R.
J. Genco. 1991. Humoral immunity of older adults with periodontal diseases to
Porphyromonas gingivalis. Infection and Immunity 59: 4363-4370
Deslauriers, M. and C. Mouton. 1992. Epitope mapping of hemagglutinating
adhesin HA-Ag2 of Porphyromonas gingivalis. Infection and Immunity 60:
2791-2799
138
Devereux, J., Haeberi, P. and O. Smithies. 1984. A comprehensive set of
sequence analysis programs for the VAX. Nucleic Acids Research 12: 387-395
Dickinson, D. P., Knbiniec, M. A., Yoshimnra, F. and R. J. Genco. 1988.
Molecular cloning and sequencing of the gene encoding the fimbrial subunit of
Bacteroides gingivalis. Journal o f Bacteriology 170: 1658-1665
Dobrossy, L., Pavelic, A. P., Vanghan, M., Porter, N. and R. J. Bemacki.
1980. Elevation of lysosomal enzymes in primary Lewis lung tumour correlated
with the intiation of metastasis. Cancer Research 40:3281-3285
Dongan, G. and D. J. Sherratt. 1977. The use of transposon I as a probe of
the structure and function of plasmid ColEl. Molecular and General Genetics
151: 151-160
Doyle and Koch. 1987. The function of autolysins in the growth and division
of Bacillus subtilis. Critical Reviews in Microbiology 15: 169-222
Drake, C. R. 1991. Thesis presented for the degree of Doctor of Philosophy.
University of Leicester
Duncan, M. J., Nakao, S., Skobe, Z. and H. Xie. 1993. Interaction of
Porphyromonas gingivalis with epithelial cells. Infection and Immunity 61:
2260-2265
Duerden, B. I. 1975. Pigment production by Bacteroides species with
reference to sub-classsification. Journal o f Medical Microbiology 8: 113-125
139
Dnsek; D. M., Progukke-Fox, A., Whidock, J. and T. A. Brown. 1993.
Isolation and characterisation of a cloned Porphyromonas gingivalis
haemagglutanin from an avirulent strain of Salmonella typhimurium. Infection
and Immunity 61: 940-946
Dwek, R. A., Edge, C. J., Harvey, D. J. and M. R. Wormland. 1993.
Analysis of glycoprotein-associated oligosaccharides. Annual Review o f
Biochemistry 62: 65-100
Dyer, D. W., Bikdis, G., Michel, J. H. and R. Malek. 1992. Coiyngal
transfer of plasmid and transposon DNA from Escherichia coli into
Porphyromonas gingivalis. Biochemical and Biophysical Reseach
Communications 186: 1012-1019
Dykes, G., Bambara, R., Marians, K. and R. Wn. 1975. On the stasdcal
significance of the primary structural features found in DNA protein interaction
sites. Nucleic Acid Research 2: 327-345
Ebersole, J. L., Tanbman, M. A., Smith, D. J. and D. E. Frey. 1985.
Human serum responses to oral microorganisms: Patterns of systemic responses
to Bacteroides species. Infection and Immunity 51: 507-513
Ebisu, S., Nakae, H., Fnkuhara, H. and H. O. Okada. 1992. The
mechanisms of Eikenella corrodens aggregation by salivary glycoprotein and
the effect of glycoprotein on oral bacterial aggregation. Journal o f Periodontal
Research 27: 615-622
140
Embery, G., Oliver, W. M. and J. B. Stanbnry. 1979. The metabolism of
proteoglycans and glycosaminoglycans in inflamed human gingiva. Journal o f
Periodontal Research 14: 512-519
EpositO; G., Faelli, A., Tnsco, M., Orenigo, M. N., De Gasperi, R. and N.
Pacces. 1983. Influence of the enteric surface coat on the unidirectional flux of
acetamide across the wall of rat small intestine. Experientia 39: 149-150
Esaiassen, M., Mymes, B. and R. Olsen. 1991. PuriGcation and some
properties of two P-N-Acetyl-hexosaminidases from the hepatopancreas of
northern shrimp Fandalus borealis. Comparitive Biochemistry and Physiology
lOlB: 513 517
Evans, R. T., Klansen, B., Sojar, H. T., Bedi, G. S., SGntescn, C.,
Ramamnrtby, N. S., Golub, L. M. and R. J. Genco. 1992. Immunisation
with Porphyromonas gingivalis fimbriae protects against periodontal
destruction. Infection and Immunity 60: 2926-2935
Falk, P., Hoskins, L. C. and G. Larson. 1990. Bacteria of the human
intestinal microbiota produce glycosidases specific for lacto-series
glycosphingolipids. Journal o f Biochemistry 108: 466-474
Falkow, §. 1988. Molecular Koch's postulates applied to microbial
pathogenicity. Reviews o f Infectious Diseases 10: S274-S276
Fein, J. E. and H. J. Rogers. 1976. Autolytic enzyme deficient mutants of
Bacillus subtilis 168. Journal o f Bacteriology 127: 1427-1442
141
Feintoerg, A. P. and B. Vogelstein, 1983. A technique for radiolabelling
restriction endonuclease fragments to a high specific activity. Analytical
Biochemistry 132: 6-13
Fem , T. 1991. Cell-cell adhesion and membrane glycosylation. Current
Opinion in Structural Biology 1: 766-770
Feizi, T. 1993. Oligosaccharides that mediate mammalian cell-cell adhesion.
Current Opinion in Structural Biology 3: 701-710
Finegold; S. M., Strong, C. A., Me Teague, M. and M. Marina. 1993. The
importance of black-pigmented Gram-negative anaerobes in human infections.
FEMS Immunology and Medical Microbiology 6: 77-82
Finlay, B. B. 1992. Molecular genetic approaches to understanding bacterial
pathogenesis. In Molecular Biology of Bacterial Infection: Current status and
future perspectives. p33-45. Hormaeche, C. E., Penn, C. W. and C. J. Smyth
(eds). The Society for General Microbiology, Symposium 49. Cambridge
University Press, U. K.
Finlay, B. B. and S. Falkow. Common themes in microbial pathogenicity.
Mirobiological Reviews 53: 210-230
Flach, J., Pilet, P. E. and P. Jolies. 1992. Whats new in chitinase research?
Experientia 42: 701-716
142
Foster, T. J. 1992. The use of mutants for defining the role of virulence
factors in vivo. In Molecular Biology of Bacterial Infection; Current status and
future perspectives. pl73-191. Hormaeche, C. E., Penn, C. W. and C. J.
Smyth (eds). The Society for General Microbiology, Symposium 49.
Cambridge University Press, U. K.
Furukawa, K. and A. Kobata. 1993. Use of structural analyses to examine
the functions of N- and O-linlced sugar chains of glycoproteins, pl-32. In Cell
Surface and Extracellular Glycoconjugates. Structure and Function. Academic
Press, Inc.
Gamper, M. and D. Haas. 1993. Processing of the Pseudomonas arcABC
mRNA requires functional RNAase E in Escherichia coli. Gene 129: 119-122
Genco, C. A., Cnder, C. W., KapczynsW, D., Maloney, K. and R. R.
Arnold. 1991. A novel mouse model to study the virulence of and host
response to Porphyromonas (Bacteroides) gingivalis. Infection and Immunity
59: 1255-1263
Georgellis, D., Barlow, T., Arvidson, S. and A. von Gabain. Retarded RNA
turnover in Escherichia coli: A means of maintaining gene expression during
anaerobiosis. Molecular Microbiology 9: 375-381
Ghnysen, J-M ,Tippler, D. J. and J. L. Stromlnger. 1966. Enzymes that
degrade their bacterial cell walls. Methods in Enzymology 8: 685-699
Gibbons, F. J. and J. B. MacDonald. 1960. Haemin and vitamin K
compounds as required factors for the cultivation of certain strains of
Bacteroides melaninogenicus. Journal o f Bacteriology 80: 164-170
143
Gibson, W. A. and H. M. Fnlbner. 1969. The Histochemistry of the
periodontal ligament IV. The glycosidases. Journal o f Periodontology 40: 470-
475
Glasgow, L. R. 1977. Systematic purification of five glycosidases from
Streptococcus pneumoniae. Journal o f Biological Chemistry 252: 8615-8623
Gold, L. and G. Stormo. 1987. Translational initiation. pl302-1307. In
Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology.
F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanilc, M. Schaechter
and H. E. Umbarger (eds). American Society for Microbiology, Washington,
D. C.
Goverman, J. M., Paresons, T. F. and J. G. Pierce. 1983. Enzymatic
deglycosylation of the subunits of human chorionic gonadotropin: Effects on
formation of tertiary structure and biological activity. Journal o f Biological
Chemistry 257: 15059-15064
Graham, T. R., Zassenhans, H. P. and A. Kaplan. 1988. Molecular cloning
of the cDNA which encodes P-N-Acetyl-hexosaminidase A from Dictyostelium
discoideum: Complete amino-acid sequence and homology with the human
enzyme. Journal o f Biological Chemistry 263: 16823-16829
Greber, U. F., Koznlic, B. and K. Mosbach. 1989. Purification of endo-N-
Acetyl-P -D-glucosaminidase H by substrate-affinity chromatography.
Carbohydrate Research 189: 289-299
144
Grenier, D. 1992a. Nutritional interactions between two suspected
periodontopathogens, Porphyromonas gingivalis and Treponema denticola.
Infection and Immunity 60: 5298-5301
Grenier, D. 1992b. Demonstration of a biomodal coaggregation reaction
between Porphyromonas gingivalis and Treponema denticola. Oral
Microbiology and Immunology 7: 280-284
Grenier, D. 1992c. Further evidence for a possible role of trypsin-like activity
in the adherence of Porphyromonas gingivalis. Canadian Journal o f
Microbiology 3d)i 1189-1192
Grenier, D. and D. Mayrand. 1987. Functional characterisation of
extracellular vesicles produced by Bacteroides gingivalis. Infection and
Immunity §Si 111-117
Gnardaü, M. C., Gnzman, C. A., Lipira, G., Piatd, G., Robbiad, F. and
C. Pmzzo. 1993. The use of monoclonal antibodies for studying the biological
properties of Staphylococcus aureus endo-p-N-acetyl-glucosaminidase. FEMS
Microbiology Letters 112; 73-80
Gnbisb, E. R., Chen, K. C. S. and T. M. Buchanan. 1982. Detection of a
gonococcal endo-P-N-acetyl-glucosaminidase and its peptidoglycan cleavage
site. Journal o f Bacteriology 151: 172-176
Haapasalo, M., Singh, U., Me Bride, B. C. and V. J. Uitto. 1991.
Sulfliydryl-dependant attachment of Treponema denticola to laminin and other
proteins. Infection and Immunity 59: 4230-4237
145
Haapasalo, M., Singh, U., Me Bride, B. C. and V. J. Uiito. 1991.
Sulfhydryl-dependant attachment of Treponema denticola to laniinin and other
proteins. Infection and Immunity 59: 4230-4237
Hakomori, S. 1993. Structure and function of shingoglycolipids in
transmembrane signaling and cell-cell interactions. Biochemical Society
Transactions 21: 583-595
Hallewall, R. A. and D. J. Sherrrat. 1976. Isolation and characterisation of
ColE2 plasmid mutants unable to kill colicin-sensitive cells. Molecular and
General Genetics 146: 239-245
Hanunond, S. M., Lambert, P. A. and A. N. Rycroft. 1984. The Bacterial
Cell Surface. The peptidoglycan layer. pl7. Kapitan Szabo Publishers,
Washington, DC
Hanazawa, S., Mnmkami, Y. and K. Hirose. 1991.
gingivalis fimbriae activate periodontal macrophages and induce expression and
production of IL-1. Infection and Immunity 59: 1972-1977
Hanover, J. A., Cohen, C. K., Willingham, M. C. and M. K. Park. 1987.
0-linked N-Acetylglucosamine is attached to proteins of the nuclear pore:
Evidence for cytoplasmic and nucleoplasmic glycoprotems. Journal o f
Biological Chemistry 262: 9887-9894
Hamada, N., Watanabe, K., Sasakawa, C., Yoshikawa, M., Yoshimnra,
F. and T. Umemoto. 1994. Construction and characterisation of afimA mutant
of Porphyromonas gingivalis. Infection and Immunity 62: 1696-1704
146
Hase, C. and R. A. Finkelstein, 1993. Bacterial extracellular zinc-containiag
metalloproteases. Microbiological Reviews 57: 823-837
Hawley, D. K. and W. R. McClnre. 1983. Compilation and analysis of
Escherichia coli promoter DNA sequences. Nucleic Acid Research 11: 2237-
2255
Hayashi, S. and H. C. Wn. 1993. Lipoproteins in bacteria. Journal o f
Bioenergetics and Biomembranes 22: 451-471
Henrissat, B. and A. Bairoch. 1993. New families in the classification of
glycosyl hydrolases based on amino acid sequence similarities. Biochemical
Journal 293: 781-788
Herron, and Schhnerlik. 1983. Glycoprotein properties of the solubilised
atrial muscarinic acetylcholine receptor. Journal o f Neurochemistry 41: 1414-
1420
Higgins, D. G., Bleasby, A. J. and R. Fnchs. 1992. Clustal-V-improved
software for multiple sequence alignment. Computer Applications in the
Biosciences 8: 189-191
Hirsch, R. S. and N. G. Clarke. 1989. Infection and periodontal diseases.
Reviews o f Infectious Diseases 11: 707-715
Hofte, H. and H. R. WMtely. 1989. Insecticidal crystal proteins of Bacillus
thuringiensis. Microbiological Reviews 53: 242-255
147
Holdeman, L. V. and W. E. C. Moore. 1974. Gram-negative anaerobic
bacteria. In Bergey’s Manual of Determinative Bacteriology. p384-404.
Buchanan, R. E. and N. E. Gibbons (eds.), Williams and Willdns, Baltimore,
MD
Holland, I. B., Casaregola, S. and V. Norris. 1990. Cytoskeletal elements
and calcium; Do they play a role in the Escherichia coli cell cycle ? Research
in Microbiology 141: 131-136
Holt, G. D., Snow, C. M ., Senior, A., Haldwanger, R. S., Gerace, L. and
G. W. Hart. 1987. Nuclear pore complex glycoproteins contam
cytoplasmically disposed N-Acetyl-glucosamine. Journal o f Cell Biology 104:
1157-1164
Holt, S. Co and T. E. Bramand. 1991. Factors in virulence expression and
their role in periodontal disease pathogenesis. Critical Reviews in Oral Biology
and Medicine 2% 177-281
Holt, S. C., Ebersole, J . , Felton, J ., Bmnsvold, M. and K. S. Korman.
1988. Implantation of Bacteroides gingivalis m non human primates initiates
progression of periodontitis. Science 239; 55-57
Holtje, J-V. and E. Z. Tuomanen. 1991. The murein hydrolases of
Escherichia coli: Properties, functions and impact on the course of infections in
vivo. Journal o f General Microbiology 137; 441-454
Holton, T. A. and M. W. Graham. 1991. A simple and effecient method for
direct cloning of PCR products using ddT-taüed vectors. Nucleic Acid Research
19: 1156-1156
148
Hoover, C. I., Ng, C. Y. and J. R. Felton. 1992b. Correlation of
haemagglutination activity with trypsin-lüce protease activity. Archives in Oral
Biology 37: 515-520
Hoskins, L. C. 1991. Bacterial glycosidases and degradation of
glycoconjugates in the human gut. p37-47. In Molecular Pathogenesis of
Gastrointestinal Infections. T. Wadstrom et al (eds). FEMS Symposium 58.
Plenum Press, NewYork and London
Hoskins, L. C., Agnstine, M., McKee, W. B., Bonlding, E. T., Kriaris, M.
and G. Neidermeyer. 1985. Mucin degradation in human colon ecosystems:
isolation and properties of faecal strains that degrade ABH blood group
antigens and oligosaccharides from mucin glycoproteins. Journal o f Clinical
Investigation 75: 944-953
Hoskins, L. C. and E. T. Bonlding. 1981. Mucin degradation in human colon
ecosystems; Evidence for the existence and role of bacterial subpopulations
producing glycosidases as extracellular enzymes. Journal o f Clinical
Investigation 1̂% 163-172
Hussain, A. M., daniel, R. C. W. and A. J. Frost. 1992. The bactericidal
effect of N-Acetyl-P-D-glucosaminidase on bacteria. Veterinary Microbiology
32: 75-80. Elsevier Science Publishers B. V.
Hynes, R. O. 1990. Interactions of fibronectins. p84-112. In Fibronectins. R.
O. Hynes (ed). Springer Series in Molecular Biology. Springer-Varlag, New
York Inc.
149
Hynes, R. O. 1990. Interactions of fibronectins. p84-112. In Fibronectins. R.
O. Hynes (ed). Springer Series in Molecular Biology. Springer-Varlag, New
York Inc.
International Union of Biochemistry Recommendations, 1976. 1978. The
nomenclature of lipids. Journal o f Lipid Research 19: 114-129
International Union of Biochemistry Recommendations, 1980. 1982.
Polysaccharide nomenclature. Journal o f Biological Chemistry 257: 3352-3354
International Union of Biochemistry Recommendations on Enzyme
Nomenclature. 1978. Academic Press, New York
Isogai; H., Isogai, E., Yoshimura, F., Suzuki, T., Kagota, W. and K.
Tanko. 1988. Specific inhibition of adherence of an oral strain of Bacteroides
gingivalis 381 to epithelial cells by monoclonal antibodies against the fimbriae.
Archives in Oral Biology 33: 479-485
Ito, H., Nak^ima, M., Okamura, Y., Nishi, K. and T. Hirota. 1989a.
Histochemical analysis of the chemical structure of blood group related
carbohydrate chains in serous cells of human submandibular glands using lectin
staining and glycosidase digestion. Journal o f Histochemistry and
Cytochemistry WJ% 1115-1124
Ito, H., Nishi, K., Nak^ima, M., Okamura, Y. and T. Hirota. 1989b.
Histochemical demonstration of 0-glycosidically linlced, type 3 based ABH
antigens in human pancrease using lectin staining and glycosidase digestion
procedures. Histochemistry 92: 307-312
150
Iwamoto, T.; Akifund, S., Asdommthee, S. and S. Nagasaki. 1993. Jack
bean p-N-Acetyl-hexosaminidase does not hydrolyse N-Acetyl-chito-
oligosaccharides. Bioscience, Biotechnology and Biochemistry 57: 841-842
Jannatiponr, M., Soto-Gil, G. W., Childers, L. C. and J. W. Zyskind.
1987. Translocation of Vibrio harveyi N, N'-Diacetylchitobiase to the outer
membrane of Escherichia coli. Journal o f Bacteriology 169: 3785-3791
Jeanloz, R. W. 1972. a^-acid glycoprotein. p565-611. In Glycoproteins 5:
Their Composition Structure and Function. A. Gottashalk (ed.). Elsevier Pub.
Comp.
Jensen, H. K, and T. Lendet. 1986. Proteolysis of arterial basement
membrane containing different amounts of carbohydrates. Thrombosis Research
44: 47-53
Joe, A. J., Murray, G. §. and B. C. Me Bride. 1994. Nucleotide sequence
of a Porphyromonas gingivalis gene encoding a surface-associated glutamate
dehydrogenase and construction of a glutamate dehydrogenase-deficient
isogenic mutant. Infection and Immunity 62: 1358-1368
Joe, A., Yamamoto, A. and B. C. Me Bride. 1993. Characterisation of
recombinant and native forms of a cell surface antigen of Porphyromonas
{Bacteroides) gingivalis. Infection and Immunity 61: 3294-3303
Joshi, S., Koziowski, M., Selvaij, G., Iyer, V. N. and R. N. Davies.
Cloning of the genes of chitin utilization regulon of Serratia liquefaciens.
Journal o f Bacteriology 170: 2984-2988
151
KalfaS; S.; Andersson, M., Edwardsson, S., Forgren, A. and A. S. Naidn.
1991. Human lactoferrin binding to Porphyromonas gingivalis, Prevotella
intermedia and Prevotella melaninogenica. Oral Microbiology and Immunology
6: 350-355
Karlson, K. A. 1989. Animal glycosphingolipids as membrane attachment sites
for bacteria. Annual Reviews o f Biochemistry 58: 309-358
ICato, To, Takahashi, N. and H. E . Knramitsn. 1992. Sequence analysis and
characterisation of the Porphyromonas gingivalis prtC gene which expresses a
novel collagenase activity. Journal o f Bacteriology 174: 3889-3895
KawagisM, S., Araki, Y. and E. Ito. 1980. Bacillus cereus autolytic
endoglucosaminidase active on cell wall peptidoglycan with N-unsubstituted
glucosamine residues. Journal o f Bacteriology 141: 137-143
Kay, H. M., Birss, A. J . and J. W. SmaUy. 1990a. Haemagglutininating and
haemolytic activity of extracellular vesicles of Bacteroides gingivalis W50.
Oral Microbiology and Immunology 5: 269-274
Kay, H. M., Birss, A. J. and J. W. Smally. 1990b. Interaction of
extracellular vesicles of Bacteroides gingivalis W50 with human
polymorphonuclear leukocytes. FEMS Microbiology Letters 72: 69-74
152
Keulers, R. A. C., Maltha, J. C., Mikx, F. H. M. and J. M. L. Wolters-
Lntgerhorst. 1993. Involvement of treponemal surface located protein and
carbohydrate moitiés in the attachment of Treponema denticola ATCC 33520 to
cultured rat palatal epithelial cells. Oral Microbiology and Immunology 8: 236-
241
Kilian, M. 1981. Degradation of immunoglobulins A l, A2 and G by suspected
principle periodontal pathogens. Infection and Immunity 34: 757-765
Kinane, D. F., Mooney, J., MacFarlane, T. W. and M. Me Donald. 1993.
Local and systemic antibody response to putative periodontopathogens in
patients with chronic periodontitis: Correlation with clinical indices. Oral
Microbiology and Immunology 8: 65-68
Kinder, S. A. and S. C. Holt. 1993. Localisation of the Fusobacterium
nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas
gingivalis T22. Journal o f Bacteriology 175: 840-850
Kirkham, J., Robinson, C., Smith, A. J. and J. A. Spence. 1992. The effect
of periodontal disease on sulphated glycosaminoglycan distribution in the sheep
periodontium. Archives in Oral Biology 37: 1031-1037
Klansen, B., Evans, R. T., Ramamnrthy, N. S., Golnb, L. M.,Sfmtescn,
C., Lee, J. Y., Bedi, G., Zambon, J. J. and R. J. Genco. 1991. Periodontal
bone level and gingival proteinase activity in gnotobiotic rats immunised with
Bacteroides gingivalis. Oral Microbiology and Immunology 6: 193-201
153
Kless, H., Sitrit, Y., Chet, I. and B. A. Oppenhehn. 1989 Cloning of the
gene for chitobiase of Serratia marcescens. Molecular and General Genetics
217: 471-47
King, G. 1993. The role of mRNA degradation in the regulated expression of
bacterial photosynthesis genes. Molecular Microbiology 9: 1-7
Knowles, B. H., Thomas, W. E. and D. J , EHar. 1984. Lectin-like binding
of Bacillus thuringiensis var. Icurstald lepidopteran-specific toxin is an initial
step in insecticidal action. FEBS Letters 168: 197-202
Kobata, A. 1979. Use of endo and exoglycosidases for structural analysis of
glycoconjugates. Analytical Biochemistry 100:1 14
Kobata, A., Mlznochi, T., Endo, T. and K. Fnmkawa. 1989. Function and
pathology of the sugar chains of human immunoglobulin G. p224-240. In
Carbohydrate Recognition in Cellular Function. Ciba Foundation Symposium
145. Wiley Interscience Pub.
Koide, N., Matso, N. and T. Mnramatso. 1977. Recognition of IgG by Fc
receptor and complement: Effects of glycosidase digestion. Biochemical and
Biophysical Research Communications 75: 838-844
Kolenbrander, P. E. 1988. Intergeneric co-aggregation among human oral
bacteria and ecology of dental plaque. Annual Reviews o f Microbiology 42:
627-656
154
Kolenbrander, P. E. 1990, Surface récognition among oral bacteria:
Multigeneric co-aggregation and their mediators. Critical Reviews in
Microbiology 17: 137-158
Kolenbrander, P. E. and J. London. 1993. Adhere today, here tomorrow:
Oral bacterial adherence. Journal o f Bacteriology 175: 3247-3252
Komelnk, R. G., Mahnran, D. J., Neote, K., Kalvinis, M. H., O'Dowd,
B. F., Tropak, M., Willard, H. F., Anderson, M^J., Lowden, J. A. and R.
A. Gravel. 1986. Isolation of the cDNA clones coding for the a-subunit of
human P-hexosaminidase. Journal o f Biological Chemistry 261: 8407-8413
Komfield, R. and S. Kornfield. 1980. Structure of glycoproteins and their
oligosaccharide units, pl-34. In The Biochemistry of Glycoproteins and
Proteoglycans. W. J. Lennarz, (ed.). Plenum Press, New York and London
Komman, K. S., Holt, S. C. and P. D. Robertson. 1981. The microbiology
of ligature-induced periodontitis in the cynomolgus monlcey. Journal o f
Periodontal Research 16: 363-371
Konichi, K. and N. Yamaji. 1992. Novel chromogenic substrates for the rate-
assay of N-Acetyl-P -D-glucosaminidase : Resorufinyl- and Resazurinyl-N-
Acetyl-P -D-glucosaminides. Analytical Sciences 8: 161-164
Kresse, H. and J. Glossl. 1987. Glycosaminoglycan degradation. Advances in
Enzymology 60: 217-311
Knlekci, G., Dnerden, B. I. and O. Ang. 1993. Black-pigmented anaerobes.
FEMS Immunology and Medical Microbiology 6: 75-75
155
Kumamoto, C. A. 1991. Molecular chaperones and protein translocation
across the Escherichia coli inner membrane. Molecular Microbiology 5: 19-22
Kusukawa, N., Yura, N. T., Ueguchi, C., Akiyama, Y. and K. Ito. 1989.
Effects of mutations in heat shock genes groEL and groES on protein export.
EMBO JoMmoZ 8: 3517-3521
Kyte, Jo and R. F. Doolitle. 1982. A simple method for displaying the
hydropathic character of a protein. Journal o f Molecular Biology 157: 105-132
Kytzia, HnJ. and K. Sandhoff. 1985. Evidence for two different active sites
on human p-hexosaminidase A. Journal o f Biological Chemistry 260: 7568-
7572
Lambster, I. B. and M. J. Novak. 1992. Host mediators in gingival
crevicular fluid: Implications for the pathogenesis of periodontal disease.
Critical Reviews in Oral Biology and Medicine 3: 31-60
Lamont, R. J., Oda, D., Persson, R. E. and G. R. Persson. 1992.
Interaction of Porphyromonas gingivalis with gingival epithelial cells
maintained in culture. Oral Microbiology and Immunology 7: 364-367
Lfmtz, M. S., Allen, R. D., Vail, T. A., Switalskl, M. and M. Hook. 1991.
Specific cell components of Bacteroides gingivalis mediate binding and
degradation of human fibrinogen. Journal o f Bacteriology 173: 495-504
156
Larson, G., Falk, P. and L. C. Hoskins. 1988. Degradation of human
glycosphingolipids by extracellular glycosidases from mucin degrading bacteria
of the human faecal flora. Journal o f Biological Chemistry 263: 10790-10798
Langhon, B. E., Syed, S. A. and W. J. Loesche. 1982. API ZYM system for
identification of Bacteroides spp., Capnocytophaga spp. and spirochetes from
oral origin. Journal o f Clinical Microbiology 15: 97-102
Leafherbarrow; R. J., Rademacher, T. W. and R. A. Dwek. 1985. Effector
functions of a monoclonal aglycosylated mouse IgG2a: Binding and activation
of complement component Cl and interaction with human monocyte Fc
receptor. Molecular Immunology 22:407-415
Lev, M., Kendell, K. C., and A. F. Milford. 1971. Succinate as a growth
factor lox Bacteroides melaninogenicus. Journal o f Bacteriology 108: 175-178
Lewy, G. A. and J. Conchie. 1966. Mammalian glycosidases and their
inhibition by aldonolactones. Methods in Enzymology 8: 571-584
Li, M. and S. Wong. 1992. Cloning and characterisation of the groESL
operon from Bacillus subtilis. Journal o f Bacteriology 174: 3981-3992
Li, S. C. and Y. T. Li. 1970. Studies on the glycosidase of jack bean meal III:
Crystalisation and properties of P-N-Acetyl-hexosaminidase. Journal o f
Biological Chemistry 245: 5153-5160
Lindhe, J., Hamp, S. E. and H. Loe. 1975. Plaque induced periodontal
disease in beagle dogs. A 4-year clinical radiographic and histological study.
Journal o f Periodontal Research 10: 243-255
157
Lipman, D. J . and W. R. Pearson. 1985. Rapid and sensitive protein
similarity searches. Science 221% 1435-1441
Loesche, W. J., Syed, S. A., Morrison, E. C., Langhon, B. and N. S.
Grossman. 1981. Treatment of periodontal infections due to anaerobic bacteria
with short term treatement with metronidazole. Journal o f Clinacal
Periodontology 8: 29-44
London, J. and J. Alien. 1989. Purification and characterisation of a
Bacteroides loeschii adhesin that interacts with prokaryotic and eukaryotic
cells. Journal o f Bacteriology 172: 2527-2530
Loos, B. G., Dyer, D. W., Whiitam, T. S. and R. K. Selander. 1992.
Genetic structure of populations of Porphyromonas gingivalis associated with
periodontitis and other oral infections. Infection and Immunity 61: 204-212
Lopatin, D. E. and E. Blackbnm. 1992. Avidity and titre of IgG subclasses
to Porphyromonas gingivalis in adult periodontitis patients. Oral Microbiology
and Immunology 7: 332-337
Lovatt, A. and I. §. Roberts. 1991. Cloning of the P-N-Acetyl-
hexosamdnidase from Porphyromonas gingivalis. Journal o f Dental Reasearch
71: 293-293
Lnndblad, G., Hnldt, G., Elander, M., Lind, J. and K. Slettengren. 1981.
P-N-Acetyl-glucosaniindase from Entamoeba histolytica. Comprehensive
Biochemistry and Physiology 68B: 71-76
158
Lundemose, A. G., Rouch, D. A., Penn, C. W. and J. H. Pearce. 1993.
The Chlamydia trachomatis Mip-like protein is a lipoprotein. Journal of
Bacteriology 175: 3669-3671
Lutkenhans, J. 1993. FtsZ ring in bacterial cytokinesis. Molecular
Microbiology 9: 403-409
Mac Donald, J. B., Socransky, S. S. and R. J. Gibbons. 1963. Aspects of
the pathogenesis of mixed infections of mucous membranes. Journal o f Dental
Research 42: 529-544
MacFarlane, G. T. and G. R. Gibson. 1991. Formation of glycoprotein
degrading enzymes by Bacteroides fragilis. FEMS Microbiology Letters 77:
289-294
MacFarlane, G. T., Hay, S. and G. R. Gibson. 1989. Influence of mucin on
glycosidase, protease and arylamidase activities of human gut bacteria grown in
a 3-stage continuous culture system. Journal o f Applied Bacteriology 66: 407-
417
Macrina, F. L. 1984. Molecular cloning of bacterial antigens and virulence
determinants. Annual Review o f Microbiology 38: 193-219
Macrina, F. L., Dertzbangh, M. T., Halnla, M. C., Krah, R. and K. R.
Ross. 1990. Genetic approaches to the study of oral microflora: A ^y iéw .
Critical Reviews in Oral Biology and Medicine 1: 207-227
159
Madden, T. E ., Thompson, T. M. and V. L. Clark. 1992. Expression of
Porphyromonas gingivalis proteolytic activity in Escherichia coli. Oral
Microbiology and Immunology 7: 349-356
Malay, J., Shoemaker, N. B. and I. S. Roberts. The introduction of colonic-
Bacteroides shuttle plasmids into Porphyromonas gingivalis: Identification of a
putative insertion-sequence element. FEMS Microbiology Letters 93: 75-82
Malek, R., Fisher, J. G., Caleca, A., Stinson, M., van Oss, C. J., Lee, J-
Y., Cho, M-I., Genco, R. J., Evans, R. T. and D. W. Dyer. 1994.
Inactivation of the Porphyromonas gingivalis flmA gene blocks periodontal
damage in gnotobiotic rats. Journal o f Bacteriology 176: 1052-1059
Maley F., Trhnble R. B ., Tarentino A. L. and T. H. Plnmmer. 1989.
Characterisation of glycoproteins and their associated oligosaccharides through
the use of endoglycosidases. Analytical Biochemistry 180: 195-204
Marsh, P. D., McKee, A. S. and A. S. McDermid. 1988. Effect of haemin
on enzyme activity and cytotoxin production by Bacteroides gingivalis W50.
FEMS Microbiology Letters 55: 87-92
Mayhall, C. W. 1970. Concerning the composition and source of the aquired
enamel pellicle of human teeth. Archives in Oral Biology 15: 1327-1341
Mayrand, D. and D. Grenier. 1985. Detection of collagenase activity in oral
bacteria. Canadian Journal o f Microbiology 31: 134-138
Mayrand, D. and S. C. Holt, 1988. Biology of asaccharolytic black-
pigmented species. Microbiological Reviews 52: 134-152
160
Me BMde, B. C., Joe, A. and U. Singh. 1990. Cloning of
gingivalis surface antigens involved in adherence. Archives in Oral Biology 35:
S59-S68
Me Carthy, J . E. G. and D. Gnalaenm. 1990. Translational control of
prokaryotic gene expression. Trends in Genetics 6: 78-65
Me Dowall, K., Hernandez, R. G., Lin-Chao, S. ans S. N. Cohen. 1993.
The ams-1 and rne-3071 temperature sensitive mutations in the ams gene are in
close proximity to each other and cause substitutions within a domain that
resembles a product of the Escherichia coli mre locus. Journal o f Bacteriology
175: 4245-4249
Me Indre, F. C. 1985. Specific surface components and microbial co
aggregation. In Molecular Basis of Oral Microbial Adhesion. pl53-163.
Mergenhagen, S. E. and B. Rosan (eds.). American Society for Microbiology,
Washington D. C.
Me Kee, A. S., Me Dermid, A. §., Baskerville, A., Dowsed, A. B.,
EUwood, Do Co and P. D. Marsh. 1986. Effect of haemin on the physiology
and virulence of Bacteroides gingivalis. Infection and Immunity 52: 349-355
Mengand, J., Vicente, M. F. and P. Cossart. 1989. Transcriptional mapping
and nucleotide sequence of the Listeria monocytogenes hlyA region reveal
structural features that may be involved in regulation. Infection and Immunity
57: 3695-3701
161
Megly:, S., Henderson, B. and M. Wilson. 1993. High titre antisera from
patients with periodontal disease inhibits bacterial capsule induced bone
breakdown. Journal o f Periodontal Researchist 115-121
Melefbrs, O., Lnndberg, U. and A. von Gabain. 1993. RNA processing and
degradation by RNAase K and RNAase E. p53-71. In Control of messenger
RNA stability. Belasco, J. and G. Brawerman (eds.). Academic Press Inc., U.
K.
Mihara, J., Yoneda, T. and S. C. Holt. Role of gmgfrafü-
derived fibroblast activating factor in bone resorption. Infection and Immunity
61: 3562-3565
Milsx, F. H. Mo and R. A. Co Kenlers. 1992. Haemagglutination activity of
Treponema denticola grown in serum-free medium in confinons culture.
Infection and Immunity 60: 1761-1766
Minhas, To and J . Greenmano 1989. Production of cell-bound and vesicle
associated trypsin-lilce protease, alkaline phosphatase and N-Acetyl-P-
glucosaminidase by Bacteroides gingivalis W50. Journal o f General
Microbiology 135: 557-564
Mirebnan, D. 1988. Protozoal cell products and their effects on the host.
pl41-154. In Surface structures of microorganisms and their interactions with
the mammalian host. E. Schrinner, M. H. Richmond, G. Seibert and U.
Schwarz (eds). Workshop Conference Hoechst 18. VCH Verlagsgesellschaft
Pub., Germany
162
M jzu o cM , T.; Taniguchi, T. and A. Sûnizn. 1982. Structural and numerical
variations of the carbohydrate moitey of IgG. Journal o f Immunology 129:
2016-2020
Moncla, B. J., Brahman, P. and S. L« Hillier, 1990. Sialidase
(neuramioidase) activity among Gram-negative anaerobic and capnophhic
bacteria. Journal o f Clinical Microbiology 28: 422-425
Mormaga, T., Kitandkado, M., Iwase, H., Li, S-C. and Y-T. Li. 1983. The
use of mannan-sepharose 4B affinity chromatography for the purification of
endo-P-N-Acetyl-glucosaminidase from Bacillus alvei. Biochimica et
Biophysica Acta 749: 211-213
Morioka, M., Hinode, D., Nagata, A., Hayashi, H., Ichhniya, S., Ueda,
M., Kido, R. and R. Nakamura. 1993. Cytotoxicity of
gingivalis toward cultured human gingival fibroblasts. Oral Microbiology and
Immunology 8: 203-207
Mndd, E. A. and C. F. Higgins. 1993. Escherichia coli endoribonuclease
RNAase E: Autoregulation of expression and site specific cleavage of mRNA.
Molecular Microbiology 9: 557-568
Murakami, Y., Hanazawa, §., Nishida, K., Iwasaka, H. and S. Kitano.
1993. N-Acetyl-galactosamine inhibits TNF-alpha gene expression m induced
peritoneal macrophages by fimbriae of Porphyromonas gingivalis, an oral
anaerobe. Biochemical and Biophysical Research Communications 192: 826-
832
163
Nakayama, K. 1994, Rapid viability loss on exposure to air in a superoxide
dismutase-deficient mutant of Pophyromonas gingivalis. Journal o f
Bacteriology 176: 1939-1943
Narberhaus, F. and H. BaM. 1992. Cloning, sequencing and molecular
analysis of the groESL operon of Clostridium acetobutylicum. Journal o f
Bacteriology 174: 3282-3289
Nagata, H., Mnrakann, E., Inoshita, E., Shiznknisbi, S. and A.
Tsimemitsuo 1990, Inhibitory effect of human plasma and saliva on co
aggregation between Bacteroides gingivalis and Streptococcus mitis. Journal o f
Dental Research 69: 1476-1479
Nageswara, R. T. and O. P. BaM. 1987. Enzymatic deglycosylation of
glycoproteins. Methods in Enzymology 138: 350-359
Naito, Yo and R. J . Gibbons. 1988. Attachment of Bacteroides gingivalis to
collagenous substrata. Journal o f Dental Research 67: 1075-1080
Naito, Y., Tohda, H., Okuda, K. and I. Takazoe. 1993. Adherence and
hydrophobicity of invasive and non-invasive strains of Porphyromonas
gingivalis. Oral Microbiology and Immunology 8: 195-202
Nanninga, N., W ienies, F. B., Mnlder, E and C. L. Woldringh. 1993.
Envelope growth in Escherichia coli: Spatial and temporal organisation. pl85-
221. In Prokaryote Structure and Function: A New Perspective. Mohan, S.,
Dow, C. and J. A. Cole (eds). The Society for General Microbiology,
Symposium 47, Cambridge University Press, U. K.
164
Nash, R. A. 1987. Fusobacterium and Bacteroides reactions in a new API
anaerobic test system (API-ATB). Fifth International Symposium on Rapid
Methods and Automation in Microbiology and Immunology. Florence
Nemeth, E ., Kulkami, G. W. and C. A. G. Me Cnlloch. 1993. Distrubances
of gingival fibroblast population homeostasis due to experimentally induced
inflammation in the cynomolgus monkey (Macaca fascicularis): Potential
mechanisms of disease progression. Journal o f Periodontal Research 28: 180-
189
Neufield, F. N. 1989. Natural history and inherited disorders of a lysosomal
enzyme, P-N-Acetyl-hexosaminiadse. Journal o f Biological Chemistry 264:
10927-10930
Nicolson, Go L. 1982. Cancer metastasis: organ colonisation and the cell
surface properties of malignant cells. Reviews in cancer. Biochimica et
Biophysica Acta €95% 113-176
Niedbatn, M. J., Madiyalakan, K., Matta, K., Ciickard, K., Shanna, M.
and R. J. BemacM. 1987. The role of glycosidases in human ovarian
carcinoma cell mediated degradation of subendothelial extracellular matrix.
Cancer Research 47: 4634-4641
Nilins, A. M., Spencer, S. C, and L. G. Simonsom. 1993. Stimulation of
invitro growth of Treponema denticola by extracellular growth factors produced
by Porphyromonas gingivalis. Journal o f Dental Research 72: 1027-1031
165
Nilsson, B., Forsberg, G., Moks, T., Hartmanis, M. and M. Uhlen. 1992.
Fusion proteins in biotechnology and structural biology. Current Opinion in
Structural Biology 2% 569-575
Nishikaia, M., Yoshimnra, F. and Y. Nodasaka. 1989. Possibility of
Bacteroides gingivalis haemagglutinin possessing protease activity revealed by
inhibition studies. Microbiology and Immunology 33: 75-80
O* Dowd, B. F., Qnan, F., Willard, H. F., Lamhonwah, A M., Korneluk,
R.G., Lowden J.A., Gravel, R. A. and D. J. Mahmnn. 1985. Isolation of
the cDNA clones coding for the P-subunit of human p-hexosaminidase.
Proceedings o f the National Academy o f Sciences o f the U. S. A. 82: 1184-1188
Oddon, P., Hartmann, H., Radecke, F. and M. Geiser. 1993.
hnmunologically unrelated Heliothis sp. and Spodoptera sp. midgut membrane
proteins bind Bacillus thuringiensis CryIA(b) 5-endotoxin. European Journal o f
Biochemistry 212: 145-150
Offenbacher, S., Collins, J. G. and R. R. Arnold. 1993. New clinical
diagnostic strategies based on pathogenesis of disease. Journal o f Periodontal
Research 28: 523-535
Okada, Y., Wachi, M., Hirata, A., Suzuki, K., Nagai, K. and M.
Matsuhashi. 1994. Cytoplasmic axial filaments in Escherichia coli cells:
Possible function in the mechanism of chromosome segragation and cell
division. Journal o f Bacteriology 176: 917-922
166
Okada, Y., Wachi, M., Nagai, K. and M. Matsnhashi. 1992. Change of the
quantity of penicülm-hmding proteins and other cytoplasmic and membrane
proteins by mutations of the cell shape-determination genes mreB, mreC and
mreD of Escherichia coli. Journal o f General and Applied Microbiology 38:
157-163
Okuda, K., Yanmamoto, A., Naito, Y., Takazoe, E., Slots, J. and R. J.
Genco. 1986. Purification and properties of haemagglutinin from culture
supernatant of Bacteroides gingivalis. Infection and Immunity 54: 659-665
Oliver, W. W. and W. B. Wherry. 1921. Notes on some bacterial parasites
of the human mucous membranes. Journal o f Infectious Diseases 28: 341-345
Oppdenakker, G., Rndd, P. M., Ponting, C. P. and R. A. Dwek. 1993.
Concepts and principles of glycobiology. The FASEB Journal 7: 1330-1337
Ortiz, J. M. 1974. Mutant of Bacillus subtilis lacking exo-P-N-Acetyl-
glucosaminidase activity. Journal o f Bacteriology 117: 909-910
Otogoto, J-I. and H. K. Karamitsn. 1993. Isolation and characterisation of
the Porphyromonas gingivalis prtT gene, coding for protease activity. Infection
and Immunity €>1% 117-123
Parekh, R. B., Dwek, R. A., Rademacher, T. W., Oppenakker, G. and J.
van Damme. 1992. Glycosylation of interleukin-6 from normal human blood
mononuclear cells. European Journal o f Biochemistry 203: 135-141
167
Park, Y. and B. C. Me Bride. 1992. Cloning of a Porphyromonas
{Bacteroides) gingivalis protease gene and characterisation of its product.
FEMS Microbiology Letters 92: 273-278
Pazzani, C. 1992. Thesis presented for the degree of Doctor of Philosophy.
p82. University of Leicester
Penn C. W. 1992. Chronic infections, latency and the carrier state. Current
status and future perspectives. pl07-125. Hormaeche, C. E., Penn, C. W. and
C. J. Smyth (eds). The Society for General Microbiology, Symposium 49.
Cambridge University Press, U. K.
Pike, P., McGraw, W., Poiempa, J. and J. Travis. 1994. Lysine and
arginine-specific proteinases from Porphyromonas gingivalis: Isolation,
characterisation and evidence for the existence of complexes with
haemagglutinins. Journal o f Biological Chemistry 269: 406-411
Pratt, J. M., Bonlnois, G. J., Darby, V., Orr, E., Wahle, E. and I. B.
Holland. 1981. Identification of gene products programmed by restriction
endonuclease DNA fragments using an E. coli in vitro system. Nucleic Acid
Research 9: 4459-4474
Prescott, L. M., Harley, J. P. and D. A. Klein. 1990. Helminfh diseases.
Appendix V. In Microbiology. W. C. Brown Pub.
Price, R. G. and N. Dance. 1972. The demonstration of multiple heat stable
forms of P-N-Acetyl-hexosaminidase in normal serum. Biochimica et
Biophysica Acta n i l 145-153
168
Pnzont, R. 1982. Degradation of intestinal glycoproteins by pathogenic
Shigella flexneri. Infection and Immunity 36: 615-620
Pugsley, A. 1991. Superfamilies of bacterial transport systems with nucleotide
binding components. p223-248. In Prokaryote Structure and Function: A new
perspective. Mohan, S., Dow, C. and J. A. Cole (eds). The Society for
General Microbiology, Symposium 47, Cambridge University Press, U. K.
Pugsley, A. 1993. The complete general secretory pathway in Gram-negative
bacteria. Microbiological Reviews 57: 50-108
Rademacher, T. W., Homans, S. W., Parekh, R. B. and R. A. Dwek.
1986. Immunoglobulin G as a glycoprotein. Biochemical Society Symposia 51;
131-148
Rademacher, T. W., Parekh, R. B. and R. A. Dwek. 1988. Glycobiology.
Annual Reviews in Biochemistry 57: 785-838
RahemtuUa, F. 1992. Proteoglycans of oral tissues. Critical Reviews in Oral
Biology and Medicine 3: 135-162
Rashid, M. H., Kuroda, A. and J. Sekiguchi. 1993. mutant
deficient in the major autolytic amidase and glucosamioidase is impaired in
motility. FEMS Microbiology Letters 112: 135-140
Ravdin, J. 3. 1986. Pathogenesis of disease caused by Entamoeba histolytica.
Studies of adherence, secreted toxins and contact dependent cytolysis. Reviews
o f Infectious Diseases 8: 247-260
169
Ready, M. S. and M. K. JeHcoat. 1993. Periodontal disease progression.
Current Opinion in Periodontology. p52-59
Rhoads, M. L. 1985. Glycosidases of Trichinella spiralis. Molecular and
Biochemical Parasitology 16: 137-148
Rhoads, M. L. 1988. Purification, characterisation and immunochemical
studies of P-N-Acetyl-hexosaminidase from the parasitic nematode Trichinella
spiralis. Molecular and Biochemical Parasitology 31: 57-70
Riordan, J. F., Me Elvany, K. D. and C. L. Boreders. 1977. Arginyl
residues: Anion recognition sites in enzymes. Science 195: 884-886
Robertson, A. M. and R. A Stanley. 1982. In vitro utilization of mucin by
Bacteroides fragilis. Applied and Environmental Microbiology 43; 325-330
Roden, L. 1980. Structure and metabolism of connective tissue proteoglycans.
p267-371. In The Biochemistry of Glycoproteins and Proteoglycans. W. J.
Lermarz, (ed.). Plenum Press, New York and London
Rogers, A. H., Gnlly, N. J., Pfennig, A. L. and P. S. Zihn. 1992. The
brealcdown and utilisation of peptides by strains of Fusobacterium nucleatum.
Oral Microbiology and Immunology 7: 299-303
Roitt, I. 1991. Autoimmune diseases. Chapter 15, Essential Immunology.
Seventh Edition. Blackwell Scientific Pub.
Roodman, G. D. 1991. Osteoclast Differentiation. Critical Reviews in Oral
Biology and Medicine 2; 389-409
170
Rosenberg, M. and D. Court. 1979. Regulatory sequences involved in the
promotion and termination of RNA transcription. Annual Review o f Genetics
13: 319-354
Ruoslahti, E. 1988. Fibronectin and its receptors. Annual Review o f
Biochemistry 57: 375-413
Ruseler van Embden, J. G. H., van der Hebn, R. and L. M. C. van
Lieshout. 1989. Degradation of intestinal glycoproteins by Bacteroides
vulgatus. FEMS Microbology Letters 58: 37-42
Saglie, R. A., Marfany, A. and P. Camargo. 1988. hitragingival occurence
of Actinobacillus actinomycetemcomitans and Bacteroides gingivalis in active
periodontal lesions. Journal o f Periodontology 59: 259-265
Saito, H. and K. I. Muria. 1963. Preparation of transforming
deoxyribonucleic acid by phenol treatment, Biochemica et Biophysica Acta 72:
619-629
Salyers, A. A., Vercellotti, J. R., West, S. E. H. and T. D. Wilkins. 1977.
Fermentation of mucin and plant polysaccharides by strains of Bacteroides from
the human colon. Applied and Environmental Microbiology 33: 319-322
Sandros, J., Papapanou, P. N. and G. Dablen. 1993.
gingivalis invades oral epithelial cells invitro. Journal o f Periodontal Research
28: 219-226
171
Sanger, F., Nicklen, S. and A. R. Coukon. 1977. DNA sequencing with
chain termination inhibitors. Proceedings o f the National Academy o f Sciences
oTfAg U. & A. 74:5463-5467
Sawyer, S. J., MacDonald, J. B. and R. Gibbons. 1962. Biochemical
characteristics of Bacteroides melaninogenicus. Archives o f Oral Biology 7:
685-691
Schenken, H. A. 1986. The effect of periodontal proteolytic Bacteroides
species on proteins of the human complement system. Journal o f Periodontal
Research 23i 187-192
Scott, C. F., Whitaker, E. J., Hammond, B. F. and R. W. Cohnan. 1993.
Purification and characterisation of a potent 70 kDa thiol lysyl-proteinase (Lys-
gingivain) from Porphyromonas gingivalis that cleaves Idninogens and
fibrinogen. Journal o f Biological Chemistry 268: 7935-7942
Segal, G and E. Z. Ron. 1993. Heat shock transcription of the groESL operon
of Agrobacterium tumifaciens may involve a hairpin loop structure. Journal o f
Bacteriology 175: 3083-3088
Shah, H. N., Bonnet, R., Matteen, B. and R. A. Williams. 1979. The
porphyrin pigmentation of subspecies of Bacteroides melaninogenicus. The
Biochemical Journal 180: 45-50
Shah, H. N and S. E. Gharbia. 1989. Lysis of erythrocytes by the secreted
poteinase of Porphyromonas gingivalis W83. FEMS Microbiology Letters 61:
213-218
172
Shah, H. N and S. E. Gharbia. 1993. Studies on the physiology and ecology
of black-pigmented Gram-negative anaerobes which may be important in
disease development. FEMS Immunology and Medical Microbiology 6: 165-172
Sharma, A., Hakimnddin, T. S., Lee, J. Y. and R. J. Genco. 1993.
Expression of a functional Porphyromonas gingivalis fimbrülin polypeptide in
Escherichia coli: Purification, physiochemical and immunochemical
characterisation and binding characteristics. Infection and Immunity 61: 3570-
3573
Shibntani, T., Nishino, W., Shiraki, M. and Y. Iwayama. 1993. ELISA
detection of glycosaminoglycan (GAG)-linlced proteoglycans in gingival
crevicular fluid. Journal o f Periodontal Research 28: 17-20
Shoemaker, N. B., Barber, R. D. and A. A. Salyers. 1989. Cloning and
characterisation of a Bacteroides conjugal tetracycline-erythromycin resistance
element by using a shuttle cosmid vector. Journal o f Bacteriology 171: 1294-
1302
Silberberg, A. 1989. Mucus glycoprotein, its biophysical and gel-forming
properties. p43-63. In Mucus and Related Topics. E. Chantier and N. A.
Ratcliffe (eds). The Society of Experimental Biology. The Company of
Biologists Ltd.
Simonson, L. G., Me Mahon, K. T., Childers, D. W. and H. E. Morton.
1992. Bacterial synergy of Treponema denticola and Porphyromonas gingivalis
in a multinational population. Oral Microbiology and Immunology 7: 111-112
173
Sisney-Durant, H. J. and R. M. Hopps. 1991. Effect of lipopolysaccharide
from Porphyromonas gingivalis on prostaglandin E2 and interleukin ip release
from rat periotoneal and human gingival fibroblasts in vitro. Oral Microbiology
and Immunology 6: 378-380
Slonnany, B. L., Mnrty, V. L. N P iotrow sW , J., Lian, Y. H. and A.
Slomiany. 1993. Glycosulphatase activity of Porphyromonas gingivalis a
bacterium associated with periodontal disease. Biochemistry and Molecular
Biology International 29: 973-980
Slots, J. and R. J. Genco. 1984. Black-pigmented Bacteroides species,
Capnocytophaga species and Actinobacillus actinomycetemcomitans in human
periodontal disease: Virulence factors in colonisation, survival and tissue
destruction. Journal o f Dental Research 63: 412-421
Smalley, J. W. and A. J. Birss. 1987. Trypsin-like enzyme activity of the
extracellular membrane vesicles of Bacteroides gingivalis W50. Journal o f
General Microbiology 133: 2883-2894
Smith, D. B. and K. S. Johnson. 1988. Single step purification of
polypeptides expressed in Escherichia coli as fusions with glutathione-S-
transferase. G ene€h 31-40
Socransky, S. S. and A. D. Haffl^ee. 1991. Microbial mechanisms in the
pathogenesis of destructive periodontal diseases: A critical assessment. Journal
o f Periodontal Research 26: 195-212
174
Sohlberg, B., Lundberg, U., Hard, F-U. and A. von Gabin. 1993.
Functional interaction of heat shock protein GroEL with an RNAase E-lilce
activity m Escherichia coli. Proceedings o f the National Academy o f Sciences o f
fAg U. & A. 90: 277-281
Sojar, H. T., Lee, J. Y., Bedi, G. S. and R. J. Genco. 1993. Purification
and characterisation of a protease from Porphyromonas gingivalis capable of
degrading salt-solubüised collagen. Infection and Immunity 61: 2369-2376
Sojar, H. T., Lee, J. Y. Bedi, G. S., Cho, M. I. and R. J. Genco. 1991.
Purification, characterisation and immunolocalisation of fimbrial protein from
Porphyromonas gingivalis. Biochemical and Biophysical Research
Communications 175: 713-719
Somerville, C. C. and R. A. Colwell. 1993. Sequence analysis of the P-N-
Acetyl-hexosaminidase gene from Vibrio vulnificus. Evidence for a common
evolutionary origin of hexosaminidases. Proceedings o f the National Academy
o f Sciences o f the U. S. A. 90: 6751-6755
Soto-Gil, R. W. and J. W. Zysldnd. 1989. N, N'-diacetylchitobiase of Vibrio
harveyi. Primary structure, processing and evolutionary relationship. Journal o f
Biological Chemistry 264: 14778-14783
Southern, E. M. 1975. Detection of specific sequences among DNA fragments
separated by gel electrophoresis. Journal o f Molecular Biology 89: 503-517
Stark, M. J. R. 1987. Multicopy expression vectors carrying the lac repressor
gene for regulated high-level expression of genes in Escherichia coli. Gene 51:
255-267
175
Stephen, J. and R. A. PietrowsM. 1986, Bacterial Toxins, 2nd Edition. Van
Nostrand Reinhold Ltd., U. K.
Stinson, M. W., Haraszthy, G. G., Zhang, X. L. and M. J. Levine. 1992.
Inhibition of Porphyromonas gingivalis adhesion to Streptococcus gordonii by
human submandibular-sublingual saliva. Infection and Immunity 60: 2598-2604
Strong, G. Jo and J. Dekker. 1992. Mucin-type glycoproteins. Critical
Reviews in Biochemistry and Molecular Biology 27: 57-92
Sngai, M., Koike, H., Hong, Y-M., Miyake, Y., Nogami, R. and H.
Snglnaka. 1989. Purification of a 51 kDa endo-P-N-Acetyl-glucosaminidase
from Staphylococcus aureus. FEMS Microbiology Letters 61: 267-272
Snndqvist, G. 1993. Pathogenicity and virulence of black-pigmented Gram-
negative anaerobes. FEMS Immunology and Medical Microbiology 6: 125-138
Snndqvist, G., Caisson, J. and L. Bbinstrom. 1987. CoUagenolytic activity
of black-pigmented Bacteroides species. Journal o f Periodontal Research 22:
300-306
Syed, S. A., Salvador, S. L. and W. J. Loesche. 1988. Enzyme profiles of
oral spirochetes in Rapid ID-ANA system. Journal o f Clinical Microbiology
26: 2226-2228
176
Tai; H.; Kobayashi, T. and K. Hara. 1993. Changes in complement and
inimunoglobulin G receptor expression of neutrophils associated with
Porphyromonas gmg/vfl/w-induced inhibition of phagocytosis. Infection and
Immunity 61: 3533-3535
Takasakm, S. and A. Kobata. 1986. Asparagine linked sugar chains:
occurence of tetrasialy/triantennary sugar chains containing the Galpl-
3GlcNAc sequence. Biochemisrty 25: 5709-5715
Tanaka, Y., Adams, D. H. and S. Shaw. 1993. Proteoglycans on endothelial
cells present adhesion-inducing cytokines to leukocytes. Immunology Today 14:
111-115
Tanignchi, T., Miznochi, T., Beale, M., Dwek, R. A., Rademacher, T. W.
and A. Kobata. 1985. Structures of the sugar chains of rabbit immunogobulin
G: Ocurrence of asparigine linlced sugar chains m the Fab fragment.
Biochemistry 24: 5551-5557
Taraseviciene, L., Mlczak, A. and D. Aphion. 1991. The gene specifying
RNase E {me) and a gene affecting mRNA stability {ams) are the same gene.
Molecular Microbiology 5: 851-855
Tews, I., Danter, Z., Oppenhehn, A. B. and C. E. Vorglas. 1992.
Crystalisation of recombinant chitobiase from Serratia marcescens. Journal o f
Molecular Biology 228: 696-697
Thomas, W. and R. Sellwood. 1993. Molecular cloning, expression and DNA
sequence of the gene that encodes the 16 kDa outer membrane lipoprotein of
Sepulina hyodysenteriae. Infection and Immunity 61: 1136-1140
177
Thomas, W., SeUwood, R. and R. J. Lysons. 1992. A 16 kDa lipoprotein of
the outer membrane of Serulina {Treponema) hyodysenteriae. Infection and
Immunity 60: 3111-3116
Tipler, L. S. and G. Embery. 1985. Glycosaminoglycan-depolymerising
enzymes produced by anaerobic bacteria isolated from the human mouth.
Archives in Oral Biology 5: 391-396
Tortora, G. and N. P. Anagnostakos. 1990. Principles of Anatomy and
Physiology. Sixth Edition. Harper and Row Pub., New York
Townsend, R. R., Hilleker, E ., Li, Y., Laine, R. A., Bell, R. A. and Y. C.
Lee. 1982. Carbohydrate structure of human fibrinogen: Use of 300MHz
^NMR to characterise glycosidase treated glycopeptides. Journal o f Biological
Chemistry 257: 9704-9710
Tucker, S. M., Pierce, R. J. and R. G. Price. 1980. Characterisation of
human P-N-Acetyl-glucosaminidase isoenzymes as an indicator of tissue
damage in disease. Clinica Chimica Acta 102: 29-40
Uitto, V. J . 1991. Extracellular matrix molecules and their receptors: An
overveiw with special emphasis on periodontal tissues. Critical Reviews in Oral
Biology and Medicine 2: 323-354
Uitto, V. J., Laqava, H., Heino, J . and T. Sorsa. 1989. A protease of
Bacteroides gingivalis degrades cell surface and matrix glycoproteins of
cultured gingival fibroblasts and induces secretion of collagenase and
fibrinogen. Infection and Immunity 57: 2213-2218
178
Valkena, S., Varaldo, P. E. and G. Satta. 1982. Purification and
charcteristion of three seperate bacteriolytic enzymes excreted by
Stapholococcus aureus. Staphylococcus simulans and Staphylococcus
saprophyticus. Journal o f Bacteriology 151: 636-647
Valisena, S., Varaldo, P. E. and G. Satta. 1991. Aqp/Q/fococcoZ endo-P-N-
Acetyl-glucosaminidase inhibits response of human lymphocytes to mitogens
and interferes with the production of antibodies in mice. Journal o f Clinical
Investigation 87:1969-1976
van Dyke, T. E., Offenbacher, §., Place, D., Dowell, V. R. and J. Jones.
1988. Rejfractiory periodontitis: mixed infection with Bacteroides gingivalis and
other unusual Bacteroides species. Journal o f Periodontology 59: 184-189
van Snick, 1990. Interleukin-6: An overveiw. Annual Review o f Immunology
8: 253-278
van Steenbergen, T. J. M., van Winkelhoff, A. J. and J. de Graff. 1993.
Classification and typing methods of black-pigmented Gram-negative
anaerobes. FEMS Immunology and Medical Microbiology 6: 83-88
van Winkelhoff, A. J., Appelmelk, B. J., Kippnw, N. and J. de Graff.
1993. K-antigens in Porphyromonas gingivalis are associated with virulence.
Oral Microbiology and Immunology 8: 259-265
van Winkelho^, A. J., van Steenbergen, T. J. M., Kippnw, N. and J. de
Graaff. 1985. Further characterisation of Bacteroides endodontalis, an
179
asaccharolytic black-pigmented Bacteroides species from the oral S cavity.
Journal o f Clinical Microbiology 22; 75-79
von Figura, K. and A. Hasilk. 1986. Lysosomal enzymes and their receptors.
Annual Review o f Biochemistry 55: 167-193
von Heijne, 6 . 1986. A new method for predicting signal cleavage sites.
Nucleic acid Research 14: 4683-4690
von Heijne, G. and L. Abrahmsen. 1989. Species-specific variation in signal
peptide design. Implications for protein secretion in foreign hosts. FEBS Letters
244: 439-446
Wachi, M., Doi, M., Okada, Y. and M. Matsuhashi. 1989. New /wg genes
mreC and mreD, responsible for the formation of the rod shape of Escherichia
coli cells. Journal o f Bacteriology 171: 6511-6516
Wachi, M., Doi, M., Ueda, T., Ueki, M., Tsuritani, K., Nagai, K. and M.
Matsuhashi. 1991. Sequence of the downstream flanking region of the shape
determining genes mreBCD of Escherichia coli. Gene 106: 135-136
Walker, J. E ., Saraste, M., Runswick, M. J. and N. J. Gay. 1982.
Distantly related sequences in the a- and P- subunits of ATP synthase, myosin,
Idnases and other ATP-requiring enzymes and a common nucleotide binding
fold. The EMBO Journal 1: 945-951
180
Washington, O. R., Deslanriers, M., Stevens, D. P., Lyfbrd, L. K.,
Haqne, S., Yan, Y. and P. M. Flood. 1993. Generation and purification of
recombinant fimbrülin from Porphyromonas gingivalis 381. Infection and
Immunity 61: 1040-1047
Watanabe, K., Yam^i, Y. and T. Umemoto. 1992. Correlation between
cell-adherent activity and surface structure in Porphyromonas gingivalis. Oral
Microbiology and Immunology 7: 357-363
Watzlawick, H., Walsh, M. T., Yoshioka, Y., Schmid, K. and R.
Brossmer. 1992. The structure of the N- and 0-glycans of the A-chain of
human plasma a 2 -HS glycoprotein as deduced from the chemical compositions
of deravitives prepared by stepwise degradation with exoglycosidases.
Biochemistry 31: 12198-12203
Weinberg, A. and S. C. Holt. 1988. Interaction of Treponema denticola TD-
4, GMl and MS25 with human gingival fibroblasts. Infection and Immunity 56:
726-728
Welply, Jo ICo 1989. Sequencing methods for carbohydrates and their
biological applications. Trends in Biotechnology 7: 5-10
Worries, E., Nebinger, P. and A. Franz. 1983. Degradation of biogene
oligosaccharides by P-N-Acetyl-hexosaminidase secreted by Entamoeba
histolytica. Molecular and Biochemical Parasitology 1% 127-140
Westerlnnd, B. and T. K. Korhonen. 1993. Bacterial proteins binding to the
extracellular matrix. Molecular Microbiology 9: 687-694
181
Wilson, K. H. and F. Pernini, 1988. Role of competition for nutrients in the
suppression of Clostridium difficile by colonic microflora. Infection and
Immunity 56: 2610-2614
Wright, N. T., Kinsella, M. G. and E. E. Qwamstrom. 1992. The role of
proteoglycans in cell adhesion, migration and proliferation. Current Opinion in
793-801
Wortman, A. T., Somervile, C. C. and R. R. Colwell. 1986. Chitinase
determinants of Vibrio vulnificus: Gene cloning and applications of a chitinase
probe. Applied and Environmental Microbiology 52: 142-145
Woynarowska, B., Wikiel, H. and R. J. Bemacki. 1989. Human ovarian
carcinoma p-N-Acetyl-glucosaminidase isoenzymes and their role in
extracellular matrix degradation. Cancer Research 49: 5598-5604
Woynarowska, B., Wikiel, H., Sharma, M., Carpenter, N., Fleet, G. W.
J. and R. J. Bemacki. 1992. Inhibition of human ovarian carcinoma cell- and
hexosaminidase mediated degradation of the extracellular matrix by sugar
analogs. Anticancer Research 12: 161-166
Wnenscher, M. D., Koehler, S., Bnhert, A., Gerike, U. and W. Goebel.
1993. The iap gene of Listeria monocytogenes is essential for cell viability, and
its gene product, has bacteriolytic activity. Journal o f Bacteriology 175: 3491-
3501
182
Yamasbita, K., Ohkura, T., Yoshima, H. and A. Kobata. 1981. Substrate
specificity of Diplococcal P-N-Acetyl-hexosaminidase, a useful enzyme for the
structural studies of complex-type asparagine-linlced sugar chains. Biochemal
and Biophysical Research Communications 100: 226-232
Yanagishita, M. 1993. Function of proteoglycans in the extracellular matrix.
Acta Pathologica Japonica 43: 283-293
Yaniscb-Perron, C., Viera, J. and J. Messing. 1985. Improved M13 phage
cloning vectors and host strains; Nucleotide sequences of M13mpl8 and
pUC 19 vectors. Gene 33% 103-119
Yasni, S., Kcjbna, T., Hata, S., Zhang, Y. J., Umeda, M. and I.
Ishikawa. 1993. Rapid identification of Porphyromonas gingivalis by bisulfite-
modified DNA probe method. Journal o f Periodontal Research 28: 98-101
Yem, D. W. and H. C. Wn. Isolation of Escherichia coli K12 mutants with
altered levels of P-N-Acetyl-glucosaminidase. Journal o f Bacteriology 125:
372-773
Yoshima, H., Matsnmoto, A., Miznochi, T., Kawasaki, T. and A. Kobata.
1981. Comparitive study of the carbohydrate moitiés of rat and human plasma
ai-acid glycoproteins. Journal o f Biological Chemistry 256: 8476-8484
Yohimnra, F., Sngano, T. Kawanami, M., Kato, H. and T. Snznki. 1987.
Detection of specific antibodies against fimbriae and membrane proteins from
the oral anaerobe Bacteroides gingivalis in patients with periodontal disease.
Microbiology and Immunology 31: 935-941
183
Yoshimnra, F., Takahashi, K., Nodasaka, Y. and T. Snznki. 1984.
Purification and characterisation of a novel type of fimbriae from the oral
anaerobe Bacteroides gingivalis. Journal o f Bacteriology 160: 949-952
Yoshimnra, F., Takahashi, K., Yoneyama, M., Tamagnchi, T., Shiokawa,
H. and T. Snznki. 1985. Fimbriae from the oral anaerobe Bacteroides
gingivalis: Physical, chemical and immunological properties. Journal o f
Bacteriology 163: 730-734
Yntaka, T., Okada, S., Kato, T. and H. Yabnnhi. 1982. Degradation of
keratan sulphate by P-N-Acetyl-hexosaminidase in GM2 gangliosidosis.
Clinical Genetics 21: 196-202
Zhn, B. C. R., Lo, J. Y., Li, Y. T., Li, S. C., Jaynes, J. M., Gildemeister,
O. S., Laine, R. A. and C. Y. On. 1992. Thermostable, salt tolerant, wide
pH range, novel chitobiase from Vibrio parahaemolyticus: Isolation,
characterisation, molecular cloning and expression. Journal o f Biochemistry
112: 163-167