MOLECULAR CHARACTERIZATION OF THE HIPPURATE HYDROLASE GENE IN HIPPURATE HYDROLASE-NEGATIVE
CAMPYLOBACTER JEJUNI ISOLATES
Tin Htwe Thin
A thesis submitted in conforrnity with the requirements for the degree of Mater of Science,
Graduate Department of Microbiology, in the University of Toronto
O Copyright by Tin Htwe Thin 1997
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Molecular Characterization of the Hippurate Hydrolase Gene in Hippurate Hydrolase-Negative Campylobacter jejuni Isolates
Degree of Mater of Science 1997 Tin Htwe Thin
Department of Microbiology University of Toronto
CampyZobacter jejuni is human entericbacterial pathogen. The present study
examined the hippurate hydrolase gene from five different hippurate hydrolase-
negative C. jejuni dinical isolates. Radiolabeled single strand confomational
polymorphic (SSCP) assay detected the polymorphism in F7 and F8 regions of the
hippurate hydrolase gene from five negative isolates compared with the respective
regions of C. jejuni TGH9011 hipO. The promoter region and the remainder coding
region revealed an identical PCR-SSCP patterns compared with the respective regions
of C. jejuni TGH9011 in five negative isolates.
The potential mutated regions were cloned and sequenced to determine the
rnolecular nature of the mutations. Most of the mutations detected were silent in
nature and they do not affect for arnino acid alteration, except for valine (Val) at
residue 250 altered to alanine (Ala). The Val-250 change was consistently present in
al1 five negative isolates. Val-250 residue may play an important role in the hippurate
hydrolase function.
Acknowledgments
My deepest thanks go to my major professor, Dr. Voo Loong (Ricky) Chan for
giving me the opportunity to participate in Campylobacter jejuni genetic studies. My
appreciation is also given to my comrnitee members Dr. A. Bognar and Dr. M.
Kregden for their advice and editorial assistance.
1 owe my sincere gratitude to my parents U Thin Tu and Daw Tin Tin Win and
my only sister, Tin 00 Thin for their permanent support through my career.
1 would like to extend rny apprteciation to my colleagues and fiiends, especialiy
to Helena Louie, for their help and encouragement.
1 aioie 01 conrenrs
Abstract Acknowledgement Table of contents List of figures List of tables List of abbreviations
Introduction
General features of the genus Campylobacter Phylogeny of Campylobacter Clinical features of Campylobacter jejuni nfect ion Virulence factors of Campylobacter jejuni Adhesins Campylobacter toxins Epidemiology of Campylobacter jejuni infection Genome size of Campylobacter jejuni Genes cloned from Campylobacter jejuni Housekeeping genes Virulence gene Antibiotics resistance gene Hippurate hydrolase of Campylobacter jejuni TGH90 1 1 Hmologous proteins belong to M40 hydrolase farnily Hippurate hydrolase-negative Campylobacter jejuni Purpose of this study
Materials and Methods
Bacterial strains Genomic DNA extraction OIigonucleotides Polymerase chah reaction Single strand conformational polymorphic(SSCP) assay and mutation detection enhancement (non denaturing) gel electrophoresis Cloning of the arnplified fragments Ligation of the DNA fragments Preparation of competent cells and transformation with
Kecombinant plasmid DNA preparation Preparation of the squencing grade plasmid DNA DNA sequencing and polyacrylarnide gel electrophoresis
Results
Detection of the hippurate hydrolase sequence heterogeneity in the hippurate hydrolase-negative C. jejuni clinical isolates Single strand conformational polymorphic analysis of naturally occuring mutant hippurate hydrolase genes Cloning of SSCP polymorphic regions and DNA sequencing Sequence comparison Predicted hydropathy profiles
Discussion
Future studies
References
Figure
Biochemical reaction of hippurate hydrolase Nucleotide sequence of C. jejuni TGH9011 hippurate hydrolase Physical map of C. jejuni TGH9011 Strategy for single Strand conformational polymorphic (SSCP) assay based on polyrnerase chain reaction PCR-SSCP analysis of F7 and F8 regions of the hippurate hydrolase gene of five different hippurate hydrolase-negative C. jejuni isolates PCR-SSCP analysis of F 1 , F2, and F3 regions of five different hippurate hydrolase-negative C. jejuni isolates Deduced amino acid sequence alignment of the mutated region of hippurate hydrolase-positive C. jejuni TGH90 1 1 and hippurate hydrolase-negative strain D835 Alignment of C. jejuni hippurate hydrolase and related proteins Comparison of the hydrophobicity profiles for arnidohydrolases of Campylobacter, Bacillus, Pseudomonas, and Synechocystis Predicted hydropathy (hydrophobicity and flexibility) profiles of C. jejuni hippurate hydrolase Comparison of the predicted secondary structure and hydrophobicity profiles of C. jejuni hippurate hydrolase and its modified sequence
Lists of tables
Table
1. Classification of the genus Campylobacter and its relatives 2 . Codon usage and molecular percentage of G+C content of C. jejuni TGH9011
hippurate hydrolase. 3 . Homologous proteins of C. jejuni hippurate hydrolase 4. Bacterial strains used in this study 5 . OIigonucleotides used in this study 6. Primer pairs used in this study 7. Summary of PCR analysis in the hippurate hydrolase gene of hippurate
hydrolase-negative C. jejuni isolates 8. Nucleotide substitutions between nucleotide 473 and 912 of the hipO gene of
C. jejuni hippurate hydrolase-negative isolates
a
P + - % ala A ~ P Arg ATP bp ' C C. CHC13 Ci CO2 CIAP CJT Ch1 CLDT cm CNW CP* CT D DNA dATP dCTP ddNTP dGTP dNTP ddTTP DMSO Dnase DTT EDTA EtBr EtOH fig g gly GTE H2
H 2 0
H2S
alpha beta positive negative percent alanine ampicillin arginine adenosine triphosphate base pair degree CeIsius Campylo bacter chloroform curie carbon dioxide calf intestinal alkaline phosphatase CampyIobucter jejuni toxin chlorarnphenicol cytolethal distension toxin centimeter catalase negative/weak counts per minute cholera toxin daltons deoxyribonucleic acid 5'-deoxyadenosine triphosphate 5'-deoxycytosine triphosphate 2',3'-dideoxynucleotide triphosphates 5'-deoxyguanine triphosphate 2'-deoxynucleotide triphophates 5'-deoxythymine triphosphate dimethy lsulphoxide deoxyribonuclease dithiothreitol ethylenediarninotetraacetic acid ethidium brornide ethanol figure gram glycine glucose, tris, EDTA buffer hydrogen water hydrogen sulfide
H 1P hr 1 A A ile IPTG K kb kDa Km L LB 1bs.lsq.in pCi Pg P 1 M Mb mg Mg MgCl2 MH min ml mM mol%G+C MOPS M r
mFWA N N-Bz- Na NaCl NaOH ng nM nt OD PAGE PCR PEG PH PI pmole PFGE phe
nlppurare nyaroiase hour Indole-3-acetic acid-arnino acid isoleucine isopropyl-P-D-thiogalactoside potassium kilobase kilodalton Michaelis constant liter Luria Bertani broth pounds per square inch microcurie microgram microliter molar megabase milligrarn magnesium magnesium chioride MuelIer Hinton minute milliliter millimolar mol percentage guanidine plus cytidine 3-[N-Morpholino]propane-sulfonic acid relative molecular mass messenger ribonucleic acid normal fi- benzoyl- sodium sodium chloride sodium hydroxide nanometer nanomolar nucleotide optical density polyacrylamide gel electrophoresis polmerase chain reaction polyethylene glycol power of hydrogen isoelectric pH picomole pulsed- field gel elctrophoresis phenylalanine resistance
KNA
RNase rRNA
SDS SSC
SPP* subspp. TBE TE TEMED tet TLC Tris R N A TS
val W
ri bonucleic acid ribonuclease ribosomal ribonucleic acid revoiution per minute sensitive sodium dodecyl sulphate sodium chloride/sodium citrate solution species species(plura1) subspecies Tris boric acid EDTA buffer Tris-EDTA N,N,N',N1-tetramethylene diamine tetracycline thin-layer chromatography tris(hydroxymethy1) benzidine transfer ribonucleic acid type strain unit microcurie ultraviolet volt valine watt weight per volume times [multiplication] times [concentration strength] 5-bromo-4-chloro-3-indoyw-D-galactopyranoside
General features of the prenus Campvlobacier
Members of the farnily Campylobacteraceae are classified as
chemoorganotrophs. They do not ferment or oxidize carbohydrates. They appear as
curved in spira-, V-, or comma-shaped or occasionally straight rods that are 0.2 to 0.9
pm wide and 0.5 to 5 pm long. They rnay occur in short or occasionally long chains.
CoIonies are usually nonpigmented and are weakIy or non-hemolytic. The cells are
Gram-negative and non-sporeforming. Motility occurs as a result of single or
occasionally multiple unsheathed flagella at one or both ends of the bacterial cells.
Campylobacters are usually microaerophilic with a respiratory metabolism. However.
some strains may grow under aerobic or anaerobic conditions. They use
menaquinones as the sole respiratory quinones. A variety of organic acids including
amino acids are used as carbon sources. Oxidation of tricarboxylic acid intermediates
and the dearnination of amino acids are the main routes through which cellular energy
is derived (Hoffman and Goodman, 1982). Optimum temperature for growth ranges
from 30' to 42'C. Biochemical tests give negative reactions in the methyl red and the
Voges-Proskauer tests and there is no production of indole. Gelatin is not liquified.
Al1 species have oxidase activity. Most species c m reduce nitrate and do not
hydrolyze hippurate (Smibert, 1 984; Penner, 1 988; Penner, 199 1 ; Ursing et al., 1 994).
Phvlogeny of Campyiobacter
Campylobacters were isolated and first named as Vibri~~fett ts due to the
- Y
Vibrio (McFadyean and Stockman, 19 1 3). Further investigations ihstrated a low
guanine plus cytosine (G+C) base composition (28 to 47 molar percentage),
microaerophilic growth requixements, nonfermentative metabolism and lack of
oxidation of carbohydrates. These Vibrio - Iike organisms were then renamed as a
new genus, Campylobacter (Sebald and Veron, 1963). Cornprehensive taxonornic
studies of Vibrio - like organisms resulted in renarning Campylobacter fetus as the
type species (with C.fetus subsp. fetus for the organisms that cause sporadic abortions
in cows and ewes and C. fetus subsp. venerealis for the organisms that are responsible
for infectious infertility), along with Campylobacter jejuni, Campylobacter coli and
Carnpylobacter sputorurn (C. sputorum subsp. sputorum, C. sputorum subsp. bubulus)
(Veron and Chatelian, 1973).
Partial 16s ribosomal ribonucleic acid sequences of Campylobacter species
were used to compare and analyze the relationships of these organisms to one another
and to other gram-negative bacteria. The genus Campylobacter was divided into three
separate ribosomal ribonucleic acid sequence homology groups. Homology group 1
contains the true Campylobacter species which are C. fetus (two subspecies), C. coli,
C. jejuni, C. laridis, C. hypointestinalis, C. concicus, C. mucosalis, C. sputorum, and
C.upsaliensis ( C N W strains). Homoiogy group II includes C. cinaedi, C. fennelliae,
C. pylori and W. succinogenes. Homology group I I I consists of C. cryaerophila and
C. nitroflgilis. These three homology groups are distantly related to the representative
of the alpha, beta, and gamma branches of the purple bacteria (Thompson et al.,
1988).
limited the genus Campylobacter to C. fetus, C. hypointestinalis, C. concisus, C.
mucosalis, C. sputorum, C. jejuni, C. coli, C. lari, and C. upsaliensis. C. cinaedi and
C. fennelliae were included in the genus Helicobacter as Helicobacter cinaedi and
Helicobacter fennelliae. Two species of the genus Carnpylobacter, C. nitrofigilis and
C. cryaerophilia were constituted as a new genus Arcobacter and were renamed as
Arcobacter nitrofigilis and Arcobacter cryaerophilus. Wolinella curva and Wolinella
recta was transferred to the genus Campylobacter as Carnpylobacter curvus and
Campylobacter rectus, respectively (Paster and Dewhirst, 1988; Vandamme et al.,
1991). Thus, Wolinella succinogenes is the only species of the genus Wolinella.
Based on the rRNA homology studies, Bacteroides gracilis and Bacteroides
ureolyticus are generically misnarned and are closely related to rnembers of the genus
Campylobacter (Vandamme et al., 1991). B. ureoZytictcs and B. graciIis are
microaerophilic and not anaerobes. Recently, B. gracilis was reclassified under the
genus CrrmpyZobacter as Campylobacter gracilis based not oniy on genotypic data, but
also on the proteolytic metabolism of respiratory quinones, protein profiles and
cellular fatty acids analysis (Vandamme et al., 1995). Presently Campylobacter hyoilei
is grouped under the genus Campylobacter and recognized as closely related to C.
jejuni and C. coli on the basis of their G+C contents, phenotypic characteristics,
hybridization with a speci'es-specific DNA probe, 16s rRNA sequence cornparison
data and DNA-DNA hybridization data (Alderton et al., 1995).
Table 1 presents the list of known species of Carnpylobacter, Helicobacter, and
Arcobacter according to the recent presentation of taxonomie position, known sources
and common diseases associated with Campylobacteria (On, 1996).
Genus Campylobacter C. coli ( Vibrio coli) C. concisus (?Pol inella curva) C. cuwus C. fetus subsp. fetus ( Vibrio fetus)
su bsp. venerealis ( Vibrio fe f us) C. gracilis C. helveticus C. hyoilei C. hyointestinalis C. jejuni subsp. jejuni ( Vibrio jejuni)
subsp. doylei C. lari C. mucosalis C. rectus C. showae C. sputorurn biovar sputoruni
biovar bubulus biovar fecalis
C. upsaliensis
Genus Arcobacter A. butderi A. cryaerophilus A. n itr0figili.s A. skirowii
Genus Bacteroides [Bacteroides] ureolyticus
Genus Helicobacter H. acirlonyx H. canis H. cinaedi H. felis H. fenrzelliae H. heilma~zrzi H. hepaticus H. muridarum H. nzustelae H. nernestrinae H. pamete~tsis H. pullorum H. pylori H. rappini
("C. butder?') (C. cryaerophila)) (C. nitrofgilis)
(C. cinaedi)
(C. fennelliae) (Gastrospirillum hominis)
(C. mustelae)
(C. pylori) (Flexispira rappini)
Gen us Wolirtella W. succinogenes
A wide spectmm of clinical features are associated with C. jejuni infections
(Skirrow and Benjamin, 1981; Allos and Blaser, 1995). The main manifestation is
diarrhea, ranging fiom watery to inflarnmatory dysentery with bloody diarrhea. Other
symptoms often present are fever, abdominal pain, nausea, headache and muscle pain
(Walker et al., 1986; Penner, 1991). Human infections with Campylobacter sp. occur
through the ingestion of contaminated food, milk and water. Generally, the most
cornmon origins of infection are uncooked poultry, beef and unpasteurized milk. The
ilhess usually occurs 2 to 5 days afier ingestion of the contarninated water or food.
The diarrhea usually continues for 2 days to a week. The organisms remain in the host
for 2 weeks to 3 months unless antibiotic therapy is taken. Early treatment with
erythromycin may reduce the length of time that infected individuals shed the bacteria
in their feces. Human feeding studies suggest that as few as 500 celis may cause
illness in some individuals. Histopathologic examination of infected colon usually
reveals diffuse inflammation of the lamina propria by neutrophils and mononuclear
cells, and injury of the surface epithelial cells. In some cases, the tissue damage
caused by C. jejuni infection resembles ulcerative colitis or Crohn's disease (Green et
al., 1984). Other abdominal complications of C. jejuni infections are gastrointestinal
hemorrhage, toxic megacolon, pseudomembranous colitis, cholecystitis, and
pancreatitis.
In addition to the gastrointestinal illness caused by C. jejuni, multi-systemic
disorders, which include septicemia, meningitis, septic abortion, Reiter's syndrome,
and reactive arthritis (Johnson et al., 1983). Most recently, Guillian-Barre syndrome
has been reported (Molnar et al., 1983; Kaldor and Speed, 1984). Guillian-Barre
etiology and pathogenesis of this illness is not completely understood. Several recent
studies (Bolton, 1995; Ho et al., 1995; Rees et al., 1995) support an earlier observation
(Kaldor, 1984) that intestinal infections with C. jejuni often precede the onset of
Guillian-Barre syndrome.
Virulence factors of Campvlobacter iejuni
Three potentially pathogenic properties have been identified for C. jejuni;
invasiveness, enterotoxin and cytotoxin production.
Adhesins
C. jejuni utilizes its adhesive ability to colonize the intestinal epithelium,
invade the intestinal mucosa and proliferate in the lamina propria and mesenteric
lymph nodes of the host. Flagella, outer membrane proteins and lipopolysaccharide
(LPS) are factors involved in bacterial adherence to epithelial cells and mucus
(McSweegan and Walker, 1986; deMe10 and Pechere, 1 990). Motility can be directed
by chemotactic factors including L-fùcose, L-aspartate, L-cystine, L-glutamate, and L-
serine, pyruvate, succinate, fumarate, citrate, malate, and oc-ketoglutarate. The C.
jejuni flagellum is able to undergo both phase and antigenic variation (Caldwell et al.,
1985; Harris et al., 1987). Both mechanisms may play a role in the ability of the
organism to evade the host immune response. The pathogen is able to revert to
flagellate forms in phase variation and can alter irnmunological specificity under
antigenic variation. Aflagellated mutants constructed by gene replacement techniques
were utilized in in vitro assays for adherence and penetration o f intestinal cells and
(Grant et al., 1993). Non invasive C. jejuni strains are also pathogenic presumably due
to the production of exotoxins that can disrupt host cells. The invasiveness of non
invasive C. jejuni strains c m be induced by coinfection with other enteroinvasive
Salmonella, Shigella, and E. coli strains (Bukholm and Kapperud, 1987).
The O side chain of LPS may be a factor contributing to the adhesive
properties for the ce11 (McSweegan and Walker, 1986). LPS has preferential binding
ability to the mucous membrane and epithelial cells.
Campvlobacters toxins
Diseases caused by C. jejuni, C. coli and C. laridis are associated with the
production of an enterotoxin or a cytotoxin (Johnson and Lior, 1986). C. jejuni
enterotoxin is a 60- to 70-kDa iron regulated heat-labile enterotoxin. It is structurally
related to cholera toxin and its 8 subunit is partially correlated with the B subunit of E.
coli heat labile enterotoxins (Ruiz-Palacios et al., 1983; Johnson and Lior, 1986;
Klipstein et al., 1986; Daikoku et al., 1990). The induction of watery diarrhea by C.
jejuni enterotoxin was postulated to be similar to cholera toxin. Like cholera toxin,
CJT elongates Chinese Hamster Ovary (CHO) cells, is detected with a GMl-based
ELISA and produces fluid accumulation in intestinal ligated loops (Fernandez et al.,
1983; Ruiz-Palacios et al., 1983; Klipstein and Engert, 1984; McCardell et al., 1984;
Walker et al., 1986; Daikoku et al., 1990).
C. jejuni strains have also been reported to produce a heat-labile protein
cytotoxin tbat is not neutralized by anti-CT, anti-Shiga, or anti-Clostridium antiserum
(Mahajan and Rodgers 1990; Guerrant et al. 1987; Johnson and Lior 1986; Walker et
i'1 -------a----, .---, A 1,
adrenal HeLa cells and the intestinal ce11 line, Int 407 (Perez-Perez et al., 1989).
Although the role of the cytotoxin in C. jejuni pathology is unknown, there is an
increase in fecal leukocytes resulting frorn epithelial ce11 damage. Another potential
toxin is a cytolethal distending toxin (CLDT) that has been identified in some C. jejuni
strains (Johnson and Lior, 1988). C. jejuni CLDT causes progressive ceIl distention
and eventually death in vitro and causes a hemorrhagic response in rat ligated
intestinal segments in vivo. The role of the C. jejuni CLDT in disease is unknown.
Recently, genes with similarity to those encoding E. coli CLDT have been isolated
from C. jejuni (Pickett et a!., 1996). A fourth potential toxin is a Shiga -1ike toxin.
Low levels of a cell-associated cytotoxic factor that is neutralized by anti-Shiga toxin
s e m have been reported from some C. jejuni strains. However, no genetic homology
has been found between the E. coli Shigu-like toxin genes and that of C. jejuni Cell-
fiee filtrates of C. jejuni have been reported to contain a heat-stable substance that
alters intestinal myoelectric activity in rabbit ileum, but no further characterization of
this factor is available (Sninsky et al., 1985).
Epidemiology of Campylobacter jejrrni infection
C. jejuni is now recognized as a common cause of diarrhea in humans. In both
the UK and USA the incidence of C. jejuni infection has been estimated to be around
1,000 per 100,000 population per year (Tauxe, 1992; Ketley, 1995). It occurs in
sporadic forrn in developed countries arnong persons living in temperate or tropical
clirnates and at a much higher incidence arnong travelers to developing countries. C.
jejuni is isolated from adults with diarrhea as ofien as Salmonella and Shigella
5------ - - 2 - - . . , - - - - - - - - - - - , - - . - , - - ------ - - -, - - . - , - - J ""- ,
1980; Young et al., 1980; Drake et al., 1981). It is the third commonest cause of
diarrhea in children of developing countries (DeMol and Bosman, 1978; Blaser et al.,
1980; Black et al., 1981 ; Ruiz-Palacios et al., l983), after enterotoxigenic E. coli, and
rotavims, and of enteritis in children of developed countries after rotavirus and
Shigella (Kapikian et al., 1976; Brunton and Heggie, 1977; Pickering et al., 1978; Pai
et al., 1979; Gurwith et al., 198 1).
Genome size of Campvlobacfer jeiuni
The genome of Campylobacter jejuni is circular and has been estimated to be
between 1.7 to 1.9 Mb by pulsed-field gel electrophoresis of digested DNA fragments
using rare cutting restriction enzymes; BssH II (g'cgcgc), Kpn 1 (ggtac'c), Nci 1
(cc'[gc]gg), Sac II (ccgc'gg), Sa1 I (g'tcgac), and Sma 1 (ccc'ggg) (Smith et al., 1988;
Chang and Taylor, 1990; Nuijten et al., 1990; Kim and Chan, 1991; Kim et al., 1992;
Taylor et al., 1991; Taylor et al., 1992). The base composition is 27 to 30 molar
percentage G+C (Owen, 1983).
Genes cloned from Can~pylobacter jeiuni
In Campylobacter jejuni, there are several chromosomal genes that have been
cloned and characterized. They play different functional roles narnely as housekeeping
genes, virulence genes or antibiotic resistance genes. Our laboratory has cloned over
30 genes from C. jejuni and completely sequenced and characterized 18 of them (Chan
et al., 1988; Kim and Chan, 1989; Chan and Bingharn, 1990; Kim and Chan, 1991;
Chan, 1994; Chan et al., 1995; Hong et al., 1995; Kim et al., 1995; Chan et al., 1997).
Housekeeping genes
Housekeeping genes are highly conserved among cross species and encode
similar functions required for the maintenance and growth of bacteria. For example,
the proA and proB genes are involved in proline biosynthesis and were isolated by
complementing proAproB mutants of E. coli (Lee et al., 1985). The proA gene
encodes gamma-glutamyl phosphate reductase which converts L-glutamate to proline.
This gene has been cloned, sequenced, and mapped on the physical map of C. jejuni
TGH9011 genome using pulse-field gel electrophoresis (PFGE) and Southern blot
hybridization (Kim et al., 1993; Louie and Chan, 1993). Furthemore, the proA gene
was expressed fiom its own promoter and the transcription start site was mapped. The
deduced arnino acid sequence of the proA gene product of C. jejuni exhibits 36.4%
and 36.0% identity to that of E. coli and Serratia marcescens respectively.
The glyA gene encoding serine hydroxymethyltransferase (SHMT) was cloned
by complementing an E. coli gEyA mutant (Chan et al., 1988). In the presence of
H4folate, SHMT catalyzes the reversible cleavage of serine to glycine and the
formation of 5,lO-CH2-H4folate which is a major contributor of CI units in ceIl
metabolism. The C. jejuni glyA gene encodes a 46 kDa protein which shows 55.6%
identity to that of E. coli. The glyA gene was mapped ont0 the C. jejuni TGH9011
chromosome (Chan and Bingham, 1991; Kim et al., 1993).
The argH gene encodes arginosuccinate lyase and was cloned by
complementing an E. coli strain deficient in ArgH activity (Hani and Chan, 1994).
argininosuccinic acid into arginine and fumarate. The C. jejuni argH gene is 1.377 kb
in size and encodes a 56 kDa protein. It was mapped ont0 the chromosome of C.
jejuni TGH9011 and lies on the Sa1 1 A, Sma 1 A, and Sac II A fragments.
The IysS encodes Iysyl-tRNA synthetase and the open reading frame overlaps 1
bp with the start codon (Met) of the glyA gene in C. jejuni (Chan and Bingham, 199 1).
The enzyme is responsible for charging the lysyl tRNA molecule with its arnino acid
moiety. The C. jejuni lysS-encoded product, LysRS shows 47.9 and 46.6% identity to
the E. coli lysS - and lysU - encoded synthetases respectively. The C. jejuni lysS gene
could complement the E. coli lysS and lysU mutations and it has been placed on C.
jejuni TGH9011 genomic map (Kim et al., 1993).
The fur ferric uptake regulatory gene is located upstream of lysS and had been
isolated by screening the genomic library with a lysS probe, characterized and mapped
ont0 the genome of C. jejuni TGH9011 (Chan et al., 1995). The C. jejuni fur gene
encodes a 18.1 kDa protein and is homologous to the E. coli fur gene. fur is
responsible for the regulation of iron-induced or repressed genes (Wooldridge et al.,
1 994).
The ileS gene encoding isoleucyl-tRNA synthetase was cloned (Hong et al.,
1995). It was completely sequenced and the deduced IleS has 91 7 arnino acids
with a molecular mass of 1 O6 kDa. Also ileS was mapped ont0 the 1360- 1 8 12 kb
region of Sma I A, Sa1 1 A and Sac II A fragments of the C. jejuni genome. The IleS is
an essential enzyme needed for the arninoacylation of isoleucyl tRNA.
The leuB, ZeuC, and leuD genes, involved in the leucine biosynthetic pathway,
were cloned by complementation of specific auxotrophs in E. coli (Labigne et al.,
The katA gene encodes catalase. The gene from C. jejuni was cloned by
functional complementation of a catalase-deficient mutant of E. coli and its nucleotide
sequence was obtained (Grant and Park, 1995). The deduced protein pxoduct of 508
amino acids with an estimated molecular mass of 59 kDa has been found to be
structurally and enzyrnatically similar to hydrogen-peroxidase from other bacterial
species.
The aroA gene encoding 5-enolpynivylshikirnate-3-phosphate (EPSP) synthase
was cloned by complementation of an E. coli auxotrophic aroA mutant. The aroA
gene has been sequenced and encodes an enzyme of 428 arnino acids which has 39%
identity to the Bacillus subtilis EPSP synthases (Wosten et al., 1996).
The cysM gene encodes O-acetylserine sulfhydrylase B which is preferentially
used for cysteine biosynthesis during anaerobic growth. It is able to utilize thiosulfate
as substrate and it has been cloned, sequenced, and expressed. The C. jejuni cysM
gene encoding a protein of 299 amino acids with a calculated molecular mass of 32
kDa was cloned by complementation of an E. coli cysteine auxotroph. The cloned C.
jejuni gene is a functional homologue of the cysM gene that codes for O-acetylserine
sulfhydrylase B in E. coli and S . îyphirnurium (Garvis et al., 1997).
Southern hybridization analysis of C. jejuni genome shows that there are 3 to 5
ribosomal RNA (rRNA) loci (Labigne-Roussel et al., 1988; Kim and Chan, 1989).
Three rRNA operons (rrn4, rrnB, and rrnC ) of C. jejuni have been isolated fiom a C.
jejuni genomic library of TGH9011 by screening with radioactively labeled C. jejuni
rRNA (Kim et al., 1993). Analysis of the structure and organization of the rRNA
genes has revealed a characteristic adjacent 16S/23S rRNA structure in the three
.,- -r"-"". "̂ ' ---- --- - - - a - - - - - - - - . - - - - - - - - - - --- -----
sites on the physical map of C. jejuni TGH9011. The isoieucine tRNA and alanine
tRNA genes were s h o w to be located in the intercistronic region between the 16s and
23s rRNA genes (Rashtchian and Shaffer, 1986; Kim et al., 1993; Kim et al., 1995).
This organization is conserved in al1 three C. jejuni rRNA operons. The rrnA rRNA
operon of C. jejuni TGH9011 was completely sequenced. The rRNAs were then
characterized by primer extension and S1 nuclease mapping analysis (Kim et al.,
1995). The comparative secondary structure analysis of the 16s and 23s rRNAs were
identified as conserved structures that are found in Proteobacteria. This finding
supports the classification of CampyIobacter in the E subdivision of the Proteobacteria
(Trust et al., 1994).
A major oxidative stress gene sodB from C. jejuni was cloned. The sodB gene
encodes an iron superoxide disrnutase (SOD) which catalyses the breakdown of
superoxide radicals to hydrogen peroxide and dioxygen which are important for
protecting the bacterial ce11 under oxidative darnage conditions (Pesci et al., 1994).
The arylsulfatase gene fiom C. jejuni has been cloned and has no sequence
similarity with other known arylsulfatase (Yao et al., 1996).
The hup gene encodes a homologue of the histone-like DNA binding protein
and the C. jejuni gene was cloned and sequenced (Konkel et al., 1994).
The C. jejuni recA gene, encoding a conserved protein involved in DNA repair
and in recombination, was cloned by PCR amplification using degenerate primers
corresponding to the conserved regions of RecA proteins found in other bacteria. The
recA gene encodes a 37 kDa protein. The terrnination codon overlaps with the
initiation codon of another open reading frarne with similarity to the E. coli enolase
a defect in generalized recombination as determined by natural transformation
fiequencies (Guerry et al., 1994).
In addition, a potential ce11 division geneflsA fiom C. jejuni was selected by
screening the C. jejuni lambda ZAP I I library with cDNA probes made by reverse
transcription of rnRNA. Although C. jejuniftsA is homologous to that of E. coli, the
structure and organization of the operon is different fiom that of E. coli The exact
fùnction of the ftsA gene product remains obscure, although together with the fstQ
gene product, it is thought to have a role in defining the site of division (Griffiths et
al., 1996).
A C. jejuni gene encoding a 29-kDa periplasmic binding protein has cloned,
sequenced, and characterized (Garvis et al., 1996). This gene has been designated as
hisJ, on the basis of complementation experiments performed with an S. fyphimurium
HisJ mutant harboring the recombinant plasmid which contains a C. jejuni
chromosomal D N A fiagment.
Furthermore, a gene conferring haemolytic activity was isolated fiom C. jejuni.
The open reading fiame encodes a protein of 36 kDa with a typical endopeptidase type
II leader sequence. The protein is modified with palmitic acid when it is processed in
E. coli, confirming it as a typical lipoprotein. The deduced gene product of 329 arnino
acids has significant homology to the group of solute binding proteins from
periplasmic-binding-protein-dependent transport systems for ferric siderophores (Park
and Richardson, 1995).
The C. jejuni tig gene encodes a 56 kDa size protein that is thought to act as a
trigger factor was cloned. It shares 31% identity to the arnino acid sequence of the
proOmpA across the cytoplasmic membrane and also serves as a potential chaperone
in ce11 division (Griffiths et al., 1995).
Virulence gene
Genetic studies have also focused on virulence deterrninants believed to play a
role in pathogenesis. Flagella play a role in internalization of C. jejuni (Grant et al.,
1993; Yao et al., 1994). ThefIaA andflrrB flagellin genes fiom C. jejuni have been
characterized (Nuitjen et al., 1990; Fisher and Nachamkin, 1991 ; Khawaja et al.,
1992). The sequence homology between the two genes in strain 8 1 1 16 is 95%. They
are expressed independently fiom two different promoters 02* and aS4 (Alm et al.,
199 1 ; Wassenaar et al., 199 1). The flagellin genes have been mapped on the C. jejuni
chromosome (Kim et al., 1993). A third flagellin (f7aC ) gene of C. jejuni has been
cloned recently (Chan et al., 1997).
Omp 18 encoding an 18-kDa outer membrane protein from C. jejuni ATCC
29428 has been isolated and expressed in E. coli. The sequence has a high degree of
similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of
Haernophilus injluenzae and the peptidoglycan-associated lipoprotein PAL of E. coli
(Burnens et al., 1995; Konkel et al., 1996).
C. jejuni MapA gene encoding 24 kDa membrane-associated protein of C.
jejuni 81 1 16 has been cloned and characterized. The 18 N-terminal arnino acid
residues constitute a signal sequence characteristic of prokaryotic membrane proteins.
In a dot blot hybridization assay with a mapA probe, 120 clinical isolates of C. jejuni
were unequivocalIy discriminated fiom 126 other Campylobacters, including 34 C.
- - - - - - - - - - - - -
related Campylobocter members based on the unique differences in antigenic
structures (Stucki et al., 1995).
TheJ7hA gene was also cloned and sequenced. It is a homologue of LcrD/FlbF
family of proteins which is involved in the regulation of virulence-related proteins
(Miller et al., 1994).
Antibiotics resistance pene
Genes encoding resistance to antibiotics, like the kanarnycin resistance (Kmr)
aminoglycoside phosphotransferase gene, aphA -7, and the tetracycline resistance (Tcr)
tet(0) genes in C. jejuni have been cloned and sequenced. Resistance to antibiotics is
usually plasmid-mediated and some plasmids carry more than one antibiotic resistance
determinant (Taylor et al., 1986; Blaser et al., 1987; Tenover et al., 2989).
Hippurate hydrolase of Campvlobacter jejuni TGH9011
The C. jejuni N -benzoyl-amino-acid amidohydrolase (hippurate hydrolase)
(EC 3.5.1.32) (hipO) gene was isolated on a pBR322 recombinant clone. pHip-O was
isolated and characterized (Hani and Chan, 1995). The exact functional roles of the
hippurate hydrolase in bacterial cells have not been defined. Amidohydrolases are
able to specifically remove N -1inked amino acids or similar substitutes from larger
substrates. Similar activities have been dernonstrated in bacteria, plants, animals, and
humans. Hippuric acid serves as a sole carbon source for the growth of some
hippurate hydrolase containing microorganisms (Miyagawa et al., 1985) and hippurate
hydrolase cataIyzes the hydrolysis of hippuric acid to release benzoic acid and glycine
differentiation of C. jejuni, a cornmon cause of human enteritis, from other
Campylobacter species (Harvey, 1980; Skirrow and Benjamin, 1980).
The hipO gene of C. jejuni TGH9011 encodes a polypeptide of 383 amino
acids with a pl of 6.0. In maxicell and Western blot analysis, hippurate hydrolase
(HipO) was shown to be a 42 kDa protein (Hani and Chan, 1995). The size of HipO is
similar to proteins of Pseudornonas sp. (Karneda et al., 1968; Watabe et al., 1992;
Ishikawa et al., 1996), Bacillus sterotherrnophilus (Sakanyan et al., 1993),
Arabidopsis thaliana (Bartel and Fink, 1995), and SuIfolobus soIfataricus (Colombo,
1995).
The nucleotide sequence of hipO C. jejuni TGH9011 has been published (Hani
and Chan, 1995) and is shown in figure 2. TAA is used for its termination codon and a
AGGAGA Shine-Dalgarno sequence is located at nt-14 to -9 upstrearn of ATG the
methionine initiation codon. The hipO region is purine rich and the molecular
percentage G+C content is 33.1%. The codon usage is similar to other C. jejuni genes
except for the rare usage of UUC (phe), CTC (leu), CTG (leu), GTC (val), TCC (ser),
CCG (pro), ACC (th) , CGA (arg), and CGG (arg). Codon usage and molecuiar
percentage G+C of the hipO open reading fiame within the insert of recombinant
plasmid pHIP-O is shown in Table 2.
Open reading frarne 2 (OTCFU2) and 3(ORFU3) are upstream of 4(ORFU4), the
hipO. These three ORFs are continuous on the genome suggesting they may be
cotranscribed as an operon.
A single genomic copy of the hipO gene is present in C. jejuni TGH9011.
Southern blot hybridization of the C. jejuni genomic DNA probed with a radiolabeled
O I I
C - NHCH, Hippurate COOH Hydrolase
+ H, O +- + NH,CH,COOH
N-benzoylglycine (Hippuric acid)
Benzoic + Glycine acid
Figure 1. Biochernical reaction of hipourate hvdrolase.
Hippuric acid is formed by condensation of benzoic acid and glycine in human and
accumulates in urine. Hippuric acid is hydrolysed ta benzoic acid and glycine in
various species of microorganisms, including C. jejuni.
hippurate hydrolase. TTT (phe) 20 TTC ( p h e ) TTA ( l e u ) TTG ( l e u ) CTT ( l e u ) CTC ( l e u ) CTA ( l e u ) CTG (leu) ATT ( i l e ) ATC ( i l e ) ATA ( i l e ) ATG (met) GTT ( v a l ) GTC ( v a l ) GTA ( v a l ) GTG ( v a l ) TCT ( s e r ) TCC ( s e r ) TCA ( s e r ) TCG ( s e r ) CCT ( p r o ) CCC ( p r o ) CCA ( p r o ) CCG ( p r o ) ACT ( t h r ) ACC ( t h r ) ACA (thr) ACG ( t h r ) GCT ( a l a ) GCC ( a l a ) GCA ( a l a ) GCG ( a l a ) TAT ( tyr ) TAC ( tyr ) TAA ( e n d ) TAG ( e n d ) CAT (his) CAC (his)
(gin) W G (gin) AAT ( a s n ) AAC ( a s n )
( l y s ) AAG ( l y s GAT ( a s p l GAC ( a s p ) GAA ( glu GAG ( g l u TGT ( w s ) TGC ( w s ) TGA (end) TGG (t-1 CGT (arg) CGC (arg) CGA ( a r g ) CGG ( a r g ) AGT (ser)
AGC (ser) 6 AGA ( a r g ) 3 AGG (arg) I GGT ( g l y ) 11 GGC ( g l y 4 GGA (@Y) 1 0 G a ( q l y ) 2 T o t a l codons 383 T o t a l base pairs 1149 M o l % G+C 33.1
Figure 2. Nucleotide sequence of C. jejrr~ti TGH9Oll hippurate hvdrolase.
The deduced arnino acid sequence of hippurate hydrolase is given in single Ietter code
above the sequence for those on the "upper" strand. Termination codons are indicated
with an asterisk (Hani and Chan, 1995).
V V S W S V D K T H S F T L G F V Y I F GGTAGTATCTTGGAGTGTTGATAAAACCCATAGTTTTACTTTGGGTTTTGTTTATATTTT -81
v A L I F I S A I L A Q F V L P R R E N F
TGCTTTGATTTTTATTTCAGCGATCTTAGCACAATTTGTTTTACCTAGMGAGAAAAT -21 ORFU4 r
I Q G E K * M N L I P E I L D L Q G E TATACAAGGAGVTAGAATGAATTTmTTCCAGAAATACTAGACTTACMZGCGAAT 4 0
F E K I R H Q I H E N P E L G F D E L C TTGAAAAAATTCGTCATCAAATTCATGAAAATCCTGAGCTTGGTTTTGATGAATATGTA 100
tiprl hor2 T A K L V A Q K L K E F G Y E V Y E E I CTGCAAAATTAGTGGCGCAAAAATTAAAAGAATTAAAAGAATTTGGTTATGAGGTTTATGAGGAAATAG 160
G K T G V V G V L K K G N S D K K I G L GAAAAACAGGCGTTGTGGGGGTTTTAAAAAAGGGGAATAGCGATAAAAAAATAGGACTTC 220
R A D M D A L P L Q E C T N L P Y K S K GTGCAGATATGGATGCTTTGCCTTTGCAAGAATGCACRAATTTGCCTTATAAAAGCAAAA 280
K E N V M H A C G H D G H T T S L L L A AAGAAAATGTAATGCATGCTTGCGGTCATGATGGACATACTACTTCTTTATTGCTTGCTG 340
A K Y L A S Q N F N G T L N L Y F Q P A CAAAGTATTTAGC~GTCAGAATTTTAATGGCACTTTMTCTTTATTTTCMCCTGCTG 400
E E G L G G A K A M I E D G L F E K F D AAGAGGGTTTGGGTGGTGCTAAGGCAATGATAGAAGATGGATTGTTTGAAAAATTTGATA 460
S D Y V F G W H N M P F G S D K K F Y L GTGATTATGTTTTTGGATGGCACAATATGCCTTTTGGTAGCGATMGMTTTTATCTTA 520
K K G A M M A S S D S Y S I E V I G R G AAAAAGGTGCGATGATGGCTTCTTCGGATAGTTATAGCÀTTGÀAGTTATTGGAAGAGGTG 580
G H G S A P E K A K D P I Y A A S L L V GTCATGGAAGTGCTCCAGWGGCWGATCCTATTTATGCTGCTTCTTTACTTGTTG 640
V A L Q S I V S R N V D P Q N S A V V S TGGCTTTACMGCATAGTATCTCGCAATGTTGTTGATCCCCRAIlATTCAGCAGTTGTAAGCA 700
I G A F N A G H A F N I I P D I V T I K TAGGAGCTTTTAATGCAGGACATGCTTTTAATATCATTCCAGATATTGTMCGATT- 760
M S V R A L D N E T R K L T E E K I Y K TGAGTGTTAGAGCATTAGATAATGAAACTAGAAAGCTIMCTGMGAnAAAATTTATAAAA 820
I C K G L A Q A N D I E I K I N K N V V TTTGTMGGTCTTGCACAGGCTAATGATATAGAGATTWTCMTWTGTTGTTG 880
A P V T M N N D E A V D F A S E V A K E C A C C A G T G A C T A T G A A T A A C G A T G A A G C T G T G G A T T T T T 940
L F G E K N C E F N H R P L M A S E D F T A T T T G G C G ~ T T G T G A A T T T A A T C A T C G T C C T T T A G T G A G G T T T T G 1000
G F F C E M K K C A Y A F L E N E N D I G h T T T T T T T G C G ~ T G ~ T G T G C C T A C T T ? T T A G T G C A C A T T T 1060
4 Y A K L A L K Y L K * ATGCGAAGCTAGCTTT~TACTTAAAATAAAAACTAATCTAGAATTTCRAGCACAATT 1180
* D L I E L V I
E S K L G K I Q I E K L K L N S P L N A
~GCTCTTTATTGTTTTTTATTTGCTTTTTTTGCACTTATGGAGACTAAAATT~~~~TA 1300 L L E K N N K I Q N K A S I S L L I G R
23s rRNA probe O IGS rRNA probe
Figure 3. Phvsical map of C. jejuni TGH9011.
C. jejuni TGH90 1 1 produced a 2.1 -kb hybridizing band. DNA hybridization detected
no hipO gene sequences in C. coli, C. lari, C. upsaliensis, C. fetus, or C. sputorum or
the former member of the Campylobacter genus- Helicobacter pylori (Hani and Chan,
1995). This is correlated with the negative hippurate hydrolase activity observed in
Helicobacter, Arcobacter, and other Campylobacter species.
Substrate specificity of the C. jejuni HipO protein is highest using N -
benzoylglycine (N -B-gly), followed by N -benzoylalanine (N -Bz- ala), N -
benzoylrnethionine (N -B-met), and N -benzoylleucine (N -B-leu) respectively. No
activity is detected using N -benzoylhistidine, N -benzoylarginine, or N -
benzoylthreonine as a substrate. The hipppurate hydrolase gene was mapped by
Southern hybridization to the Sac II-A, Sa1 LA, and Sma 1-A fragments which overlap
a 450 kb region of the 1800 kb genome (Kim et al., 1993). The hipO gene was
localized to the central region of the Smu 1-A fiagrnent by mapping a hipO
::kanamycin mutant of C. jejuni (Figure 3) . The argH and ileS genes also have been
mapped to these restriction enzyme fragments (Kim et al., 1993, Hong et al., 1995).
However the proximity of these genetic markers to each other, is currently unknown.
HomoIogous proteins belong to M40 hvdrolase family
Using a BLAST search, the deduced arnino acid sequence of C. jejuni
hippurate hydrolase shows a homology score with the SuIfolobus solfataricus
carboxypeptidase (CPSA) (Colombo et al., 1995), the Arabidopsis thdania indole-3-
acetic acid -amino acid hydrolase (ILR) (Bartel and Fink, 1995; Li et al., 1996), the
Bacillus sterothermophilus N -acyl-L-arnino acid amidohydrolase (AMA)(Sakanyan et
utilizing protein C) (HYUC) (Watabe et al., 1992; Ishikawa et al.. 1996). the
Synechocystis hypothetical protein (Kaneko et al., 1996) and the Agrobacteriurn
tumefaciens cellulose synthase (CELE) (Matthysseet al., 1995) (Table 3).
The Bacillus sterothermophilus N -acyl amino acid aminoacylase enzyme
catalyses the hydrolysis of N -acyl arnino acid to yield an arnino acid and acetic acid.
The substrate specificity of this enzyme is extremely high with N -choroacetyl-L-
phenylalanine and is also high towards N -acetyl-L-ala, N -acetyl-L-tyr, N -acetyl-L-
phe, N -acetyl-L-val, N -acetyl-L-gly, N -acetyl-L-leu, IV -acetyl-L-his, N -acetyl-L-met,
and N -benzoyl-L-phe.
N -benzoylglycine amidohydrolase from Pseudomonas putida C692-3
hydrolyzes N -benzoyl amino acids with different specificity, highest with N -Bz-gly,
follow by N -Bz-L-ala and N -Bz-L-aminobutyric acid. It has no enzyme activity
towards N-acetyl-glycine, N -acetyl-L- alanine, N -carbobenzoxyglycine, or N -
carbobenzoxy-L-alanine. Amidohydrolase are able to specifically remove N -1inked
amino acids or similar substitutes fiom larger substrates.
The Pseudomonas sp. N -carbamoyl amino acid aminoacylase catalysed
reaction yields an amino acid and carbarnate. The substrate specificity of the
benzoylamino acid arnidohydrolase of Pseudomonas sp. (strain KT801) is towards L-
amino acids and not D-amino acids. N -Bz-Dl-ala is the preferred substrate, followed
by N -Bz-DL-met, N -Bz-DL-leu, N -Bz-gly, N -Bz-DL-val, N -Bz-DL- phe, N -Bz-
DL-asp, N -Bz-DL-glu, N 432-DL-trp, and N -Bz-DL-th. No activity is observed on
the substrate acetyl-DL-alanine, or phenylacetyl-DL-alanine, indicating a considerable
effect by the acyl group (Kameda et al., 1968).
activity and c m remove amino acid residues fiom benzoyloxycarbonyl derivatives
with activity towards benzyloxycarbonyl (Cbz) substrates; Cbz-glyglyphe, Cbz-arg,
Cbz-asp, and weakly with Cbz-phe and Cbz-ala. The Arabidopsis thaliana indoxyl-3-
acetic acid amidohydrolase reaction yields an arnino acid and indole-3-acetic acid.
The substrate specificity is toward indole-3-acetic acid arnino acid conjugates: IAA-
phe, IAA-leu, and weakly towards IAA-ala, IAA-gly, and IAA-ile.
Table 3. Homologous ~roteins of C. ieiuni hippurate hydrolase.
Sequences with high homolow score Homolow Score
Campylobacter jejuni hippuricase (HipO) 2.1 e-248 Sulfolobus safataricus carboxypeptidas (CpsA) 4.8e-83 Arabidopsis thaliana indoxyl-3-acetic acid amidohydrolase (Ill 1) 7.7e-72 A rabidopsis thaliana (1112) 1.8e-70 A rabidopsis thaliana (Ilr 1 ) 1.7e-62 Synechocystis sp. hypothetical protein 4.1 e-47 Bacillus stearothermophilus N-acyl-L-amino acid amidohydrolase (Amal 1.2e-44 Bacillus subtilis (YXEP) 3.4e-62 Agrobacterium tumefaciens (CelE) 7.8e-2 1 HaernophiZus infIuenae HipO homolog 7.6e- 17 Psezrdomonas sp. hydantoin utilization protein (hyuC) 0.22 Escherichia coli hemolysin B (hlyB) 0.52
An extensive study of over 556 strains of thermophilic Cumpylobacter for
species identification was reported by Totten et al., 1987. These members were
isolated either from hurnans with diarrhea or from poultry in King County,
Washington. They were analyzed by the phenotypic test, after that the classification of
the hippurate hydrolase-negative C. jejuni strains was confirmed by a quantitative
whole-ce11 DNA hybridization test. The study showed that only 1.6% (9 of 556) of the
C. jejuni strains were hippurate hydrolase negative. Thus, hippurate hydrolase-
negative C. jejuni strains represented a small percentage (1.6%) of C. jejuni strains but
a significant portion 20% (9 of 46) of hippurate hydrolase-negative strains in that
study. These C jejuni hippurate hydrolase-negative isolates were not only hippurate
hydrolase-negative in the rapid tube test but also in more sensitive assays such as gas
liquid chromatography (GLC) and thin layer chromatography (TLC) assays.
Furthermore, serotyping was also applied to these members, yet hippurate hydrolase-
negative C. jejuni isolates show a non-random association with different serotyping
systems (Totten et al., 1987). Hippurate hydrolase-negative strains fell into four
groups by the Penner serotyping system, four groups by the Lior serotyping system,
two auxotypes, and three plasmid patterns, indicating that these strains represented a
diverse group of isolates.
Hippurate hydrolase-negative isolates of C. jejuni were also examined with
respect to the presence of h i p 0 gene sequences in the genome by Southern
hybridization analysis (Hani and Chan, 1995). Four hippurate hydrolase-negative
isolates D594, D603, D941, and D 19 16 gave a positive 2.1 kb Hind I I I hybridization
band when probed with the hipO probe. One hippurate hydrolase-negative isolate,
chromatography assay (Totten et al., 1987) and showed a hybridization band in
Southern hybridization blot with the hippurate hydrolase probe (Hani and Chan, 1995).
C. jejuni and C. coli are the most comrnon thennophilic bacteria isolated fiom
humans (Ketley, 1995). C. jejuni and C. coli share many cornmon phenotypic
characteristics. Thus, very few biochernical tests can differentiate between them
(Penner and Hennessy, 1980; Penner et al., 1983; Penner, 1988; Boltan, 1984; Patton
et al., 1992). However, they are two distinct species and show only 25 to 58%
homology relative to each other by DNA hybridization (Harvey and Greenwood, 1983;
Roop et al., 1984; Morris et al., 1985). The hippurate hydrolysis (Hippuricase) assay
is the standard bicbzhemical test used to distinguish C. jejuni, C. coli, and other
Campylobacter sp. in the clinical laboratory via the detection of the product of
hydrolysis by a color reagent system. This asssay was found to correlate closely with
species differentiation in taxonomic studies. 80% of cases of Campylobacter -
mediated enteritis in hurnans are caused by C. jejuni (Ketley, 1997). C. jejuni is the
only Campylobacter species that has the hippurate hydrolase gene and is able to
hydrolyse hippurate. Recently, the hippurate hydrolase gene from C. jejuni TGH9011
strain has been cloned and characterized (Hani and Chan, 1995). Therefore, it is
important to understand the role of the hippurate hydrolase gene in C. jejuni species.
Hippurate hydrolase-negative C. jejuni strains have also been identified (Totten
et a1.,1987). It is of interest to analyse the mutant hippurate hydrolase gene in these
natural isolates. Therefore, the major objective of this study was to determine the
molecular nature of hippurate hydrolase-negative C. jejuni clinical isolates from King
County, Washington. We used a radiolabeled single strand conformational
polymorphic assay based on polymerase chain reaction (PCR-SSCP assay) to screen
1 Y A A Y A 1 - -
hydrolase-negative C. jejtrni. The S S C P assay based on polymerase chain reaction is
a sensitive and simple technique, which is capable of distinguishing single base pair
changes between single DNA strand (Orita et al., 1989; Teleni et al., 1993). It is
successfùlly used in various studies in Mycobacteriurn tuberculosis (Orita et al., 1989,
Teleni et al., 1993), Mycobacterium leprae (Heyrn et al., 1995), Hepatitis B virus
(Yusof et al., 1994), and Parvovirus B19 (Kerr et al., 1995), etc. Oligonucleotide
primers were synthesized based on the hippurate hydrolase nucleotide sequence of
hippurate hydrolase-positive C. jejuni strain TGH9011 (Hani and Chan, 1995). The
primers were used to ampliSr eleven overlapping regions of the hippurate hydrolase
gene from each of the five hippurate hydrolase-negative C. jejuni clinical isolates. The
SSCP assay was used to detect point mutations upstream and within the hippurate
hydrolase gene sequences. The regions containing the hippurate hydrolase gene of the
five diffèrent hippurate hydrolase-negative C. jejuni isolates were cloned and
sequenced in order to characterize the exact nature of the sequence heterogeneity
within the hippurate hydrolase gene sequence of C. jejuni TGH9011 strain.
Bacterial Strains
The Campylobacter jejuni serotype reference strain for 0:3 [TGH9011 (ATCC
43431) and of Lior serotype 361 was obtained fiom Dr. J. L. Penner, University of
Toronto, Canada. C. jejuni TGH9011 was used as a hippurate hydrolase-positive
controI strain. Five different hippurate hydrolase-negative clinical strains of C. jejuni
(D594, D603, D835, D977, D1713) were received fiom Dr. C. M. Patton, Center for
Disease Control and Prevention, Atlanta, Ga, USA. These hippurate hydrolase-
negative strains were isolated either fiom humans with diarrhea or fiom poultry
(Totten et al., 1987). Campylobacter species were grown routinely on Mueller-Hinton
agar plates (Merck) or in broth for 48 to 72 hours at 42'C in a carbon dioxide incubator
set for 7% CO2.
Escherichia coli (JM101) strain [A(lac-proAB ) thi strA supE44 end4 sbcB
hsdR4 F' traD36 proA+B+ Zacl A(lacZ) Ml51 was grown routinely on Luria Bertani
media aerobically at 37'C and used for making cornpetent cells.
Genomic DNA extraction
Total genomic DNA of C. jejuni TGH9011 was extracted from cells grown on
Muller-Hinton a g a plates. A plate of C. jejuni cells was washed three times in 1 ml of
saline citrate (SSC:O. 1 SM NaCl, 0.01 5M Na citrate [pH7.0]). Suspended cells were
pelleted and resuspended in 27% sucrose in 1X SSC at a concentration not higher than
10" cells per ml. The suspended cells were digested with proteinase K to a final
Table 4. Bacterial strains used in this study.
Strains Genotvpe or Characteristics References
Campylobacter jejuni
ATCC4343 1 serotype reference strain for 0:3, TGH9011 J. L. Penner
Esclierickia coli
hippurate hydrolase negative isolate C. M. Patton
hippurate hydrolase negative isolate C. M. Patton
hippurate hydrolase negative isolate C. M. Patton
hippurate hydrolase negative isolate C. M. Patton
hippurate hydrolase negative isolate C. M. Patton
hippurate hydrolase weak isolate C. M. Patton
A(1ac-pro) thi rspL supE en& sbcB hsdR F' traD36 proAB ZacP ZAM 1 5 Yanisch-Perron et al.
ATCC:Arnerican Type Culture Collection
concentration of 0.2%. The lysate was incubated at 50'C for an hour and then
extracted three times with an equal volume of 50 mM Tris-Cl, 10 mM EDTA, pH 8.0 -
saturated phenol. The aqueous phase of the phenol-extracted lysate was removed and
extensively dialyzed in Tris-EDTA (10 mM Tris [pH 8.01-lmM EDTA). DNA was
recovered by ethanol precipitation with two volumes of 95% ethanol for 30 minutes, at
-70'C. The precipitated DNA was pelleted by centrifugation, washed with 70%
ethanol and then vacuum dried. The dried pellet was resuspended in 50 pl TE (10mM
Tris-Cl pH 8.0, 1 mM EDTA pH8.O) for further use.
Olipronucleotides
To scan the hippurate hydrolase gene for the presence of mutations by
polymerase chain reaction and single strand conformational polyrnorphism analysis,
oligonucleotides were synthesized based on the C. jejuni TGH9011 hippurate
hydrolase sequence (Hani and Chan, 1995). These primers were used in polymerase
chain reactions and sequencing reactions. The location, direction, size and sequence
of primers (PC1, PC3, PC4, H l , H2, H3, H4, H5, H6, H7, H8, H9, H 1 O, H 1 1, H 12,
H13, H14, H15) designed for the polyrnerase chain reaction, single strand
conformational polymorphism analysis and sequencing reactions are s h o w in Table 5
and 6 and Figure 4. The strategy for the amplification of the various regions of the
hippurate hydrolase gene using eleven primer sets is s h o w in Figure 4.
Table 5. Uligonucleotides usecl in mis stuay.
Oligo Sequence Purpose
5'-ATCCCGGGTGGTTTCC(A/T)TTTTTCTTTAATCT(T/A)CC-3 ' Pcr 5'-ATGTCGACAAAAAAGAAAATGToATGT(T/A)ATGCATGC/TTGCGGT-3' pcr 5'-ACCCCGGGGC(T/A)GG(T/A)TTTTATGATAAATTCTACAC-3 ' Pcr 5'-TAGGATCCTCTTGGAGTGTTGATAAAACTCAT-3 ' Pcr 5 '-ATGGATCCTTGATGGCGAATTTTTTCAAATTC-3 ' Pcr 5'-GCGGATCCGAATTTGAAAAAATTCGCCATCAA-3' Pcr 5'-TTGGATCCAGCAGTACATAATTCATCAAAACC-3' Pcr 5'-AAGGATCCAGTTCCATTAAAATTTTGACTAGC-3' Pcr 5'-TAGGATCCGCAAGTCAGAATTTTAATGGCACT-3' Pcr 5'-AAGGATCCTCCATCTTCAATCATAGCTTTAGC-3' PCr 5'-TTGGATCCTGGCACAATATGCCTTTTGGTAGC-3' pcdsequencing 5'-CTGGATCCTCCAATAACTTCAATGCTATAACTATC-3' Pcr 5'-ATGGATCCGCTCCTGAAAAAGCTAAAGATCCT-3' pcr/sequencing 5'-ACGGATCCAGCAGAATTTTGAGGATCAACATT-3' pcdsequencing 5 '-CTGGATCCTTTAATGCTGGACATGCTTTTAAT-3 ' Pcr 5'-TCGGATCCAACAGCTTCATCATTATTCATAGT-3' pcdsequencing 5'-GTGGATCCTTAATGGCTAGTGAAGATTTTGGA-3' Pcr 5'-AAGGATCCAGCATAAGCACATTTTTTCATTTC-3 ' Pcr
Table 6. Primer pairs used in this study.
Primers Repions Sizes
Chromosomal DNA of Campylobacter jejuni strains was arnplified by
polyrnerase chah reaction using Taq DNA polymerase (Boehringer, Mannheim,
Germany). The amplification was carried out in a programmable thermal cycler (Gene
Arnp PCR 9600 System, Perkin-Elmer, Norwalk, CT, USA) as follows; an initial
template denaturation step at 94.C for 5-minutes followed by 25 cycles of 1-minute 30-
seconds of denaturation at 94C, 1-minute 30-seconds primer annealing at 60C, and 2-
minutes chain extension at 72'C and then a final extension step at 72'C for 10 minutes.
The reaction (50 pl) contained 50 pmol of each primer, 1 pg of genomic DNA, 1.5
mM MgC12, 160 pM each dNTP, 2.5 U Taq DNA polyrnerase and Taq buffer. Sterile
distilled water was used to adjust the total reaction volume for the negative control.
The reaction mixture was then covered with 70 pl of minera1 oil. One tenth of the
arnplified products were electrophoresed on polyacrylarnide gel (PAGE), containing
ethidium bromide in TBE buffer (0.45 M Tris-Borate, and 10 mM EDTA). PAGE
electrophoresis was performed, as described (Maniatis, 1982). Briefly, a 10%
acrylarnide gel was cast in a Biorad Mini Protein gel caster. The DNA sarnples were
separated in running buffer at 100 V for 2 hours. Aller electrophoresis, the amplified
product was exarnined under UV light. Two rnicrograms of DNA from the phage # X
174 digested with Hae III was used as the size standard. Positive control C. jejuni
genomic DNA and negative controls: water instead of DNA, are included in every gel.
For the SSCP assay, pairs of PCR primers were chosen to generate fragment sizes of
less than 400 bp to enhanced the sensitivity of the SSCP assay.
detection enhancement (non denaturing) gel electrophoresis
For the SSCP assay, double stranded DNA was denatured into single stranded
DNA and the products were separated on a mutational detection (MDE) gel under
nondenaturation conditions. The amplification reaction was performed in a 50 pl
reaction volume containing 10 pl extracted genomic DNA (approximately 1 pg), 200
pM (each) of dTTP, dGTP, and dCTP, 100 pM dATP with 0.5 pl of [alpha-32P] ATP
(5 pCi) instead of total 200 uM dATP was added to the PCR mix, 50 pmole of each
oligonucleotide primer, 5 pl 10X Taq buffer and 2.5 U of Taq DNA polymerase
(Boehringer, Mannheim, Germany). The SSCP assays were performed as described
(Teleni et al., 1993). Briefly, 5 pl of radiolabelled arnplicon was diluted in 100 pl of
the SSCP dilution solution (10 mM EDTA, O. 1% SDS). 3 pl of diluted product was
mixed with 3 pl of formamide dye (98% formamide, 0.5% bromophenol blue, 0.5%
xylene cyanol, and 20 mM EDTA), denatured at 95'C for 5-minutes, cooled on ice, and
loaded on a nondenaturing sequencing-format 0.5 X Mutation Detection Enhancement
Acrylarnide Gel (MDE gel) (Hydrolink -MDE:AT/Biochern, Malvern, PA, USA). The
MDE gel is composed of 25 ml of MDE gel, 3 ml of 10X TBE buffer, and 22 ml of
deionized water polymerized with 300 p1 10% ammonium persulfate and 30 pl
TEMED (Bio Rad, Richmond, Calif., USA). Electrophoresis was done in 90 mM Tri-
Borate (pH 8.3) - 4 mM EDTA in a sequencing gel electrophoresis apparatus (50 by 32
by 0.04 -cm gel) (Bio Rad) at room temperature at a constant power of 8 W for 20
hours. After electrophoresis, the gel was dried for an hour, exposed 3 hours to Kodak
film, and the radioactive banding patterns were inspected. Pairs of PCR primers were
Figure 4. Strategv for single strand conformational polvmorphic (SSCP) assay
based on polymerase chah reaction.
The positions of eleven sets of primer pairs used are indicated above the hippurate
hydrolase gene which are shown as box. Oligonucleotides were synthesized based on
the published sequence of hippurate hydrolase-positive C. jejuni strain TGH90 1 1
(Hani and Chan, 1995). The eleven sets of primers were used to amplify the eleven
overlapping regions which are less than 400 bp in size. Each region was overlapping
around 100 bp.
assay and at least two SSCP runs were performed for each set of sarnples to assure
reproducibility of the assay.
Cloning of the amplified fragments
1 pg PCR product was digested in 20 pl using approximately 2 U of BamHI
enzyme per microgram of DNA in BamHI reaction buffer and cloned into a pUC19
vector. Restriction enzymes were obtained fiom Boehringer Mannheim or Pharmacia
and used as recomrnended by the manufacturers.
Ligation of DNA fragments
The arnplified product, (approximately 1.0 pg), was digested with Barn HI
restriction enzyme and purified by ethanol precipitation, as described above. 0.5 pg
pUC19 vector DNA was linearized with Barn HI and dephosphorylated using calf
intestinal alkaline phosphatase (CIAP). CIAP was used at a concentration of 1U per
pg DNA for 15-minutes at 37'C. Alkaline phosphatase activity was terminated by
heating at 65'C for 10-minutes with 20 mM EDTA and 0.5% SDS. The volume was
adjusted to 240 pl and extracted with phenol in order to remove residual alkaline
phosphatase. The top phase was extracted with ether and the DNA precipitated on ice
for 30-minutes using 0.3 M sodium acetate (NaOAc) and three volume of ethanol.
The pellet was resuspended in TloEi buffer (1 0 mM Tris-HC1, pH 8.0 and 1 mM
EDTA). The vector and 100 to 500 ng of insert DNAs were then mixed together in
ligation buffer (50 mM Tris-Cl, pH 7.6, 10 mM MgC12, 1 mM rATP) in a moiar ratio
Gerrnany).
Preparation of competent celis and transformation with recombinant plasmid
Competent cells for transformation were prepared. 20 ml of Luria broth was
inoculated with 0.5 ml of an ovemight E. coli culture. The cells were then grown at
37C with aeration to early log; OD at 600 nm which was equal to 0.13 - 0.15, taking
approximately 2 hours. The cells were pelleted by centrifugation. The pellet was
resuspended in 10 ml of solution A (10 mM 3-(N-Morpholin) propane - sulfonic acid
(MOPS) pH 7.0 and 10 m M rubidium chloride). Cells were again spun down,
resuspended in 10 ml of solution B (10 mM MOPS, pH 6.5, 10 mM rubidium chloride,
and 50 mM calcium chloride) and incubated on ice for 30-minutes. Afterward, the
cells were centrifuged and the ce11 pellet was resuspended again in 1 ml of solution B.
For transformation, 0.15 - 0.20 pg of ligated DNA, approximately 10 to 20 pl
of ligation mixture was added to 200 pl of the competent cells prepared above. It was
mixed and incubated on ice for 30-minutes. The cells were then heat-shocked at 42.C
for 90-seconds. 1 ml of Luria broth was added and the mixture was incubated at 37'C
for an hour. This allowed for the expression of the antibiotic resistant genes present
on the plasmid before transformed cells were plated ont0 selective medium. 100 pl of
the transformed cells were added per plate for selection of appropriate transformants.
Recombinant plasmid DNA preparation
S,mall scale preparation of plasmid DNA from E. coli was perforrned (Maniatis
pelleted by centrifugation at 1200 rpm for 5-minutes. The bacterial ce11 pellet was
resuspended in 100 pl of GTE-lysis buffer: 50 m M glucose, 25 mM Tris-HCl pH 8.0,
10 mM EDTA, 2 mg lysozme per ml and incubated at roorn temperature for 5-
minutes. The bacterial suspension was treated with 200 pl alkaline SDS buffer (0.2N
NaOH, 1% SDS) and mixed by inverting the tubes gently several times and placed on
ice for 5-minutes. 150 pl of acetate buffer (3 M potassium acetatelacetic acid) was
added mixed by inverting and chilled on ice for 5-minutes. The samples were
centrifuged for 10-minutes at 4'C , and the supernatant was collected. Plasmid DNA,
thus extracted was concentrated by ethanol precipitation with two volumes of 95%
ethanol and incubation at -70C for 30-minutes. DNA was pelleted by centrifugation
for 20-minutes at 4C, washed with 70% ethanol and vacuum-dried. The dried pellet
was resuspended in 50 pl TloEI (1OmM Tris-Cl - 1 mM EDTA, pH 8.0) for further
use.
Preparation of sequencing grade plasrnid DNA
Bacterial cultures were grown in Luria broth containing ampicillin to ensure
the presence of the plasmid within the culture. Afier plasmid DNA was extracted, 18
pl (-1 .O pmol) of DNA was denatured for 5-minutes with 2 pl of 2 N NaOH at room
temperature. Then, the DNA was precipitated by adding 8 pl of 5M ammonium
acetate pH 7.4 and 100 pl of absolute ethanol and incubating at -70-C for 10-minutes.
The DNA collected by centrifugation was washed twice with 70% ethanol and vacuum
dried.
supernatant (after the addition of acetate buffer and centrifugation) from the
minipreparation protocol and ethanol precipitation, as mentioned above. The DNA
pellet was then resuspended in 50 pl of TE containing RNase (1 0 pg RNse per ml) and
incubated at 37.C for 30-minutes. Then 30 pl of PEG solution (20% PEG-8000,2.5 M
NaCl) was added and incubated on ice for an hou. DNA was then centrifuged,
washed in 70% ethanol, vacuum dried, and resuspended in H20.
DNA sequencing and polyacwlamide gel electrophoresis
The sequence was determined fiom subclones prepared as rnentioned above
and by using specific primers. The DNA sequence was detennined by the dideoxy
chain termination method (Sanger, 1977). It used the Sequenase sequencing kit (US
Biochemical Corp., CleveIand, OH, USA) rnodified version of T7 DNA polmerase
with [ ~ c - ~ ~ s ] dATP for labeling. For each clone, both strands of insert were cornpletely
sequenced. The denatured DNA was annealed to a specific primer and extended
products of various lengths were obtained with the use of sequenase. The DNA (3-5
pg of denatured double-stranded DNA or 1-2 pg of the single-stranded DNA per
reaction) was mixed with an appropriate primer (0.5 pmol per reaction) in 1X
Sequenase Buffer (40 mM Tris-Cl, pH 7.5, 20 mM MgC12, 50 mM NaCl), and
annealed by heating the mixture in a capped tube at 65'C for 2-minutes followed by
slowly cooling the tube to room temperature for about 30-minutes. After annealing,
the DNA was mixed with 6.5 mM DTT. 2 nM of dGTP, dCTP. dTTP, 0.5 pl of ["SI
dATP (1000 Ci per mmol), 3 U of Sequenase enzyme and labeled by incubation at
(each mix contains 50 mM NaCI, $0 uM of dNTP/8 p M one of ddATP, ddGTP,
ddCTP, or ddTTP) was added to the labeled DNA mixes in each of the four wells, and
incubated at 37'C for 5-minutes. After the termination reaction, 4 pl of the stop
solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.5% xylene
cyan01 F F ) was added to each reaction. The sample was heated to 80'C for 5-minutes
irnmediately before loading on the 6 % denaturing polyacrylamide sequencing gel.
The polyacrylamide mix for sequencing gel was prepared as 35 gm ultrapure urea, 9
ml of a 45% acrylamide stock, 350 pl of 10% ammonium persulfate and 50 pl of
TEMED per 70 ml of polyacrylarnide gel and run at 1700 Volts. After the samples
were run on the sequencing gel, the gel was soaked in a solution of 10% acetic acid,
10% methanol, transferred ont0 3MM Whatman filter paper, and dried at 80'C for 45-
minutes. The dried sequencing gel was exposed on X-ray film overnight.
Detection of hippurate hvdrolase sequence heterogeneitv in the hippurate
hydrolase-negative C. jejurzi clinical isolates
Genomic DNA of the five hippurate hydrolase-negative C. jejuni clinical
isolates was examined by polymerase chain reaction for sequence variations. The
hippurate hydrolase-positive C. jejuni TGH9011 strain was used as a control (Hani and
Chan, 1995). The PCR strategy used in this study is shown in Figure 4. The specific
sequence primers, H l , H2, H3, H4, H5, H6, H7, H8, H9, H l0 and Hl 1, with three
degenerate primers, PCl, PC3 and PC4, were used in the polyrnerase chain reaction.
The positions of the eleven primer sets used, are indicated above the hippurate
hydrolase gene which is depicted as a box in Figure 4. The degenerate primers, PC1, 3
and 4 were previously designed with the minimal number of possible degenerate
oligonucleotides required to account for al1 variations of a particular arnino acid
residue (Hani, 1997). Primers were used to ampli@ the eleven overlapping regions of
the hippurate hydrolase gene from five different hippurate hydrolase-negative C. jejuni
clinical isolates, D594, D603, D835, D 17 13, Dg77 and hippurate hydrolase-positive C.
jejuni TGH9O 1 1. Eleven primer pairs were used in polymerase chain reactions that
covered from upstream nt -259 to nt 13 17 downstream of the hippurate hydrolase
gene. The adjacent amplified products overlapped each other by about 100 bp. The
amplified products were electrophoresed in an 8% polyacrylamide gel (PAGE) with 4
X 174 Hae III fragment as markers. The results of the amplified products from eleven
overlapping fragments of the five different C. jejuni hippurate hydrolase-negative
that were identical to that of positive control C. jejzrni TGH9011. These findings
indicate that al1 tested hippurate hydrolase-negative C. jejuni isolates do not have
major sequence variations such as gross deletions or insertions at their hippurate
hydrolase gene. However, a standard gel analysis of the arnplified products would be
unable to detect minor sequence variations within the hippurate hydrolase gene of
hippurate hydrolase-negative isolates.
Single strand conformational polvmorphic analysis of the natural mutant
hippurate hydrolase Eene
The flanking regions and the coding sequence of the hippurate hydrolase loci
from hippurate hydrolase-negative C. jejzrni isolates were analyzed by 3 2 ~ labeled
single strand conformational polymorphism (SSCP) assay based on polymerase chain
reaction.
The F7 and F8 overlapping regions spanning from nucleotides 473 to 912 of
the hippurate hydrolase gene showed polymorphisms in al1 five different hippurate
hydrolase-negative C. jejuni isolates compared with those of control C. jejuni TGH
9011 (Figure 5). Other regions, F4, F5, F6, F9, F10 and F11, showed no
polymorphism in their SSCP profiles. They were indistinguishable from each other
and also similar to those of the control C, jejuni TGH9011. The polymorphic regions,
F7 and F8, may presumably account for the hippurate hydrolase-negative phenotype of
the five negative C. jejuni isolates in the hippurate hydrolysis test.
The promoter area is covered by the overlapping amplified regions F1, F2 and
F3 spanning from nucleotides -259 to 150 bp. The SSCP analysis of promoter regions
Table 7. Surnmarv of PCR analvsis in the hippurate hvdrolase gene from the five
hippurate hydrolase-ne~ative C. jejuni isolates.
Vertically presents control C. jejuni TGH9011 strain (top) along with the hippurate
hydrolase-negative C. jejuni isolates. Horizontally presents the arnplified products of
eleven overlapping regions F1 through F11. + sign represents normal and identical
arnplified product to its respective region.
Strains PCR amplified products
Figure 5. PCR-SSCP analvsis of F7 and FS regions of the hippurate hydrolase
pene of five different hippurate hydrolase-negative C. ieiuni isolates.
Figure 5(A). The sarnples anaIyzed for F7 were lane 1, C. jejuni TGH9011; lane 2,
D594; lane 3, D603; lane 4, TGH9011; lane 5, D835; lane 6 , D977; lane 7, Dl 7 13.
Figure 5(B). The sarnples analyzed for F8 were lane 1, C.jejuni TGH9011; lane 2.
D977; lane 3, D1713; lane 4, D835; lane 5, TGH9011; lane 6, D603; lane 7,
TGH90 1 1 ; lane 8, D594; lane 9, TGH90 1 1.
Mobility shifts are found in fragments F7 and F8 of the five hippurate hydrolase-
negative C. jejuni isolates when compared to the respective regions of control C. jejuni
TGH90 1 1 .
Figure 5(A).
Figure 5 (BI.
Figure 6 (A, B, and C). PCR-SSCP analvsis of FI, F2 and F3 regions of the
hippurate hydrolase gene of five different hippurate hvdrolase-negative C. iejuni
isolates.
Figure 6(A). The sarnples analyzed for FI were lane 1, C. jejuni TGH9Oll; lane 2,
D594; lane 3, D603; lane 4, TGH90I 1; lane 5, D835; lane 6, D1713; lane 7, D977;
lane 8, TGH9011.
Figure 6(B). The sarnples analyzed for F2 were lane 1, C. jejuni TGH9011; lane 2,
D594; lane 3, D603; lane 4, D835; lane 5, D1713; lane 6, TGH9011.
Figure 6(C). The sarnples analyzed for F3 were lane 1, C. jejuni TGH9011; lane 2,
D594; lane 3, D603; lane 4, TGH9011; lane 5, D835; lane 6, D1713; lane 7,
TGH9011; lane 8, D977.
The PCR-SSCP analyses of the promoter region from the five hippurate hydrolase-
negative C. jejuni isolates which were covered by the FI, F2, and F3 amplified
fragments. The assays show unaitered strand mobility compared with that of control
C. jejuni TGH90 I 1.
Figures 6 (A).
Figures 6 (BI.
Figures 6 (C).
regions of the hippurate hydrolase-negative C. jejuni isolates. They were also identical
to the respective regions of C. jejuni TGH9011 (Figure 6).
These results suggest that the promoter regions of the hippurate hydrolase-
negative isolates are unlikely to be the site of sequence variations.
Cloning of SSCP polymorphic regions and DNA sequencing
To establish the precise molecular nature in the five hippurate hydrolase-
negative isolates, the DNA coding regions which displayed the abnormal SSCP profiles
were cloned, sequenced and compared with the hippurate hydrolase sequence from C.
jejuni TGH9011. The amplified fragments of F7 and F8 regions from the five
hippurate hydrolase-negative C. jejuni isolates were cloned into pUC 1 9 vector
(Pharmacia) and sequenced by the dideoxynucleotide chain termination procedure
using T7 sequenase (USB) with specific primers.
The nucleotide sequences of the insert in pUC19 were detennined in both
directions. The sequence of each polymorphic region was almost completely
homologous with the respective region of the C. jejuni TGH90 1 1 sequence. The
respective numbers of nucleotide substitutions from the published hippurate hydrolase
C. jejzrni TGH9011 sequence for each hippurate hydrolase-negative sample are listed in
Table 8.
Most of the mutations detected were silent in nature and they do not affect the
amino acid alterations. They were TTA + TTG at nucleotide (nt) position 633, TTA
+ TTG at nt position 648, AGC + AGT at nt position 654, CCC + CCA at nt
position 678, and TCA + TCT at nucleotide position 687. Except the nt substitution
(Val) codon to alanine (Ala) at residue 250 (Figure 7). This valine at residue 250
change was common to al1 of the hippwate hydrolase-negative C. jejuni isolates.
Sequenice comparison
The hippurate hydrolase homologous sequences were obtained from a cornputer
search of the Protein Data Base GeneBank and the Swiss Protein Database. The
Clustal 1.4W program was used to compare the deduced amidohydrolase sequence of
C. jejuni TGH9011 (HipO) with those of the Bacillus sterothermophilus arninoacylase
(Ama) (Sakanyan et al., 1993), the Pseudamonas sp. amidohydrolase hydmtoin
utilizing protein C - HyuC) (Watabe et al., 1992; Ishikawa et al., 1996) and the
Synechocystis sp. hypothetical arnidohydrolase protein (Kaneko et al., 1995) of the
AMA/HIPO/HYUC M40 hydrolase family (Figure 8). The valine at residue 250 of C.
jejuni hippurate hydrolase is located as a conserved residue in these homologous
proteins from the M40 hydrolase family.
Predicted hydrophathy profiles
A hydrophobicity plot is a current method for predicting the structure of
proteins. It is a graph displaying the distribution of polar and apolar residues along
sequences. The sequences are scanned with a moving window of a given length and
computed for each position of the window, the mean hydrophobicity of al1 residues in
the window. The predicted hydropathy and flexibility profiles of the hippurate
hydrolase protein were plotted by the Kyte-Doolittle scale with a window size of 7
Table 8. Nucleotide substitutions between nucieotide 473 and 912 of the hipO genc
of C. iejuni hippurate hvdrolase-negative isolates.
HipO Sequence from C. jejrtni TGH90 1 1
Isolates
D594
D603
D835
D977
Dl713
Nucleotide # Codon
(Hani et al., 1995)
TTA TT A AGC GTA
TT A TTA AGC CCC GTA
TT A TTA AGC GTA
TTA TCA AGC GTA
TT A TT A AGC GTA
Codon Substitutions
in isolates
TTG TTG AGT GCA
TTG TTG AGT CCA GCA
TTG TTG AGT GCA
TTG TTG TCT AGT GCA
TTG TTG AGT GCA
Phenotvpe Condition
Hippurate Hydrolysis
215
TGH 901 1 positive ALQSIVSRNVDPQNSAWSIGAFNAGHAFNI 1 PDIVRTIKMSVRALDNET 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ~ 1 1 1 1 1 1 1 1
D835 negative ALQSIVSRNVDPQNSAWSIGAFNAGHAFNI 1 PDIARTIKMSVRALDNET
Hipput-ate Hydroiysis
TGH 901 1 positive KLTEEKIYKICKGLAQANDIEIKINKNW. ................... I l l l l l l l l l l l l l t l l l l 1 1 I l I I 1 I I I
. . ............... D835 neetive KLTEEKIYKICKGLAQANDIEIKINKNWA.
Figure 7. The deduced amino acid sequence alignment of the mutated region of
hippurate hvdrolase-positive C. jejuni TGH9011 and the hippurate hvdrolase-
negative strain DS35.
A single arnino acid substitution at valine to alanine at residue 250 was found in al1 the
hippurate hydrolase-negative proteins.
Figure 8. AIignment of C. jejuni hi~purate hvdrolase and related proteins.
The complete polypeptide deduced from gene hipO for N-benzoyl-arnino acid
amidohydrolase (hippurate hydrolase) was aligned using the Clustal W (1.4) program
with the complete polpeptides of related proteins. The top line gives the C. jejuni N-
benzoyl-arnino acid amidohydrolase (HipO) protein sequence, second line shows the
Bacillus stearothermophilus N-acyl amino acid arnidohydrolase (arninoacylase) (Ama)
protein sequence and the third line is the N-carbamyl-L-amino acid amidohydrolase
(DL-hydantoinase) fiom Pseudomonas sp. (hydantoin utilizing protein C) (hyzlC ) and
lastly Synechocystic hypothetical protein. Residues involved in conserved areas are
s h o w in bold face. Identical residues are marked with stars, sirnilar residues are
indicated by dots and hyphens indicate gaps introduced to maximize alignment.
Val-250 is located as an identical residue in the sequence of the
AMNHIPOMYUC M40 hydrolase family .
CLUSTAL W (1.4) Multiple Sequence Aliqnment
H ~ P O ------- MNLIPEILDLQGEFEKIRHQIHENPELGFDELCTAKLVAQKLKEFGYEVYEEI Am= ---- MTKEEIKRLVDEVKTDVIAWRRHLHAHPELSFQEEKTAQFYETLQSFGHLELSRP HyuC MKTVTISKERLRIHIEQLGEIGKTKDKGVQRLALSKEDREATLLVSEWMREAGLTVTHDH S ~ P ------- MELKNLAQTLLPRLVEIRRHLHAHPELSGQEYQTAAWAGVLSSCGLHVEEAI . . * . .. * *
HipO GKTGVVGVLKKG--NSD-KKIGLRADMDALP-------- LQECTNLPYK--SKKENVMHA Ama TKTSVMARLIGQ--QPG-RVVAIRADMDALP-------- IQEENTFEFA--SKNPGVMMA HyuC FGN-LIGRKEGE--TPSLPSVMIGSHIDSVRNGGKFDGVIGVLAGIEIVHAISEAMNHE shp GKTGVVGQLSGKGDDP--RLLAIRTDMDALP-------- IEEMVSLPFA--SRHPGVMHA
. . * . . . .*.. . * . * HipO --------c---G--- HDGHTTSLLLAAKYLASQ--NFNGTLNLYFQPAEEGLGG-AKAM Ama --------c---G--- HDGHTAMLLGTAKIFSQLRDDIRGEIRFLFQHAEELFPGGAEEM HyuC HSIEVVAFCEEEGSRFNDGLFGSRGMVGKVKPEDLQKVDDNNVTRYEALKTFGFGIDPDF shp --------c---G--- HDIHTTLGLGTAMVLSQMGHRLPGDVRFLFQPAEEIAQG-ASWM
* . * . . * HipO IEDGLFEKFDSDYVFGWHN--MPFGSDK--KFYLKKGAMMSSDSYSIEVIGRGGH-GSA Ama VQAGVMDGVD--VVIGTHL--WSPLERG--KIGIVYGPMMAAPDRFFIRIIGKGGH-GAM HyuC THQSIREIGDIKHYFEMHIEQGPYLEKNNYPIGIVSG--IAGPSWFKVRLVGEAGHAGTV shp IQDGAMKGVS--HILGVHV--FPSIPAQ--QVGIRYGALTMDDLEIFIQGESGH-GAR
* . * * . . * ** * .
HipO PEKA-KDPIYAASLLVVALQSIVSRNVDPQNSAVISIGAF~IAGH-A~I 1 PDIVTIKMSV Ama PHQT-IDAIAIGAQVVTNLQHIVSRYVDPLEPLVLSVTQFVAGT-AHNVLPGEVEIQGTV HyuC PMSLRKDPLVGAAEVIKEVETLCMN--DPNAPWGTVGRIAAFPGGSNIIPESVEFTLDI shp PHEA-IDAIWIAAQVITALQQAISRTQNPLRPMVLSLGQISGGR-APNVIADQVR~GTV
* * . * .. * . . . . . * *. . HipO RALDNETRKLTEEKIYKICKGLAQANDIEIKINKNVVAPVTMNNDEAV-EFASEVAKELF Ama RTFDETLRRTVPQWMERIVKGITEAHGASYEFRFDYGYRPVINYDEGDPRHGGNGVRAVR H ~ u C RDIELERRNKIIEKIEEKIKLVSNTRGLEYQIEKNElAAVPVKCSENLI-NSLKQSCKEL- shp RSLHPETHAQLPQWIEGIVANVCQTYGAKYEVNYRRGVPSVQNDAQLN-KLLENAVREAW
* . . . . .
HipO GEKNCEFNHRPLMASEDFGFFCEMKKCAYAFLEN----- ENDIYLHNSSYVFNDKLLARA Ama RRGSGPLETEH--GRRRFLRLFAKSARQLFLRRRGQCRKRHRLPAPPPALYD-------- H p C -EIDAPIIVSG--AGHDAMFLAEITEIGMVFVRC------ RNGISHSPKEWAEI DDILTG ohp GESALQIIPEPSLGAEDFALYLEHAPGAMFRLGTGFGDRQMNHPLHHPRFEADEAAILTG
HipO ASYYAKLALKYLK---- Ama ----------------- HyuC TKVLYESIIKHI----- shp VVTLSYAAWQYWQNIAI
surface-exposed regions (Hopp and Woods, 198 1 ).
The predicted hydrophathy profile of C. jejuni hippurate hydrolase (HIPO) is
generally simiiar to the predicted profiles of the amidohydrolase of B.
stevothermophiIus ( AMA), Pseudornonas sp. (HYUC), and Synechocystis (Figure 9).
Hydrophilic regions are located at the outside of the protein structure, and hydrophobic
regions are buried inside the protein or within other hydrophobic environment such as
membrane. Val-250 is located in the hydrophobic region of the predicted hippurate
hydrolase protein profile of C. jejuni (Figure 10). The flexibility blot is used to identify
the specific sites that are protruding or highly mobile regions on the surface, and Val-
250 is also situated in the rigid region of the protein profile (Figure 10). When alanine
is substituted in place of valine, a change in the predicted secondary structural from
beta pleated sheet (p-slieet) to alpha helix (a-helix) and also a reduction in the
hydrophobicity of the protein were observed (Figure 1 1).
CL* LO* 1 OC L O Z L 0 1
I l L
I 1 1 100. s-
O L E 00 ' S- 00 ' fr-
Hydrophi l ic i tyWindowSize= 7 Scale = Kyte-Doo l i t t le 5.00 , 1
1 . î S ............... ......................... ........................ ................ . ....................... ........................ 1 20 - *; ; ,..................*.....; .......................*+; ......*...; ; ; ................................................... ........................ ................ ........................ i ...................*.....: ...................... *..; ; ; ........................ ......................... ..... ................ ...................... ........................ i i .....*.;
...... ......... .......... ........ ............................................. .............. ......................... ....................... ........................ ............. ......................... i.. ......................... ......................... ..............*.....**.. .............*.. . ........................ ........................ ......................... ......................... 0 85 - 6 i i 6 &
......................... .......................* ................ . ........................ ........................ ......................... ................. .........*............... 0 80 - 9 6 i & &
O . 75 1 I 1 1 l 1 1
50 1 O0 150 200 250 300 350
Figure 10. Predicted hvdropathv (hvdro~hobiciw and flexibiliw) profiles of C.
jeirrni hippurate hydrolase.
The hydropathy and flexibility profiles of the HIPO protein with the Kyte-
Doolittle scale and a window size of 7. Numbers on the bottom indicate arnino acid
positions. This profiles graph the local hydrophilicity of a protein along with its amino
acid sequence. Hydrophobic regions deflect downward (negative value). In flexibility
profile of HIPO, the region deflect downward (negative value) which represents the
rigid region.
Val-250 is predicted to locate at rigid hydrophobic region of hippurate hydrolase.
Kyet & Uoolittle Hydrophobicity Protïles
Figure 11. Corn parison of the predicted secondary structure and hvdrophobicity
profiles of C. iejuni hippurate hvdrolase (Val-250) and its modified sequence
(substitution of Ala-250).
Hippurate hydrolase is a key enzyme used to identifi C. jejuni and to
differentiate C. jejuni from other species of Campylobacter. The role of hippurate
hydrolase in C. jejuni is unclear at this moment. However, kanarnycin cassette
insertion mutants of C. jejuni hippurate hydrolase have been constructed and they
appear to grow normally in Mueller-Hinton medium (Hani, 1997). Thus, the hippurate
hydrolase gene is not essential for growth under such conditions. The prevalence of
hippurate hydrolase-negative C. jejuni in poultry and humans is very rare. The
hippurate hydrolase-negative isolates which hydrolyzed low levels of hippurate could
be detected by a sensitive method, such as gas liquid chromatography (Totten et al.,
1987). Characterization of the defective hippurate hydrolase gene and the encoded
hippurate hydrolase enzyme could provide valuable information on its structure and
fünction.
In this study, we characterized five different hippurate hydrolase-negative C.
jejuni clinical isolates (D594, D603, D835, D1713, and D941) isolated fiom chicken
and humans. These strains fell into different Lior and Penner serotypes and have
different plasmid patterns. Therefore, they represent a diverse group of isolates (Totten
et al., 1987). A radiolabeled single strand conformational polymorphic (SSCP) assay
based on polymerase chain reaction was used to detect the putative mutated regions of
the hippurate hydrolase gene from five hippurate hydrolase-negative C. jejurzi isolates.
The polymorphic regions detected in the SSCP assay from each isolate were cloned and
sequenced to determine the exact nature of the mutations. We observed that valine at
hydrolase-negative clinical C. jejuni isolates.
Although valine and alanine are both nonpolar hydrophobic arnino acids and
have alkanes as side chains, stereochemical representation shows that valine has a
larger hydrocarbon side chain than alanine. The bond between the alpha-carbon and
beta-carbon of a valine residue in a polypeptide emphasizes the steric interactions
between the peptide backbone and the carbons in the two garna-positions of this arnino
acid and, by extension, the steric interactions for each of the others. One or two
substitutes might be attached to the beta-carbon, and this places one or two substituents
at either or both of the positions of the two methyl groups of valine. This does not
happen in alanine. Therefore, the change of valine to alanine may have some impact on
both the structure and fùnction of the C. jejuni hippurate hydrolase.
Sequence cornparisons of proteins between species are frequently used to
establish conserved elements which may have importance for protein function. Multiple
sequence alignment of the C. jejuni hippurate hydrolase enzyme with homologues from
M40 hydrolase family (AMA/HIPOMYUC), identified conserved sequences within the
hydrolase proteins of different species:Bacillus sterotherrnophilus, Psedornonas
species, and Synechocystis. Homologous enzymes share the cornmon feature of the
splitting action, which is similar to the enzyme that cleaves or hydrolyzes hippurate.
Significantly, Val-250 is identified as a conserved amino acid based on sequence
alignment of the AMA/HIPO/HYUC deduced amino acids. The presence of a
particular arnino acid at a conserved location in homologous proteins across different
species suggests a functional role associated with that site. This reinforces the common
identity of the protein in that farnily. Hippurate hydrolase protein from C. jejuni is
characteristic and important positions in the structure of the C. jejuni hippurate
hydrolase protein may also be characteristic and important in the other hydrolase
members. The conservation of Val-250 in al1 these homologous proteins suggests that
it may potentially be critical for hippurate hydrolase function in C. jejuni.
Particular positions in the amino acid sequence that are buried in hydrophobic
clusters are the most invariant. Moreover, the hydrophobic effect contributes favorably
to protein folding. There are usually a number of locations in the structure of a protein
where difficulties resulting fiom the packing of the backbone of the polypeptide arise.
The preference for particular amino acids rnay reflect the constraints of an intricate,
interlocking stereochemistry in the interior of protein structure. The hydropathy
profile of the C. jejuni hippurate hydrolase protein revealed that Val-250 is located in a
hydrophobic region of the C. jejuni hippurate hydrolase protein. Furthemore, the
nearest-neighbor effects are taken into account by assigning different flexibility values
to a given kind of residue depending on whether it is surrounded by "rigid" residues or
not. The flexibility profile identified that Val-250 is located in a rigid region of the
hippurate hydrolase. The difference in standard fiee energy of folding between valine
and alanine is +5 kJ mol-' (Salahuddin and Tanford, 1970). The replacement of valine
by alanine rnay result in a change of standard free energy and this will also change the
hydrophobic effect. Therefore the change could result in destabilizing the protein.
Most likely the reason is that an empty space wiil be created in the protein, unless the
native structure is rearranged to fil1 it. Therefore, the particular residue Val-250 may be
required and essential to maintain C. jejuni hippurate hydrolase structure and enzymatic
function.
iI 1 Y 1 ir - - --
alpha helix (a-helix), in place of beta pleated sheet (P-sheet) if alanine is substituted in
the place of vaiine. Although both valine and alanine have closely related aliphatic side
chains of the building blocks, alanine with a methyl group in its side chain favors the
formation of an a-helix by a higher relative frequency of 1.29. The a-helix is tightly
coiled and it tends to reside on the periphery of the protein structure. Unlike alanine
which has one carbon side chain, valine has an additional methyl goup in its side chain
and this enhances the P-pleated sheet in a relative fiequency of 1.49 (Lim, 1974;
Garnier et al., 1978). In general, a polypeptide chain is almost fully extended in the B-
pleated sheet and tends to reside in the center of crystallographic molecular models of
proteins. This indicates that the tendency of a polypeptide to adopt a regular secondary
structure depends largely on its arnino acid composition and may have some intrinsic
preferences among the amino acids for certain secondary structures. Therefore, the
conserved arnino acids have a critical role in folding or general stability. The amino
acid substitutions may be tolerated and have no deleterious effect on the protein
structure. Yet mutation of a critical structural residue might modi@ the protein and
consequently cause the catalytic ability, and the apparent conformation and stability of
mutated protein to become less efficient.
Three continuous overlapping upstream regions of the hippurate hydrolase gene
from the five hippurate hydrolase-negative C. jejuni clinical isolates revealed no
polymorphism in the SSCP assay. This indicates that there is possibly no sequence
variations in these upstream regions. A conclusive picture wouId require nucleotide
sequencing of the upstream regions of the hippurate hydrolase genes from hippurate
multiple copies of transcripts produced by these five hippurate hydrolase-negative C.
jejuni isolates (Hani, 1997). The above study did not show a defect in transcription of
the hippurate hydrolase gene in the five hippurate hydrolase-negative C. jejuni isolates.
At this stage, the fimction of hippurate hydrolase in C. jejuni is not clear. There
is no specific information available on differences in disease severity in hurnans relative
to either hippurate hydrolase-positive or -negative C. jejuni isolates. The only
distinguishing feature of C. jejuni from a closely related farnily member, C. d i , is the
presence of a hippurate hydrolase gene and its ability to hydrolyse hippurate. In
addition, C. jejuni is fiequently isolated from cases o f human diarrhea and fiom poultry.
The active sites of hippurate hydrolase in C. jejuni need to be defined. It is a
subject for future studies. Residue substitution consequently occurring o n the catalytic
component Ieads to an enzyme with dramatically reduced activity. Valine and alanine
have closely related aliphatic amino acids with the only difference in side chain
residues being carbon chain length. Mowever, this change may affect or disturb the
folding of the enzyme structure or change the amino acid size, resulting in a
nonfunctional or reduced stability of hippurate hydrolase protein. The active sites of
hippurate hydrolase need to be determined to see whether this specific residue is
essential for hippurate hydrolase function.
Analysis of the naturally mutated hipO genes from the five different C. jejuni
isolates suggests that valine at residue 250 may potentially be a key residue of hippurate
hydrolase in C. jejuni. Therefore, we can examine if this valine at residue 250 is at the
catalytic site of hippurate hydrolysis by site-directed mutagenesis and enzyme assay.
In addition, other conserved arnino acids have been identified based on a
multiple alignment of the four amino acid sequences of the M40 hydrolase farnily. We
can also examine the putative roles of other conserved residues by site-directed
mutagenesis. The enzyrnatic properties can be exarnined using a chromogenic substrate
or the hydrolysis ability by using a color reagent system.
Our lab has constmcted an isogenic hipO mutant of C. jejuni by gene
replacement with a kanamycin-insertion hippurate hydrolase mutated gene (Hani,
1997). Therefore, the correlation between the hipO gene and C. jejuni pathogenicity
can be investigated using kmamycin-inserted isogenic mutants in animal models or the
study of invasiveness with human intestinal cell lines.
In addition, there is no report on the disease severity related to particular
Cumpylobacter species and it will be interesting to reevaluate the association of
hippurate hydrolase-negative C. jejuni strains with infection.
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