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Multiple inverted DNA repeats of Bacteroides fragilis that control polysaccharide antigenic variation are similar to the hin region inverted repeats of Salmonella typhimurium Patrick, S., Parkhill, J., McCoy, L. J., Lennard, N., Larkin, M., Collins, M., Sczaniecka, M., & Blakely, G. (2003). Multiple inverted DNA repeats of Bacteroides fragilis that control polysaccharide antigenic variation are similar to the hin region inverted repeats of Salmonella typhimurium. Microbiology (Reading, England), 149(4)(4), 915-924. https://doi.org/10.1099/mic.0.26166-0 Published in: Microbiology (Reading, England) Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal General rights Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact [email protected]. Download date:19. Jun. 2021

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  • Multiple inverted DNA repeats of Bacteroides fragilis that controlpolysaccharide antigenic variation are similar to the hin regioninverted repeats of Salmonella typhimuriumPatrick, S., Parkhill, J., McCoy, L. J., Lennard, N., Larkin, M., Collins, M., Sczaniecka, M., & Blakely, G. (2003).Multiple inverted DNA repeats of Bacteroides fragilis that control polysaccharide antigenic variation are similar tothe hin region inverted repeats of Salmonella typhimurium. Microbiology (Reading, England), 149(4)(4), 915-924.https://doi.org/10.1099/mic.0.26166-0

    Published in:Microbiology (Reading, England)

    Queen's University Belfast - Research Portal:Link to publication record in Queen's University Belfast Research Portal

    General rightsCopyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or othercopyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associatedwith these rights.

    Take down policyThe Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made toensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in theResearch Portal that you believe breaches copyright or violates any law, please contact [email protected].

    Download date:19. Jun. 2021

    https://doi.org/10.1099/mic.0.26166-0https://pure.qub.ac.uk/en/publications/multiple-inverted-dna-repeats-of-bacteroides-fragilis-that-control-polysaccharide-antigenic-variation-are-similar-to-the-hin-region-inverted-repeats-of-salmonella-typhimurium(d4c818f7-2081-45df-9c6b-0bcb99a9b8be).html

  • Multiple inverted DNA repeats of Bacteroidesfragilis that control polysaccharide antigenicvariation are similar to the hin region invertedrepeats of Salmonella typhimurium

    Sheila Patrick,1 Julian Parkhill,4 Lisa J. McCoy,1 Nicola Lennard,4

    Michael J. Larkin,2 Martin Collins,3 Matylda Sczaniecka5 and Garry Blakely5

    Correspondence

    Sheila Patrick

    [email protected]

    1,2,3Microbiology and Immunobiology1, Biology and Biochemistry2 and Food Science3, TheQueen’s University of Belfast, Grosvenor Road, Belfast BT12 6BN, UK

    4The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, CambridgeCB10 1SA, UK

    5Institute of Cell and Molecular Biology, The University of Edinburgh, Darwin Building, King’sBuildings, Edinburgh EH9 3JR, UK

    Received 2 December 2002

    Revised 15 January 2003

    Accepted 17 January 2003

    The important opportunistic pathogen Bacteroides fragilis is a strictly anaerobic Gram-negative

    bacterium and a member of the normal resident human gastrointestinal microbiota. Our earlier

    studies indicated that there is considerable within-strain variation in polysaccharide expression, as

    detected by mAb labelling. Analysis of the genome sequence has revealed multiple invertible DNA

    regions, designated fragilis invertible (fin) regions, seven of which are upstream of polysaccharide

    biosynthesis loci and are approximately 226 bp in size. Using orientation-specific PCR primers and

    sequence analysis with populations enriched for one antigenic type, two of these invertible regions

    were assigned to heteropolymeric polysaccharides with different sizes of repeating units, as

    determined by PAGE pattern. The implication of these findings is that inversion of the fin regions

    switches biosynthesis of these polysaccharides off and on. The invertible regions are bound by

    inverted repeats of 30 or 32 bp with striking similarity to the Salmonella typhimurium H flagellar

    antigen inversion cross-over (hix) recombination sites of the invertible hin region. It has been

    demonstrated that a plasmid-encoded Hin invertase homologue (FinB), present inB. fragilisNCTC

    9343, binds specifically to the invertible regions and the recombination sites have been designated

    as fragilis inversion cross-over (fix) sites.

    INTRODUCTION

    Bacteroides fragilis is the Gram-negative strictly anaerobicbacterium most frequently isolated from clinical infection,including intra-abdominal, vaginal, pilonidal, perianal andbrain abscesses. It is also the most common cause ofanaerobic bacteraemia, with a potential mortality of up to19 % (Redondo et al., 1995). The major source of theseinfections is the normal resident colonic microbiotawhere Bacteroides spp. outnumber facultatively anaerobicbacteria such as Escherichia coli by a factor of at least 102–103 (Drasar & Duerden, 1991).

    A number of factors may contribute to the virulence ofB. fragilis; however, extracellular polysaccharides (PSs) areconsidered to play a key role (Patrick, 2002). Intra-strain

    phase variation, defined as whether a given characteristicis present or not (Saunders, 1986), is evident with respectto encapsulating surface structures (Babb & Cummins,1978; Patrick & Reid, 1983). A large capsule (LC) and smallcapsule (SC) are visible by light microscopy. By electronmicroscopy, an encapsulating electron-dense layer (EDL)is visible adjacent to the outer membrane on bacterianon-capsulate by light microscopy (Patrick et al., 1986).Expression of the different capsular types is heritable;populations can be enriched by subculture from differentinterfaces of Percoll step density gradients (Patrick & Reid,1983). In addition, antigenic variation of individual cap-sular types can be demonstrated using mAb labelling;variable proportions of bacterial cells within these popula-tions label with individual mAbs (Lutton et al., 1991). TheLC and EDL phases have shared epitopes; however, the SCis antigenically different (Reid et al., 1985; Lutton et al.,1991). mAb labelling indicates that there are potentially sixdifferent high-molecular-mass polysaccharides (HMMPS)

    Abbreviations: EDL, electron-dense layer; HMMPS, high-molecular-mass polysaccharides; LC, large capsule; MBP, maltose-binding protein;PS, polysaccharide; SC, small capsule.

    0002-6166 G 2003 SGM Printed in Great Britain 915

    Microbiology (2003), 149, 915–924 DOI 10.1099/mic.0.26166-0

  • associated with both the EDL and LC. One additionalHMMPS is associated with the SC. Antigenic variation hasbeen observed in clinical isolates from a variety of anatomi-cal sites and different geographical locations, and also inbacteria grown in an in vivo model of peritoneal infection

    (Patrick et al., 1995a, b). Immunoblotting of these anti-genically variable PSs after PAGE reveals patterns char-acteristic of heteropolymeric PSs with repeating subunits.Ladders with two different sizes of steps have been observed(Lutton et al., 1991), one with a ladder pattern suggesting

    Table 1. Oligonucleotide primers used in the study

    Primer A B C

    PS1 ccagattattatctgataat attatcagataataatctgg tagaaaagacgcaccccgta

    PS2 gtatataatactcttataat attataagagtattatatac ggaacatgatcatccttcgtg

    PS3 tccaataattcattgattta taaatcaatgaattattgga cgctcgttcttgacgatgta

    PS4 ctctatatatatttgagaat attctcaaatatatatagag gccctggtaagcaggtcttt

    PS5 gtttatagatatttgattac gtaatcaaatatctataaac acggatcttacgctcaccac

    PS6 tcataaatacctgtggttta taaaccacaggtatttatga gtgcatggggatgaaattct

    PS7 agatatctatctagaaatta taatttctagatagatatct aggaaccaaaacacggacttt

    Fig. 1. Alignment of the B. fragilis NCTC9343 invertible regions of DNA upstream ofPS biosynthesis loci 1–7. Note invertedrepeats at the right and left ends (arrows)and the B. fragilis consensus promotersequence (box).

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  • that it may be similar to the O-antigen of smooth Gram-negative bacteria identified by some workers (Poxton &Brown, 1986; Delahooke et al., 1995), but not others(Lindberg et al., 1990; Comstock et al., 1999).

    If an antigenically mixed broth culture is spread onto agarplates and the resulting colonies examined microscopicallyafter immunofluorescence labelling, 90 % or more of thebacteria carry a given epitope in some colonies and 10 %or less in others. The proportion of bacteria expressing agiven epitope within a colony is maintained on subcultureof the colony into broth culture (Patrick et al., 1999).

    The possible mechanism underlying this complex variationwas entirely unknown until the B. fragilis whole-genomesequencing project being carried out at the Sanger WellcomeInstitute UK (http://www.sanger.ac.uk/Projects/B_fragilis/)revealed the presence of multiple regions of DNA withinverted repeat elements at either end that appear to bepresent in alternative orientations, within the bulk DNAsupplied for sequencing. The DNA was extracted from adefined population, enriched for the EDL/non-capsulatepopulation, but which contained different PS antigenictypes. A recent paper concluded that variation of PSexpression in B. fragiliswas probably controlled by invertibleregions bound by 19 bp inverted repeats upstream of theseven putative biosynthesis loci (Krinos et al., 2001). Wepresent evidence that the inverted repeat regions at theseloci are in fact 30 or 32 bp in length and similar to theSalmonella typhimurium H flagellar antigen inversion cross-over (hix) recombination sites of the invertible hin region ofS. typhimurium that controls biphasic flagellar antigenicvariation (Zieg et al., 1977). We also show, using mAbs, thatPS epitope expression associated with defined subpopula-tions is related to specific S. typhimurium H flagellar antigeninvertase (Hin)-like inversions and that a plasmid-encodedHin-like recombinase binds to the invertible regions.

    METHODS

    Bacterial strains and culture methods. B. fragilis NCTC 9343(National Collection of Type Cultures, Colindale Avenue, London,UK), representative of the original strain deposited in the culturecollection in 1955 and originally isolated from an abdominal abscess,was used. Identity was confirmed by partial sequencing of 16SrDNA. Bacteria were grown in defined medium (DM) broth or onDM plates (van Tassell & Wilkins, 1978) in an anaerobic cabinet[MACS Anaerobic Workstation; Don Whitley (80 % N2, 10 % CO2,10 % H2)]. Percoll density gradient enrichment was used to obtainpopulations that either were non-capsulate by light microscopy orproduced an LC detectable by light microscopy, as described pre-viously (Patrick & Larkin, 1993). Populations enriched for individualepitopes were obtained by picking individual colonies from DMagar plates and subculturing in DM broth (Patrick et al., 1999). Theproportion of bacteria labelled within a population was monitoredby double immunofluorescence labelling with mAbs and polyclonalantisera as detailed previously (Patrick & Larkin, 1993). Imageswere viewed with a Leitz Ortholux fluorescence microscope, aNikon DMX1200 digital camera and Lucia GF software. PAGE andimmunoblotting were carried out as described previously (Luttonet al., 1991).

    DNA sequencing and PCR amplification. Genomic sequencedata were produced by the Bacteroides fragilis Sequencing Group atthe Wellcome Trust Sanger Institute. These data are available fromhttp://www.sanger.ac.uk/Projects/B_fragilis/. The complete sequenceand annotation of the genome will be described elsewhere.

    DNA was extracted from populations enriched for one capsularphase and antigenic type, as determined by microscopy, using QiagenGenomic-tip 100G in accordance with the manufacturer’s instruc-tions. DNA concentration was measured spectrophotometricallyand diluted accordingly to 1 mg ml21. Oligonucleotide primer pairs(Table 1) were synthesized by Life Technologies. The PCR mixtureused was 45 ml ABgene Reddymix containing 1?5 mM MgCl2, 200 mleach dNTP, 0?025 units Thermoprime Plus DNA Polymerase, 75 mMTris/HCl (pH 8?8), 20 mM (NH4)2SO4 and 0?01 % (v/v) Tween 20.To the PCR mix described above, 2?5 ml each primer (20 mM) and2?5 ml template (1 mg ml21) were added. Primer pairs were either Aand C or B and C (Table 1). PCR amplification was performed for25 cycles of 94˚C for 1 min 30 s, 50˚C for 1 min 30 s and 72 C̊ for1 min 30 s using an MJ Research PTC-200 Peltier Thermal Cycler.Samples were then cooled to 4˚C and retained at this temperatureuntil collected. After amplification, 5 ml of the amplified product waselectrophoresed through a 1 % (w/v) agarose gel in 16Tris-acetate-EDTA. PCR product bands were detected by ethidium bromidestaining, visualized by UV light (Transilluminator TFX-35M; Gibco-BRL) and photographed using a Kodak DC290 Zoom Digital Camera

    Fig. 2. Alignment of the B. fragilis NCTC 9343 inverted repeatregions (fix1–7) at the left (top strand) and right (inversebottom strand) end of the invertible regions upstream of PSbiosynthesis loci 1–7 with the promoter in the correct orienta-tion for PS expression and top-strand left-hand inverted repeatsof the ‘Hin’ family (hix, pix, cix, gix, M. bovis pilus). Centraldinucleotides are in lower case. Bases that interact with theHin invertase in S. typhimurium are in bold. Other conservedregions are boxed.

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  • fitted to a Kodak EDAS290 Gel Imaging Hood. Gel images were

    analysed using Kodak ID Image Analysis Software Version 3.5 MAC

    USB. 16S rDNA primers were included as an internal standard. PCRproducts were purified using the MinElute PCR Purification Kit

    (Qiagen), according to the manufacturer’s instructions. The nucleo-

    tide sequences of purified PCR products were determined using theABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit

    (Applied Biosystems) and halfBD Terminator Sequencing Reagent

    (Sigma) using an ABI Prism 3100 Genetic Analyser (AppliedBiosystems), according to the manufacturer’s instructions.

    Protein purification and gel retardation. DNA fragments corre-sponding to coding sequences of the finA and finB genes, generatedby PCR using Pfu polymerase, were cloned into pMAL-c2 (NEB) toproduce translational fusions with the maltose-binding protein(MBP) encoded by malE. E. coli strain DH5a was transformed with

    Fig. 3. Immunofluorescence micrographs of mAb QUBF5-enriched population of non-capsulate B. fragilis NCTC 9343labelled with mAb QUBF5 (a), rabbit anti-B. fragilis polyclonal antiserum (b; same field of view as in a), mAb QUBF6 (c) andrabbit anti-B. fragilis polyclonal antiserum (d; same field of view as in c). The secondary antibodies are anti-mouse FITC andanti-rabbit TRITC conjugates.

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  • plasmids that expressed the invertase-MBP fusions, grown to anOD600 of 0?4 and induced with 1 mM IPTG for 1 h. Cells wereharvested by centrifugation and resuspended in lysis buffer (20 mMTris/HCl, pH 7?4, 200 mM NaCl, 1 mM EDTA, 1 mM PMSF),before addition of lysozyme (1 mg ml21) and sonication to producecell lysates. After centrifugation to remove cell debris, the invertase-MBP fusion proteins were purified by affinity chromatography onamylose resin, according to the manufacturer’s instructions (NEB).

    Gel retardation methods were carried out as detailed by Blakely et al.(1993). DNA for gel retardation analysis of the entire invertible fin1region was generated by PCR with Pfu polymerase, using primers thatflanked the fix1L and fix1R sequences shown in Fig. 1, followed byradiolabelling using [c-32P]ATP and T4 polynucleotide kinase. Bindingreactions (10 ml) containing approximately 0?03 pmol radiolabelledDNA were performed in 100 mM NaCl, 20 mM Tris/HCl, pH 8,1 mM EDTA, 10 % glycerol and 100 mg poly-dIdC ml21 at 37 C̊ for10 min before electrophoresis through a pre-run, non-denaturing4 % polyacrylamide gel. Binding gels were dried and then visualizedby autoradiography.

    RESULTS AND DISCUSSION

    An alignment of the invertible regions upstream of the PSbiosynthesis loci in B. fragilis is presented in Fig. 1. At eitherend of these invertible regions are AT-rich sequencescontaining imperfect dyad symmetry spanning 30–32 bp(inverted repeat regions). The recently proposed consensusB. fragilis promoter regions (Bayley et al., 2000) areconserved and overlap the repeat region by 2 bp.

    The inverted repeat regions, designated fragilis inversioncross-over (fixL and fixR) sites (Fig. 2) bear striking simi-larity to recombination sites found at the ends of someenteric bacterial invertible DNA sequences, for example, the995 bp hin region within the chromosome of S. typhimu-rium, the invertible regions of phage Mu (Gin system), theE. coli K-12 e14 element (Pin system) and phage P1(Cin system) (van de Putte & Goosen, 1992; Fig. 2). InS. typhimurium, the reversible expression of two antigeni-cally different flagella, H1 and H2 (Lederberg & Iino, 1956)is controlled by inversion of the hin region which carries apromoter and is upstream of the H2 gene, and a repressorfor H1. In Mu and P1 phage DNA inversion alters the tailfibres, thereby changing the phage host range (Plasterket al., 1983; Hiestand-Nauer & Iida, 1983). There is alsosome similarity with the Moraxella bovis invertible regionwhich is within the Q/I (formerly b and a) pilus gene(Fulks et al., 1990), the inversion of which mediates pilusphase and antigenic variation. There is no apparentrelationship with the 9 bp repeat of the 314 bp invertibleregion that varies expression of type 1 fimbriae in E. coliwhich is mediated by the tyrosine family recombinasesFimBE (Abraham et al., 1985).

    To test the hypothesis that orientation of these regions,with respect to the downstream PS biosynthesis operons,controls variation of different PSs associated with sub-populations that are definable using mAbs, a series ofexperiments were carried out using ‘orientation-specific’PCR primers. Primer pairs (Table 1) were designed such

    that the PCR product would bridge the right end of theinvertible region. A product from primer pair A and Cwould be obtained when the proposed B. fragilis consensuspromoter (Bayley et al., 2000) within the invertible region(Fig. 1) was in the correct orientation with respect to thePS biosynthesis operon (‘on’ position); a product fromprimer pair B and C would be expected when in the oppo-site orientation (‘off’ position). Populations enriched forreactivity with mAbs QUBF5, 6 and 7 by immunofluore-scence microscopy were prepared; a QUBF5-enrichedpopulation is illustrated in Fig. 3. Each of these mAbsreacts with antigenically variable epitopes associated withboth the LC and non-capsulate/EDL phases. After PAGEand immunoblotting, these mAbs label HMMPS withassociated ladder patterns characteristic of heteropoly-meric PS with repeating units (Lutton et al., 1991). QUBF7reacts with PS C (Coyne et al., 2000) which lacks aninvertible region upstream of the biosynthesis locus. mAbQUBF5 reacts with an HMMPS with a larger ladder step-size pattern after PAGE and immunoblotting than QUBF6and 7 (Lutton et al., 1991) (Fig. 4). Primer pairs 1A/C and5A/C yielded a product with populations enriched formAbs QUBF6 and 5, respectively (Fig. 5). None of theother A/C primer pairs yielded a similar product for thesepopulations. As the enriched populations contain low

    Fig. 4. Immunoblots of proteinase K extracts from B. fragilisNCTC 9343 after PAGE reacted with mAbs QUBF5 (lane 1),QUBF6 (lane 2) and QUBF7 (lane 3).

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  • proportions of bacteria positive for other epitopes (Fig. 5),PCR product was sometimes obtained in the ‘on’ positionfrom populations enriched for another epitope. For example,where 7–8 % of the population enriched for reactivity tomAb QUBF7 also reacted with mAb QUBF6, some PCRproduct was obtained from primer pairs 1A/C, althoughthis was considerably less than the product from primers1B/C (Fig. 5). The antigenic variation of the Bf5 HMMPScould explain the conflicting literature concerning thepresence or absence of an ‘O-antigen’ in B. fragilis (Patrick,2002).

    Sequencing of the PCR products confirmed the sequenceof the right end of the invertible region and showed thatthe inner half-sites of the fixR inverted repeats (i.e. the half-site that ‘flips’ during recombination within the invertingDNA) were in opposite orientations in the ‘on’ and ‘off’positions and confirmed that site-specific recombinationhad occurred. This pattern of PCR product was observedfor both non-capsulate/EDL and LC phases enriched forthe different mAb epitopes. This suggests that the phasechange associated with LC formation is not controlled by

    these loci. A minimum of three separate populationsenriched for each phenotype was examined. Comparisonof the sequence data indicates that mAbs QUBF5 and 6

    Fig. 5. PCR products, obtained from B. fragilis NCTC 9343 non-capsulate/EDL phase populations enriched for QUBF5,QUBF6 or QUBF7 antigenic phenotype, using primer pairs for PS biosynthesis loci 1 and 5 designed to detect fragilisinvertible (fin) regions in the ‘on’ or ‘off’ position with respect to PS expression. Note products in the ‘on’ position for primerpairs for locus 1 and QUBF6-enriched, and locus 5 and QUBF5-enriched (arrows). Lanes 1 and 24, standards starting fromthe bottom at 100 bp with 100 bp increments. The numbers underneath each lane show the proportion (percentage enriched)of populations labelled with mAbs QUBF5, QUBF6 and QUBF7 as determined by immunofluorescence microscopy.

    Fig. 6. Alignment of top-strand right-hand of fix1–7 with 19 bpinvertible regions (PS A–H) proposed by Krinos et al. (2001supplementary data Table 1, available on-line). Central dinucleo-tides of fixR are in lower case.

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  • correspond with PS E and PS D designated by Krinos et al.(2001) who suggested that the invertible repeat associatedwith the control of PS expression was 19 bp in length and ofidentical sequence in four of the seven regions (Krinos et al.,2001 supplementary data Table 1, available on-line). Theposition of the 19 bp repeat relative to the suggested 30 bprepeat is illustrated in Fig. 6. There is a variable gap betweenthe 19 bp repeat element and the consensus promoter(Bayley et al., 2000) sequence situated within the invertibleregion, whereas the fixL repeat consistently overlaps theconsensus promoter (Fig. 1). The asymmetry in the PS1 fixsites would result in the generation of different sequences ifthe DNA was cut in the 19 bp region rather than the 30 bppalindromic sequence. Examination of the sequences of thePCR product of PS 1 revealed a sequence consistent with theDNA inversion occurring as a result of a double-strandedDNA cut in the centre of the 30 bp (not illustrated). Thesesequence data were obtained with two replicate enrichedpopulations on two separate occasions.

    S. typhimurium H flagellar antigen invertase (Hin)-mediated DNA inversion occurs within a topologicallydefined synapse and proceeds by a concerted pair ofdouble-stranded staggered cuts, at the central dinucleo-tides of each recombination site, catalysed by nucleophilicattacks mediated by the active site serine from each of thefour monomers within the synapse (Heichman et al., 1991).

    Binding specificity for Hin resides in 4 amino acid residues,invariant amongst the enteric invertases, that interact withbase pairs through both the major and minor grooves ofthe recombination half-site (Feng et al., 1994). Importantbase pair interactions mediated by helix 3, within thehelix–turn–helix structural motif, through the majorgroove, include Ser174 with A10 and Arg178 with G9.Hin recognition is highly sensitive to alterations at eitherof these base pairs (Hughes et al., 1992). These bases arehighly conserved within the left half-sites of all proposedfix recombination sites, but are more variable in the righthalf-sites (Fig. 2a). Interactions of Hin with the minorgroove of hixL involve Arg140 with A 26, Gly139 with T5, Lys 187 with T 212 and Asn190 with T 210 and A 10.All these base pairs are conserved within the putative fixrecombination sites. This suggested that the fix DNAsequences would interact with recombinases related to theenteric invertase family. Two strong candidates for invert-ases have been located in B. fragilis NCTC 9343. Fragilisinvertase (Fin)A is similar to E. coli transposon Tn21resolvase TnpR (30?6 % identity in 196 aa) and FinB ishomologous to S. typhimurium Hin (46?6 % identity in191 aa). An alignment of FinA, FinB and Hin amino acidsequences is presented in Fig. 7. In addition to these, thereare 21 proteins belonging to the ‘phage integrase’ family,which includes the recombinases xerC/D and fimB/E,(Kulasekara & Blomfield, 1999; Burns et al., 2000), two

    Fig. 7. Alignment of amino acid sequencesof FinB, S. typhimurium Hin and FinA gener-ated by CLUSTAL X. The active site residueand helix–turn–helix motif, inferred from Hin,are marked with an asterisk and a bar,respectively.

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  • further members of the resolvase family and one proteinwith weak similarity to the Moraxella pilin inverting genepiv. FinB is encoded on a B. fragilis NCTC 9343 36?5 kbpclosed circular plasmid (unpublished).

    To investigate the hypothesis that the observed inversionsmight be mediated by a member of the enteric resolvase/invertase family, we partially purified both FinA and FinB asN-terminal fusions to the MBP. A 263 bp DNA fragmentthat corresponded to the invertible fin1 region was thenused to assess the binding specificity of each invertase to therecombination sites. Fig. 8 demonstrates that increasingconcentrations of FinB-MBP gave rise initially to a singleprotein/DNA complex (complex 1) that was eventuallyconverted to a slower migrating complex (complex 2). Basedon the documented attributes of the serine family of site-specific recombinases to either form dimers in solution orbind cooperatively to DNA as dimers (Spaeny-Dekkinget al., 1995; Blake et al., 1995), one simple interpretation ofthe binding pattern (Fig. 8) is that a dimer of FinB-MBP wasbound at either fix1L or fix1R to generate complex 1 and thatat higher protein concentrations both recombination siteswere occupied to give rise to complex 2. The relativeamounts of the two bands, under these conditions, suggest

    that interactions between these complexes are not coopera-tive. Further analysis of the binding specificities andstoichiometry of FinB present in higher order complexessuch as complex 2, however, is required to distinguishbetween dimer binding or formation of synaptic inter-mediates. FinB-MBP is also able to bind to a singlerecombination site derived from the fix5 locus, while theMBP fusion to FinA did not show binding to eitherinvertible fix1 or fix5 region (data not shown).

    Together these data show that expression of a significantnumber of PS biosynthetic operons in B. fragilis is directlyregulated by a DNA site-specific recombination inversionthat appears to involve a newly identified member of theresolvase/invertase family. Whether the observed level ofreversible variation of PS expression reflects the ability ofB. fragilis to colonize the hostile environment of the humanhost as an opportunistic pathogen, as a member of thenormal intestinal microbiota or both, remains to be deter-mined. Variation of the surface PSs in B. fragilis undoub-tedly occurs during natural infection by B. fragilis. Directexamination of pus drained from abdominal, perianal,ischiorectal, pilonidal, vaginal, Bartholin’s, diverticular,colostomy, groin and neoplasm abscesses as well as bloodculture bottles and labelled with six different PS-specificmAbs, revealed diverse labelling patterns. No single PS orgroup of PSs could be related to infection at one particularsite (Patrick et al., 1995b).

    The evolutionary origin of the B. fragilis recombinationsites remains open to speculation, although it has beensuggested that the Hin system in Salmonella arose as aresult of integration of a Mu-like phage (van de Putte &Goosen, 1992). The Bacteroidetes diverged early, in evolu-tionary terms, from other eubacteria, well before thedivergence of the Gram-positive bacteria from the phylumProteobacteria which contains the majority of Gram-negatives such as the enteric bacteria, E. coli and thepseudomonads (Woese, 1987). Given the relativelylimited number of other examples of reversible variationin pathogenic bacteria arising by DNA inversion, it maybe that the high number of invertible sequences inB. fragilis reflects this early evolutionary divergence. Thepresence of the invertase gene, which may be responsiblefor controlling variable expression of multiple chromo-somal loci, on a plasmid also raises a number of interest-ing evolutionary questions. For example, did the plasmidintroduce the invertase gene or did it acquire the gene as amechanism to ensure maintenance?

    In conclusion, the putative recombination sites involvedin the unprecedented level of DNA inversion observedin B. fragilis show striking similarity to those of theS. typhimurium Hin and related systems and appear toinvolve a plasmid-borne Hin-like invertase, FinB, whichwe have demonstrated binds to the invertible regions. Wetherefore propose that the invertible regions in B. fragilisare designated fin regions and the inverted repeats fixLor fixR.

    Fig. 8. Autoradiogram of in vitro binding between B. fragilisFinB-MBP and DNA, corresponding to the invertible fix1 region,analysed by gel retardation. Radiolabelled fix1 DNA was incu-bated with increasing concentrations of FinB-MBP. Lanes: 1,no FinB-MBP; 2–6, increasing concentrations of FinB-MBP.

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  • ACKNOWLEDGEMENTS

    The B. fragilis genome sequencing project is supported by WellcomeTrust Grant No. 061696 and L. J. M. by a Department for Employmentand Learning Northern Ireland PhD studentship. Short-term fundingfrom the President’s fund of the Society for General Microbiologyand the Medical Faculty QUB to enable L. J. M. to work in G. B.’slaboratory and a visiting student scholarship from the Darwin Trustof Edinburgh to M. S. are gratefully acknowledged.

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    Babb, J. L. & Cummins, C. S. (1978). Encapsulation of Bacteroidesspecies. Infection 19, 1088–1091.

    Bayley, D. P., Rocha, E. R. & Smith, C. J. (2000). Analysis of cepAand other Bacteroides fragilis genes reveals a unique promoterstructure. FEMS Microbiol Lett 193, 149–154.

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