1 Microbiology and Immunobiology, The Queen's University of Belfast, Grosvenor Road, Belfast BT12 6BN, UK
2 Biology and Biochemistry, The Queen's University of Belfast, Grosvenor Road, Belfast BT12 6BN, UK
3 Food Science, The Queen's University of Belfast, Grosvenor Road, Belfast BT12 6BN, UK
4 The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
5 Institute of Cell and Molecular Biology, The University of Edinburgh, Darwin Building, King's Buildings, Edinburgh EH9 3JR, UK
Correspondence
Sheila Patrick
s.patrick{at}qub.ac.uk
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ABSTRACT |
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INTRODUCTION |
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A number of factors may contribute to the virulence of B. fragilis; however, extracellular polysaccharides (PSs) are considered to play a key role (Patrick, 2002). Intra-strain phase variation, defined as whether a given characteristic is present or not (Saunders, 1986
), is evident with respect to encapsulating surface structures (Babb & Cummins, 1978
; Patrick & Reid, 1983
). A large capsule (LC) and small capsule (SC) are visible by light microscopy. By electron microscopy, an encapsulating electron-dense layer (EDL) is visible adjacent to the outer membrane on bacteria non-capsulate by light microscopy (Patrick et al., 1986
). Expression of the different capsular types is heritable; populations can be enriched by subculture from different interfaces of Percoll step density gradients (Patrick & Reid, 1983
). In addition, antigenic variation of individual capsular types can be demonstrated using mAb labelling; variable proportions of bacterial cells within these populations label with individual mAbs (Lutton et al., 1991
). The LC and EDL phases have shared epitopes; however, the SC is antigenically different (Reid et al., 1985
; Lutton et al., 1991
). mAb labelling indicates that there are potentially six different high-molecular-mass polysaccharides (HMMPS) associated with both the EDL and LC. One additional HMMPS is associated with the SC. Antigenic variation has been observed in clinical isolates from a variety of anatomical sites and different geographical locations, and also in bacteria grown in an in vivo model of peritoneal infection (Patrick et al., 1995a
, b
). Immunoblotting of these antigenically variable PSs after PAGE reveals patterns characteristic 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 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 agar plates and the resulting colonies examined microscopically after immunofluorescence labelling, 90 % or more of the bacteria carry a given epitope in some colonies and 10 % or less in others. The proportion of bacteria expressing a given epitope within a colony is maintained on subculture of the colony into broth culture (Patrick et al., 1999).
The possible mechanism underlying this complex variation was entirely unknown until the B. fragilis whole-genome sequencing project being carried out at the Sanger Wellcome Institute UK (http://www.sanger.ac.uk/Projects/B_fragilis/) revealed the presence of multiple regions of DNA with inverted repeat elements at either end that appear to be present in alternative orientations, within the bulk DNA supplied for sequencing. The DNA was extracted from a defined population, enriched for the EDL/non-capsulate population, but which contained different PS antigenic types. A recent paper concluded that variation of PS expression in B. fragilis was probably controlled by invertible regions bound by 19 bp inverted repeats upstream of the seven putative biosynthesis loci (Krinos et al., 2001). We present evidence that the inverted repeat regions at these loci are in fact 30 or 32 bp in length and similar to the Salmonella typhimurium H flagellar antigen inversion cross-over (hix) recombination sites of the invertible hin region of S. typhimurium that controls biphasic flagellar antigenic variation (Zieg et al., 1977
). We also show, using mAbs, that PS epitope expression associated with defined subpopulations is related to specific S. typhimurium H flagellar antigen invertase (Hin)-like inversions and that a plasmid-encoded Hin-like recombinase binds to the invertible regions.
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METHODS |
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DNA sequencing and PCR amplification.
Genomic sequence data were produced by the Bacteroides fragilis Sequencing Group at the Wellcome Trust Sanger Institute. These data are available from http://www.sanger.ac.uk/Projects/B_fragilis/. The complete sequence and annotation of the genome will be described elsewhere.
DNA was extracted from populations enriched for one capsular phase and antigenic type, as determined by microscopy, using Qiagen Genomic-tip 100G in accordance with the manufacturer's instructions. DNA concentration was measured spectrophotometrically and diluted accordingly to 1 µg ml-1. Oligonucleotide primer pairs (Table 1) were synthesized by Life Technologies. The PCR mixture used was 45 µl ABgene Reddymix containing 1·5 mM MgCl2, 200 µl each dNTP, 0·025 units Thermoprime Plus DNA Polymerase, 75 mM Tris/HCl (pH 8·8), 20 mM (NH4)2SO4 and 0·01 % (v/v) Tween 20. To the PCR mix described above, 2·5 µl each primer (20 µM) and 2·5 µl template (1 µg ml-1) were added. Primer pairs were either A and C or B and C (Table 1
). PCR amplification was performed for 25 cycles of 94 °C for 1 min 30 s, 50 °C for 1 min 30 s and 72 °C for 1 min 30 s using an MJ Research PTC-200 Peltier Thermal Cycler. Samples were then cooled to 4 °C and retained at this temperature until collected. After amplification, 5 µl of the amplified product was electrophoresed through a 1 % (w/v) agarose gel in 1x Tris-acetate-EDTA. PCR product bands were detected by ethidium bromide staining, visualized by UV light (Transilluminator TFX-35M; Gibco-BRL) and photographed using a Kodak DC290 Zoom Digital Camera 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. PCR products were purified using the MinElute PCR Purification Kit (Qiagen), according to the manufacturer's instructions. The nucleotide sequences of purified PCR products were determined using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and halfBD Terminator Sequencing Reagent(Sigma) using an ABI Prism 3100 Genetic Analyser (Applied Biosystems), according to the manufacturer's instructions.
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Gel retardation methods were carried out as detailed by Blakely et al. (1993). DNA for gel retardation analysis of the entire invertible fin1 region was generated by PCR with Pfu polymerase, using primers that flanked the fix1L and fix1R sequences shown in Fig. 1
, followed by radiolabelling using [
-32P]ATP and T4 polynucleotide kinase. Binding reactions (10 µl) containing approximately 0·03 pmol radiolabelled DNA were performed in 100 mM NaCl, 20 mM Tris/HCl, pH 8, 1 mM EDTA, 10 % glycerol and 100 µg poly-dIdC ml-1 at 37 °C for 10 min before electrophoresis through a pre-run, non-denaturing 4 % polyacrylamide gel. Binding gels were dried and then visualized by autoradiography.
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RESULTS AND DISCUSSION |
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The inverted repeat regions, designated fragilis inversion cross-over (fixL and fixR) sites (Fig. 2) bear striking similarity to recombination sites found at the ends of some enteric bacterial invertible DNA sequences, for example, the 995 bp hin region within the chromosome of S. typhimurium, the invertible regions of phage Mu (Gin system), the E. coli K-12 e14 element (Pin system) and phage P1 (Cin system) (van de Putte & Goosen, 1992
; Fig. 2
). In S. typhimurium, the reversible expression of two antigenically different flagella, H1 and H2 (Lederberg & Iino, 1956
) is controlled by inversion of the hin region which carries a promoter and is upstream of the H2 gene, and a repressor for H1. In Mu and P1 phage DNA inversion alters the tail fibres, thereby changing the phage host range (Plasterk et al., 1983
; Hiestand-Nauer & Iida, 1983
). There is also some similarity with the Moraxella bovis invertible region which is within the Q/I (formerly
and
) pilus gene (Fulks et al., 1990
), the inversion of which mediates pilus phase and antigenic variation. There is no apparent relationship with the 9 bp repeat of the 314 bp invertible region that varies expression of type 1 fimbriae in E. coli which is mediated by the tyrosine family recombinases FimBE (Abraham et al., 1985
).
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The evolutionary origin of the B. fragilis recombination sites remains open to speculation, although it has been suggested that the Hin system in Salmonella arose as a result of integration of a Mu-like phage (van de Putte & Goosen, 1992). The Bacteroidetes diverged early, in evolutionary terms, from other eubacteria, well before the divergence of the Gram-positive bacteria from the phylum Proteobacteria which contains the majority of Gram-negatives such as the enteric bacteria, E. coli and the pseudomonads (Woese, 1987
). Given the relatively limited number of other examples of reversible variation in pathogenic bacteria arising by DNA inversion, it may be that the high number of invertible sequences in B. fragilis reflects this early evolutionary divergence. The presence of the invertase gene, which may be responsible for controlling variable expression of multiple chromosomal loci, on a plasmid also raises a number of interesting evolutionary questions. For example, did the plasmid introduce the invertase gene or did it acquire the gene as a mechanism to ensure maintenance?
In conclusion, the putative recombination sites involved in the unprecedented level of DNA inversion observed in B. fragilis show striking similarity to those of the S. typhimurium Hin and related systems and appear to involve a plasmid-borne Hin-like invertase, FinB, which we have demonstrated binds to the invertible regions. We therefore propose that the invertible regions in B. fragilis are designated fin regions and the inverted repeats fixL or fixR.
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ACKNOWLEDGEMENTS |
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Received 2 December 2002;
revised 15 January 2003;
accepted 17 January 2003.
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