1 Naval Medical Research Center, Silver Spring, MD 20910, USA
2 BBSRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney Lane, Colney, Norwich NR4 7UA, UK
Correspondence
Jerry M. Wells
Jerry.Wells{at}bbsrc.ac.uk
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ABSTRACT |
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The sequence of the pTet and pCC31 plasmids have been deposited in GenBank under accession numbers AY394561 and AY394560, respectively.
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INTRODUCTION |
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Plasmids have played a major role in the ability of bacteria to exploit new environments, particularly under selective pressure, and are frequently associated with virulence attributes in pathogenic bacteria. Knowledge of plasmid genetics and the potential for conjugal transfer is therefore important for understanding the evolution and origin of transferable factors such as drug resistance genes. A survey of 688 human isolates of C. jejuni and C. coli in the USA revealed that 32 % of strains harboured plasmid DNA, estimated to range in size from 2 to 162 kb (Tenover et al., 1985). A survey of 167 poultry samples and 41 clinical isolates of Campylobacter in Taiwan revealed a high occurrence of plasmids, 91 and 44 %, respectively (Lee et al., 1994
). Of the tetracycline resistant strains surveyed, 87 % of the chicken isolates and 47 % of the clinical isolates carried the tet(O) gene conferring tetracycline resistance (TcR) on plasmids (Taylor, 1986
; Taylor et al., 1981
, 1986
; Tenover et al., 1985
). This high proportion of TcR strains may reflect the farm use of tetracycline.
The well-characterized C. jejuni strain 81-176, originally isolated from a diarrhoeal outbreak associated with the consumption of unpasteurized milk (Korlath et al., 1985), contains two large (>37 kb) plasmids, pVir and a TcR plasmid designated pTet (Bacon et al., 2000
). Strain 81-176 has been shown to cause inflammatory diarrhoea in two human feeding studies as well as disease symptoms in experimental infection models using primates and ferrets (Black et al., 1988
) (D. Tribble, unpublished, cited by Bacon et al., 2002
). The DNA sequence of the non-conjugative plasmid pVir (37 468 bp) was recently reported (Bacon et al., 2002
). This plasmid has several genes that encode orthologues of type IV secretion systems (T4SS) and show their highest level of homology to a recently described T4SS of unknown function found in Helicobacter pylori J99 (Kersulyte et al., 2003
). T4SS have been reported in numerous pathogenic bacteria and play diverse roles including DNA export, bacterial conjugation and protein secretion [for review see Cao & Saier, (2001)
]. The precise role of the T4SS carried on pVir is unknown, although mutation of several pVir genes, including some but not all, T4SS homologues, resulted in reductions of invasion into INT407 cells in vitro and, for the one mutant that was tested, a reduction in virulence in the ferret diarrhoea model (Bacon et al., 2000
, 2002
). Additionally, mutation of a subset of pVir genes affected natural transformation (Bacon et al., 2000
).
In order to gain further insight into the structure and function of Campylobacter plasmids we have completely sequenced two large TcR plasmids, the pTet plasmid (45·2 kb) from C. jejuni strain 81-176 and a plasmid pCC31 (44·7 kb) from C. coli strain CC31 that was isolated from a human case of severe gastroenteritis in the UK. Strikingly, these two plasmid sequences revealed a remarkable level of sequence identity despite the fact that the strains were isolated almost 20 years apart on different continents. Sequence analysis of the two plasmids revealed genes encoding a putative T4SS that has been shown to be involved in conjugation, and is distinct from the T4SS system found on C. jejuni virulence plasmid pVir. Both TcR plasmids also encode a number of genes whose proteins best match those found in H. pylori, including one gene from the plasticity zone of H. pylori J99 (Alm et al., 1999).
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METHODS |
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Sequencing of the pCC31 plasmid.
A basic shotgun approach was taken to sequence the plasmid isolated from C. coli strain CC31. Plasmid DNAs digested with different restriction enzymes (Sau3A, HpaII and TaqI) were ligated into pUC19 multiple cloning sites and transformed into E. coli. Colonies were selected on LB agar containing 100 µg ampicillin ml1 and 0·5 mg X-Gal plate1 to identify white colonies containing vectors with recombinant DNA inserts. These clones were then sequenced using standard universal forward and reverse primers. Following this initial phase, the physical gaps and sequence gaps were closed by primer-walking using a series of 20mer primers designed on the sequence of the contigs obtained by shotgun sequencing. In total, 829 sequence reads were used to sequence the plasmid pCC31. Sequences were assembled using Seqman 5.05 software.
Sequencing of the pTet plasmid.
Total plasmid DNA from 81-176 (comprising pTet and pVir) was digested with BglII, and pTet-specific fragments were purified from agarose gels (Bacon et al., 2000) and cloned into pBluescript (Stratagene). Clones were sequenced by a combination of primer-walking, using synthetic oligonucleotide primers, and by an in vitro transposition strategy using a previously described EZ : : TN system (Epicentre) containing a Campylobacter chloramphenicol resistance gene (Yao et al., 1993
), as previously described by Guerry et al. (2000)
. Sequence gaps were closed by using primers based on the sequences at the end of the contigs to PCR amplify linking DNA fragments. Sequences were assembled using Sequencher 4.1 software.
Synthetic oligonucleotides.
Synthetic oligonucleotides for DNA sequencing and PCR were either synthesized on an Applied Biosystems model 393 DNA synthesizer or purchased from Sigma Genosys.
Annotation.
The finished plasmid sequences were oriented starting at the first base of the tet(O) gene, and annotated manually. ORFs of greater than 50 residues were evaluated based on the presence of a suitable initiation codon with appropriate spacing to a ribosome-binding site as well as physical location to other ORFs.
Full-length nucleotide and polypeptide sequences of all plasmid-encoded ORFs greater than 50 amino acids in length were searched for matches against all available public sequence databases using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/blast/). Levels of identity and homology were calculated across the full length of the plasmid proteins by alignment of sequences in DNAMAN (Lynnon Corporation). Additionally selected polypeptide homologues were aligned and compared using CLUSTALX. Prosite (http://ca.expasy.org/prosite/) was used to identify conserved functional motifs in protein sequences. Putative promoter regions were identified using the Neural Network Promoter Prediction program (http://www.fruitfly.org/seq_tools/promoter.html). Sequences were identified based on a cut-off score of 0·8 and their location with respect to the ribosome-binding site.
Mutagenesis of pTet.
A non-polar insertion of the CAT transposon into the cmgB3/4 gene, used for DNA sequence analysis, was selected; this particular transposon insertion mapped to 1700 bp within the coding region of cmgB3/4. The clone was used to electroporate 81-176 to chloramphenicol resistance (CmR) as previously described by Bacon et al. (2000). Putative mutants were analysed by PCR using primers flanking the insertion point to confirm that a double crossover event had occurred.
Conjugations.
Conjugations between Campylobacter strains were performed by a modification of the methods previously described by Kuipers et al. (1998) and Taylor et al. (1981)
. Campylobacter strains listed in Table 1
that were used as donor stains in mating experiments were made recA-negative by crossing them with a C. jejuni strain that had a kanamycin-resistant cassette (aph3) inserted into the recA gene (Guerry et al., 1994
). Briefly, strains were grown overnight on selective MH plates, harvested in 1 ml MH broth at a density of approximately 109 c.f.u. ml1 and combined in 100 µl aliquots on MH plates without antibiotics. DNaseI (Roche) was added to the suspension at a final concentration of 10 U µl1 to prevent transfer of plasmid by natural transformation and/or transfer of counter-selectable markers from the intended recipient into the intended donor. After incubation for 1216 h at 37 °C under microaerobic conditions, bacteria were removed with a sterile swab, dispersed in 1 ml of MH broth and plated at different dilutions on MH containing the appropriate antibiotics. Transconjugants obtained were tested for the flagellin polymorphisms (Alm et al., 1993
) that distinguished donor and recipient strains to confirm conjugal transfer (data not shown).
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RESULTS |
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A cluster of five 15 bp direct repeats (ATTACATTTAAGTCA) was found in the intergenic region between cpp23 and cpp24 (bp 2088921160), as indicated by the open box in Fig. 1. Such repetitive regions are characteristic of replication origins (Konieczny, 2003
) suggesting that these sites may function as such in these TcR plasmids.
Differences between the two plasmids
The major difference between pTet and pCC31 occurs within a region of bp 1433318803 of pTet and bp 1402218699 of pCC31. Plasmid pCC31 contains a gene (cpp15) at bp 1415914887 encoding a protein with 45 % identity and 64 % similarity to a hypothetical protein from H. pylori 26695 (Tomb et al., 1997) (see below). Plasmid pTet lacks cpp15 but has an additional ORF (cpp21) at bp 1815018803 that encodes a protein with 33 % identity and 50 % similarity to JHP1408, a hypothetical protein from H. pylori J99 (Alm et al., 1999
). Additional H. pylori alleles are found on both plasmids (see below). Additionally, there is a small ORF designated gene cpp48 on pCC31 (but not pTet) that encodes a predicted protein of 6 kDa that shows no significant homology to known proteins.
The majority of matching genes contain small numbers of base substitutions generally giving rise to polypeptides which are predicted to be identical in length. In addition there are 19 cases where the alleles have different predicted lengths, e.g. in pCC31, cpp46 starts with ATGATG whereas in pTet this gene starts with only one ATG; in ssb1, cmgB7 and cmgB8 an additional 3 bp is present at the end of the gene in pTet. Similarly, genes cmgB9, cmgB10 and cpp44 have 3 or 6 bp additions at different locations in each allele. Some genes have modified 3' ends where the reading frames and stop codon of one allele appear to have been shifted by addition or deletion of bases (e.g. cpp2, 11 bp; cpp16, 7 bp; cmgB3/4, 1 bp). In addition, the ORF of some genes is shifted near to the start, including cpp32 which has several other point mutations over the entire length, and cpp33 which varies considerably between pCC31 and pTet. As the function of these genes is unknown, we cannot predict which of the frame-shifted variants is a pseudogene. The vapD homologue in pCC31 has an additional 30 bp near the 3' end, and in pTet cpp7 and cpp8 are both affected by a 299 bp section of additional DNA.
Genes encoding putative maintenance functions
A putative replication initiator protein, RepA was identified on the basis of its similarity to the rep protein on pTS1 from Treponema denticola. Similar to the broad-host-range plasmid pIPO2, this putative repA gene is embedded in putative ORFs of unknown function (Tauch et al., 2002).
Genes encoding putative conjugation and T4SS homologues
There are 10 genes in pTet and pCC31 that encode predicted proteins with homology to T4SS proteins, in a region spanning approximately 12·6 kb. T4SS are multicomponent complexes spanning the cell envelope that can translocate proteins and/or nucleoprotein complexes between bacteria (Cao & Saier, 2001). Corresponding systems found on some plasmids of Gram-negative bacteria are responsible for mating pair formation (Mpf), involving pilus assembly and initial contact to the recipient cell during conjugation and DNA transfer (Christie & Vogel, 2000). Most of the T4SS components encoded by pCC31 and pTet show their highest homologies to proteins involved in conjugation of plasmids in Gram-negative bacteria, primarily to the pVT745 plasmid from Actinobacillus actinomycetemcomitans, a periodontal pathogen (Galli et al., 2001
). Accordingly, these plasmid genes with homology to the T4SS have been designated cmg (Campylobacter mating genes) to indicate their putative role in the formation of a transfer apparatus (see below) and are numbered according to their functional homologues in the archetypal vir transfer system of Agrobacterium tumefaciens (Table 2
, Fig. 1
). Agrobacterial T-DNA transfer systems typically comprise between 10 and 15 genes in a single cluster, which encode the membrane pilus (VirB2), a trans-envelope pore complex (VirB6-10), a transfer coupling protein (VirD4) and cytoplasmic membrane ATPases (VirB4 and VirB11; Fig. 1
). Similarly, the cmg genes in pCC31 and pTet are organized in what is predicted to be a single transcription unit (Fig. 1
). The location of cmgD4 (a homologue of virD4) (Balzer et al., 1994
; Lessl et al., 1992
; Moncalian et al., 1999
), cpp44 (a homologue of cagT) and cpp45 trbM at the end of the cmg operon is, however, unusual but is also found in the conjugative plasmid pVT745 from Actinobacillus actinomycetemcomitans (Galli et al., 2001
). The trbM gene has only been found in the IncP-specific transfer operon and its role in conjugation, if any, is unknown (Pansegrau & Lanka, 1996
).
Both TcR plasmids encode a VirB2 or pilin homologue (cmgB2); this represents the first pilin gene identified in Campylobacter (Gaynor et al., 2001; Parkhill et al., 2000
). The highest homology of this predicted protein is to TraC from plasmid pIP02T, a broad-host plasmid found in a variety of plant rhizosphere bacterial symbionts (Tauch et al., 2002
). Like other pilins these Campylobacter plasmid-encoded VirB2 proteins contain putative signal peptides, predicted to be cleaved between amino acid position 18 and 19 to generate a small basic protein of 9 kDa. Generally, the signal peptides of VirB2 preproteins are longer (2550 amino acids long). Electron microscopic examination of our strains did not reveal evidence of pili, as previously reported for 81-176 (Gaynor et al., 2001
). Alignment of the C-terminal region of the cmgB2 pilin found in pTet and pCC31 with other pilins revealed that the four amino acid residues removed by the TraF protease during the cyclization of other pilins were completely conserved (Eisenbrandt et al., 2000
). However, no obvious homologue of TraF was found in pTet or pCC31. A homologue of the VirB2-associated gene VirB5 (cmgB5) is present in both pCC31 and pTet. VirB5 is reported to be a minor component of the agrobacterial T-pilus (Table 2
) (Schmidt-Eisenlohr et al., 1999
). CmgB5 also shows its strongest homology to the VirB5 homologue from Actinobacillus actinomycetemcomitans (31 % identity and 54 % similarity).
Both plasmids encode homologues of VirB6, B7, B8, B9 and B10 proteins from Actinobacillus actinomycetemcomitans, as shown in Table 2. CmgB6, the VirB6 homologue, is predicted to form five transmembrane helices and thus might form a channel in the cytoplasmic membrane. Both plasmids contain a putative, small, 5455 amino acid protein encoded by cmgB7. CmgB7 has no homology to other proteins by BLASTP analysis because of its small size, but like the small VirB7 protein of Agrobacterium and the MagB07 protein from the mating gene operon of pVT745, it contains a lipoprotein signal sequence and conserved lipid attachment site, suggesting that these genes might have a similar function (Galli et al., 2001
). In Agrobacterium VirB7 has been shown to form disulphide bonds with VirB9 and stabilize the other VirB proteins during T-pilus assembly (Anderson et al., 1996
; Spudich et al., 1996
). It is also possible that this protein plays a role in entry exclusion, as found for the small lipoprotein designated TrbK in the conjugative IncP (RP4) plasmid transfer system (Pansegrau & Lanka, 1996
). Like CmgB7 and the entry exclusion protein of the E. coli F plasmid, TrbK has a lipoprotein signal sequence at its N terminus, suggesting that it is exposed at the cell surface. TrbK mutants of RP4 lack a pilus suggesting that TrbK interacts with Mpf apparatus, although this is not essential for conjugative DNA transfer (Vergunst et al., 2000
). Interestingly, both plasmids encode a second allele of VirB7 encoded by cpp44. Cpp44 shows its highest homology to CagT, the VirB7 homologue encoded by the Cag pathogenicity island of H. pylori (Censini et al., 1996
).
The cmgB9 gene found in both TcR plasmids shares its highest homology with a VirB9-like protein identified in another C. jejuni plasmid (R. Schmidt-Ott, University of Göttingen, Germany, unpublished data; GenBank AY190285), and contains a putative signal peptide suggesting that it might be transported into the periplasm where it can interact with other components of the membrane spanning complex. Interestingly, the pTet- and pCC31-encoded proteins with homology to VirB8 and VirB10 both contain single transmembrane helices near the N terminus. This suggests that the proteins orientate such that a short N-terminal domain remains in the cytoplasm and a larger C-terminal domain is located in the periplasm. The VirB10 protein of Agrobacterium has the same predicted topological feature and the carboxy-terminal periplasmic domain is thus proposed to link the cytoplasmic and outer-membrane proteins of the mating pair channel (Beaupre et al., 1997).
Both plasmids encode homologues of the three ATPases associated with T4SS, namely VirB11 (CmgB11), VirD4 (CmgD4; a transfer coupling protein) and VirB4 (CmgB3/4; a probable lipoprotein). All three of these predicted proteins show high homology to genes from Actinobacillus actinomycetemcomitans. Like other homologues of these ATPases the Campylobacter proteins contain Walker A nucleotide-binding motifs and the conserved motifs BD that were previously shown to be essential for conjugation and phage absorption in E. coli (Krause et al., 2000; Schmidt-Eisenlohr et al., 1999
). The VirB11 homologues in pTet and pCC31 do not possess any obvious features associated with membrane- or periplasmic-located proteins and thus might interact with the cytoplasmic domains of the other components of the VirB protein channel complex, as previously suggested by Thorstenson et al. (1993)
.
Genes encoding putative DNA transfer enzymes (Dtr)
As on plasmid pSB102 (Schneiker et al., 2001), the putative genes for the processing of DNA for transfer (Dtr) and establishment of the plasmids in the recipient cell are scattered across both Campylobacter plasmids. Conjugative DNA transfer in the Enterobacteriaceae requires the formation of a nucleoprotein complex called the relaxosome (Cao & Saier, 2001
). Following cleavage by the nickase at the origin of DNA transfer (oriT), a strand replacement reaction generates a single-stranded DNA transfer intermediate (T-strand) that presumably moves with the attached proteins to dock with the DNA transfer apparatus. The Dtr processing enzymes that assemble to form the relaxosome determine the site specificity of cleavage and control the timing of DNA transfer so that it does not interfere with vegetative replication of the plasmid. The main feature of oriT is the presence of an inverted repeat adjacent to the specific cleavage site (called the nic site) of the nickase/replicase (Pansegrau & Lanka, 1996
). The non-coding region between cpp18 and cpp19 in pTet and pCC31, designated oriT in Fig. 1
, may function as the oriT region since it contains inverted DNA repeats surrounding a conserved nic site motif ATCCTG as found in other oriT sites (Fig. 2
) (Pansegrau & Lanka, 1996
). Moreover, this site lies close to the DNA processing enzymes as found in other conjugative plasmids (Fig. 1
). pVir is non-conjugative and no sequence homology was found to the oriT sites described here.
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Both plasmids encode a putative DNA nickase (cpp17) and a helicase (cpp26) involved in generating a single stand, both with closest similarity to homologues in plasmid pVT745 from Actinobacillus actinomycetemcomitans, and a single-stranded DNA-binding protein ssb1 that may coat the single-stranded DNA during transfer, as in the case of the VirE2 ssb in Agrobacterium tumefaciens (Christie et al., 1988). Cpp22 in both plasmids has significant homology to the SogL primase of E. coli plasmid R64 and possesses a functional variant of the EGYATA motif associated with the active site of other primases (Strack et al., 1992
). The SogL primase is transferred along with the transferred plasmid DNA and is thought to catalyse the synthesis of short oligonucleotides on the single-stranded template that are then elongated by the recipient replication machinery (Pansegrau & Lanka, 1996
).
Genes encoding homologues of H. pylori proteins
In addition to HP1334, JHP1408 and cagT, discussed above, there are three other homologues of H. pylori genes encoded by both pTet and pCC31. cpp14 encodes a large protein (predicted molecular mass 224 kDa) that shows 37 % identity and 55 % similarity to JHP0928, a protein encoded in the plasticity zone of H. pylori J99. Plasticity zones are regions of hypervariable genes in the chromosomes of H. pylori strains (Alm & Trust, 1999). Most of these plasticity zone genes appear to be H. pylori-specific, but several homologues have been found in C. jejuni 81-176 on pVir (Bacon et al., 2000
). Although not originally annotated as a methylase (Alm et al., 1999
), JHP0928, like Cpp14, shows homology to a putative methylase encoded by Sinorhizobium meliloti phage PBC5 (GenBank accession no. NC_003324). Genes cpp24 and cpp25 encode homologues of JHP0960 (54 % identity, 70 % similarity) and JHP0961 (70 % identity, 80 % similarity), respectively, both small proteins of unknown function from H. pylori J99.
Genes encoding other proteins of predicted function
There is a cluster of three genes transcribed in the opposite direction to the cmg operon (cpp27, cpp28 and cpp29). cpp29, which appears to be the first gene in this putative operon, encodes a predicted protein of 12 kDa that shows no homology to known proteins. cpp28 encodes a predicted protein of 1516 kDa that shows significant homology (35 % identity, 47 % similarity) to VapD2 of Rhodococcus equi, an important pulmonary pathogen of foals (Takai et al., 2000). The precise role of vapD2 and the other vap genes in virulence is not known, and the sequence does not reveal any other clues to their function. cpp27 encodes a predicted protein of 24 kDa that shows 33 % identity and 56 % similarity to an invertase from Shewanella oneidensis. Invertases and resolvases have been identified on a variety of bacterial plasmids of both Gram-negative and Gram-positive origin and have been shown to play roles in plasmid (Janniere et al., 1993
) and genomic replication (Alonso et al., 1995
; Bruand et al., 1995
).
Conjugal transfer
Although conjugative transfer of Tet O plasmids has been reported previously (Taylor et al., 1981), preliminary experiments (Bacon et al., 2000
) to determine if 81-176 could conjugally transfer either pVir or pTet were inconclusive, in large part because of problems in distinguishing conjugation from natural transformation (Bacon et al., 2000
). The ability of 81-176 to conjugally transfer pTet to several recipients was re-examined using a recA : : aph3 mutant as donor (Guerry et al., 1994
), as shown in Table 3
. A recA mutant of 81-176 was able to transfer the pTet plasmid to a recipient strain of C. jejuni (VC83) that lacked plasmids and contained a StrR chromosomal marker (Guerry et al., 1994
), at a frequency of 105106 per donor cell. A derivative of 81-176 (DB179) lacking pTet (Bacon et al., 2000
) and marked by insertion of a CmR marker into Cjp8 of pVir (Bacon et al., 2002
) was found to receive pTet from 81-176 at a frequency of 104 per recipient cell. These observed differences in frequency of conjugation are suggestive of a restriction barrier in a heterologous VC83 recipient. Interestingly, the same transfer frequency (105106) was observed for transfer of pCC31 from C. coli CC31 into C. jejuni VC83 StrR. When VC83 containing pTet was used as a donor to transfer pTet into DB179 containing the tagged version of pVir, the transfer frequency was again 104, suggesting that 81-176 did not restrict incoming DNA from the VC83 donor.
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Comparison to pVir
Although the two T4SS systems encoded by pVir and pTet in 81-176 share some homology to one another, they appear to serve distinct functions, as mentioned above. Three ORFs on pTet or pCC31 share homology to genes on pVir. One is cpp6, which encodes a predicted protein of 7 kDa with 96 % homology to Cjp19 of pVir, a protein of unknown function. Two ORFs on pTet and pCC31 designated cpp7 and cpp51 share homology to cjp20 on pVir. Analysis of the sequence homology reveals that cpp7 and cpp51 are in fact homologous to the N-terminal and C-terminal sequences of cjp20 from pVir, respectively. This suggests that the Cjp20 homologue in pTet and pCC31 was disrupted through a recombination event.
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DISCUSSION |
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Apart from the 30 ORFs of unknown function, all of the genes present in pCC31 and pTet are predicted to be involved in plasmid replication and conjugative transfer. The mating pair formation (Mpf) genes involved in conjugation share amino acid similarities to the T4SS of different Brucella species, but have the highest overall homology to the Mpf gene cluster in pVT745 from Actinobacillus actinomycetemcomitans, a periodontal pathogen. The organization of the Mpf gene cluster resembles those of other conjugative plasmids and T4SSs but is most similar to that found in pVT745. In particular, the location of cagT and TrbM homologues at the end of the T4SS gene cluster is unusual and has only been described previously in pVT745. This strongly suggested that the Mpf gene cluster in these plasmids may have originated from a common ancestor. The similarities in gene organization between pCC31, pTet and pVT745 are not apparent over the rest of the plasmid sequence, although the probable nickase (Cpp17) and a putative lipoprotein of unknown function (Cpp23) also show highest homology to genes found on Actinobacillus plasmids. The replication proteins of pCC31 and pTet showed highest similarity to Rep proteins found in plasmids of the oral spirochaete Treponema denticola (Chauhan & Kuramitsu, 2004) and Selenomonas ruminantium. The latter is a prominent and functionally diverse species found in the rumen of sheep, cows and goats. Interestingly, repA, the three upstream ORFs of unknown function and the tet(O) gene in pCC31 and pTet have a G+C content that is substantially higher than that of the rest of the plasmid sequence, suggesting that they have a different origin to the rest of the plasmid DNA. Interestingly, DNA sequences of Selenomonas ruminantium submitted to public databases have a similar G+C content, but unfortunately, little is known about the genetics of this organism or the function of the various plasmids (1·442·6 kb) that have been isolated from some strains (Fliegerova et al., 1998
). Altogether there are five genes in pCC31 and pTet that have close homologues in the chromosome of H. pylori, one of which is found in the plasticity region. Plasmids found in Helicobacter species have not yet been genetically characterized or sequenced, so it is not known whether any of the ORFs present in pCC31 and pTet have homologues on plasmids found in Helicobacter. This would be interesting, given that these two organisms are closely related, and together with the ruminant bacterium Wolinella succinogenes belong to the epsilon subclass of the proteobacteria. The genes encoding the putative enzymes involved in DNA processing and transfer such as the nickase, helicase, primase, invertase and single-stand-binding protein are all scattered across both plasmids and do not obviously have a common origin. Thus, plasmids pCC31 and pTet are true composites, with a mosaic structure comprising blocks of genes that seem to have been acquired from bacteria that inhabit the oral and intestinal tract of animals. Campylobacter has been identified as a commensal in the gastrointestinal tracts of several species of domestic animals, as well as wildlife species, and is especially abundant in avian species such as chickens, where it can reach up to 1010 c.f.u. per g caecal contents. The mosaic structure of these plasmids could reflect the recognized potential for gene transfer and recombination in the complex ecosystem of the animal host but the natural competence of Campylobacter for transformation with exogenous DNA may also be a factor contributing to their evolution and mosaic composition.
We have demonstrated that pCC31 and pTet are self-mobilizable and capable of transfer between C. jejuni and C. coli strains at frequencies of between 104 and 106, depending on the existence of restriction barriers. The full host-range of these plasmids is not known and difficult to predict as the repA gene exhibits closest homology with genes in plasmids from organisms for which genetic tools have not yet been developed. Preliminary studies with pCC31 and pTet indicated that transfer to E. coli was not possible, as reported previously for TetR plasmids in Campylobacter (Tenover et al., 1985).
Since this paper was first submitted a study was published showing that 16 out of 56 clinical isolates of C. jejuni from the area of Göttingen in Germany harbour plasmids varying in size from 6 to 66 kb (Schmidt-Ott et al., 2004). Only one of these plasmids was a homologue of pVir, the virulence plasmid previously characterized by Bacon et al. (2002)
. The relatedness of eight plasmids within a subgroup distinct from pVir was established by Southern-blot hybridization using a collection of nine PCR-amplified DNA probes from plasmid pCjA13.
Probe D used in the above study encodes the tet(O) gene, which has 94·7 % identity to that found in pCC31. The primer sequences used to amplify probes B, D, F, H and I in pCjA13 (Schmidt-Ott et al. 2004) were also present in pCC31 (allowing for 12 bp mismatches) and were predicted to amplify DNA fragments of similar length, suggesting that these regions are conserved. The sequence of six ORFs present on a 6·8 kb BglII DNA fragment of pCjA13 were submitted to the database and four of these shared closest homology with the virB8, virB9, virB10 and virB11 genes in pVT745 from Actinobacillus actinomycetemcomitans and a high degree of sequence identity to the homologues in pCC31 and pTet (e.g. 89 % amino acid identity between virB9 of pCjA13 and pCC31). However, the last two ORFs of pCjA13 that encoded genes of unknown function were not present in pCC31 or pTet.
In conclusion, it seems that a subgroup of conjugative plasmids with extensive homology to pCC31 and pTet are relatively prevalent in clinical isolates of C. jejuni (i.e. eight out of 16 strains harbouring plasmids). It is most likely that the use of tetracycline in poultry has been a contributing factor to the spread of these mobilizable plasmids, but it is also possible that there has been further selection associated with properties conferred by the many uncharacterized ORFs in pCC31 and pTet. The complete sequences of two conjugative plasmids from Campylobacter has provided us with new insights into the evolution of Campylobacter plasmids and the plasticity of the plasmid gene pool. We now intend to investigate the function of the numerous uncharacterized genes encoded in pTet and pCC31 and determine the potential for gene transfer between different bacterial species in the animal ecosystem inhabited by Campylobacter.
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ACKNOWLEDGEMENTS |
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Received 23 February 2004;
revised 9 June 2004;
accepted 30 June 2004.
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