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Nucleotide sequences and comparison of twolarge conjugative plasmids from differentCampylobacter species Roger A. Batchelor,1 Bruce M. Pearson,2 Lorna M. Friis,2 Patricia Guerry1and Jerry M. Wells2 Naval Medical Research Center, Silver Spring, MD 20910, USA 2BBSRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney Two large tetracycline resistance (TcR) plasmids have been completely sequenced, thepTet plasmid (45?2 kb) from Campylobacter jejuni strain 81-176 and a plasmid pCC31(44?7 kb) from Campylobacter coli strain CC31 that was isolated from a human case of severegastroenteritis in the UK. Both plasmids are mosaic in structure, having homologues of genesfound in a variety of different commensal and pathogenic bacteria, but nevertheless, showedstriking similarities in DNA sequence and overall gene organization. Several predicted proteinsencoded by genes involved in conjugation showed highest homology to proteins found inActinobacillus actinomycetemcomitans, a periodontal pathogen. In addition to replication- andconjugation-associated genes, both plasmids carried a tet(O) gene encoding tetracyclineresistance, a 6 kb ORF encoding a putative methylase and a number of genes of unknown function.
The pTet plasmid co-exists in C. jejuni strain 81-176 with a smaller, previously characterized,non-conjugative plasmid pVir that also encodes a type IV secretion system (T4SS) that may affect virulence. In contrast, the T4SS encoded by pTet and pCC31 are shown to mediate bacterial conjugation between Campylobacter. The possible origin and evolution of pCC31 and pTet to exploit new environments, particularly under selectivepressure, and are frequently associated with virulence Campylobacter spp. account for the majority of bacteria- attributes in pathogenic bacteria. Knowledge of plasmid related foodborne illness, with poultry and poultry products genetics and the potential for conjugal transfer is therefore being reported as the major sources of infection in deve- important for understanding the evolution and origin of loped countries (Oberhelman & Taylor, 2000). Approxi- transferable factors such as drug resistance genes. A survey mately 80 % of Campylobacter infections are caused by two of 688 human isolates of C. jejuni and C. coli in the USA species of the genus, namely, Campylobacter jejuni and revealed that 32 % of strains harboured plasmid DNA, Campylobacter coli, with the former being more frequently estimated to range in size from 2 to 162 kb (Tenover et al., associated with disease in humans. Symptoms from infec- 1985). A survey of 167 poultry samples and 41 clinical iso- tion with C. jejuni can vary from very mild diarrhoea to lates of Campylobacter in Taiwan revealed a high occurrence profuse bloody diarrhoea with mucosal damage and inflam- of plasmids, 91 and 44 %, respectively (Lee et al., 1994). Of mation, especially in the ileum and jejunum (Wassenaar & the tetracycline resistant strains surveyed, 87 % of the Blaser, 1999). In a rare number of cases infection with chicken isolates and 47 % of the clinical isolates carried the Campylobacter is associated with the peripheral neuropa- tet(O) gene conferring tetracycline resistance (TcR) on thies known as Guillain–Barre´ and Miller Fisher syndromes plasmids (Taylor, 1986; Taylor et al., 1981, 1986; Tenover et al., 1985). This high proportion of TcR strains may reflectthe farm use of tetracycline.
Plasmids have played a major role in the ability of bacteria The well-characterized C. jejuni strain 81-176, originally isolated from a diarrhoeal outbreak associated with the TcR, tetracycline resistance; T4SS, type IV secretion consumption of unpasteurized milk (Korlath et al., 1985),contains two large ( The sequence of the pTet and pCC31 plasmids have been deposited in GenBank under accession numbers AY394561 and AY394560, 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 conditions in the presence of the following antibiotics, when appro- experimental infection models using primates and ferrets priate: 20 mg tetracycline ml21, 20 mg streptomycin ml21, 20 mg (Black et al., 1988) (D. Tribble, unpublished, cited by Bacon kanamycin ml21, and/or 20 mg chloramphenicol ml21. PlasmidspUC19 and pBluescript were used as the cloning vectors and et al., 2002). The DNA sequence of the non-conjugative Escherichia coli DH5a was the host for cloning experiments. E. coli plasmid pVir (37 468 bp) was recently reported (Bacon strains were grown at 37 uC on Luria–Bertani (LB) broth supple- et al., 2002). This plasmid has several genes that encode mented with 50 mg ampicillin ml21 or 20 mg chloramphenicol ml21 as orthologues of type IV secretion systems (T4SS) and show their highest level of homology to a recently described DNA recombinant techniques. Plasmid DNAs were isolated using T4SS of unknown function found in Helicobacter pylori J99 mini-Qiagen columns as previously described by Bacon et al., (Kersulyte et al., 2003). T4SS have been reported in numer- (2000). Restriction enzymes were purchased from New England ous pathogenic bacteria and play diverse roles including Biolabs and used as recommended by the supplier. Plasmid DNA DNA export, bacterial conjugation and protein secretion samples for sequence analyses were isolated using QIAprep spin [for review see Cao & Saier, (2001)]. The precise role of the miniprep columns (Qiagen). DNA sequencing was performed using T4SS carried on pVir is unknown, although mutation of Big Dye sequencing kits (Perkin Elmer-Applied Biosystems) onApplied Biosystems 373A and 3100 DNA sequencers.
several pVir genes, including some but not all, T4SShomologues, resulted in reductions of invasion into INT407 Sequencing of the pCC31 plasmid. A basic shotgun approach cells in vitro and, for the one mutant that was tested, a was taken to sequence the plasmid isolated from C. coli strain CC31.
reduction in virulence in the ferret diarrhoea model (Bacon Plasmid DNAs digested with different restriction enzymes (Sau3A, et al., 2000, 2002). Additionally, mutation of a subset of pVir HpaII and TaqI) were ligated into pUC19 multiple cloning sitesand transformed into E. coli. Colonies were selected on LB agar con- genes affected natural transformation (Bacon et al., 2000).
taining 100 mg ampicillin ml21 and 0?5 mg X-Gal plate21 to identify In order to gain further insight into the structure and white colonies containing vectors with recombinant DNA inserts.
These clones were then sequenced using standard universal forward function of Campylobacter plasmids we have completely and reverse primers. Following this initial phase, the physical gaps sequenced two large TcR plasmids, the pTet plasmid and sequence gaps were closed by ‘primer-walking’ using a series of (45?2 kb) from C. jejuni strain 81-176 and a plasmid 20mer primers designed on the sequence of the contigs obtained by pCC31 (44?7 kb) from C. coli strain CC31 that was isolated shotgun sequencing. In total, 829 sequence reads were used to from a human case of severe gastroenteritis in the UK.
sequence the plasmid pCC31. Sequences were assembled using Strikingly, these two plasmid sequences revealed a remark- able level of sequence identity despite the fact that the Sequencing of the pTet plasmid. Total plasmid DNA from strains were isolated almost 20 years apart on different 81-176 (comprising pTet and pVir) was digested with BglII, and continents. Sequence analysis of the two plasmids revealed pTet-specific fragments were purified from agarose gels (Bacon genes encoding a putative T4SS that has been shown to et al., 2000) and cloned into pBluescript (Stratagene). Clones were be involved in conjugation, and is distinct from the T4SS sequenced by a combination of primer-walking, using syntheticoligonucleotide primers, and by an in vitro transposition strategy system found on C. jejuni virulence plasmid pVir. Both TcR using a previously described EZ : : TN system (Epicentre) containing plasmids also encode a number of genes whose proteins a Campylobacter chloramphenicol resistance gene (Yao et al., 1993), best match those found in H. pylori, including one gene as previously described by Guerry et al. (2000). Sequence gaps were from the plasticity zone of H. pylori J99 (Alm et al., 1999).
closed by using primers based on the sequences at the end of thecontigs to PCR amplify linking DNA fragments. Sequences wereassembled using Sequencher 4.1 software.
Synthetic oligonucleotides. Synthetic oligonucleotides for DNA Bacterial strains and plasmids. C. jejuni and C. coli strains used sequencing and PCR were either synthesized on an Applied this study are shown in Table 1. Campylobacter were grown on Biosystems model 393 DNA synthesizer or purchased from Sigma Mueller–Hinton (MH) medium at 42 or 37 uC under microaerobic NalR, Nalidixic acid resistant; StrR, streptomycin resistant; TcR, tetracycline resistant; cat, chloramphen-icol acetyltransferase; aph3, kanamycin resistance.
Annotation. The finished plasmid sequences were oriented starting and 29?1 % (pTet), which are only slightly lower than that of at the first base of the tet(O) gene, and annotated manually. ORFs the sequenced C. jejuni genome (30?6 %) (Parkhill et al., of greater than 50 residues were evaluated based on the presence of 2000). However, the G+C plot revealed a high G+C region a suitable initiation codon with appropriate spacing to a ribosome- incorporating the tet(O) gene (40?4 % G+C) suggesting binding site as well as physical location to other ORFs.
that this gene was horizontally transferred from another Full-length nucleotide and polypeptide sequences of all plasmid- encoded ORFs greater than 50 amino acids in length were searched formatches against all available public sequence databases using the BLAST Conjugative plasmids encoding tet(O) from Campylobacter algorithm ( Levels of identity and have been described in detail by Taylor et al. (1981) although homology were calculated across the full length of the plasmid proteins only the sequence of the Tet O determinant and limited by alignment of sequences in DNAMAN (Lynnon Corporation). Addi- adjacent DNA from pUA466 has been reported (Taylor, tionally selected polypeptide homologues were aligned and comparedusing 1986). The 45 kb pUA466 plasmid was subsequently found CLUSTALX. Prosite ( was used to identify conserved functional motifs in protein sequences. Putative to have a different restriction pattern to the plasmids promoter regions were identified using the Neural Network Promoter described in this manuscript (data not shown) (Taylor et al., (
1986). The tet(O) gene shares significant homology with the html). Sequences were identified based on a cut-off score of 0?8 and tet(M) gene of Streptococcus and is thought to have a shared their location with respect to the ribosome-binding site.
ancestry. The upstream promoter regions of tet(O) on both Mutagenesis of pTet. A non-polar insertion of the CAT trans- pTet and pCC31 are highly conserved with the published poson into the cmgB3/4 gene, used for DNA sequence analysis, was sequences (Taylor, 1986). The two plasmid tet(O) genes selected; this particular transposon insertion mapped to 1700 bp are 94?8 % homologous at the DNA level with almost all within the coding region of cmgB3/4. The clone was used to electro- differences occurring within a central 350 bp region.
porate 81-176 to chloramphenicol resistance (CmR) as previouslydescribed by Bacon et al. (2000). Putative mutants were analysed by A cluster of five 15 bp direct repeats (ATTACATTTA- PCR using primers flanking the insertion point to confirm that a AGTCA) was found in the intergenic region between cpp23 double crossover event had occurred.
and cpp24 (bp 20889–21160), as indicated by the open Conjugations. Conjugations between Campylobacter strains were box in Fig. 1. Such repetitive regions are characteristic of performed by a modification of the methods previously described by replication origins (Konieczny, 2003) suggesting that these Kuipers et al. (1998) and Taylor et al. (1981). Campylobacter strains sites may function as such in these TcR plasmids.
listed in Table 1 that were used as donor stains in mating experi-ments were made recA-negative by crossing them with a C. jejunistrain that had a kanamycin-resistant cassette (aph3) inserted into the recA gene (Guerry et al., 1994). Briefly, strains were grown over-night on selective MH plates, harvested in 1 ml MH broth at a The major difference between pTet and pCC31 occurs density of approximately 109 c.f.u. ml21 and combined in 100 ml within a region of bp 14333–18803 of pTet and bp aliquots on MH plates without antibiotics. DNaseI (Roche) was 14022–18699 of pCC31. Plasmid pCC31 contains a gene added to the suspension at a final concentration of 10 U ml21 to (cpp15) at bp 14159–14887 encoding a protein with 45 % prevent transfer of plasmid by natural transformation and/or trans- identity and 64 % similarity to a hypothetical protein from fer of counter-selectable markers from the intended recipient into H. pylori 26695 (Tomb et al., 1997) (see below). Plasmid the intended donor. After incubation for 12–16 h at 37 uC undermicroaerobic conditions, bacteria were removed with a sterile swab, pTet lacks cpp15 but has an additional ORF (cpp21) at dispersed in 1 ml of MH broth and plated at different dilutions on bp 18150–18803 that encodes a protein with 33 % identity and 50 % similarity to JHP1408, a hypothetical protein obtained were tested for the flagellin polymorphisms (Alm et al., from H. pylori J99 (Alm et al., 1999). Additional H. pylori 1993) that distinguished donor and recipient strains to confirm alleles are found on both plasmids (see below). Additionally, there is a small ORF designated gene cpp48 on pCC31 (butnot 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 A physical map comparing the two plasmids maps is shown which are predicted to be identical in length. In addition in Fig. 1 and gene annotations are presented in Table 2. The there are 19 cases where the alleles have different predicted sequence of pCC31 was determined to be 44 707 bp and lengths, e.g. in pCC31, cpp46 starts with ATGATG whereas annotation revealed 50 ORFs, 44 of which are transcribed in in pTet this gene starts with only one ATG; in ssb1, cmgB7 a clockwise orientation with respect to the tet(O) gene. The and cmgB8 an additional 3 bp is present at the end of the sequence of pTet was determined to be 45 205 bp, only 1 % gene in pTet. Similarly, genes cmgB9, cmgB10 and cpp44 larger than pCC31. Annotation of the sequence revealed 49 have 3 or 6 bp additions at different locations in each allele.
ORFs, 43 being transcribed in a clockwise orientation. The Some genes have modified 39 ends where the reading frames two plasmids are 94?3 % identical at the level of nucleotide and stop codon of one allele appear to have been shifted sequence. Approximately 90?0 % of both plasmids is coding by addition or deletion of bases (e.g. cpp2, 11 bp; cpp16, sequence, with overall G+C contents of 29?8 % (pCC31) 7 bp; cmgB3/4, 1 bp). In addition, the ORF of some genes Fig. 1. Genetic map of plasmids pTet and pCC31 showing differences in gene organization. (a) Putative promoters(designated P) and transcription terminators (designated T) identified in pCC31 are indicated, as is the inverted repeat region(filled box) containing the proposed nic site and origin of transfer (oriT ). A region of multiple direct repeats is also indicated byan open box. (b) Genes are annotated according to Table 2 and identified by their predicted function as follows: DNA transferfunctions, horizontal stripes; Campylobacter mating gene (cmg) homologues of T4SS, filled; repA, chequered; restrictionmodification, vertical stripes; tet(O), diagonal stripes; genes with unknown role, open. Genes that are only found in one of thetwo plasmids are labelled in bold. (c) G+C content. Due to the similarity of G+C content between the two plasmidsequences only that of pTet is displayed.
is shifted near to the start, including cpp32 which has several spanning approximately 12?6 kb. T4SS are multicomponent other point mutations over the entire length, and cpp33 complexes spanning the cell envelope that can translocate which varies considerably between pCC31 and pTet. As the proteins and/or nucleoprotein complexes between bacteria function of these genes is unknown, we cannot predict (Cao & Saier, 2001). Corresponding systems found on which of the frame-shifted variants is a pseudogene. The some plasmids of Gram-negative bacteria are responsible vapD homologue in pCC31 has an additional 30 bp near for mating pair formation (Mpf), involving pilus assembly the 39 end, and in pTet cpp7 and cpp8 are both affected by a and initial contact to the recipient cell during conjugation and DNA transfer (Christie & Vogel, 2000). Most of theT4SS components encoded by pCC31 and pTet show their Genes encoding putative maintenance functions highest homologies to proteins involved in conjugationof plasmids in Gram-negative bacteria, primarily to the A putative replication initiator protein, RepA was identi- pVT745 plasmid from Actinobacillus actinomycetemcomitans, fied on the basis of its similarity to the rep protein on pTS1 a periodontal pathogen (Galli et al., 2001). Accordingly, from Treponema denticola. Similar to the broad-host-range these plasmid genes with homology to the T4SS have been plasmid pIPO2, this putative repA gene is embedded in designated cmg (Campylobacter mating genes) to indicate putative ORFs of unknown function (Tauch et al., 2002).
their putative role in the formation of a transfer apparatus(see below) and are numbered according to their functional Genes encoding putative conjugation and T4SS homologues in the archetypal vir transfer system of Agro- bacterium tumefaciens (Table 2, Fig. 1). Agrobacterial T- There are 10 genes in pTet and pCC31 that encode predicted DNA transfer systems typically comprise between 10 and proteins with homology to T4SS proteins, in a region 15 genes in a single cluster, which encode the membrane Table 2. Predicted coding regions on pTet and pCC31 plasmids and the closest relationships to previously studied proteins A. denitrificans, Achromobacter denitrificans; B. cepacia, Burkholderia cepacia; C. jejuni, Campylobacter jejuni; C. crescentus, Caulobacter crescen-tus; C. hutchinsonii, Cytophaga hutchinsonii; E. faecalis, Enterococcus faecalis; G. sulfurreducens, Geobacter sulfurreducens; H. influenzae,Haemophilus influenzae; R. anatipestifer, Riemerella anatipestifer; S. typhimurium, Salmonella typhimurium; S. oneidensis, Shewanella oneidensis;T. denticola, Treponema denticola; W. succinogenes, Wolinella succinogenes; Y. pestis, Yersinia pestis. For genes cpp32, ssb, cmgB8, cmgB9,cmgB10 and cmgB11, the highest homology hits were to C. jejuni genes/proteins with GenBank accession numbers AY190288, NC_005012,AY190284, AY190285, AY190286 and AY190287, respectively.
Homologous protein (heterologous species, GenBank/Entrez accession no.) (% identity/% similarity) to pTet; pCC31, respectively Tetracycline resistance (C. jejuni, P10952) (100/100 %); (C. jejuni, AAO38916) (93/95 %) ORF6, Tn916 conjugative transposon (E. faecalis, AAB60023) (68/82 %); none Hypothetical protein TDE1306 (T. denticola, AAS11833) (29/50 %); (27/52 %) Rep (T. denticola, AAG50423) (38/56 %); (38/56 %) Cjp19 (C. jejuni, AAN46914) (97/97 %); (97/97 %) Cjp20 (C. jejuni, AAN46915) (33/52 %); (39/60 %) Cjp38 (C. jejuni, AAN46931) (39/61 %); (38/59 %) ParA (Synechocystis sp., BAD02093) (23/51 %); (20/48 %) (B. cepacia, NZ_AAEH01000008) (42/62 %); (42/62 %) None; HP1334 (H. pylori, AAD08389) (45/64 %) MagA2 nickase (A. actinomycetemcomitans, AAG24403) (33/54 %); (33/54 %) Primase SogL (E. coli, AAQ17618) (32/53 %); (31/52 %) Lipoprotein (A. actinomycetemcomitans, AAG24432) (32/55 %); (31/56 %) Hypothetical protein (H. pylori, AAD06533) (53/69 %); (54/71 %) JHP0961 (H. pylori, AAD06534) (64/75 %); (66/75 %) (C. crescentus, AAK23638) (12/32 %); ATP-dependent serine protease RadA (C. jejuni, Hypothetical site-specific recombinase (W. succinogenes, CAE10935) (37/61 %); (41/64 %) Virulence associated protein 2 (R. anatipestifer, AAC27553) (34/48 %); (35/47 %) TraC (plasmid pSB102, CAC79181) (34/59 %); (33/60 %) MagB03 ATPase (A. actinomycetemcomitans, AAG24434) (41/61 %); (40/61 %) Putative anti-repressor (S. typhimurium, AAL25919) (31/53 %); (26/49 %) (G. sulfurreducens, AAR35527) (35/53 %); (33/50 %) MagB04 (A. actinomycetemcomitans, AAG24433) (31/53 %); (30/52 %) Type IV secretion pathway VirB6 component (Y. pestis AAS58617) (21/44 %); (25/44 %) Tag8 (A. actinomycetemcomitans, AAK19531) (43/65 %); (44/68 %) Tag9 (A. actinomycetemcomitans, AAK19532) (46/68 %); (46/65 %) Tag10 (A. actinomycetemcomitans, AAK19533) (38/56 %); (38/54 %) Tag11 (A. actinomycetemcomitans, AAK19534) (49/69 %); (48/68 %) MagB12 ATPase (A. actinomycetemcomitans, AAG24425) (43/60 %); (44/61 %) Cag island protein (H. pylori 26695, NC_000915) (24/42 %); (24/45 %) TraQ (pIPo2T, CAC82764) (35/50 %); YggA-like (H. influenzae, AAM64136) (39/57 %) Homologous protein (heterologous species, GenBank/Entrez accession no.) (% identity/% similarity) to pTet; pCC31, respectively TraE (A. denitrificans, AAS49467) (42/61 %); (42/61 %) Cjp20 (C. jejuni, AAN46915) (44/60 %); (44/61 %) pilus (VirB2), a trans-envelope pore complex (VirB6-10), might form a channel in the cytoplasmic membrane. Both a transfer coupling protein (VirD4) and cytoplasmic plasmids contain a putative, small, 54–55 amino acid membrane ATPases (VirB4 and VirB11; Fig. 1). Similarly, protein encoded by cmgB7. CmgB7 has no homology to the cmg genes in pCC31 and pTet are organized in what other proteins by BLASTP analysis because of its small size, is predicted to be a single transcription unit (Fig. 1). The but like the small VirB7 protein of Agrobacterium and the location of cmgD4 (a homologue of virD4) (Balzer et al., MagB07 protein from the mating gene operon of pVT745, 1994; Lessl et al., 1992; Moncalian et al., 1999), cpp44 it contains a lipoprotein signal sequence and conserved lipid (a homologue of cagT) and cpp45 trbM at the end of the attachment site, suggesting that these genes might have a cmg operon is, however, unusual but is also found in the similar function (Galli et al., 2001). In Agrobacterium VirB7 conjugative plasmid pVT745 from Actinobacillus actino- has been shown to form disulphide bonds with VirB9 and mycetemcomitans (Galli et al., 2001). The trbM gene has only stabilize the other VirB proteins during T-pilus assembly been found in the IncP-specific transfer operon and its (Anderson et al., 1996; Spudich et al., 1996). It is also role in conjugation, if any, is unknown (Pansegrau & Lanka, possible that this protein plays a role in entry exclusion, as found for the small lipoprotein designated TrbK in theconjugative IncP (RP4) plasmid transfer system (Pansegrau Both TcR plasmids encode a VirB2 or pilin homologue & Lanka, 1996). Like CmgB7 and the entry exclusion protein (cmgB2); this represents the first pilin gene identified in of the E. coli F plasmid, TrbK has a lipoprotein signal Campylobacter (Gaynor et al., 2001; Parkhill et al., 2000).
sequence at its N terminus, suggesting that it is exposed at The highest homology of this predicted protein is to TraC the cell surface. TrbK mutants of RP4 lack a pilus suggesting from plasmid pIP02T, a broad-host plasmid found in a that TrbK interacts with Mpf apparatus, although this is variety of plant rhizosphere bacterial symbionts (Tauch not essential for conjugative DNA transfer (Vergunst et al., et al., 2002). Like other pilins these Campylobacter plasmid- 2000). Interestingly, both plasmids encode a second allele encoded VirB2 proteins contain putative signal peptides, of VirB7 encoded by cpp44. Cpp44 shows its highest predicted to be cleaved between amino acid position 18 homology to CagT, the VirB7 homologue encoded by the and 19 to generate a small basic protein of 9 kDa. Gener- Cag pathogenicity island of H. pylori (Censini et al., 1996).
ally, the signal peptides of VirB2 preproteins are longer(25–50 amino acids long). Electron microscopic examina- The cmgB9 gene found in both TcR plasmids shares its tion of our strains did not reveal evidence of pili, as pre- highest homology with a VirB9-like protein identified in viously reported for 81-176 (Gaynor et al., 2001). Alignment another C. jejuni plasmid (R. Schmidt-Ott, University of the C-terminal region of the cmgB2 pilin found in pTet of Go¨ttingen, Germany, unpublished data; GenBank and pCC31 with other pilins revealed that the four amino AY190285), and contains a putative signal peptide suggest- acid residues removed by the TraF protease during the ing that it might be transported into the periplasm where cyclization of other pilins were completely conserved it can interact with other components of the membrane (Eisenbrandt et al., 2000). However, no obvious homologue spanning complex. Interestingly, the pTet- and pCC31- of TraF was found in pTet or pCC31. A homologue of the encoded proteins with homology to VirB8 and VirB10 both VirB2-associated gene VirB5 (cmgB5) is present in both contain single transmembrane helices near the N terminus.
pCC31 and pTet. VirB5 is reported to be a minor com- This suggests that the proteins orientate such that a short ponent of the agrobacterial T-pilus (Table 2) (Schmidt- N-terminal domain remains in the cytoplasm and a larger Eisenlohr et al., 1999). CmgB5 also shows its strongest C-terminal domain is located in the periplasm. The VirB10 homology to the VirB5 homologue from Actinobacillus protein of Agrobacterium has the same predicted topological actinomycetemcomitans (31 % identity and 54 % similarity).
feature and the carboxy-terminal periplasmic domain isthus proposed to link the cytoplasmic and outer-membrane Both plasmids encode homologues of VirB6, B7, B8, B9 and proteins of the mating pair channel (Beaupre et al., 1997).
B10 proteins from Actinobacillus actinomycetemcomitans,as shown in Table 2. CmgB6, the VirB6 homologue, is Both plasmids encode homologues of the three ATPases predicted to form five transmembrane helices and thus associated with T4SS, namely VirB11 (CmgB11), VirD4 (CmgD4; a transfer coupling protein) and VirB4 (CmgB3/4;a probable lipoprotein). All three of these predicted proteinsshow high homology to genes from Actinobacillus actino-mycetemcomitans. Like other homologues of these ATPasesthe Campylobacter proteins contain Walker A nucleotide-binding motifs and the conserved motifs B–D that werepreviously shown to be essential for conjugation and phageabsorption in E. coli (Krause et al., 2000; Schmidt-Eisenlohret al., 1999). The VirB11 homologues in pTet and pCC31 donot possess any obvious features associated with membrane-or periplasmic-located proteins and thus might interact Fig. 2. The proposed oriT sequence of pCC31 and pTet are with the cytoplasmic domains of the other components of aligned to the conserved nic regions of IncP and Ti plasmids the VirB protein channel complex, as previously suggested (Zechner et al., 2000) as well as the putative oriT of plasmids pIPO2, pSB102 and pXF51. Nucleotide sequences that arecompletely conserved are underlined. Bold type indicatesnucleotide positions that are at least 70 % conserved among Genes encoding putative DNA transfer enzymes the aligned sequences. The arrow shows the position of the nic site determined for the IncP transfer system.
As on plasmid pSB102 (Schneiker et al., 2001), the putativegenes for the processing of DNA for transfer (Dtr) and Both plasmids encode a putative DNA nickase (cpp17) establishment of the plasmids in the recipient cell are and a helicase (cpp26) involved in generating a single stand, scattered across both Campylobacter plasmids. Conjugative both with closest similarity to homologues in plasmid DNA transfer in the Enterobacteriaceae requires the forma- pVT745 from Actinobacillus actinomycetemcomitans, and a tion of a nucleoprotein complex called the relaxosome single-stranded DNA-binding protein ssb1 that may coat (Cao & Saier, 2001). Following cleavage by the nickase at the single-stranded DNA during transfer, as in the case of the origin of DNA transfer (oriT), a strand replacement the VirE2 ssb in Agrobacterium tumefaciens (Christie et al., reaction generates a single-stranded DNA transfer inter- 1988). Cpp22 in both plasmids has significant homology mediate (T-strand) that presumably moves with the to the SogL primase of E. coli plasmid R64 and possesses attached proteins to dock with the DNA transfer apparatus.
a functional variant of the EGYATA motif associated with The Dtr processing enzymes that assemble to form the the active site of other primases (Strack et al., 1992). The relaxosome determine the site specificity of cleavage and SogL primase is transferred along with the transferred control the timing of DNA transfer so that it does not plasmid DNA and is thought to catalyse the synthesis of interfere with vegetative replication of the plasmid. The short oligonucleotides on the single-stranded template that main feature of oriT is the presence of an inverted repeat are then elongated by the recipient replication machinery adjacent to the specific cleavage site (called the ‘nic’ site) of the nickase/replicase (Pansegrau & Lanka, 1996). Thenon-coding region between cpp18 and cpp19 in pTet andpCC31, designated ‘oriT’ in Fig. 1, may function as the oriT region since it contains inverted DNA repeats sur- rounding a conserved ‘nic’ site motif ATCCTG as foundin other oriT sites (Fig. 2) (Pansegrau & Lanka, 1996).
In addition to HP1334, JHP1408 and cagT, discussed Moreover, this site lies close to the DNA processing enzymes above, there are three other homologues of H. pylori as found in other conjugative plasmids (Fig. 1). pVir is genes encoded by both pTet and pCC31. cpp14 encodes a non-conjugative and no sequence homology was found to large protein (predicted molecular mass 224 kDa) that shows 37 % identity and 55 % similarity to JHP0928, aprotein encoded in the plasticity zone of H. pylori J99.
The pCC31 and pTet CmgD4 proteins share homology Plasticity zones are regions of hypervariable genes in the with the transfer coupling proteins VirD4 (Ti plasmid) chromosomes of H. pylori strains (Alm & Trust, 1999). Most and TraG (F plasmid) that are required for recruiting the of these plasticity zone genes appear to be H. pylori-specific, relaxosome nucleoprotein complex and coupling it to the but several homologues have been found in C. jejuni 81- Mpf DNA transfer apparatus in the cell envelope (Zechner 176 on pVir (Bacon et al., 2000). Although not originally et al., 2000). T4SS coupling proteins are required for DNA annotated as a methylase (Alm et al., 1999), JHP0928, like or protein transfer in Agrobacterium tumefaciens, H. pylori Cpp14, shows homology to a putative methylase encoded and bacterial conjugation systems (Cabezon et al., 1994; by Sinorhizobium meliloti phage PBC5 (GenBank accession Covacci et al., 1999; Moncalian et al., 1999; Vergunst et al., no. NC_003324). Genes cpp24 and cpp25 encode homo- 2000). However, some T4SS are devoted to export of logues of JHP0960 (54 % identity, 70 % similarity) and proteins such as the Bordetella pertussis toxin and so lack JHP0961 (70 % identity, 80 % similarity), respectively, both small proteins of unknown function from H. pylori J99.
Table 3. Mating frequency of Campylobacter strains harbouring pTet or pCC31 Chl, Chloramphenicol; Str, streptomycin; Tet, tetracycline.
81-176 recA : : aph3 (pVir, pTet/cmgD4 : : cat) Genes encoding other proteins of predicted of pCC31 from C. coli CC31 into C. jejuni VC83 StrR. When VC83 containing pTet was used as a donor to transfer pTetinto DB179 containing the tagged version of pVir, the There is a cluster of three genes transcribed in the opposite transfer frequency was again 1024, suggesting that 81-176 direction to the cmg operon (cpp27, cpp28 and cpp29). cpp29, did not restrict incoming DNA from the VC83 donor.
which appears to be the first gene in this putative operon,encodes a predicted protein of 12 kDa that shows no The kinetics of conjugal transfer between C. coli CC31 and homology to known proteins. cpp28 encodes a predicted C. jejuni VC83 were monitored at various times from 0?5 protein of 15–16 kDa that shows significant homology to 24 h. Peak mating frequency between these strains was (35 % identity, 47 % similarity) to VapD2 of Rhodococcus observed to take place between 8 and 16 h of incubation equi, an important pulmonary pathogen of foals (Takai et al., prior to plating on selective media (Fig. 3), although 2000). The precise role of vapD2 and the other vap genes significant transfer was detected as early as 30 min.
in virulence is not known, and the sequence does not revealany other clues to their function. cpp27 encodes a predicted A site-specific mutation of the cmgB3/4 gene of pTet in 81- protein of 24 kDa that shows 33 % identity and 56 % 176 recA : : aph3 was constructed as described in Methods.
similarity to an invertase from Shewanella oneidensis.
When this strain was used as donor in a cross with VC83 Invertases and resolvases have been identified on a variety StrR, no transconjugants were detected, indicating that the of bacterial plasmids of both Gram-negative and Gram- cmgB3/4 gene is required for conjugation proficiency. Since positive origin and have been shown to play roles in the donor strain carried pVir, it appears that pVir cannot plasmid (Janniere et al., 1993) and genomic replication complement the cmgB3/4 defect, possibly due to the low (Alonso et al., 1995; Bruand et al., 1995).
overall homology between cmgB3/4 and the virB4-like genepresent in the T4SS of pVir. This indicates that the T4SS Although conjugative transfer of Tet O plasmids has beenreported previously (Taylor et al., 1981), preliminaryexperiments (Bacon et al., 2000) to determine if 81-176could conjugally transfer either pVir or pTet were incon-clusive, in large part because of problems in distinguishingconjugation from natural transformation (Bacon et al.,2000). The ability of 81-176 to conjugally transfer pTet toseveral recipients was re-examined using a recA : : aph3mutant as donor (Guerry et al., 1994), as shown in Table 3.
A recA mutant of 81-176 was able to transfer the pTetplasmid to a recipient strain of C. jejuni (VC83) that lackedplasmids and contained a StrR chromosomal marker(Guerry et al., 1994), at a frequency of 1025–1026 perdonor cell. A derivative of 81-176 (DB179) lacking pTet(Bacon et al., 2000) and marked by insertion of a CmRmarker into Cjp8 of pVir (Bacon et al., 2002) was foundto receive pTet from 81-176 at a frequency of 1024 per recipient cell. These observed differences in frequency of (pCC31) and C. jejuni VC83. The number of transconjugants conjugation are suggestive of a restriction barrier in a represent the number of colonies present on counter-selective heterologous VC83 recipient. Interestingly, the same media (MH agar with tetracycline and streptomycin) per ml of transfer frequency (1025–1026) was observed for transfer inoculum divided by the total number of recipient cells per ml.
carried by pTet and pCC31 is required for conjugation and G+C content that is substantially higher than that of the is functionally distinct from the T4SS carried on pVir.
rest of the plasmid sequence, suggesting that they have adifferent origin to the rest of the plasmid DNA. Interest- ingly, DNA sequences of Selenomonas ruminantium sub-mitted to public databases have a similar G+C content, Although the two T4SS systems encoded by pVir and pTet but unfortunately, little is known about the genetics of in 81-176 share some homology to one another, they this organism or the function of the various plasmids appear to serve distinct functions, as mentioned above.
Three ORFs on pTet or pCC31 share homology to genes (1?4–42?6 kb) that have been isolated from some strains on pVir. One is cpp6, which encodes a predicted protein (Fliegerova et al., 1998). Altogether there are five genes of 7 kDa with 96 % homology to Cjp19 of pVir, a protein in pCC31 and pTet that have close homologues in the of unknown function. Two ORFs on pTet and pCC31 chromosome of H. pylori, one of which is found in the designated cpp7 and cpp51 share homology to cjp20 on plasticity region. Plasmids found in Helicobacter species pVir. Analysis of the sequence homology reveals that cpp7 have not yet been genetically characterized or sequenced, so and cpp51 are in fact homologous to the N-terminal and it is not known whether any of the ORFs present in pCC31 C-terminal sequences of cjp20 from pVir, respectively. This and pTet have homologues on plasmids found in Helico- suggests that the Cjp20 homologue in pTet and pCC31 was bacter. This would be interesting, given that these two disrupted through a recombination event.
organisms are closely related, and together with theruminant bacterium Wolinella succinogenes belong to theepsilon subclass of the proteobacteria. The genes encoding the putative enzymes involved in DNA processing and Plasmids pCC31 (44?7 kb) and pTet (45?2 kb) are both transfer such as the nickase, helicase, primase, invertase and tetracycline resistance plasmids isolated from clinical iso- single-stand-binding protein are all scattered across both lates of C. coli and C. jejuni, respectively. Although these plasmids and do not obviously have a common origin. Thus, two strains were isolated around 20 years apart and on plasmids pCC31 and pTet are true composites, with a different continents they showed a remarkable similarity in mosaic structure comprising blocks of genes that seem overall sequence (94?3 % identity) and genomic organiza- to have been acquired from bacteria that inhabit the oral tion (Fig. 1). Only three genes of unknown function are and intestinal tract of animals. Campylobacter has been uniquely found on one of the plasmids, two of which have identified as a commensal in the gastrointestinal tracts of several species of domestic animals, as well as wildlifespecies, and is especially abundant in avian species such Apart from the 30 ORFs of unknown function, all of the as chickens, where it can reach up to 1010 c.f.u. per g caecal genes present in pCC31 and pTet are predicted to be contents. The mosaic structure of these plasmids could involved in plasmid replication and conjugative transfer.
reflect the recognized potential for gene transfer and The mating pair formation (Mpf) genes involved in recombination in the complex ecosystem of the animal conjugation share amino acid similarities to the T4SS of host but the natural competence of Campylobacter for different Brucella species, but have the highest overall transformation with exogenous DNA may also be a factor homology to the Mpf gene cluster in pVT745 from Actino- contributing to their evolution and mosaic composition.
bacillus actinomycetemcomitans, a periodontal pathogen.
The organization of the Mpf gene cluster resembles those We have demonstrated that pCC31 and pTet are self- of other conjugative plasmids and T4SSs but is most mobilizable and capable of transfer between C. jejuni and similar to that found in pVT745. In particular, the loca- C. coli strains at frequencies of between 1024 and 1026, tion of cagT and TrbM homologues at the end of the depending on the existence of restriction barriers. The full T4SS gene cluster is unusual and has only been described host-range of these plasmids is not known and difficult to previously in pVT745. This strongly suggested that the Mpf predict as the repA gene exhibits closest homology with gene cluster in these plasmids may have originated from a genes in plasmids from organisms for which genetic tools common ancestor. The similarities in gene organization have not yet been developed. Preliminary studies with between pCC31, pTet and pVT745 are not apparent over pCC31 and pTet indicated that transfer to E. coli was not the rest of the plasmid sequence, although the probable possible, as reported previously for TetR plasmids in nickase (Cpp17) and a putative lipoprotein of unknown Campylobacter (Tenover et al., 1985).
function (Cpp23) also show highest homology to genesfound on Actinobacillus plasmids. The replication proteins Since this paper was first submitted a study was published of pCC31 and pTet showed highest similarity to Rep pro- showing that 16 out of 56 clinical isolates of C. jejuni from teins found in plasmids of the oral spirochaete Treponema the area of Go¨ttingen in Germany harbour plasmids varying denticola (Chauhan & Kuramitsu, 2004) and Selenomonas in size from 6 to 66 kb (Schmidt-Ott et al., 2004). Only one ruminantium. The latter is a prominent and functionally of these plasmids was a homologue of pVir, the virulence diverse species found in the rumen of sheep, cows and goats.
plasmid previously characterized by Bacon et al. (2002). The Interestingly, repA, the three upstream ORFs of unknown relatedness of eight plasmids within a subgroup distinct function and the tet(O) gene in pCC31 and pTet have a from pVir was established by Southern-blot hybridization using a collection of nine PCR-amplified DNA probes from Alonso, J. C., Weise, F. & Rojo, F. (1995). The Bacillus subtilis histone-like protein Hbsu is required for DNA resolution and DNAinversion mediated by the b recombinase of plasmid pSM19035.
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pCC31 (allowing for 1–2 bp mismatches) and were pre- Bacon, D. J., Alm, R. A., Burr, D. H., Hu, L., Kopecko, D. J., Ewing, dicted to amplify DNA fragments of similar length, sug- C. P., Trust, T. J. & Guerry, P. (2000). Involvement of a plasmid gesting that these regions are conserved. The sequence in virulence of Campylobacter jejuni 81-176. Infect Immun 68,4384–4390.
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