Diversity within the Campylobacter jejuni type I restriction–modification loci

William G. Miller1, Bruce M. Pearson2, Jerry M. Wells3, Craig T. Parker1, Vladimir V. Kapitonov4 and Robert E. Mandrell1

1 Produce Safety and Microbiology Research Unit, Agricultural Research Service, US Department of Agriculture, Albany, CA 94710, USA
2 BBSRC Institute of Food Research, Norwich Research Park, Colney, Norwich, UK
3 University of Amsterdam, Swammerdam Institute for Life Sciences, Amsterdam, The Netherlands
4 Genetic Information Research Institute, Mountain View, CA 94043, USA

Correspondence
William G. Miller
bmiller{at}pw.usda.gov


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The type I restriction–modification (hsd) systems of 73 Campylobacter jejuni strains were characterized according to their DNA and amino acid sequences, and/or gene organization. A number of new genes were identified which are not present in the sequenced strain NCTC 11168. The closely related organism Helicobacter pylori has three type I systems; however, no evidence was found that C. jejuni strains contain multiple type I systems, although hsd loci are present in at least two different chromosomal locations. Also, unlike H. pylori, intervening ORFs are present, in some strains, between hsdR and hsdS and between hsdS and hsdM. No definitive function can be ascribed to these ORFs, designated here as rloA–H (R-linked ORF) and mloA–B (M-linked ORF). Based on parsimony analysis of amino acid sequences to assess character relatedness, the C. jejuni type I R–M systems are assigned to one of three families: ‘IAB’, ‘IC’ or ‘IF’. This study confirms that HsdM proteins within a family are highly conserved but share little homology with HsdM proteins from other families. The ‘IC’ hsd loci are >99 % identical at the nucleotide level, as are the ‘IF’ hsd loci. Additionally, whereas the nucleotide sequences of the ‘IAB’ hsdR and hsdM genes show a high degree of similarity, the nucleotide sequences of the ‘IAB’ hsdS and rlo genes vary considerably. This diversity suggests that recombination between ‘IAB’ hsd loci would lead not only to new hsdS alleles but also to the exchange of rlo genes; five C. jejuni hsd loci are presumably the result of such recombination. The importance of these findings with regard to the evolution of C. jejuni type I R–M systems is discussed.


Abbreviations: ARD, amino-proximal recognition domain; BA, Brucella agar; CRD, carboxy-proximal recognition domain; GBS, Guillain–Barré syndrome; LA, Luria–Bertani agar; LB, Luria–Bertani broth; R–M, restriction–modification; TRD, target recognition domain

The GenBank accession numbers for the sequences reported in this paper are listed in a supplementary table with the online version of this paper (at http://mic.sgmjournals.org). Restriction endonuclease and methyltransferase nomenclature follows the recommendations of Roberts et al. (2003a).


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Restriction–modification (R–M) systems are common in bacteria and are generally believed to provide a barrier against foreign DNA and bacteriophages (for general reviews of R–M systems, see Bickle & Kruger, 1993; Murray, 2000; Redaschi & Bickle, 1996; Wilson & Murray, 1992). In classical R–M systems, foreign DNA is cleaved, or restricted, by endonucleases. Host cell DNA avoids restriction through the methylation, or modification, of certain adenine or cytosine residues in the target sequence. Based on subunit composition, cofactor requirements, and position of the DNA cleavage site, R–M systems have been classified into four distinct groups, namely, type I, type II, type III and type IV.

The type I enzyme is a bifunctional, multisubunit complex containing products of the hsdR, hsdM and hsdS genes (host specificity for DNA). HsdS interacts with the target sequence as a component of the restriction and modification complexes. HsdS is composed of four domains: two variable target recognition domains (TRDs), a central-conserved domain, and a conserved C-terminus (Chen et al., 1995; Fuller-Pace et al., 1984; Fuller-Pace & Murray, 1986; Gubler et al., 1992; Nagaraja et al., 1985). The type I target sequence is asymmetric and composed of two half-sites: a 5' half-site, of 3–4 bp, and a 3' half-site, of 4–5 bp, separated by a non-specific spacer of 6–8 bp. In the HsdS subunit, each TRD recognizes one half-site while the conserved domains are thought to interact with the HsdR and HsdM proteins in the complex (Murray, 2000; Weiserova & Firman, 1998; Weiserova et al., 2000). HsdS and HsdM are sufficient for methyltransferase activity but all three subunits are required for endonuclease activity.

Based on complementation studies, DNA sequence similarity and immunological cross-reactivity, type I R–M systems have been divided into four families. The first type I R–M systems to be described, EcoKI and EcoBI, are members of the type IA family (Murray, 2000). The type IB family, first described in Escherichia coli, is represented by the EcoAI and EcoEI systems (Fuller-Pace et al., 1985; Suri & Bickle, 1985). The type IC family, represented by EcoR124I, includes both plasmid-encoded (Firman et al., 1983; Skrzypek & Piekarowicz, 1989) and chromosomally encoded (Kong et al., 2000; Piekarowicz et al., 2001; Tyndall et al., 1994) members. A fourth family, type ID, was first identified in Salmonella enterica serovar Blegdam (Titheradge et al., 1996). HsdM and HsdR proteins within the same family are highly conserved, with any pairwise amino acid sequences having >90 % identity; however, sequence identity between HsdM proteins from different families is usually very low (<35 %) (Sharp et al., 1992; Titheradge et al., 2001).

Many of the type I R–M systems originally described were from well-characterized strains of S. enterica and E. coli, within the Enterobacteriaceae. However, functional type I systems have been described in a wide variety of bacterial taxa, such as Helicobacter pylori (Kong et al., 2000), Neisseria gonorrhoeae (Piekarowicz et al., 2001), Lactococcus lactis (Schouler et al., 1998) and Mycoplasma pulmonis (Dybvig & Yu, 1994). With the genomes of ever more bacteria being sequenced, it is likely that additional type I R–M systems will be identified. The genomic sequence of the human pathogen Campylobacter jejuni was determined recently (NCTC 11168; Parkhill et al., 2000) and a putative type I locus (genes Cj1549Cj1553) was identified. We have noted, in molecular studies of Campylobacter in our laboratory, that plasmid DNA purified from any given transformed C. jejuni strain could be electroporated readily into an untransformed isolate of the same strain; however, this same plasmid DNA could only rarely be electroporated into heterologous strains. Additionally, preliminary data indicated that the type I R–M locus of C. jejuni strain 81116 (which could not be electroporated by plasmid DNA purified from NCTC 11168) was substantially different from the type I locus of NCTC 11168, suggesting a possible genetic basis for the differences in electroporation efficiency.

In this paper, we report the analysis of the type I R–M systems from the commonly used laboratory strains NCTC 11168, 81116 and 81-176, and an additional 70 C. jejuni strains. Based on parsimony analysis for relatedness of nucleotide sequences, and the presence of characteristic spacer repeats within hsdS, some C. jejuni hsd systems were assigned to the type IC family. Two additional type I R–M families, termed here type ‘IAB’ and type ‘IF’, were identified. The family structure of the C. jejuni type I R–M systems, as well as the effect of lateral transfer on both family affiliations and type I diversity, are discussed.


   METHODS
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INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains, growth conditions and chemicals.
Bacterial strains used in this study are listed in Table 1. E. coli was cultured routinely at 37 °C on Luria–Bertani agar (LA). Where applicable, the medium was supplemented with either 50 µg kanamycin ml–1 or 150 µg ampicillin ml–1. All C. jejuni strains, except strain 30, were cultured routinely at 42 °C under microaerophilic conditions (5 % O2, 10 % CO2 and 85 % N2) on Brucella agar (BA) supplemented with 0·025 % (w/v) FeSO4.7H2O, 0·025 % (w/v) sodium metabisulfite (anhydrous) and 0·025 % (w/v) sodium pyruvate (anhydrous). C. jejuni strain 30 was cultivated on BD Campylobacter Skirrow agar plates containing (per litre) 15 g proteose peptone, 2·5 g liver digest, 5 g Bacto yeast extract, 5 g NaCl, 12 g Bacto agar, 10 mg vancomycin, 2500 units polymyxin B and 5 mg trimethoprim. Strain 30 was grown under microaerophilic conditions (see above) in a DW Scientific MACS-MG-1000 anaerobic workstation incubator at 42 °C with 80 % relative humidity.


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Table 1. C. jejuni strains used in this study

 
Restriction and modifying enzymes were purchased from New England Biolabs. All chemicals were purchased from Sigma-Aldrich Chemicals or Fisher Scientific. DNA sequencing chemicals and capillaries were purchased from Applied Biosystems.

Preparation of C. jejuni genomic DNA.
C. jejuni was streaked onto a BA plate and grown overnight at 42 °C. Cells were scraped from the plate and resuspended in 1·5 ml 10 % (w/v) sucrose, 50 mM Tris (pH 8·0), to which was added 250 µl of a 10 mg ml–1 lysozyme solution (in 250 mM Tris, pH 8·0), followed by 600 µl 0·1 M EDTA. The suspension was incubated for 10 min on ice, then 300 µl of a 5 % (w/v) SDS solution was added and the mixture was vortexed briefly to clarify the solution. Then 25 µl RNaseA (1 mg ml–1) and 10 µl proteinase K (10 mg ml–1) were added, sequentially, and the lysates incubated for 30 min and 1 h at 37 °C, respectively. Sodium acetate (0·1 vol.) and ethanol (2 vols) were added and DNA was removed by spooling onto a hooked Pasteur pipette. DNA was resuspended in Tris/EDTA (pH 8·0), extracted twice with phenol/chloroform (1 : 1, v/v), once with chloroform, and concentrated by ethanol precipitation. Strain 30 genomic DNA was isolated from bacteria grown on Campylobacter Skirrow agar plates using the DNeasy Tissue Kit (Qiagen).

Construction of hsd clones.
The hsd locus was amplified from the genomic DNA of C. jejuni strains RM1221, 81-176, RM1046, RM1047, RM1847 and RM1849 using the flanking oligonucleotides HSDF1 and HSDF2 as primers (Fig. 1, Table 2). Amplifications were performed with the Expand Long Template PCR system (Roche Molecular Biochemicals) on an Applied Biosystems 9700 thermocycler with the following thermal programme: 10 s at 94 °C; 30 s at 60 °C; 12 min at 68 °C (10 cycles), and 10 s at 94 °C; 30 s at 60 °C; 12 min at 68 °C (20 cycles with the elongation period extended by 20 s for each successive cycle). The amplification products were gel-purified using the Sephaglas BandPrep kit (Amersham Pharmacia Biotech) and cloned into the pCR-XL-TOPO cloning vector (Invitrogen). The hsd clones (p1221, p81176, p1046, p1047, p1847, p1849) were verified by restriction analysis.



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Fig. 1. Gene organization of selected hsd loci. The hsd loci of strains RM1049, RM1846, RM1860 and RM1861 are similar to the hsd locus of 81116. The hsd locus of strain RM2240 is similar to the hsd locus of 1852. The hsd loci of strains RM1046, RM1847, RM1849 and 81-176 are similar to the hsd locus of strain NCTC 11168. Large arrows represent ORFs. Identical gene designations or allele numbers were assigned the same colour. R1–R3, hsdR alleles; S1–S10, hsdS alleles; M1–M3, hsdM alleles. ORFs 1–9 represent C. jejuni genes Cj1548, Cje1725, Cje1727, Cje1728, Cj1555, putP (Cj1502), Cj1501, Cj1500 and purA (Cj1498), respectively. Small arrows represent flanking oligonucleotide primers used for amplification and sequencing. The arrows underneath ORFs 1, 5, 6 and 9, and underneath the intergenic regions upstream of hsdR and downstream of hsdM, represent primers HSDF1, HSDF2, HSCJ1502c, HSCJ1498c, ss_hsdR5 and hsd8M3, respectively. Cje gene names are from Fouts, D.E., Mongodin, E.F., Mandrell, R.E., Miller, W.G., Rasko, D.A., Ravel, J., Brinkac, L.M., DeBoy, R.T., Parker, C.T., Daugherty, S.C., Dodson, R.J., Durkin, A.S., Madupu, R., Sullivan, S.A., Shetty, J.U., Ayodeji, M.A., Shvartsbeyn, A., Schatz, M.C., Badger, J.H., Fraser, C.M. & Nelson, K.E., unpublished data.

 

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Table 2. Oligonucleotides used in this study

 
DNA sequencing.
Cycle sequencing reactions were performed on an Applied Biosystems 9700 thermocycler using the ABI PRISM BigDye terminator cycle sequencing kit (version 1.0 or version 3.0) and standard protocols as recommended by the manufacturer. All extension products were purified on Centri-Sep spin columns (Princeton Separations). DNA sequencing was performed on an ABI PRISM 310 Genetic Analyser, an ABI PRISM 373 DNA sequencer, or an ABI PRISM 3100 Genetic Analyser (Applied Biosystems) using the POP-6 polymer and ABI PRISM Genetic Analyser Data Collection and ABI PRISM Genetic Analyser Sequencing Analysis software. Sequencing oligonucleotide primers were purchased from Oligos Etc., Integrated DNA Technologies, Sigma-Genosys or Operon Technologies.

Sequencing of hsd clones and hsd genes.
Nested deletion mutagenesis was performed on p1221 using the Erase-a-Base kit according to the protocol recommended by the manufacturer (Promega). The sequences were completed by primer walking. The hsd loci of RM1046, RM1047, RM1847, RM1849 and 81-176 were sequenced using the hsd clones and primers based on the published sequence of the NCTC 11168 hsd locus. The sequence of the C. jejuni strain 81116 hsd locus was determined by first amplifying the locus and then sequencing the amplicon with RM1221 hsd primers or sequencing ApoI subclones derived from the amplicon with M13 sequencing primers. Additional hsd loci were sequenced using existing hsd primers, followed by primer walking. For all loci, sequencing of both strands was accomplished with a final coverage of 2·7–3·3x. Sequence discrepancies were resequenced at least once on each strand to verify the correct base at that position.

The hsd locus of C. jejuni strain 30 was amplified from genomic DNA using the oligonucleotides HSCJ1498c and HSCJ1502c (Fig. 1, Table 2). Reactions were performed with the TaqPlus Precision PCR System (Stratagene) on an Eppendorf Mastercycler thermocycler (Brinkmann) with the following settings: 30 s at 94 °C; 30 s at 50 °C; 5 min at 72 °C (30 cycles). The product was gel-purified from a 1 % (w/v) agarose gel using the Qiagen gel extraction protocol. The product was first sequenced using the original PCR primers and completed by primer walking. The hsdS genes from several C. jejuni type ‘IAB’, ‘IC’ and ‘IF’ loci were amplified using the HSDSF1+HSDSR, 30F8+30R9 and hsdR13+hsdR30 primer sets (Table 2), respectively. Additionally, the rlo genes from several C. jejuni type ‘IAB’ loci were amplified using the 8R19 and 8S1 primers (Table 2). The PCR products were purified, then sequenced using the amplification primers. The sequences were completed by primer walking.

Amplification using hsdR primer sets.
A portion of hsdR was amplified in a multiplex PCR using the oligonucleotide primers 30F2, 30R3, hsdR7, hsdR20, 1221contig1A and 8R16 (Table 2). PCRs were carried out with 30 cycles of 1 min at 94 °C, 2 min at 50 °C, and 3 min at 72 °C using 50 ng of each C. jejuni genomic DNA as the template. In each experiment, RM1221, C. jejuni 30 and NCTC 11168 genomic DNA were used as positive controls.

Characterization of the 1516hsdS homopolymeric tract.
The hsdS locus of strain RM1516 was amplified from genomic DNA using Pfu Turbo (Stratagene) and the oligonucleotide primers 30F8 and 30R9 (Table 2). Amplifications were carried out at 35 cycles of 1 min at 94 °C, 1 min at 50 °C and 3 min at 68 °C using 1 : 10 dilutions of RM1516 genomic DNA as template. An aliquot of the PCR product was then ligated to the pCR4Blunt-TOPO vector (Invitrogen). Plasmid DNA was isolated from 16 positive clones, diluted 1 : 50, and amplified with Pfu Turbo using the oligonucleotide primers 30F12 and 30R14 (Table 2). Amplifications were carried out as above except that a 2 min extension time was used. The 16 PCR products were purified using the QIAQuick PCR purification kit (Qiagen), then sequenced using the 30F12 and 30R14 primers.

Phylogenetic analysis.
DNA and amino acid sequences were aligned using the CLUSTALW module of MEGALIGN (DNASTAR v. 5.0). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.1 (Kumar et al., 2001). The phylogenetic tree was constructed using the neighbour-joining method. For DNA alignments, evolutionary distances were estimated using the Kimura two-parameter (gamma) distance model with a gamma shape parameter of 2·25. Other distance models (e.g. Tamura three-parameter) resulted in trees with similar topology. For amino acid alignments, evolutionary distances were estimated using the gamma distance model with a gamma shape parameter of 2·25. Other distance models (e.g. Poisson correction) resulted in trees with similar topology.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Organization and chromosomal location of the C. jejuni type I R–M loci
Type I R–M systems have been found in approximately 100 different bacterial species (Roberts et al., 2003b; http://rebase.neb.com). In this study, we describe the type I R–M systems of C. jejuni. The type I R–M (hsd) loci of 17 C. jejuni strains are illustrated in Fig. 1 as inferred from PCR amplification and/or DNA sequencing data (data not shown); the gene organization in NCTC 11168 is from the genomic sequence (accession no. AL111168). In all characterized C. jejuni hsd loci, the hsdS gene is located between the hsdR and hsdM genes; however, the gene order is hsdR-hsdS-hsdM in 15 strains (e.g. RM1221, 81116 and 30) and hsdM-hsdS-hsdR in strains RM1047 and NCTC 11168.

A distinct feature of the C. jejuni hsd loci is the presence, in many cases, of intervening ORFs between the hsdR and hsdS genes and between the hsdS and hsdM genes. The ORFs between hsdR and hsdS are designated here as rloA through rloH (R-linked ORF) and the ORFs between hsdS and hsdM are designated here as mloA and mloB (M-linked ORF). rlo and mlo gene designation does not imply gene function but solely reflects the level of similarity between the predicted Rlo and Mlo proteins; rlo or mlo genes predicted to encode proteins with >70 % amino acid identity were assigned the same gene designation. An rlo gene is present in all of the C. jejuni strains characterized in this study, with the exception of strains 30, RM1050, RM1516 and RM3200, which contain neither an rlo nor an mlo gene, and RM1160, which has an rloH deletion that extends into hsdR (data not shown). Several strains (e.g. 81116 and RM1052) contain two rlo genes between hsdR and hsdS. Whereas an mlo gene is present in the majority of C. jejuni hsd loci (45/74; 61 %), the mloA gene is absent in all 19 loci which contain the hsdS1 and hsdS5 alleles (e.g. 81116 and RM1167) and the mloB gene is absent in six of the 26 loci containing the hsdS8 allele (e.g. RM1047).

The hsd loci in most Salmonella and E. coli strains are located in the same relative chromosomal position and are closely linked to the serB locus (Arber & Wauters-Willems, 1970; Bickle, 1993; Daniel et al., 1988; Kannan et al., 1989). The hsd locus in C. jejuni strain NCTC 11168 consists of the genes Cj1549Cj1553. Primer sets designed to the regions flanking the NCTC 11168 hsd locus successfully amplified the genomic DNA from several different C. jejuni strains; however, the genomic DNA from many strains (e.g. 81116 and RM1503) could not be amplified. During an investigation of recombination hot-spots in C. jejuni (B. M. Pearson & J. M. Wells, unpublished), another hsd locus was found in C. jejuni strain 30 between the Cj1501 and Cj1502 ORFs. No evidence exists for an hsd locus in strain 30 at the same chromosomal location as the NCTC 11168 hsd locus. Additionally, amplification using primers CJ1498 and CJ1502 indicates that the hsd loci from strains 81116 and RM1503 are not located between Cj1501 and Cj1502 (data not shown). Therefore, at least two chromosomal locations exist for C. jejuni hsd genes (i.e. Cj1501Cj1502 and Cj1549Cj1553).

The presence of hsd loci in two or more chromosomal locations raised the possibility that some strains may contain multiple hsd loci. The existence of multiple hsd loci in one strain is not unusual: both sequenced H. pylori strains (Alm et al., 1999; Tomb et al., 1997) as well as the plant pathogen Xylella fastidiosa 9a5c (Simpson et al., 2000) contain three hsd loci. However, no evidence was found for the existence of multiple hsd loci in C. jejuni. For the 93 C. jejuni strains characterized in this study, only one amplification product was obtained by multiplex PCR with a three-family hsdR primer set (data not shown). Similar results were obtained when 100+ C. jejuni strains were genomotyped using C. jejuni microarrays (see below). These results suggest that these C. jejuni strains only contain one hsd locus, but they do not eliminate the possibility that they contain additional hsd loci dissimilar to existing C. jejuni hsd loci, nor do they eliminate the possibility that orphan hsdS genes (Donahue & Peek, 2001), i.e. hsdS genes that are unlinked to both hsdR and hsdM, are present in some strains. One such orphan hsdS gene appears to be present in strain RM3200. Strain RM3200 contains a complete type ‘IC’ locus. However, it also contains a unique hsdS allele, hsdS11. No ‘IAB’, ‘IC’ or ‘IF’ hsdR, hsdM, rlo or mlo gene is associated with this hsdS gene (data not shown).

Sequence analysis of the ‘IAB’ hsd loci identified a putative RpoD promoter sequence (Petersen et al., 2003) upstream of hsdR. A rho-independent transcriptional terminator was identified between hsdS and mloA, followed by another putative promoter sequence. Therefore, presumably, in most type ‘IAB’ hsd loci, hsdR, hsdS and the intervening rlo gene(s) are co-transcribed, and mloA and hsdM are co-transcribed; in those ‘IAB’ hsd loci which do not contain mloA (and therefore do not contain the terminator or promoter between hsdS and mloA), all hsd genes would be co-transcribed. No promoter sequences were found in the ‘IC’ or ‘IF’ hsd loci. As described above, the type ‘IC’ hsd loci are located between Cj1501 and Cj1502 (putP) and are thus downstream of the proline utilization (putPA) operon; therefore, ‘IC’ hsd transcription would presumably be driven by the putPA promoter. Additional analysis will be necessary to determine the location of promoters within the type ‘IF’ loci.

Family classification of the C. jejuni hsd loci
Amino acid sequence comparisons of hsd subunits (e.g. HsdM) have been used to assign R–M systems into one of four discrete families (Titheradge et al., 2001). The amino acid sequences of several HsdM proteins from eubacteria and archaea are available in the GenBank database. The phylogenetic relationship of the HsdM polypeptides of the available bacteria and three HsdM polypeptides determined in this study was analysed (Fig. 2). Because of near identity, the HsdM amino acid sequence from strain NCTC 11168 was used as the exemplary sequence for strains 81-176, RM1046, RM1047, RM1847 and RM1849. Similarly, the HsdM amino acid sequence from strain RM1221 served as the exemplary sequence for strains 81116, RM1049, RM1170, RM1852, RM1861, RM2227, RM2232 and RM2240. While HsdM11168 is nearly identical to other C. jejuni HsdM proteins (Table 3: e.g. HsdM81–176, approx. 100 % identity), it shows very little similarity to several others (e.g. HsdM1221, 16 % identity; or HsdM30, 26 % identity). However, it is very similar to two HsdM polypeptides from the cyanobacterium Nostoc sp. PCC 7120 (61 % identity) and the archeon Methanosarcina mazei (67 % identity), and somewhat similar (49 % identity) to type ID HsdM proteins. Also, while HsdM30 is essentially identical to the HsdM subunit from another C. jejuni strain (HsdM1516; approx. 100 % identity) and shows some similarity to HsdM subunits from members of the type IC hsd family [EcoR124II HsdM (56 % identity), NgoAV HsdM (53 % identity), and Hpy850 HsdM (62 % identity)], it has little similarity to other HsdM proteins. HsdM1221 has no significant similarity to the HsdM proteins of strain NCTC 11168 or strain 30 (19·6 % identity). Among the other HsdM proteins, >25 % identity exists only with members of the type IB hsd family [EcoA HsdM (34 % identity) and EcoE HsdM (34 % identity)].



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Fig. 2. Phylogenetic tree depicting the similarities among 35 HsdM polypeptides. Bootstrap values are based on 1000 replicates and are indicated as percentages; values below 75% are not shown. Scale bar, 20 substitutions/100 bases. Labels in red, blue, violet or green represent Type IA, IB, IC or ID HsdM polypeptides, respectively (Titheradge et al., 2001). HsdM polypeptides labeled with a star indicate that the associated HsdS subunits are predicted to contain spacer repeats. In addition to the C. jejuni HsdM polypeptides characterized in this study, the tree consists of polypeptides from the following strains: Helicobacter pylori J99 (GenBank accession no. AE001439.1), H. pylori 26695 (AE000511.1), Haemophilus influenzae RdKw20 (L42023.1), Nitrosomonas europaea ATCC 19718 (NC_004757), Pasteurella multocida (AE006190·1), Mycobacterium tuberculosis H37RV (AL008962.1), Xylella fastidiosa 9a5c (AE003849), Chlorobium tepidum TLS (NC002932.3), Salmonella enterica serovar Blegdam (CAA68057, Klebsiella pneumoniae M5A1 (U93843), Nostoc sp. PCC7120 (NC003272.1), C. jejuni NCTC 11168 (AL111168), Methanosarcina mazei Goe1 (NC003901.1), Methanococcus jannaschii (NC001732), Lactococcus lactis subsp. lactis pND861 (T09460), Staphylococcus aureus subsp. aureus N315 (AP003130.2), L. lactis subsp. lactis IL403 (AEE006298_2), Mycoplasma pneumoniae M129 (U00089), E. coli pR124/3 (P10484), Neisseria gonorrhoeae FA1090 (AE004969), E. coli K12 (P08957), Salmonella enterica serovar Potsdam (P07989), Streptococcus pneumoniae R6 (AE008425.1), E. coli A58 (Q47282), E. coli 15T (A47200) and Salmonella enterica serovar Kaduna (CAA71895. The hsdM allele from Burkholderia cenocepacia was obtained from the uncompleted genome. These sequence data were produced by the Microbial Pathogen Sequencing Group at the Sanger Institute and can be obtained from http://www.sanger.ac.uk/Projects/B_cenocepacia/.

 

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Table 3. Pairwise comparisons of selected HsdM proteins from C. jejuni and other taxa

Numbers represent percentage amino acid identity. Values above 50 % are in bold; values above 30 % are in bold italic. HsdM designations are from Fig. 2. ‘AB’, ‘C’, ‘F’, A, B, C and D represent hsd family classifications; NA, hsd family not assigned.

 
Based on the similarities and differences between the C. jejuni HsdM proteins and the organization of the hsd loci, it was clear that the C. jejuni hsd loci could be classified into at least three families. Based on both amino acid sequence similarity and the presence of the characteristic four amino acid spacer repeat (Murray, 2000; Piekarowicz et al., 2001; Price et al., 1989; Tyndall et al., 1994) in the central-conserved region of HsdS7, strains 30 and RM1516 are members of the IC family. However, the other hsd loci showed no significant similarity to any of the other type I families (IA, IB or ID). The similarity between the HsdM proteins of the strains represented by HsdM1221, and the enteric type IB HsdM proteins was not extensive; therefore, the hsd loci from these strains could not be given the IB designation. From phylogenetic analyses we inferred that the HsdM proteins of these strains were in a clade sister to the IB family (Fig. 2). The IA family was, in turn, sister to a clade which included the C. jejuni RM1221 group plus the IB family. In view of the fact that the C. jejuni RM1221 group cannot be discretely placed in either the IA or IB families, but has a sister affiliation to both, we propose that the hsd family represented by RM1221 be designated ‘IAB’. Similarly, while the hsd locus from strain NCTC 11168 was somewhat similar in its amino acid sequence to members of the type ID hsd family, it was inferred to be in a sister group (which includes the hsd loci from Nostoc sp. and M. mazei) to the clade of the ID family. Therefore, the hsd family represented by strain NCTC 11168 was given the putative designation of ‘IF’ (as were RM1046hsd, RM1047hsd, RM1847hsd, RM1849hsd and 81-176hsd). We propose that the Nostoc sp. and M. mazei hsd loci are also representatives of the type ‘IF’ family. Finally, the genomic DNA from 20 C. jejuni strains (e.g. RM1503) did not amplify with the ‘IAB’, ‘IC’ or ‘IF’ hsdR primer sets (data not shown). To test whether ‘IAB’, ‘IC’ and ‘IF’ hsd loci were present in these strains but could not be amplified because of sequence diversity at the primer binding site(s), genomic DNAs from many strains classified as ‘unknown hsd family’ were hybridized to microarrays containing all five NCTC 11168 hsd genes, as well as hsdR genes from three type ‘IAB’ strains (RM1852, RM1221 and 81116) and two type ‘IC’ strains (30 and RM1516). No hybridization was detected at any of the hsd spots (data not shown). Although this implies that the hsd loci from these strains are not in the ‘IAB’, ‘IC’ or ‘IF’ family and that additional hsd families may be present in C. jejuni, it is also possible that these strains do not contain a type I R–M system.

The hsd family designation was unrelated to either the source (i.e. chicken, human or bovine) or the location (Table 1) from which the strains were obtained. However, all of the strains containing ‘rloDhsd loci were poultry isolates and all of the strains containing ‘rloEGhsd loci were clinical isolates (Fig. 3). Also, whereas many of the Guillain–Barré syndrome (GBS) isolates were classified as ‘IAB’ and were strongly associated with the ‘rloF’ clade (Fig. 3), several GBS isolates were classified as either ‘IF’ (strains RM3264, RM3266) or ‘unknown’ (strains RM3148, RM3149, RM3193, RM3196, RM3197, RM3198, RM3211 and RM3265) (Table 1).



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Fig. 3. Phylogenetic tree depicting evolutionary relatedness of C. jejuni taxa based on 64 hsdS alleles. Bootstrap values are based on 1000 replicates and are indicated as percentages; values below 75% are not shown. Scale bar, 20 substitutions per 100 bases. hsdS alleles labelled in blue represent clinical isolates; clinical isolates labelled with a star represent GBS isolates. hsdS alleles labelled in black represent non-human isolates. Boxed hsdS alleles (hsdS4 and hsdS9) are hybrid. Letters in parentheses represent the rlo gene associated with each hsdS allele. The hsdS11 allele does not appear to be linked to any additional hsd, rlo or mlo genes.

 
Titheradge et al. (2001) used an HsdM identity value of 45 % to place novel HsdM polypeptides into the four existing families of type I enzymes; HsdM proteins with >45 % identity were placed into the same family. Based on this sole criterion, many of the newly characterized type I R–M systems can be assigned to one of four families identified in the Enterobacteriaceae (Fuller-Pace et al., 1985; Price et al., 1987; Suri & Bickle, 1985; Titheradge et al., 1996, 2001). However, only 17 % of the pairwise combinations between the 32 HsdM proteins in Fig. 2 show >45 % identity (data not shown). While it is clear that the type I R–M loci of C. jejuni represent three distinct families, only one group of C. jejuni hsd loci described in this study (e.g. Cje30, Cje1516) can clearly be assigned as a member of a monophyletic group of an existing family (type IC). The placement of this group in the type IC family is based not only on sequence similarity to and immediate phylogenetic affiliation with other type IC enzymes, but also on the presence of IC-specific motifs, such as the four amino acid repeat in HsdS30 and HsdS1516. The other two groups of C. jejuni hsd loci do not clearly belong to any of the existing type I R–M families. Indeed, according to the phylogenetic tree presented in Fig. 2, at least eight new families might exist if the level of HsdM homology was the sole standard of classification. Therefore, classification of HsdM families based on amino acid sequence similarity alone has the potential drawback of eventually generating an enormous number of type I R–M families. Analyses based on other criteria, such as allelic complementation and immunological cross-reactivity (Fuller-Pace et al., 1985; Murray et al., 1982; Suri & Bickle, 1985), may be able to place distantly related type I systems (e.g. the type ID and type ‘IF’ systems) into the same family; however, analyses performed on such diverse taxa may be problematic.

Hsd coding repeats
Our study found that the HsdR subunit encoded by 1221hsdR is truncated due to a frameshift mutation and, therefore, presumably non-functional. This frameshift mutation is probably caused by slipped-strand mispairing within an upstream coding repeat, in this case a ‘TATA’ repeat. A similar suggestion has been made for the hsdR1 genes from H. pylori J99 and H. pylori 26695, in which a homopolymeric C-tract precedes a frameshift mutation (Donahue & Peek, 2001). In addition to the H. pylori hsdR1 genes, several examples of coding repeats can be found among other H. pylori and C. jejuni R–M genes: one C. jejuni type II R–M gene (Cj0031/0032; Parkhill et al., 2000), one H. pylori type II restriction gene (HP1471 or JHP1364; Donahue & Peek, 2001), and two type III modification genes in H. pylori (mod-2 and mod-4a/4b; Donahue & Peek, 2001). In all four cases, a homopolymeric G-tract precedes the frameshift mutation. Additionally, the frameshift mutation in 1167hsdS is preceded by an 8 base homopolymeric A-tract. The hsdS7 genes contain a homopolymeric G-tract. In 30hsdS7 and 3200hsdS7, the G-tract is 9 bp in length (G9) and in 1050hsdS7 it is 7 bp in length (G7). When 1516hsdS7 was amplified from genomic DNA and sequenced, sequences downstream of the G-tract in either direction could not be obtained accurately due to overlapping peaks in the sequencing read, suggesting that two populations of cells existed in the culture used to make the genomic DNA: cells containing a presumably non-functional G8 hsdS7 gene or cells containing a functional G9 hsdS7 gene. To verify this, the region surrounding the homopolymeric tract was amplified, cloned and sequenced. The results showed that 62·5 % (10/16) of the clones were G8 and 37·5 % (6/16) were G9, confirming that a minority of the strain RM1516 cells produced a functional HsdS7 protein. These data indicate that strains like RM1516 would be mixtures of cells with different frequencies of HsdS expression and, thus, restriction activity. Overlapping peaks in the sequencing reads were not seen after sequencing similar amplicons from the essentially identical 30hsdS7 gene, suggesting that polymorphisms in amplicons containing homopolymeric G-tracts are not PCR artifacts. Similar results were obtained by Wassenaar et al. (2002) after amplifying across multiple homopolymeric G-tracts in the NCTC 11168 genome.

Although many homopolymeric tracts have been described in C. jejuni, only a fraction of these have been shown to be hypervariable. These contingency genes encode not only R–M proteins but proteins involved in lipooligosaccharide and capsule biosynthesis. Contingency gene variants, caused by slipped-strand mispairing, appear at a much higher frequency in C. jejuni and H. pylori than in other taxa, e.g. Neisseria spp. (Alm et al., 1999; Parkhill et al., 2000). The cause of this high phase-variation frequency is not known, but it may be due to deficiencies in DNA repair functions (Parkhill et al., 2000). The hsdS locus of strain 30 does not show the same variation as 1516hsdS. Therefore, comparative genomics of these two strains may reveal genetic differences important in either enhancement or repression of mutations caused by slipped-strand mispairing. It is intriguing to note that RM1516 is the only ‘IC’ strain isolated from an animal (bovine) source. Perhaps, in some strains, unique conditions present within such a host increase variation at this homopolymeric tract. Recent evidence suggests that, under certain conditions, coding repeat variations might be enhanced. Rasmussen & Bjorck (2001) found that variations of a pentanucleotide coding repeat in the Streptococcus pyogenes sclB gene occurred when the organism was grown in fresh human blood but not during growth in medium.

Sequence analysis of the hsdS, rlo and mlo genes
hsdS.
The hsdS gene encodes the specificity protein of the type I R–M system. HsdS interacts with the target DNA sequence as part of both the methylation and restriction complexes. To assess the potential diversity present in C. jejuni at this locus, a phylogenetic tree was constructed using the hsdS sequences from 64 C. jejuni strains (Fig. 3). Several monophyletic groups (clades) were inferred. While the hsdS genes within each clade are essentially identical (>98 % nucleotide identity), similarities between clades range between 4 % and 84 % nucleotide identity. The hsdS genes in each clade were assigned a different allele designation; however, those hsdS genes with a high degree of similarity (e.g. 1167hsdS5 and 2232hsdS3, 84 % identity) might still be considered to be the same allele. As described above, HsdS is composed of four domains: two variable domains (the amino-proximal recognition domain (ARD; Chen et al., 1995) and the carboxy-proximal recognition domain (CRD; Chen et al., 1995), a central-conserved domain, and a conserved C-terminus. hsdS genes encoding the same ARD and CRD would most likely result in HsdS subunits that recognize the same target sequence and would be considered to be the same hsdS allele; hsdS genes encoding a different ARD and/or a different CRD would represent different hsdS alleles. Therefore, to verify that each of the clades in Fig. 3 represents a group of taxa possessing a unique hsdS allele, the ARD or CRD regions encoded by each of the 64 hsdS genes were aligned. The nucleotide identities for each pairwise comparison of the ARD or CRD regions were either >90 % or <40 % (data not shown). ARD or CRD regions with >90 % nucleotide identity were assigned the same type designation. Seven different ARD types and eight different CRD types are present among the 64 hsdS genes (Table 4). As expected, each hsdS allele contains a unique set of variable domains. Therefore, 1167hsdS5 (ARD#1, CRD#3) and 2232hsdS3 (ARD#1, CRD#2) are different alleles even though they show 84 % nucleotide identity across the entire gene. Finally, four distinct types of central-conserved regions are seen with approximately 80 % sequence identity between type 1 and type 2 (Table 4). 1170hsdS4 contains a hybrid central-conserved region in which the 5' half is type 1 and the 3' half is type 2.


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Table 4. Domain composition of the hsdS alleles

 
rlo and mlo.
Comparison of the predicted amino acid sequences of Rlo and Mlo polypeptides to the SWISS-PROT database using the BLASTP program revealed only a few, low-scoring, sequence pairs to proteins of known function. However, RloA and RloB were moderately (E=10–45) to weakly (E=10–05) similar to putative phage-resistance proteins in the cyanobacterium Nostoc sp. PCC 7120 (Kaneko et al., 2001) and a putative transporter in Fusobacterium nucleatum subsp. nucleatum (ATCC 25586; Kapatral et al., 2002). RloA and RloB are also similar to the AbiLi and AbiLii proteins of Lactococcus lactis biovar diacetylactis plasmid pND861 (Deng et al., 1999), respectively. In this organism, the AbiL proteins confer resistance to the phage phi 712 and partial resistance to the phage phi c2. The predicted coding sequence of rloH in strain NCTC 11168 shows only partial similarity to a class of ATP/GTP-binding proteins; however, no function can be attributed to any of the other rlo or mlo genes.

Whereas rlo and mlo homologues are absent from H. pylori hsd loci, ORFs similar to C. jejuni rlo and mlo genes are found in the Helicobacter hepaticus hsd locus; the H. hepaticus protein HH1422 is 66 % identical to MloA. Proteins similar to MloA are also found in other taxa. It is noteworthy that in three instances, the genes encoding these proteins are adjacent to methyltransferases (XCC0214, Xanthomonas campestris pv. campestris; hsdM2, Shewanella oneidensis) or hsdS (hsdS1, Chlorobium tepidum), reflecting the linkage of mloA with hsdM and hsdS. Association with a particular class of hsd genes is also seen with the rlo genes. RloB proteins are predicted to form two classes; RloB proteins from different classes show only 73 % amino acid identity. One class of rloB genes (e.g. 1048rloB) is associated with the hsdS6 and S6-hybrid hsdS9 alleles while the other class of rloB genes (e.g. 1049rloB) is associated with the hsdS1 allele. In general, rlo genes predicted to encode identical Rlo proteins are associated with the same hsdS allele.

Rearrangement within the C. jejuni hsd loci
One feature of the ‘IAB’ hsd loci is the presence of hybrid hsdS genes (e.g. 1850hsdS9), in which the 5' half and the 3' half of the hybrid gene is derived from two different ‘parental’ hsdS genes (in this instance, 1551hsdS6 and 1221hsdS2). Hybrid hsdS genes often have an altered specificity with respect to the two ‘parental’ specificities. Following an experiment to P1-transduce the S. enterica serovar Potsdam StySPI hsd genes into cells with a StySBI specificity, a new specificity, designated SQ, was seen in one of the transductants (Bullas et al., 1976). Later, it was determined that recombination between the hsd genes had occurred and the ‘SQ’ hsdS gene was a hybrid consisting of the 5' half of the ‘SP’ hsdS gene and the 3' half of the ‘SB’ hsdS gene (Fuller-Pace et al., 1984; Gann et al., 1987; Nagaraja et al., 1985). The ‘SQ’ target sequence consisted of the 5' half-site of StySPI and the 3' half-site of StySBI.

In C. jejuni, the hsdS4 and hsdS9 alleles appear to be hybrids from two separate ancestral sources (Fig. 4a, Table 4). A possible set of ‘parental’ hsdS genes for each hybrid allele is presented in Fig. 4(a). In the C. jejuni hsdS genes, significant sequence conservation occurs only within the central and 3' terminus of the gene. Very little sequence similarity is present at the 5' end. Therefore, while the CRD-encoding region could be exchanged readily by recombination at the central-conserved region and at the 3' conserved region (or at either the mlo or the hsdM locus), the ARD-encoding region would be exchanged primarily via recombination at the central-conserved region and at a site upstream of hsdS. Since the rlo loci are so diverse in the ‘IAB’ family, this upstream site would, in most cases, have to be within hsdR. Therefore, exchange of the ARD-encoding region would often entail a concomitant exchange of the rlo locus. An example of this is illustrated in Fig. 4(b). In this instance, a fragment of genomic DNA from strain RM1551 recombines with an ‘RM1221-like’ hsd locus in strain RM1850, both creating a hybrid hsdS allele (hsdS9) and exchanging the rloC gene with the rloA and rloB genes. Similar recombination events can be constructed for the RM1048 and RM1170 hsd loci (data not shown). However, 2227hsdS4 probably originated from recombination at the 5' and central-conserved regions of hsdS. While formation of hybrid hsdS alleles suggests recombination events that exchange entire genes or domains, it is likely that smaller recombination events occur at the C. jejuni hsd locus. However, these recombination events are probably obscured by accumulation of spontaneous mutations. Additionally, since ‘IC’ hsd loci are essentially identical to each other, as are ‘IF’ hsd loci, detection of ‘IC’ or ‘IF’ hsd recombination events would not be possible.



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Fig. 4. The hybrid nature of RM1170hsdS, RM2227hsdS and RM1850hsdS. (a) The hsdS sequences of strains RM2232, RM2227 and RM3145, the hsdS sequences of strains RM1437, RM1170 and RM1221, and the hsdS sequences of RM1551, RM1850 and RM1221 were aligned using CLUSTALW. The polymorphic sites in each alignment were collected using the program PSFIND. A graphical output of these sites was generated with the program HAPPLOT. ARD, amino-proximal recognition domain; CC, central-conserved domain; CRD, carboxy-proximal recognition domain. (b) Hypothetical derivation of the RM1850 hsd locus. Numbers within hsdS refer to the ARD and CRD types listed in Table 3. The central-conserved domains are identical for all three loci and are therefore not shown. Dotted lines indicate putative recombination events.

 
The shuffling of HsdS domains was proposed as a factor in the evolution of type I R–M specificities (Murray, 2000). Five ARD types and six CRD types are present in the type ‘IAB’ hsd loci (Table 4). If each ARD and each CRD recognizes different half-sites, then our findings suggest that there may be at least 30 different type I target sequences present in C. jejuni. Two additional target sequences are present in the ‘IC’ and ‘IF’ families but low sequence similarity between the three hsd families would make inter-family recombination events unlikely. The large amount of potential target sequences plus variation at the rlo locus implies a high degree of diversity at the type ‘IAB’ hsd locus. This diversity is enhanced by the ability of C. jejuni to take up fragments of genomic DNA through natural transformation (Wang & Taylor, 1990). Also, deBoer et al. (2002) demonstrated that interstrain genomic exchange occurs in C. jejuni in vivo. Lateral transfer, therefore, probably plays a major role in the evolution of the C. jejuni hsd locus, not only in genetic exchange between two C. jejuni strains, but in facilitating recombination between C. jejuni hsd loci and hsd loci from different taxa. These recombination events could potentially introduce new rlo genes, novel hsdS domains or even new hsd families into C. jejuni. This type of lateral transfer and recombination obviously explains the distribution of C. jejuni hsd loci into a number of different families and further explains the rather remarkable direct affiliation of strain NCTC 11168 with the Archaea. Perhaps of greater interest will be to determine the exact function of hsd-encoded proteins possessing variable domains, and whether the function is related to the variable ability of C. jejuni strains to survive in different environments.


   ACKNOWLEDGEMENTS
 
We thank S. Abbott, M. Englen, P. Guerry, W. Johnson, A. Lastovica, R. Meinersmann, I. Nachamkin, M. Nicholson, L. Stanker, I. Wesley and D. Woodward for providing strains and plasmids. We thank D. Woodward for Campylobacter serotyping. We also thank B. Campbell, R. Meinersmann and members of this unit for their critical reading of the manuscript, and S. Horn for sequencing some of the ‘IAB’ hsdS genes. This work was funded by the United States Department of Agriculture Agricultural Research Service CRIS project 5325-42000-041. We gratefully acknowledge the BBSRC core strategic grant funding for B. M. P. and J. M. W.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 13 May 2004; revised 28 October 2004; accepted 12 November 2004.



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