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
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ABSTRACT |
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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).
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INTRODUCTION |
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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 34 bp, and a 3' half-site, of 45 bp, separated by a non-specific spacer of 68 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 RM systems have been divided into four families. The first type I RM 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 RM 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 RM 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 RM 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 RM 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 RM families, termed here type IAB and type IF, were identified. The family structure of the C. jejuni type I RM systems, as well as the effect of lateral transfer on both family affiliations and type I diversity, are discussed.
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METHODS |
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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 ml1 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 ml1) and 10 µl proteinase K (10 mg ml1) 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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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 2·73·3x. 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.
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RESULTS AND DISCUSSION |
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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 RM 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. HsdM81176, 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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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 rloD hsd loci were poultry isolates and all of the strains containing rloEG hsd loci were clinical isolates (Fig. 3
). Also, whereas many of the GuillainBarré 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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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 RM genes: one C. jejuni type II RM 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 RM 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 RM 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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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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ACKNOWLEDGEMENTS |
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REFERENCES |
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Arber, W. & Wauters-Willems, D. (1970). Host specificity of DNA produced by Escherichia coli. XII. The two restriction and modification systems of strain 15T. Mol Gen Genet 108, 203217.[Medline]
Bickle, T. A. (1993). The ATP-dependent restriction enzymes. In Nucleases (Cold Spring Harbor Monograph Series no. 25), 2nd edn, pp. 89109. Edited by S. M. R. S. L. Linn & R. J. Roberts. Plainview, NY: Cold Spring Harbor Laboratory.
Bickle, T. A. & Kruger, D. H. (1993). Biology of DNA restriction. Microbiol Rev 57, 434450.[Medline]
Black, R. E., Levine, M. M., Clements, M. L., Hughes, T. P. & Blaser, M. J. (1988). Experimental Campylobacter jejuni infection in humans. J Infect Dis 157, 472479.[Medline]
Bullas, L. R., Colson, C. & Van Pel, A. (1976). DNA restriction and modification systems in Salmonella. SQ, a new system derived by recombination between the SB system of Salmonella typhimurium and the SP system of Salmonella potsdam. J Gen Microbiol 95, 166172.[Medline]
Chang, N. & Taylor, D. E. (1990). Use of pulsed-field agarose gel electrophoresis to size genomes of Campylobacter species and to construct a SalI map of Campylobacter jejuni UA580. J Bacteriol 172, 52115217.[Medline]
Chen, A., Powell, L. M., Dryden, D. T., Murray, N. E. & Brown, T. (1995). Tyrosine 27 of the specificity polypeptide of EcoKI can be UV crosslinked to a bromodeoxyuridine-substituted DNA target sequence. Nucleic Acids Res 23, 11771183.[Abstract]
Daniel, A. S., Fuller-Pace, F. V., Legge, D. M. & Murray, N. E. (1988). Distribution and diversity of hsd genes in Escherichia coli and other enteric bacteria. J Bacteriol 170, 17751782.[Medline]
de Boer, P., Wagenaar, J. A., Achterberg, R. P., Putten, J. P., Schouls, L. M. & Duim, B. (2002). Generation of Campylobacter jejuni genetic diversity in vivo. Mol Microbiol 44, 351359.[CrossRef][Medline]
Deng, Y. M., Liu, C. Q. & Dunn, N. W. (1999). Genetic organization and functional analysis of a novel phage abortive infection system, AbiL, from Lactococcus lactis. J Biotechnol 67, 135149.[CrossRef][Medline]
Donahue, J. P. & Peek, R. M. (2001). Restriction and modification systems. In Helicobacter pylori: Physiology and Genetics, pp. 269276. Edited by H. L. T. Mobley, G. L. Mendz & S. L. Hazell. Washington, DC: American Society for Microbiology.
Dybvig, K. & Yu, H. (1994). Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis. Mol Microbiol 12, 547560.[Medline]
Engberg, J., Nachamkin, I., Fussing, V., McKhann, G. M., Griffin, J. W., Piffaretti, J. C., Nielsen, E. M. & Gerner-Smidt, P. (2001). Absence of clonality of Campylobacter jejuni in serotypes other than HS : 19 associated with Guillain-Barré syndrome and gastroenteritis. J Infect Dis 184, 215220.[CrossRef][Medline]
Firman, K., Creasey, W. A., Watson, G., Price, C. & Glover, S. W. (1983). Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3. Mol Gen Genet 191, 145153.[CrossRef][Medline]
Fuller-Pace, F. V. & Murray, N. E. (1986). Two DNA recognition domains of the specificity polypeptides of a family of type I restriction enzymes. Proc Natl Acad Sci U S A 83, 93689372.[Abstract]
Fuller-Pace, F. V., Bullas, L. R., Delius, H. & Murray, N. E. (1984). Genetic recombination can generate altered restriction specificity. Proc Natl Acad Sci U S A 81, 60956099.[Abstract]
Fuller-Pace, F. V., Cowan, G. M. & Murray, N. E. (1985). EcoA and EcoE: alternatives to the EcoK family of type I restriction and modification systems of Escherichia coli. J Mol Biol 186, 6575.[Medline]
Gann, A. A., Campbell, A. J., Collins, J. F., Coulson, A. F. & Murray, N. E. (1987). Reassortment of DNA recognition domains and the evolution of new specificities. Mol Microbiol 1, 1322.[Medline]
Gubler, M., Braguglia, D., Meyer, J., Piekarowicz, A. & Bickle, T. A. (1992). Recombination of constant and variable modules alters DNA sequence recognition by type IC restriction-modification enzymes. EMBO J 11, 233240.[Abstract]
Kaneko, T., Nakamura, Y., Wolk, C. P. & 19 other authors (2001). Complete genomic sequence of the filamentous nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120. DNA Res 8, 205213, 227253.
Kannan, P., Cowan, G. M., Daniel, A. S., Gann, A. A. & Murray, N. E. (1989). Conservation of organization in the specificity polypeptides of two families of type I restriction enzymes. J Mol Biol 209, 335344.[Medline]
Kapatral, V., Anderson, I., Ivanova, N. & 22 other authors (2002). Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586. J Bacteriol 184, 20052018.
Kong, H., Lin, L. F., Porter, N., Stickel, S., Byrd, D., Posfai, J. & Roberts, R. J. (2000). Functional analysis of putative restriction-modification system genes in the Helicobacter pylori J99 genome. Nucleic Acids Res 28, 32163223.
Korlath, J. A., Osterholm, M. T., Judy, L. A., Forfang, J. C. & Robinson, R. A. (1985). A point-source outbreak of campylobacteriosis associated with consumption of raw milk. J Infect Dis 152, 592596.[Medline]
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2 - molecular evolutionary genetics analysis software. Bioinformatics 17, 12441245.
Miller, W. G., Bates, A. H., Horn, S. T., Brandl, M. T., Wachtel, M. R. & Mandrell, R. E. (2000). Detection on surfaces and in Caco-2 cells of Campylobacter jejuni cells transformed with new gfp, yfp, and cfp marker plasmids. Appl Environ Microbiol 66, 54265436.
Murray, N. E. (2000). Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle). Microbiol Mol Biol Rev 64, 412434.
Murray, N. E., Gough, J. A., Suri, B. & Bickle, T. A. (1982). Structural homologies among type I restriction-modification systems. EMBO J 1, 535539.[Medline]
Nachamkin, I., Engberg, J., Gutacker, M. & 10 other authors (2001). Molecular population genetic analysis of Campylobacter jejuni HS : 19 associated with Guillain-Barre syndrome and gastroenteritis. J Infect Dis 184, 221226.[CrossRef][Medline]
Nagaraja, V., Shepherd, J. C. & Bickle, T. A. (1985). A hybrid recognition sequence in a recombinant restriction enzyme and the evolution of DNA sequence specificity. Nature 316, 371372.[Medline]
Parkhill, J., Wren, B. W., Mungall, K. & 18 other authors (2000). The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences. Nature 403, 665668.[CrossRef][Medline]
Penner, J. L., Hennessy, J. N. & Congi, R. V. (1983). Serotyping of Campylobacter jejuni and Campylobacter coli on the basis of thermostable antigens. Eur J Clin Microbiol 2, 378383.[Medline]
Petersen, L., Larsen, T. S., Ussery, D. W., On, S. L. & Krogh, A. (2003). RpoD promoters in Campylobacter jejuni exhibit a strong periodic signal instead of a 35 box. J Mol Biol 326, 13611372.[CrossRef][Medline]
Piekarowicz, A., Klyz, A., Kwiatek, A. & Stein, D. C. (2001). Analysis of type I restriction modification systems in the Neisseriaceae: genetic organization and properties of the gene products. Mol Microbiol 41, 11991210.[CrossRef][Medline]
Price, C., Pripfl, T. & Bickle, T. A. (1987). EcoR124 and EcoR124/3: the first members of a new family of type I restriction and modification systems. Eur J Biochem 167, 111115.[Abstract]
Price, C., Lingner, J., Bickle, T. A., Firman, K. & Glover, S. W. (1989). Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes. J Mol Biol 205, 115125.[Medline]
Rasmussen, M. & Bjorck, L. (2001). Unique regulation of SclB a novel collagen-like surface protein of Streptococcus pyogenes. Mol Microbiol 40, 14271438.[CrossRef][Medline]
Redaschi, N. & Bickle, T. A. (1996). DNA restriction and modification systems. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn, pp. 773781. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Roberts, R. J., Belfort, M., Bestor, T. & 44 other authors (2003a). A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res 31, 18051812.
Roberts, R. J., Vincze, T., Posfai, J. & Macelis, D. (2003b). REBASE: restriction enzymes and methyltransferases. Nucleic Acids Res 31, 418420.
Schouler, C., Clier, F., Lerayer, A. L., Ehrlich, S. D. & Chopin, M. C. (1998). A type IC restriction-modification system in Lactococcus lactis. J Bacteriol 180, 407411.
Sharp, P. M., Kelleher, J. E., Daniel, A. S., Cowan, G. M. & Murray, N. E. (1992). Roles of selection and recombination in the evolution of type I restriction-modification systems in enterobacteria. Proc Natl Acad Sci U S A 89, 98369840.
Simpson, A. J., Reinach, F. C., Arruda, P. & 113 other authors (2000). The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis. Nature 406, 151157.[CrossRef][Medline]
Skrzypek, E. & Piekarowicz, A. (1989). The EcoDXX1 restriction and modification system: cloning the genes and homology to type I restriction and modification systems. Plasmid 21, 195204.[Medline]
Suri, B. & Bickle, T. A. (1985). EcoA: the first member of a new family of type I restriction-modification systems. Gene organization and enzymatic activities. J Mol Biol 186, 7785.[Medline]
Titheradge, A. J., Ternent, D. & Murray, N. E. (1996). A third family of allelic hsd genes in Salmonella enterica: sequence comparisons with related proteins identify conserved regions implicated in restriction of DNA. Mol Microbiol 22, 437447.[Medline]
Titheradge, A. J., King, J., Ryu, J. & Murray, N. E. (2001). Families of restriction enzymes: an analysis prompted by molecular and genetic data for type ID restriction and modification systems. Nucleic Acids Res 29, 41954205.
Tomb, J. F., White, O., Kerlavage, A. R. & 22 other authors (1997). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539547.[CrossRef][Medline]
Tyndall, C., Meister, J. & Bickle, T. A. (1994). The Escherichia coli prr region encodes a functional type IC DNA restriction system closely integrated with an anticodon nuclease gene. J Mol Biol 237, 266274.[CrossRef][Medline]
Wang, Y. & Taylor, D. E. (1990). Natural transformation in Campylobacter species. J Bacteriol 172, 949955.[Medline]
Wassenaar, T. M., Wagenaar, J. A., Rigter, A., Fearnley, C., Newell, D. G. & Duim, B. (2002). Homonucleotide stretches in chromosomal DNA of Campylobacter jejuni display high frequency polymorphism as detected by direct PCR analysis. FEMS Microbiol Lett 212, 7785.[CrossRef][Medline]
Weiserova, M. & Firman, K. (1998). Isolation of a non-classical mutant of the DNA recognition subunit of the type I restriction endonuclease R.EcoRI24I. Biol Chem 379, 585589.[Medline]
Weiserova, M., Dutta, C. F. & Firman, K. (2000). A novel mutant of the type I restriction-modification enzyme EcoRI24I is altered at a key stage of the subunit assembly pathway. J Mol Biol 304, 301310.[CrossRef][Medline]
Wenman, W. M., Chai, J., Louie, T. J., Goudreau, C., Lior, H., Newell, D. G., Pearson, A. D. & Taylor, D. E. (1985). Antigenic analysis of Campylobacter flagellar protein and other proteins. J Clin Microbiol 21, 108112.[Medline]
Wilson, G. G. & Murray, N. E. (1992). Restriction and modification systems. Annu Rev Genet 25, 585628.[CrossRef]
Received 13 May 2004;
revised 28 October 2004;
accepted 12 November 2004.
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