Diversity within the Campylobacter jejuni type I restriction–modification loci William G. Miller, 1 Bruce M. Pearson, 2 Jerry M. Wells, 3 Craig T. Parker, 1 Vladimir V. Kapitonov 4 and Robert E. Mandrell 1 Correspondence William G. Miller bmiller@pw.usda.gov 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 Received 13 May 2004 Revised 28 October 2004 Accepted 12 November 2004 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. INTRODUCTION 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 modifica- tion 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 59 half-site, of 3–4 bp, and a 39 half-site, of 4–5 bp, separated Abbreviations: ARD, amino-proximal recognition domain; BA, Brucella agar; CRD, carboxy-proximal recognition domain; GBS, Guillain–Barre ´ 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). 0002-7327 G 2005 SGM Printed in Great Britain 337 Microbiology (2005), 151, 337–351 DOI 10.1099/mic.0.27327-0