BMC Genomics 2007, 8:110 http://www.biomedcentral.com/1471-2164/8/110 plexes, have a more restricted distribution amongst differ- ent host animals, including humans [4,7]. The complete genome sequences have been published for the C. jejuni strains NCTC11168 [8], RM1221 [9] and C. jejuni strain 81–176 [10], including its pTet and pVir plas- mids [11]. Genome sequences have also been published for strains of C. lari, C. coli and C. upsaliensis [9] and fur- ther genome sequence projects for several other Campylo- bacter strains are ongoing [12]. Although unpublished, the genome of C. fetus is also complete. In addition, strain- specific DNA sequences have been identified for C. jejuni ATCC43431 [13]. There has been considerable interest in characterising genetic variation between isolates of C. jejuni with a view to identifying those genes relevant to severity of disease or host colonisation. Inter-strain variations in loci such as those encoding lipooligosaccharide (LOS) [14], capsule [14] or restriction-modification (RM) systems [15] have already been characterised. Comparative genome analyses using microarrays, based largely upon the strain NCTC11168 genome sequence [16-19], indicate high lev- els of genome diversity but low levels of genome plasticity in C. jejuni [20]. Recently it has been suggested that this kind of approach can help to identify genetic markers pre- dictive of the source of an infection [21]. Since these studies were mostly restricted to those genes present in a single strain, NCTC11168, further identifica- tion of such markers would be greatly facilitated by the construction of microarrays containing all potentially var- iable genes. Genes contributing to plasticity amongst C. jejuni populations have been identified from the genomes of strains such as RM1221, 81–176 and ATCC43431 [9,11,13,16]. However, it is not clear whether the widely accessible nucleotide and protein sequence databases are representative of the variable genes that occur in C. jejuni. Suppression subtractive hybridisation (SSH) is a method designed to identify sequences present in one strain (the TESTER) but absent from a reference strain (the DRIVER) [22,23] and has been applied previously to identify genetic differences between two C. jejuni human isolates with different colonisation potentials [24]. In this study we used several rounds of SSH between and within C. jejuni MLST clonal complexes, using isolates from various sources, in order to gain a better understanding of the genomic variability that remains uncharacterised in pop- ulations of C. jejuni. Furthermore, we tested the hypothe- sis that the distribution of variable genes involved in the use of alternative terminal electron acceptors may be related to host preference. Results and discussion SSH libraries Our initial choice of restriction enzymes for the digestion of DNAs in the SSH was based on a previous study of C. jejuni [24]. A summary of all of the SSH sequences obtained is shown in Table 1. The SSH libraries obtained with AluI/DraI-digested DNAs were dominated by very short DNA sequences (92% were <400 bp). In order to obtain longer sequences we carried out two further sub- tractions using RsaI-digested DNAs. This reduced the pro- portion of sequences <400 bp in length. The majority of subtracted sequences from strains 670, 504 and 1967 matched sequences previously found in C. jejuni strains RM1221 or 81–176. Thus, in an attempt to enrich for sequences not previously reported, the final two subtrac- tions were carried out with strain 1967 (ST-42 complex) as an additional driver to strain NCTC11168 (ST-21 com- plex). This proved successful in that the majority of SSH sequences no longer matched strains RM1221 or 81–176. However, the overall proportion of SSH sequences match- ing previously reported Campylobacter sp. was not reduced significantly. The combination of enrichment and the choice of an enzyme giving longer SSH fragments resulted in fewer SSH sequences overall (Table 1). In total, 195 subtracted sequences were obtained. Details of these are given in Additional File 1. These SSH sequences were all confirmed as absent from the driver strain C. jejuni NCTC11168, even though occasionally the best BLASTX match in the database was against this strain. Furthermore, the second driver strain, strain 1967, was PCR-negative for nine of 11 sequences tested from the two dual driver subtractions. Of the 195 SSH sequences obtained only two (1%) had a best BLASTX match with proteins from outside the genus Campylobacter. A further three SSH sequences (2%) had no significant BLASTX match. Thus, the vast majority of SSH sequences matched within the genus Campylobacter. However, it should be noted that eight SSH sequences (4%), although having a best BLASTX match with a Campylobacter sp. protein, shared less than 80% identity with the matching protein. Those SSH sequences matching outside the genus Campy- lobacter, matching Campylobacter sequences with <80% identity, or used in distribution analysis are shown in Table 2. Both the mean and the median % GC content for the 195 subtracted sequences was 29%, with a range of 18–42%. The mean % GC contents varied little between the sub- tractions (28.9–30.8%). Thus, the average % GC contents for the subtracted sequences was only slightly below the values reported for the genomes of the C. jejuni strains NCTC11168 (30.6%) [8] or RM1221 (30.3%) [9]. Page 2 of 11 (page number not for citation purposes)