BMC Genomics 2007, 8:110 http://www.biomedcentral.com/1471-2164/8/110 Table 2: Summary of selected SSH sequences SSH Length (bp) Best BLASTX match/comments [GenBank accession number] %ID Length (AA) E-value 629-D8 629-D9 670-H5 670-2 1967-D4 1967-F11 629-E2 961-E7 961-HG5 961-D5 1967-H9 1967-C2 629-C10 1967-A5 961-A9 670-D7 1116 >502 116 365 272 369 1001 750 160 385 382 310 1213 364 678 466 Best match with another genus extracellular solute-binding protein, family 1/ABC transporter (Rhodopseudomonas palustris BisB5) [ABE40377] phosphodiesterase (Delftia acidovorans) [AAN52089] Best match with C. jejuni (<80% identity) Outer membrane lipoprotein MapA CJE1173 (C. jejuni RM1221) [AAW35499] conserved hypothetical protein CJE0262 (C. jejuni RM1221) [AAW34855] flagellar hook subunit protein (C. jejuni NCTC 11168) [CAB73715] Putative membrane protein CJE0032 (C. jejuni RM1221) [AAW34630] putative periplasmic protein (C. jejuni 81–176) [EAQ73077] capsular polysaccharide biosynthesis protein CJE1602 (C. jejuni RM1221) [AAW36035] putative sugar transferase, capsule-related (C. jejuni 176.83) [CAI39731] putative glycosyltransferase, LOS-related (C. jejuni ATCC 43446) [AAX33825] Metabolism using alternative terminal electron acceptors anaerobic dimethyl sulfoxide reductase chain B, DmsB, Cju35 (C. jejuni 81–176) [ABF83738] probable pyridine nucleotide-disulfide oxidoreductase YkgC (C. jejuni 84-25) [EAQ95476] cytochrome C biogenesis protein, Cju05 (C. jejuni 81–176) [ABF61591]; gamma- glutamyl transpeptidase, Cju06 (C. jejuni 81–176) [ABF61592] Other SSH sequences used in the distribution analysis serine protease, subtilase family (C. jejuni 81–176) [AEQ72903] dTDP-glucose pyrophosphorylase (C. jejuni subsp. doylei 269.97) [EAU03120] putative nickase, cpp17, plasmid pTet/pCC31 – related (C. jejuni HB93-13) [EAQ59637] 38 42 70 76 60 56 52 29 47 52 100 100 100 100 100 96 99 234 107 31 75 86 107 209 240 51 126 109 102 120 154 155 211 118 3e-36 7e-16 4e-4 2e-27 3e-18 6e-20 1e-36 3e-17 9e-6 4e-31 8e-62 1e-51 e-108 1e-92 8e-32 3e-74 5e-77 SSH sequences matching outside the genus Campylobacter, sharing less than 80% identity in BLASTX searches with Campylobacter proteins, or used in distribution analysis, were included in the table. A full list of SSH sequences is included in Additional file 1. %ID; % protein sequence identity increased [22]. Because of our use of single restriction digestion conditions for each subtraction, we cannot claim the extent of coverage achieved by Agron et al. [22], but the increased proportion of repeat sequences obtained with the dual driver subtractions is indicative of a reduc- tion in the overall pool of tester-specific sequences in these subtractions. It is our belief that we have detected a significant proportion of the genetic variation between the strains and that our data indicate that there are very few genes present in C. jejuni strains with best BLASTX matches outside the genus rather than with already sequenced genes/proteins from within the genus. SSH sequences matching outside the genus Campylobacter The two sequences matching outside the genus Campylo- bacter were a putative transport protein (SSH629-D8) and a putative phosphodiesterase (SSH629-D9) (Table 2). These SSH sequences have been submitted to GenBank (EF076761 and EF076762). The predicted protein sequence derived from SSH629-D8 matched a transport protein from Rhodopseudomonas palustris, predicted to be a cytoplasmic component of an ABC-type sugar transport system. The gene encoding the phosphodiesterase enzyme from Delftia acidovorans matching SSH629-D9 has been cloned and expressed in Escherichia coli. It shares sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activ- ity on cAMP in vivo [25]. Using dot-blot hybridization, we determined the distribution of these two SSH sequences amongst a panel of C. jejuni isolates representative of common MLST clonal complexes and various sources (Table 3). The sequences SSH629-D8 and SSH629-D9 were found only in the ST-45 complex (Tables 3 and 4). It should be noted that SSH629-D8 appears to be part of a pseudogene containing a frame-shift mutation. We used PCR amplification to amplify this region from four of the C. jejuni isolates testing positive for SSH629-D8, includ- ing strain 629, and confirmed that this mutation is genu- inely present and not an artefact of the SSH procedure. Genomic islands and mobile elements The distributions of SSH sequences according to the func- tion of the matching proteins varied considerably between strains, reflecting the divergence amongst those strains chosen (Table 1). The SSH data suggest that the ST- Page 4 of 11 (page number not for citation purposes)