BMC Genomics 2007, 8:110 http://www.biomedcentral.com/1471-2164/8/110 tains most of the diversity within this species. However, it should also be noted that genes currently associated with other closely related species, such as C. coli, can also make a contribution to the diversity within C. jejuni. ST-257 and ST-61) and different sources were used to study the distribution of subtracted sequences (Table 3). The bacteria were cultured on blood agar at 37°C under microaerophilic conditions. It is clear from our subtractions between ST-21 complex isolates that variation in gene content occurs within as well as between clonal complexes. However, our survey of strain-variable sequences, including four associated with genes involved in the use of alternative terminal electron acceptors, indicated a distribution according to clonal complex rather than host source. Thus, we found no evi- dence that the presence or absence of such genes plays a role in the host preferences of C. jejuni strains. Methods Bacterial strains The bacterial strains used in this study are listed in Table 3 and were isolated in a previous study [7]. Strains for SSH were chosen to enable comparisons within (ST-21 com- plex) and between MLST clonal complexes. In addition, isolates from diverse animal host sources were chosen (bird, cattle and rabbit; Table 1). These differed from the sources of previously sequenced strains of C. jejuni (human or chicken). A panel of isolates representing com- mon MLST clonal complexes (ST-21, ST-42, ST-45, ST-48, Table 5: Oligonucleotide primers used in this study Primer 1967-H9F 1967-H9R 1967-C2F 1967-C2R 264F2 264R2 629-C10F 629-C10R 629-D8F 629-D8R 629-D9F2 629-D9R 961-A9F 961-A9R 670-D7F 670-D7R FlaA1 FlaA2 Sequence (5' to 3') GGCGCACCTCAATTTAAC TCCATGTTCTTCTCTCAG GCCTTAGGGATTGCTACA CTTATGCTCCATTTAGGC ACTCACGTATCTTGCTTC ACTCGCTGCATTTTGAAG CAAGTATTATGGCCGGAA ATCCTATAACACTCGCAC AACTACGGATCCACCTACG AGCGGGGTGTATTTTGGAG TTGAGTAAAACGACAGC TGGCCTTAGAGCTATGGA CCAAATCCTCAGGTGTTC ACGAGGTTTAGATGGTTC TACTGATAATCCGCATTGC CTTGCATATTCACCCCTT GCATTTCGTATTAACACACAAATGGTGC CTGTAGTAATCTTAAAACATTTTG A.T.; annealing temperature. MLST MLST alleles, STs and clonal complexes were assigned using the Campylobacter PubMLST database [37] with sequences submitted for allele designation as appropriate. Construction and screening of subtraction libraries Genomic DNA for SSH was isolated from C. jejuni strains 670, 504, 1967, 629, 961 and NCTC11168 as described previously [38]. SSH was carried out using the CLON- TECH PCR-Select™ Bacterial Genome Subtraction Kit (Clontech) essentially as recommended by the supplier but with a hybridisation temperature of 58°C. In the first three hybridisations, DNAs from C. jejuni strains 670, 504 and 1967 respectively were used as tester and DNA from C. jejuni NCTC11168 was used as the driver. All DNAs were digested with AluI and DraI. A further two hybridisa- tions, using RsaI-digested DNA from C. jejuni strains 629 and 961 respectively as tester, were carried out with dual RsaI-digested driver DNAs from strains 1967 and NCTC11168. PCR amplicons obtained following SSH were cloned into pGEM-T (Invitrogen). The subtraction Amplicon size (bp) A.T. (°C) Target 157 172 859 459 339 249 326 414 1700 50 50 50 50 50 50 55 50 55 1967-H9 (Cju35) [dmsB] 1967-C2 Cj0264c 629-C10 (Cju05) [cytC locus] 629-D8 629-D9 961-A9 670-D7 flaA Page 9 of 11 (page number not for citation purposes)