Table 1 Allelic variation in 8 housekeeping genes Locus Polymorphic see more sites GC% content (mol%) d N d S d N /d S * carB 4 44.09% 0.0100 0.2852 0.0349 groEL 5 46.24% 0.0000 0.0556 0.0000 murC 9 44.90% 0.0077 0.2467 0.0313 pheS 5 45.26% 0.0012 0.0900 0.0130 pyrG 8 43.12% 0.0016 0.1356 0.0114 recA 3 48.31% 0.0025 0.2399 0.0104 rpoB 7 43.97% 0.0018 0.0715 0.0245 uvrC 6 43.68% 0.0028 0.2684 0.0103 *The ratio of non-synonymous (d N ) and
synonymous (d S ) substitutions is indicative of selective pressure on loci. Table 2 Genes and sequencing primers used Gene Protein PCR primers Amplicon size (bp) Location* pyrG CTP synthase 5′-AGCAAACACCCAAGAACG-3′ 598 481322 to 482935 5′-TGGTGAAGCGAAGACAAA-3′ rpoB DNA-directed RNA polymerase subunit beta 5′-CACTGTGCGGTCGTCTTCC-3′ 608 1798123 to 1801731 5′-GCGTTCTCCTGGTATCTATT-3′ groEL Chaperonin GroEL 5′-CGGTGATAAGGCTGCTGT-3′ 892 1734716 to 1736335 5′-TTTGTTGGGTCCACGATA-3′ recA Recombinase A 5′-GGAGTCGTTTCTGGGTTAC-3′ 550 555064 to 556221 5′-GTTGCTTTAGGCGTTGGTG-3′ uvrC Excinuclease ABC subunit C 5′-AGAAATACAAGCCGTACTACAA-3′ 560 483053 to 484852 5′-TCTTCATCAGCGGAACCAA-3′ carB Carbamoyl phosphate synthase large subunit 5′-ATGGGTTGTGGGAGTTGTA-3′ 833 1202174 SC75741 in vitro to 1205353 5′-ACTTGTTGCGTCGTGGTGT-3′ murC UDP-N-acetylmuramate-L-alanine ligase 5′-TTTCATAGGCGAACTCAT-3′
619 679802 to 681136 5′-GTGCCATTGTTTGGTCAG-3′ pheS Phenylalanyl-tRNA selleckchem synthetase subunit alpha 5′-TTTCTTAGGTTTAGGCTTTG-3′ 665 406737 to 407813 5′-CCTTTCGGTTAAATTGTGA-3′ *Positions correspond to the complete genome sequence of Leu. mesenteroides subsp. mesenteroides ATCC 8293. Recombination in L. lactis The level of linkage disequilibrium between all alleles of the isolates evaluated was high as the calculated I A S was 0.4264 (p = 0.000) and significantly different from the I A S value of 0 expected for a population Florfenicol with linkage equilibrium, indicating the genes investigated in this study were close to linkage disequilibrium. Split decomposition analysis to examine evolutionary relationships amongst the isolates revealed different structures in the split
graphs for all eight loci (Figure 1A). In the split graphs for murC, pheS, pyrG and uvrC, the parallelogram-shaped structures detected indicated that intergenic recombination had occurred during the evolution of these four genes. The split graphs obtained for carB, groEL, recA and rpoB loci revealed tree-like structures, suggesting that the descent of these genes was clonal and not significantly affected by intergenic recombination. The split graphs of the recA and carB genes were a polygonal line and columnar respectively because only three (recA) or four (carB) alleles were analysed.The combined split graph of alleles for all eight MLST loci displayed a network-like structure (Figure 1B). The 20 STs representing all isolates were divided into two main subpopulations and each subpopulation was completely disconnected.