Further analysis of the locus was undertaken for 7 of these strains distributed in 5 clusters. Amplification obtained with primers designed on the basis of the L. sakei 23 K genome outside of sigH suggested that the genetic context is conserved in all these strains (see position of primers AML50 and AML58 in Figure 1). Polymorphism analysis of the sigH sequences brought additional information. As shown in Figure 3, 29 polymorphic sites were identified in the sigH CDS, of which only 9 were involved in 7 aa changes,

mostly conservative. Thus, SigH function Src inhibitor and coding gene location appear to be conserved in the L. sakei species. Figure 3 Polymorphic nucleotide sites of sigH sequences in L. sakei. The entire CDS sequence (561 nt) was analyzed with MEGA software http://​www.​megasoftware.​net/​. Only nucleotide residues different from the upper line sequence are written. The site numbers at the top are in vertical format. Letter-code genetic cluster according to Chaillou et al. [20] is indicated for each strain and reported subspecies are shaded differently. Polymorphic deduced aa are indicated under the sequence. L. lactis subspecies lactis and cremoris exhibit two comX allelic

types whose nucleotide divergence is at most 27.5% [21]. In contrast, sigH divergence (4.5% maximum divergence) was incongruent with the previously reported genotypic classification of L. sakei strains [20], PF299 and its two proposed subspecies (Figure 3). This discrepancy may be explained either by a particular evolutionary history of that gene in L. sakei or by the possibility second that the classification based on the flexible gene pool does not reflect the phylogenetic relationships between strains which remain to be established. High nucleotide divergence between species, contrasted with generally higher conservation within species, was also observed for sigH loci in the genus AR-13324 price Staphylococcus [22]. The reason for such high inter-species polymorphism

is unknown. However, rapid evolution after species divergence rather than lateral gene transfer may be responsible, as the phylogeny of sigH genes was reported to be concordant with species phylogeny in staphylococci [22]. As reported in this paper, functional studies were further conducted on RV2002, a derivative of L. sakei strain 23 K, for which genome data is available, and in which the endogenous β-galactosidase encoding gene is inactivated, thus enabling the use of a lacZ reporter gene [23]. Temporal transcription of sigH In B. subtilis, sigH Bsu transcription increases from mid-exponential to stationary phase [24]. We used quantitative PCR (qPCR) following reverse transcription to determine if sigH Lsa expression in L. sakei is also temporally regulated. L. sakei was cultivated in chemically defined medium (MCD) at 30°C and total RNA was extracted from cells 2 h after inoculation and every hour from 4 to 8 h.