In N315 and Mu50, the ssl8 levels were similar to each other,
Lumacaftor but in a negligible amount when compared with the Newman strain (Fig. 1). When the expression levels of ssl5 and ssl8 were compared, they were found to be similar in RN6390 and FPR3757, but ssl8 expression was fourfold higher in the Newman strain compared with ssl5. Interestingly, MW2 had twofold higher ssl8 levels compared with ssl5, whereas MSSA476 showed sevenfold higher ssl5 levels compared with ssl8 levels. In contrast, Mu50 and N315 showed 17- and 10-fold higher ssl5 levels, respectively, compared with their ssl8 expression levels (Fig. 1). The differential expression of both ssl5 and ssl8 in different strains prompted us to see whether different haplotypes of ssl5 and ssl8 are present in these strains and whether they correlated with their differential expression. We sequenced ssl5, ssl8 and their 100 bp upstream regions from the seven clinical strains and various Newman mutant strains used in this study. Because the Newman strain had the highest expression of both ssl5 and ssl8 compared with the other clinical strains tested, the ssl5, ssl8 and their 100 bp upstream sequences obtained were compared with the respective genes of this strain to determine any allelic differences. Based on the respective comparison of ssl5 and ssl8 coding sequences of the seven
strains tested (Table 1), three haplotypes emerged. Haplotype A included Newman, FPR3757, and RN6390 strains; haplotype B included MW2 Seliciclib and MSSA476 strains; and
haplotype C included Mu50 and N315 strains (Figs 2a and 3a). For the ssl5 or ssl8 upstream sequence comparative analysis, three allelic forms were identified for each one. For both ssl5 and ssl8, allelic type A included the same three strains: Newman, FPR3757, and RN6390. However, for ssl5, allelic type B included MW2, MSSA476, and N315, whereas allelic type C included next Mu50 (Fig. 2b). For ssl8, allelic type B included MW2, Mu50, and N315, whereas allelic type C included MSSA476 (Fig. 3b). The ssl5 and ssl8 coding and promoter sequences showed several single nucleotide polymorphisms (SNPs) (Figs 2a, b and 3a, b). These SNPs and the corresponding amino acid change in the coding region were described in Supporting Information, Tables S1 and S2. There was no correlation between haplotypes or allelic types relative to ssl5 or ssl8 expression. The differential expressions of ssl5 and ssl8 within a haplotype with identical upstream sequences in strains such as Newman, RN6390, and FPR3757 suggested that their expression was influenced by additional factors (Fig. 1). Using Newman as the model strain because of its highest expression of ssl5 and ssl8, we determined the role of known regulatory elements, Agr, Sae, and SigB, in their expression.