The sequence of the stkP gene from 50 clinical isolates and 6 reference strains was determined. The stkP gene in each strain was amplified by PCR using oligonucleotides complementary to sequences at -10 and +1997 Birinapant of the gene. In each case, a 2007 bp DNA fragment was obtained and the nucleotide sequences confirmed that

they corresponded to stkP. There were 61 segregating sites (S) with a rate of segregating sites per site (pS) of 0.033, resulting in 27 allelic variants with an average of 10.26 nucleotides substitutions per sequence. Analysis of the encoded amino-acid sequences revealed 11 segregating sites (S) and a rate of segregating sites per site (pS) of 0.020, resulting in 12 allelic variants (including strain R6) with an average of 1.37 amino acid substitution per sequence (Additional file 1: Table ST1 and Figure 1). Thus, Dasatinib mouse the full-size StkP protein is well conserved in invasive and colonising clinical isolates and independent of their penicillin-resistance character. Figure 1 Inference of phylogenetic history of StkP from 56 strains using the Maximum Parsimony method. A number was given to each branch corresponding to the StkP alleles. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates)

are shown next to the branches. We considered PASTA domains and kinase domains individually: nucleotide divergence was higher in the 5′ terminal part of the

gene encoding the kinase module (d = 0.0072; S.E.: 0.0013) than in the 3′ part of the gene encoding the PASTA modules (d = 0.0048; S.E.: 0.0011). By contrast, GBA3 amino acid divergence was higher in the PASTA domains (d = 0.0037; S.E.: 0.0011) than in kinase domain (d = 0.0012; S.E.: 0.0007). The distribution of the amino acid allelic variants of StkP into penicillin-resistance classes was assessed (Figure 1): alleles 2, 3, 5, 6, 7, 8, 10 and 11 were found in penicillin-susceptible strains and alleles 1, 4, 9 and 12 were found both in penicillin-resistant and -sensitive strains (Additional file 1: Table ST1). The StkP amino acid sequence divergence was similar among penicillin-susceptible strains (d = 0.0027; S.E.: 0.0009), penicillin-intermediate strains (d = 0.0015; S.E.: 0.0009) and highly resistant strains (d = 0.0017; S.E.: 0.0011). To evaluate the effects of the StkP mutations on its kinase, a model of the enzymatic domain, amino acid 4 to 274, based on the sequence of the strain R6 was developed (Accession number: NP_359169) (Figure 2). The mutations carried by the various alleles were located outside of the catalytic site and appeared unlikely to affect the ATP binding site. Thus, these clinical isolates are unlikely to carry loss of kinase function mutations. Figure 2 Predicted structure of the kinase catalytic domain of StkP. (A) Image of backbone with oxygens of the StkP kinase domain (4–274).