For P1sarA, the phosphorylation of SarA by Stk1 (Fig. 3d), and, to a lesser extent, by SA0077 (data not shown) led to a delayed shift [2 and 0.8 μg required, respectively, for a complete shift vs. 0.5 μg when SarA is not phosphorylated (Fig. 3c)]. Concerning PfnbA and Prot, phosphorylation of SarA by SA0077 induced a delayed shift (Fig. 4b and d) compared with the unphosphorylated SarA conditions (Fig. 4a and c), whereas phosphorylation by Stk1 had no effect (data not shown). SarA was also incubated with Stk1-K39 and SA0077-K152, two mutants that are unable to autophosphorylate, and thus, are unable to phosphorylate
any substrate. As expected, no difference was observed between unphosphorylated SarA and SarA incubated with each of these two mutant kinases, showing that neither Stk1 nor SA0077 interacts with the different promoters tested. The main result of this study CB-839 datasheet is the demonstration, for the first time, that the S. aureus virulence regulator SarA is phosphorylated both in vivo and in vitro. SarA is a global transcriptional regulator of numerous virulence determinants produced by S. aureus (Wolz et al., 2000; Cheung et al., 2004, 2008b), which is expressed constitutively (Bayer et al., 1996; Manna et al., 1998; Blevins et al., 1999). SarA activates Fnb expression during the buy Neratinib exponential growth phase.
These data suggest that SarA is mainly activated in early growth. Wolz et al. (2000) hypothesized that a post-translational modification of SarA may occur during various stages of
the growth cycle. It would therefore be interesting to know in which growth phase Stk1 or SA0077 is expressed. Although SarA regulates virulence genes in a growth-phase-dependent manner, especially in the late exponential phase and in the beginning of the stationary phase, still the intracellular amount of SarA remains constant throughout growth (Blevins et al., 1999). In any case, the mechanism that controls its own activity remains to be determined. In this regard, two different hypotheses have been proposed recently, suggesting that SarA activity is controlled by either its binding to some specific protein or its post-translational modification (Blevins et al., 1999; Wolz et al., 2000; Schumacher et al., 2001; 3-oxoacyl-(acyl-carrier-protein) reductase Bronner et al., 2004). Our data support the latter hypothesis by showing that SarA is phosphorylated in vivo. Furthermore, it is unable to autophosphorylate, but can be intensely phosphorylated in vitro by both Stk1, mainly at threonine residues, and SA0077, mainly at serine residues. These results strongly suggest that there exists a tight and selective regulation of SarA by phosphorylation catalyzed by both Ser/Thr kinases of S. aureus. MS was used to determine the phosphorylated sites on SarA, but no significant result could be obtained. However, phosphorylation on seryl residues by SA0077 led to a decreased ability of SarA to bind DNA.