This protein was then purified to learn more homogeneity by affinity chromatography using an immobilized Zn2+ matrix. The purified fusion protein was subjected to specific cleavage by thrombin. The resulting tag-free SarA (14.7 kDa) was analyzed by gel electrophoresis and used

in all further experiments. Previous work in the laboratory (Debarbouille et al., 2009) showed that analysis of the genome sequence of S. aureus revealed the presence of two Ser/Thr kinases in S. aureus, which were overproduced and purified. The phosphorylating activity of SarA was first assayed by incubating the pure protein in the presence of radioactive ATP. After gel migration and autoradiography, no labeled band was detected, indicating that His6-SarA was unable to autophosphorylate (Fig. 2a and b, lane 2). In contrast, when Stk1 or SA0077 was added to the incubation medium, His6-SarA was intensely labeled (Fig. 2a and b, lanes

4 and 7). Furthermore, to ensure that the 16-kDa band was actually SarA, the histidine tag was previously removed from SarA, and the tag-free protein was then incubated with both the kinases. Once more, we did observe a shifted band (14 kDa) corresponding to this protein (Fig. 2a and b, lanes 5 and 8), indicating that the native SarA was effectively phosphorylated. The phosphorylation of SarA by Stk1 or SA0077 was then studied in more detail by analyzing its phosphoamino acid content. Under the conditions used, only acid-resistant phosphoamino acids were analyzed because a number of other phosphorylated compounds, such as phosphohistidine, phosphoarginine and phosphoaspartate, are known to be labile in acid. Interestingly, Galunisertib mw two-dimensional analysis of an acid hydrolysate of SarA showed that this protein was mainly phosphorylated at threonine residues by Stk1 [Fig. 2c(1)], whereas it was phosphorylated essentially at serine residues by SA0077 [Fig. 2c(2)]. SarA was previously described to bind several promoter regions to regulate different

genes involved in the virulence of S. aureus including accessory regulator gene promoter (P1sar) (Cheung et al., 2008a), accessory gene regulator promoter Sclareol (P2agr) (Chien & Cheung, 1998), regulator gene promoter (Prot) (Manna & Ray, 2007; Hsieh et al., 2008) and staphylococcal fibronectin gene promoter (PfnbA) (Wolz et al., 2000). To further investigate whether phosphorylated SarA interacts differently from nonphosphorylated SarA with its promoters, comparative gel shift assays were performed on promoters P2agr, PfnbA, Prot and P1sarA, with either unphosphorylated SarA or SarA phosphorylated by Stk1 (Fig. 3) or by SA0077 (Fig. 4). Concerning P2agr, a striking difference emerged between unphosphorylated SarA, which must be added in the amount of 8 μg to obtain a complete shift (Fig. 3a), and SarA phosphorylated by Stk1, where only 2 μg was sufficient to obtain the same shift (Fig. 3b).