However, ZnSO4 and CoCl2 did not cause substantial up-regulation

However, ZnSO4 and CoCl2 did not cause substantial up-regulation of the four genes. A bacterial two-hybrid system was used to determine whether there was an interaction of McsA protein with McsB protein or with CtsR protein (Borloo et al., 2007). pB2H∆ω-mcsA and pB2H∆α-mcsB, or pB2H∆α-ctsR was cotransformed into E. coli MC1061 and then tested for β-galactosidase activity. Sirolimus Escherichia coli MC1061 with plasmid pB2HΔω-mcsA co-expressed with pB2HΔα-ctsR gave activity of 1218.5 ± 55 nmol

of ONP formed min−1 mg−1 of protein. Escherichia coli MC1061 with plasmid pB2HΔω-mcsA co-expressed with pB2HΔα-mcsB gave an activity of 1088.3 ± 204 nmol ONP formed min−1 mg−1 of protein. β-Galactosidase activity was not found in E. coli MC1061 co-expressed with pB2HΔα/pB2HΔω Veliparib concentration (negative control). Lower β-galactosidase activity was detected when pB2HΔω-ΔmcsA

co-expressed with pB2HΔα-ctsR (105 ± 10 nmol ONP formed min−1 mg−1 of protein) and pB2HΔω-ΔmcsA co-expressed with pB2HΔα-mcsB (70.5 ± 36 nmol ONP formed min−1 mg−1 of protein). In this study, McsA from S. aureus containing four CXXC metal-binding motifs was investigated. Previous studies with others proteins have shown that the paired cysteine residues in the metal-binding motifs of various organisms are involved in heavy metal binding and transporting (Nash & Mowatt, 1993; Walker et al., 2002, 2004; Sitthisak et al., 2007; Agarwal et al., 2010). The CXXC motif in the metal-binding domain from CopA and CopZ of S. aureus can bind specifically to copper, cobalt, and cadmium (Sitthisak et al., 2007). The CXXC motif in McsA bound

copper, cobalt, cadmium, and zinc, which is consistent with previous reports. Six of the eight conserved cysteines in the CXXC motifs of McsA protein were changed into alanine. Our data demonstrated that copper still binds to the nonmutated CXXC motif (C87XXC90) in the ΔMcsA. These data are in agreement with previous studies that the determination of metal-binding activity by a mutated pheromone CXXC shows that the CXXC domain requires two conserved cysteine ligands provided by one CXXC motif to bind copper ions (Lutsenko et al., 1997), while four conserved cysteine ligands provided by two CXXC motifs are required to bind zinc ions (Allen et al., 2006; Zimmermann et al., 2009). Gene expression of the ctsR operon was induced by heat, cold, osmotic pressure, and disulfide stress (Derre et al., 1999; Anderson et al., 2006; Bore et al., 2007; Fiocco et al., 2010; Elsholz et al., 2011). DNA microarray analyses showed that copper shock in S. aureus and disulfide stress in B. subtilis induces the expression of the genes in ctsR regulon (Leichert et al., 2003; Baker et al., 2010). In this study, qRT-PCR analyses showed that copper and cadmium induced expression of all four genes of the ctsR operon (Table 2). These data indicated that genes in the ctsR operon are heavy metal inducible. Metal ions are an important component in several regulatory proteins (Berg, 1990).

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