These mutants were used to determine the precise roles ofMsr proteins in survival of S. aureus under a variety of stress conditions. The presented data suggest that MsrA2 and MsrA3 play little or no role in staphylococcal protection fromoxidative stress or inmice.However, the role of the msrA1/msrB locus is complex.While lack of MsrA1 increases the sensitivity of S. aureus to oxidative stress and host immune defense, the lack of MsrB, to some extent, is actually beneficial to the bacterial organism under these conditions. To construct a mutation in msrA1 and msrB genes simultaneously, flanking regions were PCR amplified and ligated. Briefly, primer pairs P1 and P2 were used to amplify a 1449 bp DNA fragment. Another set of primers P3 and P4 were used to amplify an 841 bp DNA fragment. These two fragments were ligated in vector pTZ18R which simultaneously engineered a unique BamHI site between the ligated fragments to which a 1.7 kb kanamycinresistance cassette was cloned. This fragment was used to construct a deletion CHIR-99021 mutant in S. aureus utilizing the methodology described previously for the construction of individual msrA1 and msrA2 mutants. To construct an msrA3 mutant, primers P5 and P6 were used to amplify a 1084 bp DNA fragment upstream of msrA3. Another set of primers, P7 and P8, were used to amplify a 1047 bp msrA3 downstream fragment. These two fragments were ligated together in vector pTZ18R to generate a unique BamHI restriction site between the fragments to which a 1.4 kb erythromycin-resistance cassette was cloned. The above construct was used as a suicidal plasmid to construct a mutation in the msrA3 gene utilizing a method described previously. For in vitro and in vivo studies, the S. aureus strain SH1000, which is a sigB positive derivative of the S. aureus strain RN450, was used. Since most MRSA strains are naturally resistant to tetracycline and or erythromycin, a S. aureus MRSA strain BB270 was used to combine msr mutations for antibiotic resistance studies. The individual msr mutants were combined in these two S. aureus strains to generate a triple and a quadruple mutant. S. aureus produces three different MsrA proteins and one MsrB protein. MsrA1 and MsrB production in S. aureus are induced by cell wall-active antibiotics. In the presence of these ABT-199 clinical trial antibiotics, the cell wall is likely destabilized and the oxidizing agents have easy access to bacterial membrane and cytosolic compartments. In response, the staphylococcal cells produce a higher level of MsrA1 and MsrB; however, oxidative stress has not been shown to induce the synthesis of these proteins in S. aureus. In addition to these four Msr proteins, there is an additional gene in S. aureus that codes for a protein that reduces the free methionine sulfoxide. Although the structural and biochemical properties of this protein have been determined, its physiological relevance is unclear. The extent of expression of S. aureus fRMsr is also not clear. The fRMsr gene in S. aureus may be expressed at a very low level since there was no detectable Msr activity in the msrAB quadruple mutant. Studies with the individual msr gene mutants make it clear that the MsrA2 and MsrA3 contribute little to cellular Msr activities, play a little to no role in protecting S. aureus from oxidative stress and neutrophils, and have no impact on bacterial survival in mice. Using promoter fusion experiments, we have previously shown that msrA2 and msrA3 are expressed at significantly lower levels compared to the expression of the msrA1-msrB locus in S. aureus.