, 2008). MsrR clusters in the main LCP subfamily (F1), which also contains Psr from enterococci (Rice et al., 2001); Vincristine price SA0908 and SA2103, both group in subfamily F2, together with BrpA from S. mutans (Wen et al., 2006) and LytR from B. subtilis (Lazarevic et al., 1992; Hubscher et al., 2008). In this study, we analysed the impact of each of the three S. aureus LCP proteins on various envelope-related characteristics and determined the extent to which these proteins can complement each other. The strains and plasmids used are listed in Table 1. Strains were grown in Luria–Bertani broth at 37 °C unless stated otherwise. Erythromycin (10 mg L−1), tetracycline (10 mg L−1), chloramphenicol (10 mg L−1)
or ampicillin (100 mg L−1) was added when http://www.selleckchem.com/products/bmn-673.html appropriate. Markerless sa0908 and sa2103 deletions were generated using the pKOR1 counter selection system (Bae & Schneewind, 2006), to obtain RH53 and PS47, respectively. The primers used are shown in Supporting Information, Table S1. The Δsa0908/ΔmsrR and Δsa2103/ΔmsrR double mutants, RH72 and PS60, were obtained by phage 85-mediated transduction of ΔmsrR∷ermB from strain JH100 into the corresponding single mutants. The Δsa2103/Δsa0908
double mutant PS110 was constructed by sequential markerless deletion of the genes. Transduction of ΔmsrR∷ermB into PS110 yielded the triple mutant PS111. Correct gene deletion profiles were confirmed by Southern blot and sequencing. The absence of major genomic rearrangements was demonstrated by pulsed-field gel electrophoresis. The sa0908 ORF and promoter region was amplified using the primers sa0908-compF PFKL and sa0908-compR (Table S1), digested with EcoRI and cloned into plasmid pGC2, to create plasmid pGC2sa0908. Primers sa2103-compF and sa2103-compR were used to amplify the sa2103 gene and promoter region, which was ligated into the SmaI site of pGC2 to create pGC2sa2103. Plasmid inserts were confirmed by sequencing. Total RNA isolation and Northern hybridization were performed as described previously (Hubscher et al.,
2009). The primers used for probe amplification are listed in Table S1. Primer extension reactions were performed as described in (McCallum et al., 2010). Reactions included 20 μg of total RNA from MSSA1112 that was grown to OD600 nm 1.0 and induced with 1 mg L−1 of oxacillin for 30 min and the 5′-biotin-labelled primers sa0908-pe1 and sa2103-pe1 (Table S1). RNA samples used for primer extension were harvested from cultures induced with oxacillin as this is known to induce the cell wall stress stimulon, hence increasing the transcript abundance of sa0908 and sa2103 (Dengler et al., 2011). The promoter regions of msrR, sa0908 and sa2103 were amplified using the primer pairs JR13/JR14 (Rossi et al., 2003), sa0908.lucF/sa0908.lucR (Dengler et al., 2011) and sa2103.lucF/sa2103.lucR (Table S1), respectively. Promoter fragments were digested with Asp718 and NcoI and ligated directly upstream of the promoterless luciferase (luc+) gene in vector pSP−luc+(Promega).