We have to postulate therefore that CB-5083 order SA1665 may modulate β-lactam resistance in a mecA-independent manner, by controlling cellular functions affecting resistance levels. Experiments to determine the SA1665 regulon are ongoing. The impact of deleting SA1665 in MRSA was extremely strain specific, underlining the importance of the genetic background in governing the final methicillin resistance levels of MRSA,

and demonstrating www.selleckchem.com/products/tpx-0005.html the large genomic variability between different strain lineages. Conclusion SA1665 is a previously uncharacterised DNA-binding protein that has a negative effect on β-lactam resistance in MRSA. The SA1665 protein was identified in a DNA-binding protein purification assay, SB525334 molecular weight in which it bound to a DNA fragment covering the mec operator region. However, while nonpolar deletion of SA1665

was shown to increase oxacillin resistance levels in several heterogeneously resistant MRSA, its deletion had no effect on mecA transcription or PBP2a production. Therefore the negative impact of SA1665 on methicillin resistance is most likely to be through the regulation of other chromosomal factors or cellular functions required for methicllin resistance. Methods Strains and growth conditions Strains and plasmids used in this study are listed in Table 1. Clinical isolates are from the IMM collection in Zurich, Switzerland. Strains were grown at 37°C in Luria Bertani (LB) broth, shaking at 180 rpm, or on LB agar. Media were supplemented with the following antibiotics when appropriate: 25 or 50 μg/ml kanamycin, 10 μg/ml chloramphenicol, 5 or 10 μg/ml tetracycline, 100 μg/ml ampicillin. Concentrations of cefoxitin used for transcriptional induction were either sub-inhibitory (4 μg/ml) or inhibitory (120 μg/ml). Table 1 Strains and plasmids used in this study. Strain/plasmid Relevant genotype a Reference/source S. aureus        CHE482 clinical MRSA isolate, CC45/ST45, SCCmec N1, blaZ (pBla) [23, 24]    ΔCHE482 CHE482 ΔSA1665 this study    ZH37 clinical MRSA isolate, CC45/ST45, SCCmec type IV, blaZ [24]

   ΔZH37 G protein-coupled receptor kinase ZH37 ΔSA1665 this study    ZH44 clinical MRSA isolate, CCT8/ST8, SCCmec type II, aac-aph [24]    ΔZH44 ZH44 ΔSA1665 this study    ZH73 clinical MRSA isolate, CC22/ST22, SCCmec type IV, blaZ [24]    ΔZH73 ZH73 ΔSA1665 this study    RN4220 NCTC8325-4, restriction negative [38] E. coli        DH5α restriction-negative strain for cloning Invitrogen    BL21 (DE3) F- ompT hsdSB(rB -mB -) gal dcm (DE3) Novagen Plasmids        pBUS1 S. aureus-E. coli shuttle vector, tetL [37]    pAW17 S. aureus-E. coli shuttle vector, aac-aph [37]    pKOR1 S. aureus-E. coli shuttle vector, cat, bla [34]    pME17 pKOR1-SA1664/SA1666, cat this study    pET28nHis6 E. coli protein expression vector, with n-terminal His6 tag, aac-aph D.