A novel DNA-binding protein modulating methicillin resistance in Staphylococcus aureus

Abstract

Background: Methicillin resistance in Staphylococcus aureus is conferred by the mecA-encoded penicillin-binding protein PBP2a. Additional genomic factors are also known to influence resistance levels in strain specific ways, although little is known about their contribution to resistance phenotypes in clinical isolates. Here we searched for novel proteins binding to the mec operator, in an attempt to identify new factor(s) controlling methicillin resistance phenotypes.

Results: Analysis of proteins binding to a DNA fragment containing the mec operator region identified a novel, putative helix-turn-helix DNA-binding protein, SA1665. Nonpolar deletion of SA1665, in heterogeneously methicillin resistant S. aureus (MRSA) of different genetic backgrounds, increased methicillin resistance levels in a strain dependent manner. This phenotype could be fully complemented by reintroducing SA1665 in trans. Northern and Western blot analyses, however, revealed that SA1665 had no visible influence on mecA transcription or amounts of PBP2a produced.

Conclusion: SA1665 is a new chromosomal factor which influences methicillin resistance in MRSA. Although SA1665 bound to the mecA promoter region, it had no apparent influence on mecA transcription or translation, suggesting that this predicted DNA-binding protein modulates resistance indirectly, most likely through the control of other genomic factors which contribute to resistance.

Figure 1

DNA-binding protein purification assay using mec operator DNA region as a bait. A, Silver stained SDS-polyacrylamide protein gel containing the elutions from DNA-binding protein capture assays performed with either DNA-coated (+) or uncoated (-) streptavidin magnetic beads. One protein band, indicated by the arrow, was only captured by the DNA-coated beads, indicating that it bound specifically to the mec operator DNA. The protein size marker (M) is shown on the left. B, Organisation of the genomic region surrounding SA1665. The regions used to construct the deletion mutants are indicated by lines framed by inverted arrow, which represent the positions of primers used for their amplification. The chromosomal organisation, after deletion of SA1665 is shown beneath. The position of the SA1665 transcriptional terminator, which remained intact after SA1665 markerless deletion is indicated (⫯).

Figure 2

Electromobility shift of mec operator DNA by SA1665. A, Gel shift using biotinylated DNA (6 ng) and crude protein extracts. Lane 1, DNA only control; lanes 2 and 3, DNA incubated with 200 ng and 500 ng of crude protein extract from E. coli BL21 pET28nHis₆, respectively; lanes 4 and 5, DNA incubated with 200 ng and 500 ng of crude protein extract from E. coli BL21 pME20, expressing SA1665, respectively. B, Gel shift of biotinylated DNA (6 ng) with purified SA1665 protein. Lane 1, DNA only control; lanes 2–7 DNA incubated with 10, 40, 75, 150, 200 and 250 ng protein, respectively; lane 8, DNA incubated with 250 ng of protein in the presence of a 130-fold excess of unlabelled specific competitor DNA; lane 9, DNA incubated with 250 ng of protein in the presence of unspecific competitor (herring sperm) DNA.

Figure 3

Effect of SA1665 deletion on oxacillin resistance. A, Growth of MRSA strains and their SA1665 deletion mutants, containing empty plasmid vector pAW17 or pBUS1, and trans complemented mutants, containing pME26 or pME27, was compared on plates containing appropriate oxacillin gradients, as indicated. Plates were supplemented with either kanamycin (25 μg/ml) or tetracycline (5 μg/ml) to ensure plasmid maintainence. B, Representative population analysis profiles of MRSA strains CHE482, ZH37, ZH44, and ZH73 and their corresponding mutants. Wildtype strains are indicated by squares and mutants by triangles. x- and y-axis show the oxacillin concentrations (μg/ml) and the cfu/ml, respectively. Oxacillin concentrations used were two-fold dilutions ranging from 0.1–256 μg/ml for strains CHE482 and ZH37 and 1–1024 μg/ml for strains ZH44 and ZH73. C, Growth curves of wildtype strains (solid lines, closed symbols) and their corresponding SA1665 mutants (dashed lines, open symbols); CHE482 (diamonds), ZH37 (triangles), ZH44 (circles), ZH73 (squares).

Figure 4

Primer extension analysis of SA1665. A, Lanes A, C, G, T show the dideoxy-terminator sequencing ladder and lane RT the reverse transcription products obtained using primer me97. Two potential transcriptional start sites (TSS) were identified, as indicated by arrows (◀). B, Sequence of the SA1665 promoter region. TSS (+1) are shown in bold, putative -10 and -35 promoter sequences are underlined, the predicted ribosome binding site (rbs) is framed and the translational start (ATG) of SA1665 is highlighted in grey.

Figure 5

Northern and Western blot analyses. A, Transcription of SA1665 over growth in CHE482, with RNA harvested at the OD_{600 nm} values indicated. B, Transcription of SA1665 from CHE482 grown to OD_{600 nm} 0.25 and either left uninduced (-) or induced with either 4 or 120 μg/ml of cefoxitin fo 0', 10' and 30'. C, Transcriptional profiles of SA1664, SA1665, SA1666 and SA1667 in CHE482 and ΔCHE482, grown to OD_{600 nm} 0.25 and either uninduced or induced with cefoxitin 4 μg/ml for 0', 10' or 30'. Approximate sizes of transcripts, in kb, are indicated on the right of the blots. D, Transcription of mecA and mecR1 in CHE482 and ΔCHE482, grown to OD 0.25 and either left uninduced or induced with cefoxitin (4 μg/ml) and sampled after 0', 10' and 30'. Ethidium bromide stained 16S rRNA bands from all Northern gels are shown as a comparative indication of RNA loading. E, Western blots showing amounts of PBP2a in ZH44 and ZH73 and their respective SA1665 deletion mutants, before (0') and after induction with 4 μg/ml of cefoxitin for 10' and 30'.