Regulation of the expression of the β-lactam antibiotic-resistance determinants in methicillin-resistant Staphylococcus aureus (MRSA)

Abstract

β-Lactam antibiotics have faced obsolescence with the emergence of methicillin-resistant Staphylococcus aureus (MRSA). A complex set of events ensues upon exposure of MRSA to these antibiotics, which culminates in proteolysis of BlaI or MecI, two gene repressors, and results in the induction of resistance. We report studies on the mechanism of binding of these gene repressors to the operator regions by fluorescence anisotropy. Within the range of in vivo concentrations for BlaI and MecI, these proteins interact with their regulatory elements in a reversible manner, as both a monomer and a dimer.

Figure 1

Transcription of the genes for antibiotic-resistance determinants PBP2a and PC1 β-lactamase is regulated by MecI and BlaI. Red, blue, and green arrows represent the blaI/mecI, blaR1/mecR1, and blaZ/mecA genes, respectively. The gene repressor binding sites in each operon are highlighted in gray.

Figure 2

(A) MALDI mass spectrum of MecI at 12 μM. (B) Electrophoresis mobility shift assay of mec double-stranded DNA (150 nM) in the presence of increasing concentrations of MecI.

Figure 3

Fluorescence anisotropy measurements for (A) binding of MecI to the mec (●) and bla (○) operators and (B) binding of BlaI to the mec operator (●). The lines show the fit to the model given as the inset of panel A.