Figure 1.
The mecA regulatory locus is a three-component system.
(A) Genetic organization of the mecA regulatory locus in major SCCmec types I–V and prototype MRSA strains used in this study. The magnified DNA sequence shows the two mecR2 start codons in SCCmec type III and III (boxed), the stop codon (boxed), the four-thymine deletion in SCCmec type II (underlined), and the putative terminator (inverted arrows). (B) Multiple sequence alignment between the repressor of the xylose operon (XylR) and MecR2 from prototype SCCmec types II and III strains. Green – identical residues; red – similar residues; white – divergent residues. The figure was prepared using “The Sequence Manipulation Suite” freely available at http://www.bioinformatics.org.
Figure 2.
mecR2 interferes with the mecI-mediated repression of β-lactam resistance.
(A) Co-overexpression of mecI and mecR2 region (COL+mecI-mecR2) reverted the effect of mecI overexpression (COL+mecI) on the oxacillin-resistance phenotype in strain COL, as evaluated with diffusion disks containing 1 mg of oxacillin. (B) Northern blotting analysis of mecA transcription shows that in the presence of mecR2 locus the repressor effect of mecI is reverted.
Figure 3.
Reconstruction of the mecA regulatory locus in prototype strain COL.
Reconstruction of the mecR1-mecI locus in the chromosome of strain COL (COL::RI) causes a decrease of the resistance level to oxacillin, which can be reverted by the reconstruction of the full mecA regulatory locus, mecR1-mecI-mecR2 (COL::RI-R2). Control experiments with mecI overexpressed in trans (COL::RI+mecI and COL::RI-R2+mecI) demonstrate that the functions of mecR1 and mecR2 are not affected by high levels of MecI. For comparative purposes the overexpression of mecI in parental strain COL (COL+mecI) is also shown. The oxacillin-resistance levels were evaluated by diffusion disks containing 1 mg of oxacillin (left) or by population analysis profiles (PAP's) (right).
Figure 4.
Role of mecR2 on the optimal expression of β-lactam resistance.
(A) Deletion of mecR2 from the chromosome of strain N315 (N315::ΔmecR2) causes a decrease on the resistance level to oxacillin, which can be reverted upon complementation with mecR2 expressed from an inducible promoter (N315::ΔmecR2+spac::mecR2) in the presence of the inducer (IPTG 100 µM). (B) The poor expression of oxacillin resistance by recombinant strain COL::RI, can also be reverted upon complementation with mecR2 expressed from an inducible promoter (COL::RI+spac::mecR2) in the presence of the inducer (IPTG 100 µM). The oxacillin-resistance levels were evaluated by diffusion disks containing 1 mg of oxacillin (left) and by population analysis profiles (PAP's) (right).
Figure 5.
Effect of mecR2 on the induction of mecA transcription.
(A) Northern blot and (B) qRT-PCR analysis of the mecA induction profile in parental strain N315, mecR2 null-mutant (N315::ΔmecR2) and complemented mutant (N315::ΔmecR2+spac::mecR2, IPTG 100 µM). Cultures were induced with a sub-MIC concentration of oxacillin (0.05 mg/L) and samples were taken at 0′, 5′, 10′ 30′ and 60′.
Figure 6.
qRT-PCR analysis of the mecR2 induction profile in parental strain N315 and its complemented mecR2 mutant (N315::ΔmecR2+spac::mecR2, IPTG 100 µM). Cultures were induced with a sub-MIC concentration of oxacillin (0.05 mg/L).
Figure 7.
mecR2 is essential for the optimal expression of β-lactam resistance in strains with functional mecI-mecR1 regulatory locus.
(A) Deletion of mecR2 from the chromosome of prototype epidemic strains USA100, USA200 and USA600 harboring SCCmec type II causes a decrease on the resistance level to oxacillin, which can be reverted upon complementation with mecR2 expressed from an inducible promoter (spac::mecR2) in the presence of the inducer (IPTG 100 µM). (B) Northern blot analysis of the mecA induction profile in parental strains USA100, USA200 and USA600 and respective mecR2 null-mutants. Cultures were induced with a sub-MIC concentration of oxacillin (0.05 mg/L) and samples were taken at 0′, 10′ and 60′. For comparative purposes the profile of parental strain N315 and mecR2 null-mutant were also repeated. Note that film was exposed for 4 h whereas in Figure 5A it was exposed for 48 h.
Figure 8.
The mecR2 function is not dependent of mecR1 neither of the β-lactamase locus.
(A) Prototype strain HT0350 is negative for mecR1-mecI and for the β-lactamase locus. Co-overexpression of mecI and mecR2 in strain HT0350 (HT0350+mecI-mecR2), reverts the effect of mecI overexpression (HT0350+mecI). (B) The strategy used to delete mecR2 in prototype strain N315 generated an intermediate mutant that has lost the β-lactamase plasmid. The chromosomal mecR2 deletion was then transduced back to the parental strain generating a β-lactamase positive mecR2 null mutant. In both variants, the deletion of mecR2 caused a sharp decrease of the resistance level to oxacillin. (C) Prototype strain HU25 is positive for mecR2 and the β-lactamase locus and has a truncated non-functional MecI and, as such, the mecA expression is under the exclusive control of bla regulatory genes. Deletion of mecR2 in strain HU25 (HU25::ΔmecR2) has no effect on the phenotypic expression of oxacillin resistance.
Figure 9.
MecR2 interacts directly with MecI, interfering with the binding of MecI to the mecA promoter and fostering the proteolysis of MecI.
(A) In vivo analysis of the MecR2::MecI interaction using the bacterial two-hybrid strategy. This strategy is based on the restoration of the adenylate cyclase (CyaA) activity of E. coli, which activates a specific reporter gene, lacZ. Interactions between protein fusions were evaluated in liquid cultures through the hydrolysis of the chromogenic X-gal substrate by the activated β-galoctasidase. The MecR2::MecI interaction was evaluated using the eight possible combinations: fusions either with T25 or T18 fragments of CyaA at either the N′ or C′ terminals. Tube 1, T18-MecR2::MecI-T25; tube 2, T18-MecR2::T25-MecI; tube 3, MecR2-T18::MecI-T25; tube 4, MecR2-T18::T25-MecI; tube 5, MecR2-T25::T18-MecI; tube 6, T25-MecR2::T18-MecI; tube 7, MecR2-T25::MecI-T18; tube 8, T25-MecR2::MecI-T18; tube 9, positive control provided by the manufacturer (Zip-T25::Zip-T18); tube 10, negative control (T25::T18); tube 11, “in-house” positive control testing the MecI-MecI interaction T18-MecI::T25-MecI. (B) Electrophoretic mobility shift assay (EMSA) of the binding of purified MecI to a labeled 212 bp DNA fragment encompassing the mecA promoter in the presence of purified MecR2. MecI concentration was constant in all binding reactions (0.05 µg). Lane 1, negative control, labeled DNA only; lane 2, 8-fold excess of MecI; lane 3, 4-fold excess of MecI; lane 4, binding control, MecI only; lane 5, 2-fold excess of MecI; lane 6, equimolar amounts of MecI and MecR2; lane 7, 2-fold excess of MecR2; lane 8, control for specific binding, MecI with a 125 molar excess of unlabelled DNA. (C) Western blotting analysis of MecI cleavage in total protein extracts (60–80 mg/lane). Lane 1, prototype strain N315; lane 2, mecR2 null-mutant (N315::ΔmecR2); lane 3, strain HT0350 co-overexpressing MecI and MecR2 (HT0350+mecImecR2); lane 4, strain HT0350 overexpressing MecI (HT0350+mecI). Cultures of N315 and N315::ΔmecR2 cultures were induced with a sub-MIC concentration of oxacillin (0.05 mg/L).
Figure 10.
Model for the mecA induction by MecR1-MecI-MecR2.
In the presence of a β-lactam antibiotic, MecR1 is activated and rapidly induces the expression of mecA and mecR1-mecI-mecR2. The anti-repressor activity of MecR2 is essential to sustain the mecA induction since it promotes the inactivation of MecI by proteolytic cleavage. In the absence of β-lactams, MecR1 is not activated and a steady state is established with stable MecI-dimers bound to the mecA promoter and residual copies of MecR1 at the cell membrane.