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Minimal functional system required for expression of erythromycin resistance by msrA in Staphylococcus aureus RN4220
Abstract:
Previous studies have suggested that inducible erythromycin (Er) resistance in staphylococci mediated by the plasmid-borne ABC-transporter msrA is dependent on additional unidentified chromosomally encoded transmembrane (TM) domains. The requirement for two S. aureus candidate sequences, stpC and smpC, highly similar to sequences adjacent to msrA on the original S. epidermidis plasmid was investigated. Deletion of the sequences by allelic replacement was accomplished by electroporation of S. aureus RN4220 with a nonreplicating suicide vector. S. aureus strains carrying a delta(stpC-smpC) mutation showed an identical ErR phenotype to those arising from single crossover events and unmutated RN4220 containing msrA. This proves that neither stpC nor smpC is required for ErR. To further define the minimal functional unit required for MSR, the control region within the leader sequence of msrA was deleted. This resulted in constitutive resistance to Er and type B streptogramins (Sg), proving that SgR does not require the presence of Er. Deletion constructs containing the N- or C-terminal ABC regions of MsrA did not confer ErR in RN4220 singly or in combination.
Insights
Chromosomal genes stpC and smpC are not required for inducible erythromycin resistance in staphylococci. Deleting the msrA leader sequence confers constitutive resistance to erythromycin and streptogramins.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Inducible erythromycin resistance in staphylococci is mediated by the plasmid-borne ABC-transporter msrA.
- Previous studies suggested involvement of unidentified chromosomally encoded transmembrane domains.
- Candidate genes stpC and smpC, similar to sequences near msrA, were investigated.
Purpose of the Study:
- To investigate the requirement of S. aureus sequences stpC and smpC for erythromycin resistance.
- To determine the minimal functional unit of MsrA required for resistance.
- To clarify the relationship between erythromycin and streptogramin resistance.
Main Methods:
- Allelic replacement using electroporation of S. aureus RN4220 with a suicide vector to create delta(stpC-smpC) mutations.
- Deletion of the control region within the leader sequence of msrA.
- Construction and testing of deletion constructs of MsrA's ABC regions.
Main Results:
- Deletion of stpC and smpC did not affect the erythromycin resistance phenotype.
- Deletion of the msrA leader sequence resulted in constitutive resistance to erythromycin and type B streptogramins.
- Deletion constructs of MsrA's ABC regions did not confer erythromycin resistance.
Conclusions:
- The stpC and smpC genes are not required for inducible erythromycin resistance in staphylococci.
- Erythromycin resistance mediated by msrA does not require additional chromosomally encoded factors.
- Constitutive resistance and streptogramin resistance are separable from inducible erythromycin resistance and do not require the presence of erythromycin.