Mutation of RNA polymerase beta subunit (rpoB) promotes hVISA-to-VISA phenotypic conversion of strain Mu3

Abstract

The clinical vancomycin-intermediate Staphylococcus aureus (VISA) strain Mu50 carries two mutations in the vraSR and graRS two-component regulatory systems (TCRSs), namely, vraS(I5N) and graR(N197S) (hereinafter designated graR). The clinical heterogeneously vancomycin-intermediate S. aureus (hVISA) strain Mu3 shares with Mu50 the mutation in vraS that encodes the VraS two-component histidine kinase. Previously, we showed that introduction of the plasmid pgraR, carrying the mutated two-component response regulator graR, converted the hVISA strain Mu3 into VISA (vancomycin MIC = 4 mg/liter). Subsequently, however, we found that the introduction of a single copy of graR into the Mu3 chromosome by a gene replacement method did not confer on Mu3 the VISA phenotype. The gene-replaced strain Mu3graR thus obtained remained hVISA (MIC ≤ 2 mg/liter), although a small increase in vancomycin MIC was observed compared to that of the parent strain Mu3. Reevaluation of the Mu3 and Mu50 genomes revealed the presence of another mutation responsible for the expression of the VISA phenotype in Mu50. Here, we demonstrate that in addition to the two regulator mutations, a third mutation found in the Mu50 rpoB gene, encoding the RNA polymerase β subunit, was required for Mu3 to achieve the level of vancomycin resistance of Mu50. The selection of strain Mu3graR with rifampin gave rise to rpoB mutants with various levels of increased vancomycin resistance. Furthermore, 3 (33%) of 10 independently isolated VISA strains established from the heterogeneous subpopulations of Mu3graR were found to possess rpoB mutations with or without an accompanying rifampin-resistance phenotype. The data indicate that a sizable proportion of the resistant hVISA cell subpopulations is composed of spontaneous rpoB mutants with various degrees of increased vancomycin resistance.

Fig. 1.

Effect of graR(N197S) mutation (graR*) on the vancomycin-resistant subpopulations of Mu3. The numbers of colonies on BHI agar plates containing various concentrations of vancomycin were counted after 48 h of incubation at 37°C. Mu3graR* is a Mu3-derived mutant strain with the graR* mutation introduced by a gene replacement method. Mu3(pgraR*) is a transformant of Mu3 with the plasmid pgraR*. L, liter.

Fig. 2.

Effect of rpoB(H481Y) mutation on the vancomycin-resistant subpopulations of Mu3 and Mu3graR*. (A) Mu3RP1-3 is a rifampin-resistant mutant of Mu3 having the rpoB(H481Y) mutation. Mu3graR*RP1-33 is a rifampin-resistant mutant of Mu3graR* having the rpoB(H481Y) mutation. (B) Mu3rpoB(H481Y) and Mu3graR*rpoB(H481Y) are derivative strains of Mu3 and Mu3graR*, respectively, with the rpoB(H481Y) mutation introduced by a gene replacement method.