Analysis of a Cell Wall Mutant Highlights Rho-Dependent Genome Amplification Events in Staphylococcus aureus

In a study of antibiotic resistance in Staphylococcus aureus, specific cell wall mutants were previously generated for the peptidoglycan biosynthesis gene murF, by the insertion of an integrative plasmid. A collection of 30 independent mutants was obtained, and all harbored a variable number of copies of the inserted plasmid, arranged in tandem in the chromosome. Of the 30 mutants, only 3, F9, F20 and F26, with a lower number of plasmid copies, showed an altered peptidoglycan structure, lower resistance to β-lactams and a different loss-of-function mutation in rho gene, that encodes a transcription termination factor. The rho mutations were found to correlate with the level of oxacillin resistance, since genetic complementation with rho gene reestablished the resistance and cell wall parental profile in F9, F20 and F26 strains. Furthermore, complementation with rho resulted in the amplification of the number of plasmid tandem repeats, suggesting that Rho enabled events of recombination that favored a rearrangement in the chromosome in the region of the impaired murF gene. Although the full mechanism of reversion of the cell wall damage was not fully elucidated, we showed that Rho is involved in the recombination process that mediates the tandem amplification of exogeneous DNA fragments inserted into the chromosome. IMPORTANCE The cell wall of bacteria, namely, peptidoglycan, is the target of several antibiotic classes such as β-lactams. Staphylococcus aureus is well known for its capacity to adapt to antibiotic stress and develop resistance strategies, namely, to β-lactams. In this context, the construction of cell wall mutants provides useful models to study the development of such resistance mechanisms. Here, we characterized a collection of independent mutants, impaired in the same peptidoglycan biosynthetic step, obtained through the insertion of a plasmid in the coding region of murF gene. S. aureus demonstrated the capacity to overcome the cell wall damage by amplifying the copy number of the inserted plasmid, through an undescribed mechanism that involves the Rho transcription termination factor.