Reduced Expression of therplU-rpmARibosomal Protein Operon inmexXY-Expressing Pan-Aminoglycoside-Resistant Mutants of Pseudomonas aeruginosa

Abstract

ABSTRACTPan-aminoglycoside-resistantPseudomonas aeruginosamutants expressing themexXYcomponents of the aminoglycoside-accommodating MexXY-OprM multidrug efflux system but lacking mutations in themexZgene encoding a repressor of this efflux system and in themexXYpromoter have been reported (S. Fraud and K. Poole, Antimicrob. Agents Chemother. 55:1068–1074, 2011). Genome sequencing of one of these mutants, K2966, revealed the presence of a mutation within the predicted promoter region of therplU-rpmAoperon encoding ribosomal proteins L21 and L27, consistent with an observed 2-fold decrease in expression of this operon in the mutant relative to wild-typeP. aeruginosaPAO1. Moreover, correction of the mutation restoredrplU-rpmAexpression and, significantly, reversed the elevatedmexXYexpression and pan-aminoglycoside resistance of the mutant. ReducedrplU-rpmAexpression was also observed in a secondmexXY-expressing pan-aminoglycoside-resistant mutant, K2968, which, however, lacked a mutation in therplU-rpmApromoter region. Restoration ofrplU-rpmAexpression in the K2968 mutant following chromosomal integration of therplU-rpmAoperon derived from wild-typeP. aeruginosafailed, however, to reverse the elevatedmexXYexpression and pan-aminoglycoside resistance of this mutant, although it did so for K2966, suggesting that the mutation impactingrplU-rpmAexpression in K2968 also impacts othermexXY-related genes. IncreasedmexXYexpression owing to reducedrplU-rpmAexpression in K2966 and K2968 was dependent on PA5471, whose expression was also elevated in these mutants. Thus, mutational disruption of ribosome function, by limiting expression of ribosomal constituents, promotes recruitment ofmexXYand does so via PA5471, reminiscent ofmexXYinduction by ribosome-disrupting antimicrobial agents. Interestingly, reducedrplU-rpmAexpression was also observed in amexXY-expressing pan-aminoglycoside-resistant clinical isolate, suggesting that ribosome-perturbing mutations have clinical relevance in the recruitment of the MexXY-OprM aminoglycoside resistance determinant.