RpoN Promotes Pseudomonas aeruginosa Survival in the Presence of Tobramycin

Abstract

Pseudomonas aeruginosa has developed diverse strategies to respond and adapt to antibiotic stress. Among the factors that modulate survival in the presence of antibiotics, alternative sigma factors play an important role. Here, we demonstrate that the alternative sigma factor RpoN (σ54) promotes survival in the presence of tobramycin. The tobramycin-sensitive phenotype of logarithmic phase ΔrpoN mutant cells is suppressed by the loss of the alternative sigma factor RpoS. Transcriptional analysis indicated that RpoN positively regulates the expression of RsmA, an RNA-binding protein, in the P. aeruginosa stationary growth phase in a nutrient-rich medium. The loss of RpoS led to the upregulation of gacA expression in the nutrient-limited medium-grown stationary phase cells. Conversely, in the logarithmic growth phase, the ΔrpoS mutant demonstrated lower expression of gacA, underscoring a regulatory role of RpoS for GacA. Supplementation of tobramycin to stationary phase ΔrpoN mutant cells grown in nutrient-rich medium resulted in decreased expression of gacA, relA, and rpoS without altering the expression of rsmA relative to wild-type PAO1. The observed downregulation of gacA and relA in the ΔrpoN mutant in the presence of tobramycin could be reversed through the mutation of rpoS in the ΔrpoN mutant background. The tobramycin-tolerant phenotype of the ΔrpoNΔrpoS mutant logarithmic phase cells may be associated with the expression of relA, which remained unresponsive upon addition of tobramycin. The logarithmic phase ΔrpoS and ΔrpoNΔrpoS mutant cells demonstrated increased expression of gacA in response to tobramycin. Together, these results suggest that a complex regulatory interaction between RpoN, RpoS, the Gac/Rsm pathway, and RelA modulates the P. aeruginosa response to tobramycin.

Blocking the alternative sigma factor RpoN reduces virulence ofPseudomonas aeruginosaisolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain

“…We employed a clinical laboratory assay for testing bacterial susceptibility or resistance to antibiotics, and found that RpoN* improved antibiotic susceptibility at least two-fold for five different antibiotics, including imipenem. This agrees with work by others that showed RpoN is involved in P. aeruginosa tolerance of carbapenems, quinolones, and tobramycin (34–36). Unfortunately, the commercial assay uses predetermined antibiotic concentrations in a 96-well plate, limiting the scope of the molecular roadblock’s effects.…”

“…We previously reported that RpoN* alters transcription of several genes involved in multidrug efflux pumps that confer natural resistance (38). Additionally, RpoN is implicated in tolerance to various classes of antibiotics (34–36). We evaluated the effects of RpoN* on antibiotic susceptibility using a MicroScan Neg MIC 43 panel.…”

“…The alternative sigma factor, σ 54 or RpoN, regulates nitrogen assimilation, quorum sensing, motility, and biofilm formation (28–33). RpoN regulation was recently linked to P. aeruginosa tolerance to several antibiotics (34–36). RpoN binds to specific promoters with conserved −24, −12 sequences upstream of RpoN-regulated genes throughout the genome and is a key virulence regulator (37).…”

Response regulator VemR regulates the transcription of flagellar rod gene flgG by interacting with σ54 factor RpoN2 in Xanthomonas citri ssp. citri

“…Flagellar motility governs the translational movement towards favourable environments in response to physical or chemical attractants (Ottamann and Miller, ). Moreover, flagellar‐dependent cell motility is essential for the establishment of a mature biofilm, and is thus involved in the ability to attach to host surfaces and to elicit canker lesions on citrus (Li and Wang, ; Malamud et al ., ; Viducic et al ., ; Yaryura et al ., ). In addition, the filament flagellin has been shown to be a pathogen‐associated molecular pattern, inducing innate immune responses in host plants (Zipfel et al ., ).…”

RpoN-Dependent Direct Regulation of Quorum Sensing and the Type VI Secretion System in Pseudomonas aeruginosa PAO1

“…Additionally, the expression of oprE is controlled by RpoN under aerobic conditions (20). RpoN also regulates the susceptibility to tobramycin (21), quinolones, and carbapenems (22,23) in P. aeruginosa.…”