Overexpression of the Small RNA PA0805.1 in Pseudomonas aeruginosa Modulates the Expression of a Large Set of Genes and Proteins, Resulting in Altered Motility, Cytotoxicity, and Tobramycin Resistance

Pseudomonas aeruginosa is a motile species that initiates swarming motility in response to specific environmental cues, i.e., a semisolid surface with amino acids as a nitrogen source (relevant to the human lung). Swarming is an intricately regulated process, but to date posttranscriptional regulation has not been extensively investigated. Small noncoding RNAs (sRNAs) are hypothesized to play posttranscriptional regulatory roles, largely through suppression of translation, and we previously demonstrated 20 sRNA species that were dysregulated under swarming conditions. One of these, sRNA PA0805.1 (which was 5-fold upregulated under swarming conditions), when cloned, transformed into wild-type (WT) PAO1, and overexpressed, led to broad phenotypic changes, including reduced swarming, swimming, and twitching motilities, as well as increased adherence, cytotoxicity, and tobramycin resistance. A ΔPA0805.1 deletion mutant was more susceptible to tobramycin than the WT under swarming conditions. The strain overexpressing PA0805.1 was compared to the empty-vector strain by transcriptome sequencing (RNA-Seq) and proteomics under swarming conditions to determine sRNA targets. Broad transcriptional and proteomic profiles showed 1,121 differentially expressed genes and 258 proteins with significantly different abundance. Importantly, these included 106 transcriptional regulators, two-component regulatory systems, and sigma and anti-sigma factors. Downstream of these regulators were found downregulated type IV pilus genes, many upregulated adherence and virulence factors, and two multidrug efflux systems, mexXY and mexGHI-opmD. Therefore, the sRNA PA0805.1 appears to be a global regulator that influences diverse bacterial lifestyles, most likely through a regulatory cascade. IMPORTANCE P. aeruginosa is an opportunistic pathogen of humans. With roughly 10% of its genes encoding transcriptional regulators, and hundreds of small noncoding RNAs (sRNAs) interspersed throughout the genome, P. aeruginosa is able to fine-tune its response to adapt and survive in the host and resist antimicrobial agents. Understanding mechanisms of genetic regulation is therefore crucial to combat pathogenesis. The previously uncharacterized sRNA PA0805.1 was overexpressed in P. aeruginosa strain PAO1, resulting in decreased motility, increased adherence, cytotoxicity, and tobramycin resistance. In contrast, a ΔPA0805.1 deletion mutant had increased susceptibility to tobramycin under swarming conditions. Omic approaches uncovered 1,121 transcriptomic and 258 proteomic changes in the overexpression strain compared with the empty-vector strain, which included 106 regulatory factors. Downstream of these regulators were upregulated adherence factors, multidrug efflux systems, and virulence factors in both transcriptomics and proteomics. This study provides insights into the role of the sRNA PA0805.1 in modulating bacterial adaptations.