Regulatory roles of an sRNA derived from the 5´ UTR and sequence internal to lapA in Pseudomonas aeruginosa PAO1.

Several key virulence factors of Pseudomonas aeruginosa are regulated by quorum-sensing systems, small noncoding RNAs (sRNAs), and environmental stress, leading to a high mortality rate. Our previous studies indicated that the alkaline phosphatase LapA regulated P. aeruginosa PAO1 biofilm formation in a chronic wound model established with ex vivo porcine skin explants. Notably, one particular sRNA located upstream of the lapA gene was highly expressed in the model. Therefore, the sRNA was further characterized via northern blotting and rapid amplification of cDNA ends. The results revealed that the sRNA we named LapS is 197 nucleotides in length and is derived from the 5´ UTR and sequence internal to the lapA gene. Next, LapS mutation, overexpression, and complementation strains were constructed from the PAO1 strain, and phenotypic experiments associated with lapA were performed and compared with those of the ΔlapA and wild-type strains. The results indicated that LapS is involved in regulating swarming motility, rhamnolipid and alkaline phosphatase production, las/rhl quorum-sensing systems, and biofilm formation by controlling the level of lapA mRNA under phosphate-depleted conditions. Therefore, this LapS-lapA signaling cascade is beneficial for balancing the virulence regulation of P. aeruginosa. Additionally, an in vitro study indicated that LapS directly and post-transcriptionally regulated at least one unlinked gene, putA, which encodes bifunctional proline dehydrogenase, a virulence factor of P. aeruginosa. Collectively, our findings reveal a previously unstudied regulatory sRNA and advance the understanding of the roles of sRNAs in the pathogenicity of P. aeruginosa. IMPORTANCE: Pseudomonas aeruginosa is a common nosocomial pathogen that contains hundreds of virulence factors regulated by quorum-sensing systems and environmental stress. Small noncoding RNAs (sRNAs) involved in virulence regulation have been identified in P. aeruginosa. Recently, several potential sRNAs were identified in P. aeruginosa using transcriptome sequencing. However, some of these novel sRNAs have been functionally characterized. In this study, a previously uncharacterized sRNA, LapS, in P. aeruginosa PAO1 was identified as a novel sRNA. LapS is involved in regulating swarming motility, rhamnolipid production, and alkaline phosphatase production during phosphate-depleted stress by controlling the level of lapA mRNA. Furthermore, LapS deletion also reduced the mortality rate of Caenorhabditis elegans in a fast-kill assay. Additionally, LapS directly suppressed PutA, a virulence factor of P. aeruginosa. This study highlights the role of LapS in modulating P. aeruginosa virulence during phosphate-depleted stress.