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.