A <i>rhlI</i> 5′ UTR-Derived sRNA Regulates RhlR-Dependent Quorum Sensing in Pseudomonas aeruginosa

Abstract

<i>N</i>-Acyl homoserine lactone (AHL) quorum sensing (QS) controls expression of over 200 genes in <i>Pseudomonas aeruginosa</i>. There are two AHL regulatory systems: the LasR-LasI circuit and the RhlR-RhlI system. We mapped transcription termination sites affected by AHL QS in <i>P. aeruginosa</i>, and in doing so we identified AHL-regulated small RNAs (sRNAs). Of interest, we noted that one particular sRNA was located within the <i>rhlI</i> locus. We found that <i>rhlI</i>, which encodes the enzyme that produces the AHL <i>N</i>-butanoyl-homoserine lactone (C4-HSL), is controlled by a 5' untranslated region (UTR)-derived sRNA we name RhlS. We also identified an antisense RNA encoded opposite the beginning of the <i>rhlI</i> open reading frame, which we name asRhlS. RhlS accumulates as wild-type cells enter stationary phase and is required for the production of normal levels of C4-HSL through activation of <i>rhlI</i> translation. RhlS also directly posttranscriptionally regulates at least one other unlinked gene, <i>fpvA</i>. The asRhlS appears to be expressed at maximal levels during logarithmic growth, and we suggest RhlS may act antagonistically to the asRhlS to regulate <i>rhlI</i> translation. The <i>rhlI</i>-encoded sRNAs represent a novel aspect of RNA-mediated tuning of <i>P. aeruginosa</i> QS.<b>IMPORTANCE</b> The opportunistic human pathogen <i>Pseudomonas aeruginosa</i> possesses multiple quorum sensing systems that regulate and coordinate production of virulence factors and adaptation to different environments. Despite extensive research, the regulatory elements that play a role in this complex network are still not fully understood. By using several RNA sequencing techniques, we were able to identify a small regulatory RNA we named RhlS. RhlS increases translation of RhlI, a key enzyme in the quorum sensing pathway, and represses the <i>fpvA</i> mRNA encoding one of the siderophore pyoverdine receptors. Our results highlight a new regulatory layer of <i>P. aeruginosa</i> quorum sensing and contribute to the growing understanding of the role regulatory RNAs play in bacterial physiology.