Dataset Information

Negative Control of RpoS Synthesis by the sRNA ReaL in Pseudomonas aeruginosa.

ABSTRACT: Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen, able to resist host defense mechanisms and antibiotic treatment. In Pae, the master regulator of stress responses RpoS (?S) is involved in the regulation of quorum sensing and several virulence genes. Here, we report that the sRNA ReaL translationally silences rpoS mRNA, which results in a decrease of the RpoS levels. Our studies indicated that ReaL base-pairs with the Shine-Dalgarno region of rpoS mRNA. These studies are underlined by a highly similar transcription profile of a rpoS deletion mutant and a reaL over-expressing strain.

SUBMITTER: Thi Bach Nguyen H

PROVIDER: S-EPMC6215814 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Publications

Negative Control of RpoS Synthesis by the sRNA ReaL in Pseudomonas aeruginosa.

Thi Bach Nguyen Hue H Romero A David D Amman Fabian F Sorger-Domenigg Theresa T Tata Muralidhar M Sonnleitner Elisabeth E Bläsi Udo U

Frontiers in microbiology 20181029

Pseudomonas aeruginosa (Pae) is an opportunistic human pathogen, able to resist host defense mechanisms and antibiotic treatment. In Pae, the master regulator of stress responses RpoS (σS) is involved in the regulation of quorum sensing and several virulence genes. Here, we report that the sRNA ReaL translationally silences rpoS mRNA, which results in a decrease of the RpoS levels. Our studies indicated that ReaL base-pairs with the Shine-Dalgarno region of rpoS m ...[more]

PMID: 30420839

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Project description:At low temperatures the Escherichia coli rpoS mRNA, encoding the stationary phase sigma factor RpoS, forms an intramolecular secondary structure (iss) that impedes translation initiation. Under these conditions the small RNA DsrA, which is stabilzed by Hfq, forms a duplex with rpoS mRNA sequences opposite of the ribosome-binding site (rbs). Both the DEAD box helicase CsdA and Hfq have been implicated in DsrA·rpoS duplex formation. Hfq binding to A-rich sequences in the rpoS leader has been suggested to restructure the mRNA, and thereby to accelerate DsrA·rpoS duplex formation, which, in turn, was deemed to free the rpoS rbs and to permit ribosome loading on the mRNA. Several experiments designed to elucidate the role of Hfq in DsrA-mediated translational activation of rpoS mRNA have been conducted in vitro. Here, we assessed RpoS synthesis in vivo to further study the role of Hfq in rpoS regulation. We show that RpoS synthesis was reduced when DsrA was ectopically overexpressed at 24 °C in the absence of Hfq despite of DsrA·rpoS duplex formation. This observation indicated that DsrA·rpoS annealing may not be sufficient for efficient ribosome loading on rpoS mRNA. In addition, a HfqG29A mutant protein was employed, which is deficient in binding to A-rich sequences present in the rpoS leader but proficient in DsrA binding. We show that DsrA·rpoS duplex formation occurs in the presence of the HfqG29A mutant protein at low temperature, whereas synthesis of RpoS was greatly diminished. RNase T1 footprinting studies of DsrA·rpoS duplexes in the absence and presence of Hfq or HfqG29A indicated that Hfq is required to resolve a stem-loop structure in the immediate coding region of rpoS mRNA. These in vivo studies corroborate the importance of the A-rich sequences in the rpoS leader and strongly suggest that Hfq, besides stabilizing DsrA and accelerating DsrA·rpoS duplex formation, is also required to convert the rpoS mRNA into a translationally competent form.

Project description:The global stress response controlled by the alternative sigma factor RpoS protects enteric bacteria from a variety of environmental stressors. The role of RpoS in other, nonenteric bacteria, such as the opportunistic pathogen Pseudomonas aeruginosa, is less well understood. Here, we employed experimental social evolution to reveal that cooperative behavior via secreted public goods is an important function in the RpoS response of P. aeruginosa Using whole-genome sequencing, we identified rpoS loss-of-function mutants among isolates evolved in a protein growth medium that requires extracellular proteolysis. We found that rpoS mutants comprise up to 25% of the evolved population and that they behave as social cheaters, with low fitness in isolation but high fitness in mixed culture with the cooperating wild type. We conclude that rpoS mutants cheat because they exploit an RpoS-controlled public good produced by the wild type, the secreted aminopeptidase PaAP, and because they do not carry the metabolic costs of expressing PaAP and many other gene products in the large RpoS regulon. Our results suggest that PaAP is an integral part of a proteolytic sequence in P. aeruginosa that permits the utilization of protein as a nutrient source. Our work broadens the scope of stress response functions in bacteria.IMPORTANCE Bacterial stress responses are generally considered protective measures taken by individual cells. Enabled by an experimental evolution approach, we describe a contrasting property, collective nutrient acquisition, in the RpoS-dependent stress response of the opportunistic human pathogen P. aeruginosa Specifically, we identify the secreted P. aeruginosa aminopeptidase (PaAP) as an essential RpoS-controlled function in extracellular proteolysis. As a secreted "public good," PaAP permits cheating by rpoS mutants that save the metabolic costs of expressing RpoS-controlled genes dispensable under the given growth conditions. Proteolytic enzymes are important virulence factors in P. aeruginosa pathogenesis and constitute a potential target for antimicrobial therapy. More broadly, our work contributes to recent findings in higher organisms that stress affects not only individual fitness and competitiveness but also cooperative behavior.

Project description:N-Acyl homoserine lactone (AHL) quorum sensing (QS) controls expression of over 200 genes in Pseudomonas aeruginosa. 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 P. aeruginosa, and in doing so we identified AHL-regulated small RNAs (sRNAs). Of interest, we noted that one particular sRNA was located within the rhlI locus. We found that rhlI, which encodes the enzyme that produces the AHL N-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 rhlI 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 rhlI translation. RhlS also directly posttranscriptionally regulates at least one other unlinked gene, fpvA. The asRhlS appears to be expressed at maximal levels during logarithmic growth, and we suggest RhlS may act antagonistically to the asRhlS to regulate rhlI translation. The rhlI-encoded sRNAs represent a novel aspect of RNA-mediated tuning of P. aeruginosa QS.IMPORTANCE The opportunistic human pathogen Pseudomonas aeruginosa 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 fpvA mRNA encoding one of the siderophore pyoverdine receptors. Our results highlight a new regulatory layer of P. aeruginosa quorum sensing and contribute to the growing understanding of the role regulatory RNAs play in bacterial physiology.

Dataset Information

Negative Control of RpoS Synthesis by the sRNA ReaL in Pseudomonas aeruginosa.

Publications

Negative Control of RpoS Synthesis by the sRNA ReaL in <i>Pseudomonas aeruginosa</i>.

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