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The GacS/GacA signal transduction system of Pseudomonas aeruginosa acts exclusively through its control over the transcription of the RsmY and RsmZ regulatory small RNAs.

ABSTRACT: We report here the results of an analysis of the regulatory range of the GacS/GacA two-component system in Pseudomonas aeruginosa. Using microarrays, we identified a large number of genes that are regulated by the system, and detected a near complete overlap of these genes with those regulated by two small RNAs (sRNAs), RsmY and RsmZ, suggesting that the expression of all GacA-regulated genes is RsmY/Z-dependent. Using genome-wide DNA-protein interaction analyses, we identified only two genomic regions that associated specifically with GacA, located upstream of the rsmY and rsmZ genes. These results demonstrate that in P. aeruginosa, the GacS/GacA system transduces the regulatory signals to downstream genes exclusively by directly controlling the expression of only two genes rsmY and rsmZ. These two sRNAs serve as intermediates between the input signals and the output at the level of mRNA stability, although additional regulatory inputs can influence the levels of these two riboregulators. We show that the A+T-rich DNA segment upstream of rsmZ is bound and silenced by MvaT and MvaU, the global gene regulators of the H-NS family. This work highlights the importance of post-transcriptional mechanisms involving sRNAs in controlling gene expression during bacterial adaptation to different environments.

SUBMITTER: Brencic A

PROVIDER: S-EPMC2761719 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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The GacS/GacA signal transduction system of Pseudomonas aeruginosa acts exclusively through its control over the transcription of the RsmY and RsmZ regulatory small RNAs.

Brencic Anja A McFarland Kirsty A KA McManus Heather R HR Castang Sandra S Mogno Ilaria I Dove Simon L SL Lory Stephen S

Molecular microbiology 20090709 3

We report here the results of an analysis of the regulatory range of the GacS/GacA two-component system in Pseudomonas aeruginosa. Using microarrays, we identified a large number of genes that are regulated by the system, and detected a near complete overlap of these genes with those regulated by two small RNAs (sRNAs), RsmY and RsmZ, suggesting that the expression of all GacA-regulated genes is RsmY/Z-dependent. Using genome-wide DNA-protein interaction analyses, we identified only two genomic ...[more]

PMID: 19602144

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Project description:Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCE CsrA homologs are present in many bacteria. The opportunistic pathogen Pseudomonas aeruginosa uses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activity in vivo Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activity in vivo and in vitro are more stringent than those for RsmA.

Project description:In Pseudomonas aeruginosa, the complex multisensing regulatory networks RetS-GacS/GacA have been demonstrated to play key roles in controlling the switch between planktonic and sessile lifestyles. However, whether this multisensing system is involved in the regulation of phage infection has not been investigated. Here, we provide a link between the sensors RetS/GacS and infection of phages vB_Pae_QDWS and vB_Pae_W3. Our data suggest that the sensors kinases RetS and GacS in Pseudomonas aeruginosa play opposite regulatory functions on phage infection. Mutation in retS increased phage resistance. Cellular levels of RsmY and RsmZ increased in PaΔretS and were positively correlated with phage resistance. Further analysis demonstrated that RetS regulated phage infection by affecting the type IV pilus (T4P)-mediated adsorption. The regulation of RetS on phage infection depends on the GacS/GacA two-component system and is likely a dynamic process in response to environmental signals. The findings offer additional support for the rapid emergence of phage resistance. IMPORTANCE Our knowledge on the molecular mechanisms behind bacterium-phage interactions remains limited. Our study reported that the complex multisensing regulatory networks RetS-GacS/GacA of Pseudomonas aeruginosa PAO1 play key roles in controlling phage infection. The main observation was that the mutation in RetS could result in increased phage resistance by reducing the type IV pilus-mediated phage adsorption. The bacterial defense strategy is generally applicable to various phages since many P. aeruginosa phages can use type IV pilus as their receptors. The results also suggest that the phage infection is likely to be regulated dynamically, which depends on the environmental stimuli. Reduction of the signals that RetS favors would increase phage resistance. Our study is particularly remarkable for uncovering a signal transduction system that was involved in phage infection, which may help in filling some knowledge gaps in this field.

Dataset Information

The GacS/GacA signal transduction system of Pseudomonas aeruginosa acts exclusively through its control over the transcription of the RsmY and RsmZ regulatory small RNAs.

Publications

The GacS/GacA signal transduction system of Pseudomonas aeruginosa acts exclusively through its control over the transcription of the RsmY and RsmZ regulatory small RNAs.

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