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The distal ExsA-binding site in Pseudomonas aeruginosa type III secretion system promoters is the primary determinant for promoter-specific properties.

ABSTRACT: Transcription of the Pseudomonas aeruginosa type III secretion system is controlled by ExsA, a member of the AraC/XylS family of regulators. Each ExsA-dependent promoter contains two adjacent binding sites for monomeric ExsA. The promoter-proximal site (binding site 1) consists of highly conserved GnC and TGnnA sequences that are individually recognized by the two helix-turn-helix (HTH) DNA-binding motifs of an ExsA monomer. While the GnC and TGnnA sequences are important for binding to site 1, the promoter-distal binding sites (site 2) lack obvious similarity among themselves or with binding site 1. In the present study, we demonstrate that site 2 in the P(exsC) promoter region contains a GnC sequence that is functionally equivalent to the GnC in site 1 and recognized by the first HTH motif of an ExsA monomer. Likewise, the second HTH interacts with an adenine residue in binding site 2. Although several candidate GnC sequences are also present in site 2 of the P(exsD), P(exoT), and P(pcrG) promoters, the GnC sequences were not required for ExsA-dependent transcription or ExsA binding. A comparison of hybrid promoters composed of binding site 2 from one promoter fused to binding site 1 derived from another promoter indicates that ExsA-binding affinity, promoter strength, and the degree of promoter bending are properties that are largely determined by binding site 2. Based on these data, we propose that the manner in which ExsA interacts with binding site 2 at the P(exsC) promoter is distinct from the interactions occurring at other promoters.

SUBMITTER: Brutinel ED

PROVIDER: S-EPMC3347217 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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The distal ExsA-binding site in Pseudomonas aeruginosa type III secretion system promoters is the primary determinant for promoter-specific properties.

Brutinel Evan D ED King Jessica M JM Marsden Anne E AE Yahr Timothy L TL

Journal of bacteriology 20120309 10

Transcription of the Pseudomonas aeruginosa type III secretion system is controlled by ExsA, a member of the AraC/XylS family of regulators. Each ExsA-dependent promoter contains two adjacent binding sites for monomeric ExsA. The promoter-proximal site (binding site 1) consists of highly conserved GnC and TGnnA sequences that are individually recognized by the two helix-turn-helix (HTH) DNA-binding motifs of an ExsA monomer. While the GnC and TGnnA sequences are important for binding to site 1, ...[more]

PMID: 22408165

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Project description:UnlabelledThe Pseudomonas aeruginosa cyclic AMP (cAMP)-Vfr system (CVS) is a global regulator of virulence gene expression. Regulatory targets include type IV pili, secreted proteases, and the type III secretion system (T3SS). The mechanism by which CVS regulates T3SS gene expression remains undefined. Single-cell expression studies previously found that only a portion of the cells within a population express the T3SS under inducing conditions, a property known as bistability. We now report that bistability is altered in avfr mutant, wherein a substantially smaller fraction of the cells express the T3SS relative to the parental strain. Since bistability usually involves positive-feedback loops, we tested the hypothesis that virulence factor regulator (Vfr) regulates the expression of exsA ExsA is the central regulator of T3SS gene expression and autoregulates its own expression. Although exsA is the last gene of the exsCEBA polycistronic mRNA, we demonstrate that Vfr directly activates exsA transcription from a second promoter (PexsA) located immediately upstream of exsA PexsA promoter activity is entirely Vfr dependent. Direct binding of Vfr to a PexsA promoter probe was demonstrated by electrophoretic mobility shift assays, and DNase I footprinting revealed an area of protection that coincides with a putative Vfr consensus-binding site. Mutagenesis of that site disrupted Vfr binding and PexsA promoter activity. We conclude that Vfr contributes to T3SS gene expression through activation of the PexsA promoter, which is internal to the previously characterized exsCEBA operon.ImportanceVfr is a cAMP-dependent DNA-binding protein that functions as a global regulator of virulence gene expression in Pseudomonas aeruginosa Regulation by Vfr allows for the coordinate production of related virulence functions, such as type IV pili and type III secretion, required for adherence to and intoxication of host cells, respectively. Although the molecular mechanism of Vfr regulation has been defined for many target genes, a direct link between Vfr and T3SS gene expression had not been established. In the present study, we report that Vfr directly controls exsA transcription, the master regulator of T3SS gene expression, from a newly identified promoter located immediately upstream of exsA.

Project description:The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence factor important for phagocytic avoidance, disruption of host cell signaling, and host cell cytotoxicity. ExsA is the master regulator of T3SS transcription. The expression, synthesis, and activity of ExsA is tightly regulated by both intrinsic and extrinsic factors. Intrinsic regulation consists of the well-characterized ExsECDA partner-switching cascade, while extrinsic factors include global regulators that alter exsA transcription and/or translation. To identify novel extrinsic regulators of ExsA, we conducted a transposon mutagenesis screen in the absence of intrinsic control. Transposon disruptions within gene PA2840, which encodes a homolog of the Escherichia coli RNA-helicase DeaD, significantly reduced T3SS gene expression. Recent studies indicate that E. coli DeaD can promote translation by relieving inhibitory secondary structures within target mRNAs. We report here that PA2840, renamed DeaD, stimulates ExsA synthesis at the posttranscriptional level. Genetic experiments demonstrate that the activity of an exsA translational fusion is reduced in a deaD mutant. In addition, exsA expression in trans fails to restore T3SS gene expression in a deaD mutant. We hypothesized that DeaD relaxes mRNA secondary structure to promote exsA translation and found that altering the mRNA sequence of exsA or the native exsA Shine-Dalgarno sequence relieved the requirement for DeaD in vivo. Finally, we show that purified DeaD promotes ExsA synthesis using in vitro translation assays. Together, these data reveal a novel regulatory mechanism for P. aeruginosa DeaD and add to the complexity of global regulation of T3SS.Although members of the DEAD box family of RNA helicases are appreciated for their roles in mRNA degradation and ribosome biogenesis, an additional role in gene regulation is now emerging in bacteria. By relaxing secondary structures in mRNAs, DEAD box helicases are now thought to promote translation by enhancing ribosomal recruitment. We identify here an RNA helicase that plays a critical role in promoting ExsA synthesis, the central regulator of the Pseudomonas aeruginosa type III secretion system, and provide additional evidence that DEAD box helicases directly stimulate translation of target genes. The finding that DeaD stimulates exsA translation adds to a growing list of transcriptional and posttranscriptional regulatory mechanisms that control type III gene expression.

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Dataset Information

The distal ExsA-binding site in Pseudomonas aeruginosa type III secretion system promoters is the primary determinant for promoter-specific properties.

Publications

The distal ExsA-binding site in Pseudomonas aeruginosa type III secretion system promoters is the primary determinant for promoter-specific properties.

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