ExsA and LcrF Recognize Similar Consensus Binding Sites, but Differences in Their Oligomeric State Influence Interactions with Promoter DNA

King, Jessica M.; Bartra, Sara Schesser; Plano, Gregory V.; Yahr, Timothy L.

doi:10.1128/jb.00990-13

Cited by 20 publications

(34 citation statements)

References 49 publications

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“…Based on the defect exhibited by ExsA L140A,L148A , the corresponding mutations recently were introduced into Yersinia pestis LcrF, a close homolog of ExsA that exists as a dimer. As expected, the LcrF L136A,L144A mutant is largely monomeric, preferentially binds to a single site in electrophoretic mobility shift assays, and is significantly impaired for T3SS promoter activation (32).…”

Section: Discussionmentioning

confidence: 88%

Self-Association Is Required for Occupation of Adjacent Binding Sites in Pseudomonas aeruginosa Type III Secretion System Promoters

Marsden¹,

Schubot²,

Yahr³

2014

Journal of Bacteriology

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ExsA is a member of the AraC/XylS family of transcriptional regulators and is required for expression of the Pseudomonas aeruginosa type III secretion system (T3SS). All P. aeruginosa T3SS promoters contain two adjacent binding sites for monomeric ExsA. The amino-terminal domain of ExsA (NTD) is thought to mediate interactions between the ExsA monomers bound to each site. Threading the NTD onto the AraC backbone revealed an ␣-helix that likely serves as the primary determinant for dimerization. In this study, we performed alanine scanning mutagenesis of the ExsA ␣-helix (residues 136 to 152) to identify determinants required for self-association. Residues L137, C139, L140, K141, and L148 exhibited self-association defects and were required for maximal activation by ExsA. Disruption of self-association resulted in decreased binding to T3SS promoters, particularly loss of binding by the second ExsA monomer. Removing the NTD or increasing the space between the ExsA-binding sites restored the ability of the second ExsA monomer to bind the P exsC promoter. This finding indicated that, in the absence of self-association, the NTD prevents binding by a second monomer. Similar findings were seen with the P exoT promoter; however, binding of the second ExsA monomer in the absence of self-association also required the presence of a high-affinity site 2. Based on these data, ExsA self-association is necessary to overcome inhibition by the NTD and to compensate for low-affinity binding sites, thereby allowing for full occupation and activation of ExsA-dependent promoters. Therefore, ExsA self-association is indispensable and provides an attractive target for antivirulence therapies.

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Section: Discussionmentioning

confidence: 88%

Self-Association Is Required for Occupation of Adjacent Binding Sites in Pseudomonas aeruginosa Type III Secretion System Promoters

Marsden¹,

Schubot²,

Yahr³

2014

Journal of Bacteriology

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“…However, these proposed LcrF binding sites were highly variable in terms of distance from the transcriptional start site, directionality, and distance of the half-sites from each other (17). In a more recent analysis, King et al observed that the carboxy-terminal domain of LcrF is nearly identical to the DNA binding region of the homologous AraC-like master regulator ExsA of the Pseudomonas aeruginosa T3SS (18). The authors further showed that Y. pestis LcrF binds and activates ExsA-dependent promoters in P. aeruginosa.…”

Section: Lcrf History Structure and Functionmentioning

confidence: 98%

“…The authors further showed that Y. pestis LcrF binds and activates ExsA-dependent promoters in P. aeruginosa. Similarly, ExsA was able to induce expression of T3SS genes in Y. pestis in the absence of LcrF (18). LcrF and ExsA were shown to interact with a common nucleotide sequence motif (AaAAAnwnMygrCynnnmYTGyaAk), which is also recognized by activators of T3SS genes from Photorhabdus luminescens, Aeromonas hydrophilus, and Vibrio parahaemolyticus (W stands for A or T, M for A or C, Y for C or T, R for A or G, and K for G or T [with uppercase letters representing more highly conserved residues]) (18,21,22).…”

Section: Lcrf History Structure and Functionmentioning

confidence: 99%

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Yersinia Type III Secretion System Master Regulator LcrF

et al. 2016

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c Many Gram-negative pathogens express a type III secretion (T3SS) system to enable growth and survival within a host. The three human-pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, encode the Ysc T3SS, whose expression is controlled by an AraC-like master regulator called LcrF. In this review, we discuss LcrF structure and function as well as the environmental cues and pathways known to regulate LcrF expression. Similarities and differences in binding motifs and modes of action between LcrF and the Pseudomonas aeruginosa homolog ExsA are summarized. In addition, we present a new bioinformatics analysis that identifies putative LcrF binding sites within Yersinia target gene promoters. There are three Yersinia species pathogenic to humans. Y. pestis is the causative agent of bubonic and pneumonic plague and is transmitted through a flea vector or through airborne transmission from one mammalian host to another (1). In contrast, the enteropathogenic Yersinia species Y. enterocolitica and Y. pseudotuberculosis grow in the environment but can be transmitted to mammalian hosts through ingestion of contaminated food or water (1). Y. enterocolitica and Y. pseudotuberculosis cause typically self-limiting mesenteric lymphadenitis or gastroenteritis in otherwise healthy individuals but can cause a serious blood-borne infection in people with iron overload disorders such as hereditary hemochromatosis (2). In addition, sequelae following enteropathogenic Yersinia infection, such as erythema nodosum and reactive arthritis, have also been reported (3-5).Human-pathogenic Yersinia species share a virulence plasmid, called pCD1 in Y. pestis and pYV in enteropathogenic yersiniae, encoding the Ysc type III secretion system (T3SS) essential for causing disease (6). These 70-kb plasmids carry dozens of T3SS structural genes and encode five or six T3SS effector proteins called Yops and their dedicated chaperones, as well as genes encoding proteins involved in regulating expression and function of the T3SS. One of these regulatory proteins, LcrF, serves as the Yersinia T3SS master regulator, controlling transcription of a large number of plasmid-borne genes. Several environmental cues influence expression of LcrF itself, possibly enabling Yersinia to control T3SS expression during transitions from one niche to another. Recent reviews have highlighted important advances in our understanding of T3SS structure and modulation of the innate immune response by T3SS effector proteins (7). In this review, we focus on factors controlling LcrF expression, the target genes LcrF regulates, and how LcrF activity influences Yersinia pathogenesis. LcrF HISTORY, STRUCTURE, AND FUNCTIONIt has long been appreciated that human-pathogenic Yersinia carrying T3SS genes requires millimolar concentrations of calcium to grow at 37°C, and this phenomenon was termed the low-calcium response, or Lcr (8, 9). The absence of calcium, in combination with a shift to 37°C, triggers secretion of T3SS effector proteins, mimicking t...

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“…We took advantage of the fact that ExsA orthologs from Aeromonas hydrophila (AxsA), Photorhabdus luminescens (PxsA), Yersinia pestis (LcrF), and Vibrio parahaemolyticus (ExsA Vp ) each complement an exsA mutant for P exsD-lacZ reporter activity (60,61). Although these proteins are highly conserved in their C-terminal DNA-binding domains, their DNA sequences are less conserved ranging from 67 to 75% sequence identity relative to exsA.…”

Section: Figmentioning

confidence: 99%

The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

et al. 2015

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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 wellcharacterized 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. IMPORTANCEAlthough 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. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that expresses a large array of virulence factors to cause both acute and chronic infections (1). P. aeruginosa infections generally initiate as acute infections that can range from self-limiting folliculitis to life-threatening ventilator-associated pneumonia (1). P. aeruginosa utilizes a type III secretion system (T3SS) to promote host cell cytotoxicity and avoid phagocytosis (2, 3). The T3SS is a multiprotein complex that spans the cell envelope and functions by directly ...

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ExsA and LcrF Recognize Similar Consensus Binding Sites, but Differences in Their Oligomeric State Influence Interactions with Promoter DNA

Cited by 20 publications

References 49 publications

Self-Association Is Required for Occupation of Adjacent Binding Sites in Pseudomonas aeruginosa Type III Secretion System Promoters

Self-Association Is Required for Occupation of Adjacent Binding Sites in Pseudomonas aeruginosa Type III Secretion System Promoters

Yersinia Type III Secretion System Master Regulator LcrF

The RNA Helicase DeaD Stimulates ExsA Translation To Promote Expression of the Pseudomonas aeruginosa Type III Secretion System

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