The Distal ExsA-Binding Site in Pseudomonas aeruginosa Type III Secretion System Promoters Is the Primary Determinant for Promoter-Specific Properties

Brutinel, Evan D.; King, Jessica M.; Marsden, Anne E.; Yahr, Timothy L.

doi:10.1128/jb.00106-12

Cited by 12 publications

(17 citation statements)

References 19 publications

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“…This finding seemed contrary to our previous data indicating that occupation of both binding sites is required for activation by ExsA (18). We previously proposed that, in some cases, site 2 occupation involves nonspecific protein-DNA interactions, because site 2 is poorly conserved among the 10 ExsA-dependent promoter regions (20). Based on these combined observations, we propose a model that takes into consideration the relative affinity of ExsA for itself (i.e., self-association) and for site 2 binding.…”

Section: Figcontrasting

confidence: 56%

“…In addition to self-association, protein-DNA interactions also contribute to occupation of the second binding site in the P exsC promoter (20). In other ExsAdependent promoters, however, binding site 2 is poorly defined, but the available data suggest that ExsA has a lower affinity for binding site 2 than site 1 (18).…”

Section: Figmentioning

confidence: 93%

“…Binding site 2 in the P exoT promoter is poorly defined but is thought to represent a low-affinity site for ExsA (18,20). Unlike ExsA, binding by both ExsA L140A,L148A and CTD to the P exoT probe resulted in preferential formation of shift product 1 ( Fig.…”

Section: Discussionmentioning

confidence: 99%

“…On at least one of the T3SS promoters (P exoT ), occupation of the binding sites occurs in an ordered fashion whereby the first ExsA monomer binds to site 1 and then recruits a second ExsA monomer to binding site 2 (18). Whereas binding site 1 of all T3SS promoters contains conserved GnC and TGnnA sequences, functionally equivalent sites have been identified only in binding site 2 for the P exsC promoter (19,20). Occupation of site 2 in other promoters is not well understood but may depend on nonspecific interactions between ExsA and site 2 and/or self-association with the monomer bound to site 1.…”

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confidence: 99%

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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: Figcontrasting

confidence: 56%

Section: Figmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

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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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“…5 monomer binds to a consensus sequence within site 1 (5'-tAaAAAnwnMyGrCynnnmYTGayAk) and subsequently recruits a second monomer to the adjacent site 2 (50, 51). Site 2 has no recognizable sequence similarity to site 1, but seems to influence whether high-or low-affinity interactions occur at T3SS promoters (51,52). Once bound to both promoter sites, ExsA facilitates transcription by enhancing recruitment of RNA polymerase (53).…”

Section: Intrinsic Regulation Of the Type III Secretion Systemmentioning

confidence: 99%