Structure/Function Analysis of the<i>Pantoea stewartii</i>Quorum-Sensing Regulator EsaR as an Activator of Transcription

Schu, Daniel J.; Carlier, Aurélien; Jamison, Katherine P.; Bodman, Susanne von; Stevens, Ann M.

doi:10.1128/jb.00994-09

Cited by 33 publications

(45 citation statements)

References 39 publications

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“…EsaR has previously been shown to respond to AHL in E. coli in the same manner as in its native host (20,28). EsaR was equally stable in the absence or presence of AHL for up to 60 min after synthesis of the protein (Fig.…”

Section: Resultsmentioning

confidence: 84%

“…In the absence of AHL, EsaR is capable of repressing transcription from the esaR and rcsA promoters (6,14,15) and activating transcription of an sRNA (20) at low cell density. AHL hinders the ability of EsaR to regulate transcription at high cell density.…”

Section: Discussionmentioning

confidence: 99%

“…EsaR also positively regulates expression of a small RNA (sRNA) native to P. stewartii (20). In P. stewartii the LuxI homologue EsaI synthesizes N-3-oxo-hexanoyl-L-homoserine lactone (2), and at high cell densities this AHL interferes with the ability of EsaR to regulate gene expression (15,28).…”

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confidence: 99%

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Probing the Impact of Ligand Binding on the Acyl-Homoserine Lactone-Hindered Transcription Factor EsaR of Pantoea stewartii subsp. stewartii

et al. 2011

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The quorum-sensing regulator EsaR from Pantoea stewartii subsp. stewartii is a LuxR homologue that is inactivated by acyl-homoserine lactone (AHL). In the corn pathogen P. stewartii, production of exopolysaccharide (EPS) is repressed by EsaR at low cell densities. However, at high cell densities when high concentrations of its cognate AHL signal are present, EsaR is inactivated and derepression of EPS production occurs. Thus, EsaR responds to AHL in a manner opposite to that of most LuxR family members. Depending on the position of its binding site within target promoters, EsaR serves as either a repressor or activator in the absence rather than in the presence of its AHL ligand. The effect of AHL on LuxR homologues has been difficult to study in vitro because AHL is required for purification and stability. EsaR, however, can be purified without AHL enabling an in vitro analysis of the response of the protein to ligand. Western immunoblots and pulse-chase experiments demonstrated that EsaR is stable in vivo in the absence or presence of AHL. Limited in vitro proteolytic digestions of a biologically active His-MBP tagged version of EsaR highlighted intradomain and interdomain conformational changes that occur in the protein in response to AHL. Gel filtration chromatography of the full-length fusion protein and cross-linking of the N-terminal domain both suggest that this conformational change does not impact the multimeric state of the protein. These findings provide greater insight into the diverse mechanisms for AHL responsiveness found within the LuxR family.

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Section: Resultsmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

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Probing the Impact of Ligand Binding on the Acyl-Homoserine Lactone-Hindered Transcription Factor EsaR of Pantoea stewartii subsp. stewartii

et al. 2011

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“…Of note, we detected five particularly interesting apparently noncoding RNAs that were induced by pC-HSL and RpaR, including an rpaR antisense transcript. These RNAs deserve further study, particularly because small regulatory RNAs (sRNAs) have been implicated in the mediation of quorum-controlled gene activation in other bacteria (36,42). We note a recent report of antisense psyR (a luxR homolog) transcripts in Pseudomonas syringae (10).…”

Section: Discussionmentioning

confidence: 99%

Activity of the Rhodopseudomonas palustris p-Coumaroyl-Homoserine Lactone-Responsive Transcription Factor RpaR

et al. 2011

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The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorumsensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His 6 -RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.Many bacteria control subsets of genes in a cell densitydependent manner. This coordinated group behavior is known as quorum sensing and response. More than 100 species of Proteobacteria contain acyl-homoserine lactone (acyl-HSL) quorum-sensing (QS) circuits (12, 45). Acyl-HSLs can diffuse into and out of cells, and once a threshold concentration is reached, acyl-HSLs bind specific transcriptional regulators that control target genes. A variety of genes are controlled by QS depending on the bacterial species, including protease genes, conjugal transfer genes, antibiotic synthesis genes, and bioluminescence genes (12, 45). Many QS-regulated gene products are "public goods," exoproducts that can be shared by all of the individuals in a group.Two types of genes are involved in most acyl-HSL-type QS systems: luxI-and luxR-type genes. LuxI proteins are QS signal synthases that catalyze amide bond formation between an acyl group on an appropriate side chain donor (most often acyl-acyl carrier protein) and S-adenosylmethionine (SAM) resulting in the final acyl-HSL product (22,26,34,35). For fatty acyl-HSLs, signal specificity is conferred by the length (4 to 18 carbons) and side chain modifications of the fatty acyl group. LuxR homologs are homodimeric transcription factors, with each monomer consisting of two domains: an N-terminal acyl-HSL binding domain and a C-terminal DNA-binding domain that contains a helix-turn-helix motif (5,6,13,50). Genes controlled by LuxR homologs often have specific inverted repeat DNA sequences in their promoter regions. These elements are known as lux box-like sequences. The lux box is a 20-bp palindromic sequence centered at bp Ϫ42.5 from the transcription start of the Vibrio fischeri lux operon, which encodes...

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“…Most studies of these regulatory RNAs have focused on trans-encoded small RNAs (sRNAs) known to control a range of functions. The list of sRNA-controlled functions includes examples of AHL signal receptors (18,19). With the advent of RNAseq methods, there have been reports showing cis-encoded asRNAs can be quite common in bacteria (10,11).…”

Section: Discussionmentioning

confidence: 99%

Antisense RNA that affects Rhodopseudomonas palustris quorum-sensing signal receptor expression

Hirakawa

Harwood

Pechter

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Quorum sensing in the bacterium Rhodopseudomonas palustris involves the RpaI signal synthase, which produces p -coumaroyl-homoserine lactone ( p C-HSL) and RpaR, which is a p C-HSL–dependent transcriptional activator. There is also an antisense rpaR transcript (as rpaR ) of unknown function. Recent RNAseq studies have revealed that bacterial antisense RNAs are abundant, but little is known about the function of these molecules. Because as rpaR expression is quorum sensing dependent, we sought to characterize its production and function. We show that as rpaR is approximately 300–600 bases and is produced in response to p C-HSL and RpaR. There is an RpaR-binding site centered 51.5 bp from the mapped as rpaR transcript start site. We show that as rpaR overexpression reduces RpaR levels, rpaI expression, and p C-HSL production. We also generated an as rpaR mutant, which shows elevated RpaR levels, and elevated rpaI expression. Thus, as rpaR inhibits rpaR translation, and this inhibition results in suppression of RpaR-dependent rpaI expression and, thus, p C-HSL production. The R. palustris as rpaR represents an antisense RNA for which an activity can be measured and for which a distinct regulatory circuit related to a function is elucidated. It also represents yet another subtle regulatory layer for acyl-homoserine lactone quorum-sensing signal-responsive transcription factors.

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Structure/Function Analysis of thePantoea stewartiiQuorum-Sensing Regulator EsaR as an Activator of Transcription

Cited by 33 publications

References 39 publications

Probing the Impact of Ligand Binding on the Acyl-Homoserine Lactone-Hindered Transcription Factor EsaR of Pantoea stewartii subsp. stewartii

Probing the Impact of Ligand Binding on the Acyl-Homoserine Lactone-Hindered Transcription Factor EsaR of Pantoea stewartii subsp. stewartii

Activity of the Rhodopseudomonas palustris p-Coumaroyl-Homoserine Lactone-Responsive Transcription Factor RpaR

Antisense RNA that affects Rhodopseudomonas palustris quorum-sensing signal receptor expression

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