Molecular Basis of Transcriptional Antiactivation

Qin, Yinping; Su, Shiming; Farrand, Stephen K.

doi:10.1074/jbc.m703332200

Cited by 29 publications

(21 citation statements)

References 37 publications

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“…Taken together, these findings suggest that the extensive and complex intermolecular hydrogen bond patterns observed in NGR234 should be general to the TraR-TraM interaction in related systems. These findings provide structural information that largely corroborates the extensive mutational analyses of TraR and TraM proteins from A. tumefaciens (8,13,14). Some of the corresponding residues are clearly implicated in the NGR234 antiactivation complex structure, whereas others may play transient roles in complex formation or additional ancillary functions.…”

Section: Resultssupporting

confidence: 67%

“…It is clear that TraM can associate with TraR that is free in solution, as well as with TraR that is preassociated at a DNAbinding site (8,10,11,13). For free TraR, our findings with the NGR234 complex suggest a relatively simple mechanism.…”

Section: Discussionmentioning

confidence: 65%

“…This mechanism suggests that a transient ternary complex (TraM-TraR 2 -DNA) may form, although this may be very short lived. This ternary intermediate has been detected in A. tumefaciens when incubating TraM At with the TraR At -DNA complex (13).…”

Section: Discussionmentioning

confidence: 99%

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Structural basis for antiactivation in bacterial quorum sensing

Chen

Jeffrey

Fuqua

et al. 2007

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

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Bacteria can communicate via diffusible signal molecules they generate and release to coordinate their behavior in response to the environment. Signal molecule concentration is often proportional to bacterial population density, and when this reaches a critical concentration, reflecting a bacterial quorum, specific behaviors including virulence, symbiosis, and horizontal gene transfer are activated. Quorum-sensing regulation in many Gramnegative bacteria involves acylated homoserine lactone signals that are perceived through binding to LuxR-type, acylated-homoserine-lactone-responsive transcription factors. Bacteria of the rhizobial group employ the LuxR-type transcriptional activator TraR in quorum sensing, and its activity is further regulated through interactions with the TraM antiactivator. In this study, we have crystallographically determined the 3D structure of the TraR-TraM antiactivation complex from Rhizobium sp. strain NGR234. Unexpectedly, the antiactivator TraM binds to TraR at a site distinct from its DNA-binding motif and induces an allosteric conformational change in the protein, thereby preventing DNA binding. Structural analysis reveals a highly conserved TraR-TraM interface and suggests a mechanism for antiactivation complex formation. This structure may inform alternative strategies to control quorumsensing-regulated microbial activity including amelioration of infectious disease and antibiotic resistance. In addition, the structural basis of antiactivation presents a regulatory interaction that provides general insights relevant to the field of transcription regulation and signal transduction.crystal structure ͉ TraR-TraM complex ͉ allosteric mechanism ͉ protein-protein interaction ͉ signal transduction

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Section: Resultssupporting

confidence: 67%

Section: Discussionmentioning

confidence: 65%

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Structural basis for antiactivation in bacterial quorum sensing

Chen

Jeffrey

Fuqua

et al. 2007

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

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“…It is interesting that in some A. tumefaciens reporter strains which contain the wild-type Ti plasmid there was an 8-h delay in gene activation when AHLs were added (24). This was found to be due to the presence of a negative regulator protein, TraM, whose expression is located on the Ti plasmid (18,25). TraM tightly binds to TraR and inhibits activation of target genes of TraR (16,33).…”

Section: Confirmation Of ␤-Galactosidase Expression Upon Addition Of mentioning

confidence: 99%

“…Previously, the regulator protein TraR was thought to be loosely associated with the inner leaflet of the cytoplasmic membrane when it was not complexed to an AHL (25). Complexation of TraR with a cognate AHL converts this transcription factor to an active form (18).…”

Section: Confirmation Of ␤-Galactosidase Expression Upon Addition Of mentioning

confidence: 99%

Rapid Screening of Quorum-Sensing Signal N -Acyl Homoserine Lactones by an In Vitro Cell-Free Assay

Kawaguchi

Chen

Norman

et al. 2008

Appl Environ Microbiol

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A simple, sensitive, and rapid cell-free assay system was developed for detection of N-acyl homoserine lactone (AHL) autoinducers involved in bacterial quorum sensing (QS). The present approach improves upon previous whole-cell biosensor-based approaches in its utilization of a cell-free assay approach to conduct bioassays. The cell-free assay was derived from the AHL biosensor bacterium Agrobacterium tumefaciens NTL4(pCF218)(pCF372), allowing the expression of ␤-galactosidase upon addition of exogenous AHLs. We have shown that ␤-galactosidase expression is possible in cell-free solution [lysate from Agrobacterium tumefaciens NTL4(pCF218)(pCF372) culture]. Assay detection limits with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) ranged from approximately 100 nM to 300 nM depending on the specific AHL. Replacement (of X-Gal) with the luminescent substrate Beta-Glo increased sensitivity to AHLs by 10-fold. A major advantage of the cell-free assay system is elimination of time-consuming steps for biosensor cell culture conditioning, which are required prior to whole-cell bioassays. This significantly reduced assay times from greater than 24 h to less than 3 h, while maintaining high sensitivity. Assay lysate may be prepared in bulk and stored (؊80°C) over 6 months for future use. Finally, the present protocol may be adapted for use with other biosensor strains and be used in high-throughput AHL screening of bacteria or metagenomic libraries.Quorum sensing (QS) has been an emerging research focus in health and environmental sciences during the past decade (2, 5, 9, 37). QS is the population-dependent ability of bacteria to communicate and regulate gene expression through the production, release, and concentration-dependent sensing of signal molecules called autoinducers (9,12,38). A wide range of bacterial processes are now known to be influenced by QS and include bioluminescence, cell density control, toxin production, cell differentiation, exopolysaccharide production, motility, biofilm formation, and virulence factor production (20, 40).Autoinducers are released by cells, diffuse through the extracellular environment, and are "detected" by neighboring cells, often resulting in concentration-dependent changes in gene expression. A major class of autoinducers is the N-acyl homoserine lactones (AHLs) (20). To date, many qualitative and quantitative approaches have been developed to detect AHLs. These include whole-cell-based bioassays using AHL-specific biosensors, thinlayer chromatography, gas chromatography-mass spectrometry (MS), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), isotopic labeling, and absorbance-based assays (1,6,14,19,21,26,27,34,36,39,41,42,43).A very useful and often applied approach for QS screening is the whole-cell bioassay, which utilizes specific bacterial biosensors (31). It is relatively sensitive and does not require extensive research instrumentation, such as HPLCs and LC-MS.The ␤-galactosidase e...

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Quorum‐dependent transfer of the opine‐catabolic plasmid pAoF64/95 is regulated by a novel mechanism involving inhibition of the TraR antiactivator TraM

et al. 2018

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We previously described a plasmid of Agrobacterium spp ., pAoF 64/95, in which the quorum‐sensing system that controls conjugative transfer is induced by the opine mannopine. We also showed that the quorum‐sensing regulators TraR, TraM, and TraI function similarly to their counterparts in other rep ABC plasmids. However, traR , unlike its counterpart on Ti plasmids, is monocistronic and not located in an operon that is inducible by the conjugative opine. Here, we report that both traR and traM are expressed constitutively and not regulated by growth with mannopine. We report two additional regulatory genes, mrtR and tmsP , that are involved in a novel mechanism of control of TraR activity. Both genes are located in the distantly linked region of pAoF 64/95 encoding mannopine utilization. MrtR, in the absence of mannopine, represses the four‐gene mocC operon as well as tmsP , which is the distal gene of the eight‐gene motA operon. As judged by a bacterial two‐hybrid analysis, TmsP, which shows amino acid sequence relatedness with the TraM‐binding domain of TraR, interacts with the antiactivator. We propose a model in which mannopine, acting through the repressor MrtR, induces expression of TmsP which then titrates the levels of TraM thereby freeing TraR to activate the tra regulon.

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Molecular Basis of Transcriptional Antiactivation

Abstract: Conjugative transfer of

Cited by 29 publications

References 37 publications

Structural basis for antiactivation in bacterial quorum sensing

Structural basis for antiactivation in bacterial quorum sensing

Rapid Screening of Quorum-Sensing Signal N -Acyl Homoserine Lactones by an In Vitro Cell-Free Assay

Quorum‐dependent transfer of the opine‐catabolic plasmid pAoF64/95 is regulated by a novel mechanism involving inhibition of the TraR antiactivator TraM

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