Journal articles: 'TraR'

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He, Xuesong, William Chang, Deanne L. Pierce, Laura Ort Seib, Jennifer Wagner, and Clay Fuqua. "Quorum Sensing in Rhizobium sp. Strain NGR234 Regulates Conjugal Transfer (tra) Gene Expression and Influences Growth Rate." Journal of Bacteriology 185, no. 3 (February 1, 2003): 809–22. http://dx.doi.org/10.1128/jb.185.3.809-822.2003.

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ABSTRACT Rhizobium sp. strain NGR234 forms symbiotic, nitrogen-fixing nodules on a wide range of legumes via functions largely encoded by the plasmid pNGR234a. The pNGR234a sequence revealed a region encoding plasmid replication (rep) and conjugal transfer (tra) functions similar to those encoded by the rep and tra genes from the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens, including homologues of the Ti plasmid quorum-sensing regulators TraI, TraR, and TraM. In A. tumefaciens, TraI, a LuxI-type protein, catalyzes synthesis of the acylated homoserine lactone (acyl-HSL) N-3-oxo-octanoyl-l-homoserine lactone (3-oxo-C8-HSL). TraR binds 3-oxo-C8-HSL and activates expression of Ti plasmid tra and rep genes, increasing conjugation and copy number at high population densities. TraM prevents this activation under noninducing conditions. Although the pNGR234a TraR, TraI, and TraM appear to function similarly to their A. tumefaciens counterparts, the TraR and TraM orthologues are not cross-functional, and the quorum-sensing systems have differences. NGR234 TraI synthesizes an acyl-HSL likely to be 3-oxo-C8-HSL, but traI mutants and a pNGR234a-cured derivative produce low levels of a similar acyl-HSL and another, more hydrophobic signal molecule. TraR activates expression of several pNGR234a tra operons in response to 3-oxo-C8-HSL and is inhibited by TraM. However, one of the pNGR234a tra operons is not activated by TraR, and conjugal efficiency is not affected by TraR and 3-oxo-C8-HSL. The growth rate of NGR234 is significantly decreased by TraR and 3-oxo-C8-HSL through functions encoded elsewhere in the NGR234 genome.

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Kataoka, Masakazu, Takeshi Tanaka, Toshiyuki Kohno, and Yusuke Kajiyama. "The Carboxyl-Terminal Domain of TraR, a Streptomyces HutC Family Repressor, Functions in Oligomerization." Journal of Bacteriology 190, no. 21 (August 22, 2008): 7164–69. http://dx.doi.org/10.1128/jb.00843-08.

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ABSTRACT Efficient conjugative transfer of the Streptomyces plasmid pSN22 is accomplished by regulated expression of the tra operon genes, traA, traB, and spdB. The TraR protein is the central transcriptional repressor regulating the expression of the tra operon and itself and is classified as a member of the HutC subfamily in the helix-turn-helix (HTH) GntR protein family. Sequence information predicts that the N-terminal domain (NTD) of TraR, containing an HTH motif, functions in binding of DNA to the cis element; however, the function of the C-terminal region remains obscure, like that for many other GntR family proteins. Here we demonstrate the domain structure of the TraR protein and explain the role of the C-terminal domain (CTD). The TraR protein can be divided into two structural domains, the NTD of M1 to R95 and the CTD of Y96 to E246, revealed by limited proteolysis. Domain expression experiments revealed that both domains retained their function. An in vitro pull-down assay using recombinant TraR proteins revealed that TraR oligomerization depended on the CTD. A bacterial two-hybrid system interaction assay revealed that the minimum region necessary for this binding is R95 to P151. A mutant TraR protein in which Leu121 was replaced by His exhibited a loss of both oligomerization ability and repressor function. An in vitro cross-linking assay revealed preferential tetramer formation by TraR and the minimum CTD. These results indicate that the C-terminal R95-to-P151 region of TraR functions to form an oligomer, preferentially a tetramer, that is essential for the repressor function of TraR.

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Chen, Guozhou, Chao Wang, Clay Fuqua, Lian-Hui Zhang, and Lingling Chen. "Crystal Structure and Mechanism of TraM2, a Second Quorum-Sensing Antiactivator of Agrobacterium tumefaciens Strain A6." Journal of Bacteriology 188, no. 23 (September 22, 2006): 8244–51. http://dx.doi.org/10.1128/jb.00954-06.

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ABSTRACT Quorum sensing is a community behavior that bacteria utilize to coordinate a variety of population density-dependent biological functions. In Agrobacterium tumefaciens, quorum sensing regulates the replication and conjugative transfer of the tumor-inducing (Ti) plasmid from pathogenic strains to nonpathogenic derivatives. Most of the quorum-sensing regulatory proteins are encoded within the Ti plasmid. Among these, TraR is a LuxR-type transcription factor playing a key role as the quorum-sensing signal receptor, and TraM is an antiactivator that antagonizes TraR through the formation of a stable oligomeric complex. Recently, a second TraM homologue called TraM2, not encoded on the Ti plasmid of A. tumefaciens A6, was identified, in addition to a copy on the Ti plasmid. In this report, we have characterized TraM2 and its interaction with TraR and solved its crystal structure to 2.1 Å. Like TraM, TraM2 folds into a helical bundle and exists as homodimer. TraM2 forms a stable complex (Kd = 8.6 nM) with TraR in a 1:1 binding ratio, a weaker affinity than that of TraM for TraR. Structural analysis and biochemical studies suggest that protein stability may account for the difference between TraM2 and TraM in their binding affinities to TraR and provide a structural basis for L54 in promoting structural stability of TraM.

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Su, Shengchang, Sharik R. Khan, and Stephen K. Farrand. "Induction and Loss of Ti Plasmid Conjugative Competence in Response to the Acyl-Homoserine Lactone Quorum-Sensing Signal." Journal of Bacteriology 190, no. 13 (January 18, 2008): 4398–407. http://dx.doi.org/10.1128/jb.01684-07.

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ABSTRACT Conjugative transfer of the Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of TraR and its signal N-(3-oxo-octanoyl)-l-homoserine lactone. This system is, in turn, controlled by the conjugative opines produced by crown gall tumors induced on plants by the bacteria. Using nonpolar traI mutants, we examined the kinetics of induction of conjugative transfer in response to exogenous acyl-homoserine lactone. In the absence of the antiactivator TraM, onset of induction of transfer requires about 30 min, 15 to 20 min of which is needed for expression and construction of the conjugative apparatus. TraM delays the onset of conjugation by 30 min. While the rate of development of conjugative competence was not significantly affected by levels of TraR, maximum efficiencies of transfer were correlated with amounts of the activator in the donors. Donors harboring Ti plasmids lacking TraM were fully induced by the quormone at concentrations as low as 100 pM. TraM raised the concentration of signal required for maximum activity to 1 nM. Donors grown in batch culture retained conjugative competence following signal removal, even when in stationary phase. However, donors kept in balanced growth rapidly lost transfer ability following signal removal. Loss of transfer was mirrored by a decrease in levels of active TraR. Decreases in TraR activity and conjugative competence could be accounted for by dilution associated with cell division, suggesting that while induction of Ti plasmid conjugation is an active process, the cells lack a mechanism for disassembling the conjugative apparatus when signals become limiting.

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Zhu, Jun, John W. Beaber, Margret I. Moré, Clay Fuqua, Anatol Eberhard, and Stephen C. Winans. "Analogs of the Autoinducer 3-Oxooctanoyl-Homoserine Lactone Strongly Inhibit Activity of the TraR Protein ofAgrobacterium tumefaciens." Journal of Bacteriology 180, no. 20 (October 15, 1998): 5398–405. http://dx.doi.org/10.1128/jb.180.20.5398-5405.1998.

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ABSTRACT The TraR and TraI proteins of Agrobacterium tumefaciensmediate cell-density-dependent expression of the Ti plasmidtra regulon. TraI synthesizes the autoinducer pheromoneN-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL), while TraR is an 3-oxo-C8-HSL-responsive transcriptional activator. We have compared the abilities of 3-oxo-C8-HSL and 32 related compounds to activate expression of a TraR-regulated promoter. In a strain that expresses wild-type levels of TraR, only 3-oxo-C8-HSL was strongly stimulatory, four compounds were detectably active only at high concentrations, and the remaining 28 compounds were inactive. Furthermore, many of these compounds were potent antagonists. In contrast, almost all of these compounds were stimulatory in a congenic strain that overexpresses TraR and no compound was a potent antagonist. We propose a model in which autoinducers enhance the affinity of TraR either for other TraR monomers or for DNA binding sites and that overexpression of TraR potentiates this interaction by mass action. Wild-type A. tumefaciens released a rather broad spectrum of autoinducers, including several that antagonize induction of a wild-type strain. However, under all conditions tested, 3-oxo-C8-HSL was more abundant than any other analog, indicating that other released autoinducers do not interfere with tra gene induction. We conclude that (i) in wild-type strains, only 3-oxo-C8-HSL significantly stimulates tra gene expression, while many autoinducer analogs are potent antagonists; (ii) TraR overexpression increases agonistic activity of autoinducer analogs, allowing sensitive biodetection of many autoinducers; and (iii) autoinducer stimulatory activity is potentiated by TraR overproduction, suggesting that autoinducers may shift an equilibrium between TraR monomers and dimers or oligomers. When autoinducer specificities of other quorum-sensing proteins are tested, care should be taken not to overexpress those proteins.

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6

White, Catharine E., and Stephen C. Winans. "Cell–cell communication in the plant pathogen Agrobacterium tumefaciens." Philosophical Transactions of the Royal Society B: Biological Sciences 362, no. 1483 (March 13, 2007): 1135–48. http://dx.doi.org/10.1098/rstb.2007.2040.

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The plant pathogen Agrobacterium tumefaciens induces the formation of crown gall tumours at wound sites on host plants by directly transforming plant cells. This disease strategy benefits the bacteria as the infected plant tissue produces novel nutrients, called opines, that the colonizing bacteria can use as nutrients. Almost all of the genes that are required for virulence, and all of the opine uptake and utilization genes, are carried on large tumour-inducing (Ti) plasmids. The observation more than 25 years ago that specific opines are required for Ti plasmid conjugal transfer led to the discovery of a cell–cell signalling system on these plasmids that is similar to the LuxR–LuxI system first described in Vibrio fischeri . All Ti plasmids that have been described to date carry a functional LuxI-type N -acylhomoserine lactone synthase (TraI), and a LuxR-type signal receptor and transcriptional regulator called TraR. The traR genes are expressed only in the presence of specific opines called conjugal opines. The TraR–TraI system provides an important model for LuxR–LuxI-type systems, especially those found in the agriculturally important Rhizobiaceae family. In this review, we discuss current advances in the biochemistry and structural biology of the TraR–TraI system.

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7

Luo, Zhao-Qing, Shengchang Su, and Stephen K. Farrand. "In Situ Activation of the Quorum-Sensing Transcription Factor TraR by Cognate and Noncognate Acyl-Homoserine Lactone Ligands: Kinetics and Consequences." Journal of Bacteriology 185, no. 19 (October 1, 2003): 5665–72. http://dx.doi.org/10.1128/jb.185.19.5665-5672.2003.

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ABSTRACT Conjugal transfer of Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of the transcriptional activator TraR and its acyl-homoserine lactone quormone N-(3-oxo-octanoyl)-l-homoserine lactone (3-oxo-C8-HSL). The population density dependence of quorum-sensing systems can often be circumvented by addition of the quormone to cultures at low cell numbers. However, the quorum-dependent activation of Ti plasmid conjugal transfer exhibited a lag of almost 8 h when the quormone was added to donor cells at low population densities (Piper and Farrand, J. Bacteriol. 182:1080-1088, 2000). As measured by activation of a TraR-dependent traG::lacZ reporter fusion, TraR in cells exposed to the cognate signal for 5 min showed detectable activity, while exposure for 15 min resulted in full activity. Thus, the lag in activation is not due to some intrinsic property of TraR. Cells exposed to the agonistic analog N-(3-oxo-hexanoyl)-l-homoserine lactone (3-oxo-C6-HSL) exhibited similar induction kinetics. However, activation of the reporter in cells exposed to the poorly effective alkanoyl acyl-HSL N-hexanoyl-l-homoserine lactone (C6-HSL) required the continued presence of the signal. As measured by an in vivo repressor assay, TraR activated by 3-oxo-C6-HSL or by 3-oxo-C8-HSL remained active for as long as 8 h after removal of exogenous signal. However, TraR activated by the alkanoyl quormone C6-HSL rapidly lost activity following removal of the signal. In quormone retention assays, which measure signal binding by TraR, cells grown with either of the two 3-oxo-acyl-HSL quormones retained the ligand after washing, while cells grown with C6-HSL lost the alkanoyl-HSL concomitant with the rapid loss of TraR activity. We conclude that TraR rapidly binds its quormone and that, once bound, the cognate signal and its close homologs are tightly retained. Moreover, in the absence of other regulatory factors, activated TraR remains functional after removal of the signal. On the other hand, poorly active signals are not tightly bound, and their removal by washing leads to rapid loss of TraR activity.

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8

Tun-Garrido, Cristina, Patricia Bustos, Víctor González, and Susana Brom. "Conjugative Transfer of p42a from Rhizobium etli CFN42, Which Is Required for Mobilization of the Symbiotic Plasmid, Is Regulated by Quorum Sensing." Journal of Bacteriology 185, no. 5 (March 1, 2003): 1681–92. http://dx.doi.org/10.1128/jb.185.5.1681-1692.2003.

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ABSTRACT Rhizobium etli CFN42 contains six plasmids. Only one of them, p42a, is self-conjugative at high frequency. This plasmid is strictly required for mobilization of the symbiotic plasmid (pSym). To study the transfer mechanism of p42a, a self-transmissible cosmid clone containing its transfer region was isolated. Its sequence showed that most of the tra genes are highly similar to genes of Agrobacterium tumefaciens pTiC58 and other related plasmids. Four putative regulatory genes were identified; three of these (traI, traR, and cinR) belong to the LuxR-LuxI family. Mutagenesis of these genes confirmed their requirement for p42a transfer. We found that the conjugative transfer of p42a is dependent on quorum sensing, and consequently pSym transfer also was found to be similarly regulated, establishing a complex link between environmental conditions and pSym transfer. Although R. etli has been shown to produce different N-acyl-homoserine lactones, only one of them, a 3-oxo-C8-homoserine lactone encoded by the traI gene described here, was involved in transfer. Mutagenesis of the fourth regulatory gene, traM, had no effect on transfer. Analysis of transcriptional fusions of the regulatory genes to a reporter gene suggests a complex regulation scheme for p42a conjugative transfer. Conjugal transfer gene expression was found to be directly upregulated by TraR and the 3-oxo-C8-homoserine lactone synthesized by TraI. The traI gene was autoregulated by these elements and positively regulated by CinR, while cinR expression required traI. Finally, we did not detect expression of traM, indicating that in p42a TraM may be expressed so weakly that it cannot inhibit conjugal transfer, leading to the unrepressed transfer of p42a.

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Luo, Zhao-Qing, and Stephen K. Farrand. "The Agrobacterium tumefaciens rndHomolog Is Required for TraR-Mediated Quorum-Dependent Activation of Ti Plasmid tra Gene Expression." Journal of Bacteriology 183, no. 13 (July 1, 2001): 3919–30. http://dx.doi.org/10.1128/jb.183.13.3919-3930.2001.

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ABSTRACT Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-l-homoserine lactone. We isolated four Tn5-induced mutants of A. tumefaciens C58 deficient in TraR-mediated activation oftra genes on pTiC58ΔaccR. These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn5 was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by rnd ofEscherichia coli, an RNase known to be involved in tRNA processing. The wild-type allele of the rnd homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of cya2, surround A. tumefaciens rnd, but none of these genes exerted a detectable effect on the expression of the tra reporter. In the mutant,traR was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of tra, but not the growth rate, was partially restored by increasing the copy number of traR or by disrupting traM, a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in rnd also slightly reduced expression of two tested vir genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect intra gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D.

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Chai, Yunrong, and Stephen C. Winans. "The Chaperone GroESL Enhances the Accumulation of Soluble, Active TraR Protein, a Quorum-Sensing Transcription Factor from Agrobacterium tumefaciens." Journal of Bacteriology 191, no. 11 (March 27, 2009): 3706–11. http://dx.doi.org/10.1128/jb.01434-08.

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ABSTRACT TraR of Agrobacterium tumefaciens is a LuxR-type quorum-sensing transcription factor that regulates genes required for replication and conjugation of the tumor-inducing (Ti) plasmid. TraR requires its cognate autoinducer N-3-oxooctanoyl-homoserine lactone (OOHL) for resistance of proteolysis in wild-type bacteria and for correct protein folding and solubility when overexpressed in E. coli. In this study, we ask whether GroESL might also play a role in TraR folding, as this molecular chaperone assists many proteins in attaining their native tertiary structure. Expression of E. coli GroESL in a strain expressing TraR increases the solubility of TraR and increases transcriptional activity of a TraR-dependent promoter. Both solubility and activity still require OOHL. We also studied the folding of TraR in the closely related bacterium Sinorhizobium meliloti. A mutation in one groEL gene slightly decreased the expression of a TraR-dependent promoter, strongly decreased the accumulation of TraR in Western immunoblot assays, and also strongly influenced the fate of pulse-labeled TraR.

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Arutyunov, Denis, Barbara Arenson, Jan Manchak, and Laura S. Frost. "F Plasmid TraF and TraH Are Components of an Outer Membrane Complex Involved in Conjugation." Journal of Bacteriology 192, no. 6 (January 15, 2010): 1730–34. http://dx.doi.org/10.1128/jb.00726-09.

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ABSTRACT F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH.

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Chai, Yunrong, and Stephen C. Winans. "Amino-Terminal Protein Fusions to the TraR Quorum-Sensing Transcription Factor Enhance Protein Stability and Autoinducer-Independent Activity." Journal of Bacteriology 187, no. 4 (February 15, 2005): 1219–26. http://dx.doi.org/10.1128/jb.187.4.1219-1226.2005.

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ABSTRACT TraR of Agrobacterium tumefaciens is a member of the LuxR family of quorum-sensing transcription factors and regulates genes required for conjugation and vegetative replication of the tumor-inducing (Ti) plasmid in the presence of the autoinducer 3-oxooctanoyl-homoserine lactone (OOHL). In the absence of OOHL, TraR is rapidly destroyed by proteolysis, suggesting that this ligand is required for TraR folding. To date, no TraR variant has been found that is active in the absence of OOHL. In this study, we conducted whole-cell and plasmid mutagenesis experiments to search for constitutive mutations of traR and identified two constitutive alleles. Surprisingly, neither contained a point mutation within the traR gene, but rather, both encoded fusion proteins between TraR and the N-terminal domain of an aminoglycoside N-acetyltransferase, encoded by a plasmid-borne antibiotic resistance gene present in the original strain. Data from Western immunoblot assays, pulse-chase assays, and immunoprecipitation assays show that these fusion proteins are far more stable to proteolysis than native apo-TraR. We also constructed a library of traR alleles encoding random amino-terminal fusions and selected for constitutive TraR activity. Five independent fusion proteins were identified by this approach. These fusion proteins accumulated to far higher levels than wild-type TraR in the absence of OOHL. One of these fusions was overexpressed in Escherichia coli and showed detectable tra box binding in the absence of OOHL. These data suggest that the native amino terminus of TraR may signal proteolysis and that fusing it to other proteins might sequester it from intracellular proteases.

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Gopalkrishnan, Saumya, Wilma Ross, Albert Y. Chen, and Richard L. Gourse. "TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp." Proceedings of the National Academy of Sciences 114, no. 28 (June 26, 2017): E5539—E5548. http://dx.doi.org/10.1073/pnas.1704105114.

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TheEscherichia coliF element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the β′ rim helices that contribute to the ppGpp binding site in the DksA–ppGpp–RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements.

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Piper, Kevin R., and Stephen K. Farrand. "Quorum Sensing but Not Autoinduction of Ti Plasmid Conjugal Transfer Requires Control by the Opine Regulon and the Antiactivator TraM." Journal of Bacteriology 182, no. 4 (February 15, 2000): 1080–88. http://dx.doi.org/10.1128/jb.182.4.1080-1088.2000.

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ABSTRACT Conjugal transfer of the Ti plasmids from Agrobacterium tumefaciens is controlled by autoinduction via the transcriptional activator TraR and the acyl-homoserine lactone ligand,Agrobacterium autoinducer (AAI). This control process is itself regulated by opines, which are small carbon compounds produced by the crown gall tumors that are induced by the bacteria. Opines control autoinduction by regulating the expression of traR. Transfer of pTiC58 from donors grown with agrocinopines A and B, the conjugal opines for this Ti plasmid, was detected only after the donors had reached a population level of 107 cells per cm2. Donors incubated with the opines and AAI transferred their Ti plasmids at population levels about 10-fold lower than those incubated with opines only. Transcription of the traregulon, as assessed by monitoring atraA::lacZ reporter, showed a similar dependence on the density of the donor population. However, even in cultures at low population densities that were induced with opines and AAI, there was a temporal lag of between 15 and 20 h in the development of conjugal competence. Moreover, even after this latent period, maximal transfer frequencies required several hours to develop. This lag period was independent of the population density of the donors but could be reduced somewhat by addition of exogenous AAI. Quorum-dependent development of conjugal competence required control by the opine regulon; donors harboring a mutant of pTiC58 deleted for the master opine responsive repressor accR transferred the Ti plasmid at maximum frequencies at very low population densities. Similarly, an otherwise wild-type derivative of pTiC58 lackingtraM, which codes for an antiactivator that inhibits TraR activity, transferred at high frequency in a population-independent manner in the absence of the conjugal opines. Thus, while quorum sensing is dependent upon autoinduction, the two phenomena are not synonymous. We conclude that conjugal transfer of pTiC58 is regulated in a quorum-dependent fashion but that supercontrol of the TraR-AAI system by opines and by TraM results in a complex control process that requires not only the accumulation of AAI but also the expression of TraR and the synthesis of this protein at levels that overcome the inhibitory activity of TraM.

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Acosta-Jurado, Sebastián, Cynthia Alías-Villegas, Andrés Almozara, M. Rosario Espuny, José-María Vinardell, and Francisco Pérez-Montaño. "Deciphering the Symbiotic Significance of Quorum Sensing Systems of Sinorhizobium fredii HH103." Microorganisms 8, no. 1 (January 2, 2020): 68. http://dx.doi.org/10.3390/microorganisms8010068.

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Quorum sensing (QS) is a bacterial cell-to-cell signaling mechanism that collectively regulates and synchronizes behaviors by means of small diffusible chemical molecules. In rhizobia, QS systems usually relies on the synthesis and detection of N-acyl-homoserine lactones (AHLs). In the model bacterium Sinorhizobium meliloti functions regulated by the QS systems TraI-TraR and SinI-SinR(-ExpR) include plasmid transfer, production of surface polysaccharides, motility, growth rate and nodulation. These systems are also present in other bacteria of the Sinorhizobium genus, with variations at the species and strain level. In Sinorhizobium fredii NGR234 phenotypes regulated by QS are plasmid transfer, growth rate, sedimentation, motility, biofilm formation, EPS production and copy number of the symbiotic plasmid (pSym). The analysis of the S. fredii HH103 genomes reveal also the presence of both QS systems. In this manuscript we characterized the QS systems of S. fredii HH103, determining that both TraI and SinI AHL-synthases proteins are responsible of the production of short- and long-chain AHLs, respectively, at very low and not physiological concentrations. Interestingly, the main HH103 luxR-type genes, expR and traR, are split into two ORFs, suggesting that in S. fredii HH103 the corresponding carboxy-terminal proteins, which contain the DNA-binding motives, may control target genes in an AHL-independent manner. The presence of a split traR gene is common in other S. fredii strains.

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Chu, Chishih, Cheng-Hsun Chiu, Chi-Hong Chu, and Jonathan T. Ou. "Nucleotide and Amino Acid Sequences of oriT-traM-traJ-traY-traA-traL Regions and Mobilization of Virulence Plasmids of Salmonella enterica Serovars Enteritidis, Gallinarum-Pullorum, and Typhimurium." Journal of Bacteriology 184, no. 11 (June 1, 2002): 2857–62. http://dx.doi.org/10.1128/jb.184.11.2857-2862.2002.

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ABSTRACT The virulence plasmid of Salmonella enterica serovar Gallinarum-Pullorum (pSPV) but not those of Salmonella enterica serovars Enteritidis (pSEV) and Typhimurium (pSTV) can be readily mobilized by an F or F-like conjugative plasmid. To investigate the reason for the difference, the oriT-traM-traJ-traY-traA-traL regions of the three salmonella virulence plasmids (pSVs) were cloned and their nucleotide and deduced amino acid sequences were examined. The cloned fragments were generally mobilized more readily than the corresponding full-length pSVs, but the recombinant plasmid containing the oriT of pSPV was, as expected, more readily mobilized, with up to 100-fold higher frequency than the recombinant plasmids containing the oriT of the other two pSVs. The nucleotide sequences of the oriT-traM-traJ-traY-traA-traL region of pSEV and pSTV were almost identical (only 4 bp differences), but differed from that of pSPV. Major nucleotide sequence variations were found in traJ, traY, and the Tra protein binding sites sby and sbm. sby of pSPV showed higher similarity than that of pSEV or pSTV to that of the F plasmid. The reverse was true for sbm: similarity was higher with pSEV and pSTV than with pSPV. In the deduced amino acid sequences of the five Tra proteins, major differences were found in TraY: pSEV's TraY was 75 amino acids, pSTV's was 106 amino acids, and pSPV's was 133 amino acids; and there were duplicate consensus βαα fragments in the TraY of pSPV and F plasmid, whereas there was only a single βαα fragment in that of pSEV and pSTV.

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Oger, Philippe, and Stephen K. Farrand. "Two Opines Control Conjugal Transfer of an Agrobacterium Plasmid by Regulating Expression of Separate Copies of the Quorum-Sensing Activator Gene traR." Journal of Bacteriology 184, no. 4 (February 15, 2002): 1121–31. http://dx.doi.org/10.1128/jb.184.4.1121-1131.2002.

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ABSTRACT Conjugal transfer of Ti plasmids from Agrobacterium spp. is controlled by a hierarchical regulatory system designed to sense two environmental cues. One signal, a subset of the opines produced by crown gall tumors initiated on plants by the pathogen, serves to induce production of the second, an acyl-homoserine lactone quorum-sensing signal, the quormone, produced by the bacterium itself. This second signal activates TraR, and this transcriptional activator induces expression of the tra regulon. Opines control transfer because the traR gene is a member of an operon the expression of which is regulated by the conjugal opine. Among the Ti plasmid systems studied to date, only one of the two or more opine families produced by the associated tumor induces transfer. However, two chemically dissimilar opines, nopaline and agrocinopines A and B, induce transfer of the opine catabolic plasmid pAtK84b found in the nonpathogenic Agrobacterium radiobacter isolate K84. In this study we showed that this plasmid contains two copies of traR, and each is associated with a different opine-regulated operon. One copy, traR noc, is the last gene of the nox operon and was induced by nopaline but not by agrocinopines A and B. Mutating traR noc abolished induction of transfer by nopaline but not by the agrocinopines. A mutation in ocd, an upstream gene of the nox operon, abolished utilization of nopaline and also induction of transfer by this opine. The second copy, traR acc, is located in an operon of four genes and was induced by agrocinopines A and B but not by nopaline. Genetic analysis indicated that this gene is required for induction of transfer by agrocinopines A and B but not by nopaline. pAtK84b with mutations in both traR genes was not induced for transfer by either opine. However, expression of a traR gene in trans to this plasmid resulted in opine-independent transfer. The association of traR noc with nox is unique, but the operon containing traR acc is related to the arc operons of pTiC58 and pTiChry5, two Ti plasmids inducible for transfer by agrocinopines A-B and C-D, respectively. We conclude that pAtK84b codes for two independently functioning copies of traR, each regulated by a different opine, thus accounting for the activation of the transfer system of this plasmid by the two opine types.

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Grace, Elicia D., Saumya Gopalkrishnan, Mary E. Girard, Matthew D. Blankschien, Wilma Ross, Richard L. Gourse, and Christophe Herman. "Activation of the σE-Dependent Stress Pathway by Conjugative TraR May Anticipate Conjugational Stress." Journal of Bacteriology 197, no. 5 (December 22, 2014): 924–31. http://dx.doi.org/10.1128/jb.02279-14.

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Horizontal gene transfer by conjugation plays a major role in bacterial evolution, allowing the acquisition of new traits, such as virulence and resistance to antibacterial agents. With the increased antibiotic resistance in bacterial pathogens, a better understanding of how bacteria modulate conjugation under changing environments and the genetic factors involved is needed. Despite the evolutionary advantages conjugation may confer, the process can be quite stressful for the donor cell. Here, we characterize the ability of TraR, encoded on the episomal F′ plasmid, to upregulate the σEextracytoplasmic stress pathway inEscherichia coli. TraR, a DksA homolog, modulates transcription initiation through the secondary channel of RNA polymerase. We show here that TraR activates transcription directly; however, unlike DksA, it does so without using ppGpp as a cofactor. TraR expression can stimulate the σEextracytoplasmic stress response independently of the DegS/RseA signal transduction cascade. In the absence of TraR, bacteria carrying conjugative plasmids become more susceptible to external stress. We propose that TraR increases the concentrations of periplasmic chaperones and proteases by directly activating the transcription of σE-dependent promoters; this increased protein folding capacity may prepare the bacterium to endure the periplasmic stress of sex pilus biosynthesis during mating.

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Luo, Zhao-Qing, Audra J. Smyth, Ping Gao, Yinping Qin, and Stephen K. Farrand. "Mutational Analysis of TraR." Journal of Biological Chemistry 278, no. 15 (February 4, 2003): 13173–82. http://dx.doi.org/10.1074/jbc.m210035200.

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Miyazaki, Ryo, Yoshiyuki Ohtsubo, Yuji Nagata, and Masataka Tsuda. "Characterization of the traD Operon of Naphthalene-Catabolic Plasmid NAH7: a Host-Range Modifier in Conjugative Transfer." Journal of Bacteriology 190, no. 19 (August 1, 2008): 6281–89. http://dx.doi.org/10.1128/jb.00709-08.

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ABSTRACT Pseudomonas putida G7 carries a naphthalene-catabolic and self-transmissible plasmid, NAH7, which belongs to the IncP-9 incompatibility group. Adjacent to the putative origin of conjugative transfer (oriT) of NAH7 are three genes, traD, traE, and traF, whose functions and roles in conjugation were previously unclear. These three genes were transcribed monocistronically and thus were designated the traD operon. Mutation of the three genes in the traD operon resulted in 10- to 105-fold decreases in the transfer frequencies of the plasmids from Pseudomonas to Pseudomonas and Escherichia coli and from E. coli to E. coli. On the other hand, the traD operon was essential for the transfer of NAH7 from E. coli to Pseudomonas strains. These results indicated that the traD operon is a host-range modifier in the conjugative transfer of NAH7. The TraD, TraE, and TraF proteins were localized in the cytoplasm, periplasm, and membrane, respectively, in strain G7 cells. Our use of a bacterial two-hybrid assay system showed that TraE interacted in vivo with other essential components for conjugative transfer, including TraB (coupling protein), TraC (relaxase), and MpfH (a channel subunit in the mating pair formation system).

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Aneskievich, B. J., and E. Fuchs. "The A/B domain of truncated retinoic acid receptors can block differentiation and promote features of malignancy." Journal of Cell Science 108, no. 1 (January 1, 1995): 195–205. http://dx.doi.org/10.1242/jcs.108.1.195.

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Recently, we discovered that stable introduction of a carboxyl-terminally truncated retinoic acid receptor gamma (tRAR gamma) into an epidermal keratinocyte line blocked the ability of these cells to differentiate, as judged by their failure to express late markers of squamous differentiation. We now demonstrate a correlation between the level of residual endogenous RAR activity of tRAR gamma-expressing keratinocyte lines and degree of terminal differentiation. Mutagenesis studies localize the effects to the A/B subdomain of the truncated receptor. Despite tRAR gamma's capacity to interfere with RAR-mediated transactivation of retinoic acid response elements (RAREs) in keratinocytes, the effects of the truncated receptor are independent of its ability to bind DNA and directly interact with endogenous RARs. tRAR alpha also inhibits RARE-mediated gene expression in keratinocytes, even though its full-length counterpart enhances RARE activity in these cells. Intriguingly, both tRAR gamma and RAR gamma suppress keratin promoter activity in epidermal cells, although for tRAR gamma, the effect is mediated through the A/B domain whereas for RAR gamma, the effects require DNA binding. Taken together, these findings suggest that the truncation allows for new and aberrant interactions with transcriptional proteins/cofactors that participate in governing RARE activity. This discovery may have relevance in tumorigenesis, where genetic lesions can result in mutant RARs or in loss of receptor expression.

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Pérez, Daniela, Maicol Ahumedo, Eileen Herrera, Catalina Vivas-Gomez, and Ricardo Vivas-Reyes. "Computational study of the interactions among structural analogues of acyl homoserine lactones (AHLs) and the Agrobacterium tumefaciens TraR binding site." F1000Research 8 (December 5, 2019): 2062. http://dx.doi.org/10.12688/f1000research.20793.1.

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Background: In the present investigation, relationships between a set of 34 analogues of N-acyl-L-homoserine lactones (AHL) and the TraR receptor were studied. The aim was to use molecular modeling as a strategy for elucidating important aspects of the mechanism of chemical signaling in the Gram-negative bacteria Agrobacterium tumefaciens, with the idea of identifying some of analogues’ structural characteristics and molecular interactions with the active site of the TraR receptor. Methods: For this purpose, we combine two molecular modeling strategies: molecular docking and three-dimensional quantitative structure-activity relationship (3D-QSAR). First, the molecular docking methodology was applied to a series of 34 analogues of AHL on the TraR transcriptional receptor to simulate the binding of analogues at the active TraR site. Secondly, 3D-QSAR models were generated to describe the correlation with the experimental biological activity using partial least squares (PLS) calculations and steric and electrostatic properties, which theoretically predict the activity of the 34 AHL analogues through statistical parameters and evaluate the prediction of the models obtained. Two alignment models were constructed; one using the optimized structures of the 34 analogues (ligand-based model) and another using the conformations of the best poses generated in the docking with TraR (receptor-based model). Results: The outcomes obtained for each protein-ligand complex showed that the Aspartic acid 70 and Threonine 129 residues are residues that participate in the formation of hydrogen bonds, while residues Alanine 38, Leucine, 40, Tyrosine 53, Glutamine 58, Tyrosine 61, Phenylalanine 62 and Valine 72 form hydrophobic interactions. These interactions are important in determining the antagonistic activity of the analogues under study against TraR. Conclusions: The ligand-based model produces better statistical results expressed in terms of several rigorous evaluation criteria, such as Q2 and R2 for the data sets than those of the receptor-based model.

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Chen, Guozhou, James W. Malenkos, Mee-Rye Cha, Clay Fuqua, and Lingling Chen. "Quorum-sensing antiactivator TraM forms a dimer that dissociates to inhibit TraR." Molecular Microbiology 52, no. 6 (May 14, 2004): 1641–51. http://dx.doi.org/10.1111/j.1365-2958.2004.04110.x.

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Li, Pei-Li, and Stephen K. Farrand. "The Replicator of the Nopaline-Type Ti Plasmid pTiC58 Is a Member of the repABC Family and Is Influenced by the TraR-Dependent Quorum-Sensing Regulatory System." Journal of Bacteriology 182, no. 1 (January 1, 2000): 179–88. http://dx.doi.org/10.1128/jb.182.1.179-188.2000.

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ABSTRACT The replicator (rep) of the nopaline-type Ti plasmid pTiC58 is located adjacent to the trb operon of this conjugal element. Previous genetic studies of this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1034–1041, 1985) identified functions involved in partitioning, origin of replication and incompatibility, and copy number control. In this study, we determined the nucleotide sequence of a 6,146-bp segment that encompasses the rep locus of pTiC58. The region contained four full open reading frames (ORFs) and one partial ORF. The first three ORFs, oriented divergently from the traI-trb operon, are closely related to the repA, repB, andrepC genes of the octopine-type Ti plasmid pTiB6S3 as well as to other repA, -B, and -C genes from the Ri plasmid pRiA4b and three large plasmids fromRhizobium spp. The fourth ORF and the partial ORF are similar to y4CG and y4CF, respectively, of the Sym plasmid pNGR234a. The 363-bp intergenic region betweentraI and repA contained two copies of thetra box which is the cis promoter recognition site for TraR, the quorum-sensing activator of Ti plasmid conjugal transfer. Expression of the traI-trb operon from thetra box II-associated promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an intact tra box III. On the other hand, the region containing the two tra boxes was required for maximal expression of repA, and this expression was enhanced slightly by TraR and AAI. Copy number of a minimal repplasmid increased five- to sevenfold in strains expressingtraR but only when AAI also was provided. Consistent with this effect, constitutive expression of the quorum-sensing system resulted in an apparent increase in Ti plasmid copy number. We conclude that Ti plasmid copy number is influenced by the quorum-sensing system, suggesting a connection between conjugal transfer and vegetative replication of these virulence elements.

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Khan, Sharik R., Jennifer Gaines, R. Martin Roop, and Stephen K. Farrand. "Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing." Applied and Environmental Microbiology 74, no. 16 (July 7, 2008): 5053–62. http://dx.doi.org/10.1128/aem.01098-08.

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ABSTRACT Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI q-lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha- and gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZα. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens, a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.

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Marketon, Melanie M., and Juan E. González. "Identification of Two Quorum-Sensing Systems in Sinorhizobium meliloti." Journal of Bacteriology 184, no. 13 (July 1, 2002): 3466–75. http://dx.doi.org/10.1128/jb.184.13.3466-3475.2002.

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ABSTRACT Sinorhizobium meliloti is a free-living soil bacterium which is capable of establishing a symbiotic relationship with the alfalfa plant (Medicago sativa). This symbiosis involves a network of bacterium-host signaling, as well as the potential for bacterium-bacterium communication, such as quorum sensing. In this study, we characterized the production of N-acyl homoserine lactones (AHLs) by two commonly used S. meliloti strains, AK631 and Rm1021. We found that AK631 produces at least nine different AHLs, while Rm1021 produces only a subset of these molecules. To address the difference in AHL patterns between the strains, we developed a novel screening method to identify the genes affecting AHL synthesis. With this screening method, chromosomal groEL (groELc) was shown to be required for synthesis of the AHLs that are unique to AK631 but not for synthesis of the AHLs that are made by both AK631 and Rm1021. We then used the screening procedure to identify a mutation in a gene homologous to traM of Agrobacterium tumefaciens, which was able to suppress the phenotype of the groELc mutation. A traR homolog was identified immediately upstream of traM, and we propose that its gene product requires a functional groELc for activity and is also responsible for inducing the synthesis of the AHLs that are unique to AK631. We show that the traR/traM locus is part of a quorum-sensing system unique to AK631 and propose that this locus is involved in regulating conjugal plasmid transfer. We also present evidence for the existence of a second quorum-sensing system, sinR/sinI, which is present in both AK631 and Rm1021.

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Gu, Jiqing, Chao Song, Wenjun Jiang, Xiaomin Wang, and Ming Liu. "Enhancing Personalized Trip Recommendation with Attractive Routes." Proceedings of the AAAI Conference on Artificial Intelligence 34, no. 01 (April 3, 2020): 662–69. http://dx.doi.org/10.1609/aaai.v34i01.5407.

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Personalized trip recommendation tries to recommend a sequence of point of interests (POIs) for a user. Most of existing studies search POIs only according to the popularity of POIs themselves. In fact, the routes among the POIs also have attractions to visitors, and some of these routes have high popularity. We term this kind of route as Attractive Route (AR), which brings extra user experience. In this paper, we study the attractive routes to improve personalized trip recommendation. To deal with the challenges of discovery and evaluation of ARs, we propose a personalized Trip Recommender with POIs and Attractive Route (TRAR). It discovers the attractive routes based on the popularity and the Gini coefficient of POIs, then it utilizes a gravity model in a category space to estimate the rating scores and preferences of the attractive routes. Based on that, TRAR recommends a trip with ARs to maximize user experience and leverage the tradeoff between the time cost and the user experience. The experimental results show the superiority of TRAR compared with other state-of-the-art methods.

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Karl, Wolfgang, Martina Bamberger, and Ellen L. Zechner. "Transfer Protein TraY of Plasmid R1 Stimulates TraI-Catalyzed oriT Cleavage In Vivo." Journal of Bacteriology 183, no. 3 (February 1, 2001): 909–14. http://dx.doi.org/10.1128/jb.183.3.909-914.2001.

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ABSTRACT The effect of TraY protein on TraI-catalyzed strand scission at the R1 transfer origin (oriT) in vivo was investigated. As expected, the cleavage reaction was not detected in Escherichia coli cells expressing tral and the integration host factor (IHF) in the absence of other transfer proteins. The TraM dependence of strand scission was found to be inversely correlated with the presence of TraY. Thus, the TraY and TraM proteins could each enhance cleaving activity at oriT in the absence of the other. In contrast, no detectable intracellular cleaving activity was exhibited by TraI in an IHF mutant strain despite the additional presence of both TraM and TraY. An essential role for IHF in this reaction in vivo is, therefore, implied. Mobilization experiments employing recombinant R1 oriT constructions and a heterologous conjugative helper plasmid were used to investigate the independent contributions of TraY and TraM to the R1 relaxosome during bacterial conjugation. In accordance with earlier observations,traY was dispensable for mobilization in the presence oftraM, but mobilization did not occur in the absence of bothtraM and traY. Interestingly, although the cleavage assays demonstrate that TraM and TraY independently promote strand scission in vivo, TraM remained essential for mobilization of the R1 origin even in the presence of TraY. These findings suggest that, whereas TraY and TraM function may overlap to a certain extent in the R1 relaxosome, TraM additionally performs a second function that is essential for successful conjugative transmission of plasmid DNA.

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Chai, Yunrong, Jun Zhu, and Stephen C. Winans. "TrlR, a defective TraR-like protein of Agrobacterium tumefaciens, blocks TraR function in vitro by forming inactive TrlR:TraR dimers." Molecular Microbiology 40, no. 2 (April 2001): 414–21. http://dx.doi.org/10.1046/j.1365-2958.2001.02385.x.

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Koch, B., T. Liljefors, T. Persson, J. Nielsen, S. Kjelleberg, and M. Givskov. "The LuxR receptor: the sites of interaction with quorum-sensing signals and inhibitors." Microbiology 151, no. 11 (November 1, 2005): 3589–602. http://dx.doi.org/10.1099/mic.0.27954-0.

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The function of LuxR homologues as quorum sensors is mediated by the binding of N-acyl-l-homoserine lactone (AHL) signal molecules to the N-terminal receptor site of the proteins. In this study, site-directed mutagenesis was carried out of the amino acid residues comprising the receptor site of LuxR from Vibrio fischeri, and the ability of the L42A, L42S, Y62F, W66F, D79N, W94D, V109D, V109T and M135A LuxR mutant proteins to activate green fluorescent protein expression from a PluxI promoter was measured. X-ray crystallographic studies of the LuxR homologue TraR indicated that residues Y53 and W57 form hydrogen bonds to the 1-carbonyl group and the ring carbonyl group, respectively, of the cognate AHL signal. Based on the activity and signal specificity of the LuxR mutant proteins, and on molecular modelling, a model is suggested in which Y62 (corresponding to Y53 in TraR) forms a hydrogen bond with the ring carbonyl group rather than the 1-carbonyl group, while W66 (corresponding to W57 in TraR) forms a hydrogen bond to the 1-carbonyl group. This flips the position of the acyl side chain in the LuxR/signal molecule complex compared to the TraR/signal molecule complex. Halogenated furanones from the marine alga Delisea pulchra and the synthetic signal analogue N-(sulfanylacetyl)-l-homoserine lactone can block quorum sensing. The LuxR mutant proteins were insensitive to inhibition by N-(propylsulfanylacetyl)-l-homoserine lactone. In contrast, the mutations had only a minor effect on the sensitivity of the proteins to halogenated furanones, and the data strongly suggest that these compounds do not compete in a ‘classic’ way with N-3-oxohexanoyl-l-homoserine lactone for the binding site. Based on modelling and experimental data it is suggested that these compounds bind in a non-agonist fashion.

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Zhu, Jun, and Stephen C. Winans. "Activity of the quorum-sensing regulator TraR of Agrobacterium tumefaciens is inhibited by a truncated, dominant defective TraR-like protein." Molecular Microbiology 27, no. 2 (February 1998): 289–97. http://dx.doi.org/10.1046/j.1365-2958.1998.00672.x.

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Harris, Robin L., and Philip M. Silverman. "Tra Proteins Characteristic of F-Like Type IV Secretion Systems Constitute an Interaction Group by Yeast Two-Hybrid Analysis." Journal of Bacteriology 186, no. 16 (August 15, 2004): 5480–85. http://dx.doi.org/10.1128/jb.186.16.5480-5485.2004.

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ABSTRACT Using yeast two-hybrid screens, we have defined an interaction group of six Tra proteins encoded by the F plasmid and required by F+ cells to elaborate F pili. The six proteins are TraH, TraF, TraW, TraU, TrbI, and TrbB. Except for TrbI, these proteins were all identified as hallmarks of F-like type IV secretion systems (TFSSs), with no homologues among TFSS genes of P-type or I-type systems (T. Lawley, W. Klimke, M. Gubbins, and L. Frost, FEMS Microbiol. Lett. 224:1-15, 2003). Also with the exception of TrbI, which is an inner membrane protein, the remaining proteins are or are predicted to be periplasmic. TrbI consists of one membrane-spanning segment near its N terminus and an 88-residue, hydrophilic domain that extends into the periplasm. Hence, the proteins of this group probably form a periplasmic cluster in Escherichia coli. The interaction network identifies TraH as the most highly connected node, with two-hybrid links to TrbI, TraU, and TraF. As measured by transcriptional activation of lacZ, the TrbI-TraH interaction in Saccharomyces cerevisiae requires the TraH amino acid segment from residues 193 to 225. The TraU and TraF interactions are localized to C-terminal segments of TraH (amino acids 315 to 458 for TraF and amino acids 341 to 458 for TraU). The TrbI-TraH interaction with full-length (less the signal peptide) TraH is weak but increases 40-fold with N-terminal TraH deletions; the first 50 amino acids appear to be critical for inhibiting TrbI binding in yeast. Previous studies by others have shown that, with the exception of trbB mutations, which do not affect the elaboration or function of F pili under laboratory conditions, a mutation in any of the other genes in this interaction group alters the number or length distribution of F pili. We propose a model whereby one function of the TraH interaction group is to control F-pilus extension and retraction.

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Ramsay, Joshua P., Laura G. L. Tester, Anthony S. Major, John T. Sullivan, Christina D. Edgar, Torsten Kleffmann, Jackson R. Patterson-House, et al. "Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing–activated transfer of a mobile genetic element." Proceedings of the National Academy of Sciences 112, no. 13 (March 18, 2015): 4104–9. http://dx.doi.org/10.1073/pnas.1501574112.

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Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSymR7A is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNAphe from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSymR7A, suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSymR7A-encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSymR7A excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSymR7A transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSymR7A transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels.

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Vannini, Alessandro, Cinzia Volpari, and Stefania Di Marco. "Crystal Structure of the Quorum-sensing Protein TraM and Its Interaction with the Transcriptional Regulator TraR." Journal of Biological Chemistry 279, no. 23 (March 24, 2004): 24291–96. http://dx.doi.org/10.1074/jbc.m401855200.

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Vannini, A., C. Volpari, and S. Di Marco. "Crystal structure of the quorum sensing protein TraM and its interaction with the transcriptional regulator TraR." Acta Crystallographica Section A Foundations of Crystallography 60, a1 (August 26, 2004): s137. http://dx.doi.org/10.1107/s0108767304097296.

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Fuqua, Clay, and Stephen C. Winans. "Localization of OccR-activated and TraR-activated promoters that express two ABC-type permeases and the traR gene of Ti plasmid pTiR10." Molecular Microbiology 20, no. 6 (June 1996): 1199–210. http://dx.doi.org/10.1111/j.1365-2958.1996.tb02640.x.

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Grahn, A. Marika, Jana Haase, Dennis H. Bamford, and Erich Lanka. "Components of the RP4 Conjugative Transfer Apparatus Form an Envelope Structure Bridging Inner and Outer Membranes of Donor Cells: Implications for Related Macromolecule Transport Systems." Journal of Bacteriology 182, no. 6 (March 15, 2000): 1564–74. http://dx.doi.org/10.1128/jb.182.6.1564-1574.2000.

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ABSTRACT During bacterial conjugation, the single-stranded DNA molecule is transferred through the cell envelopes of the donor and the recipient cell. A membrane-spanning transfer apparatus encoded by conjugative plasmids has been proposed to facilitate protein and DNA transport. For the IncPα plasmid RP4, a thorough sequence analysis of the gene products of the transfer regions Tra1 and Tra2 revealed typical features of mainly inner membrane proteins. We localized essential RP4 transfer functions to Escherichia coli cell fractions by immunological detection with specific polyclonal antisera. Each of the gene products of the RP4 mating pair formation (Mpf) system, specified by the Tra2 core region and by traF of the Tra1 region, was found in the outer membrane fraction with one exception, the TrbB protein, which behaved like a soluble protein. The membrane preparation from Mpf-containing cells had an additional membrane fraction whose density was intermediate between those of the cytoplasmic and outer membranes, suggesting the presence of attachment zones between the twoE. coli membranes. The Tra1 region is known to encode the components of the RP4 relaxosome. Several gene products of this transfer region, including the relaxase TraI, were detected in the soluble fraction, but also in the inner membrane fraction. This indicates that the nucleoprotein complex is associated with and/or assembled facing the cytoplasmic site of the E. coli cell envelope. The Tra1 protein TraG was predominantly localized to the cytoplasmic membrane, supporting its potential role as an interface between the RP4 Mpf system and the relaxosome.

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Khan, Sharik R., and Stephen K. Farrand. "The BlcC (AttM) Lactonase of Agrobacterium tumefaciens Does Not Quench the Quorum-Sensing System That Regulates Ti Plasmid Conjugative Transfer." Journal of Bacteriology 191, no. 4 (November 14, 2008): 1320–29. http://dx.doi.org/10.1128/jb.01304-08.

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ABSTRACT The conjugative transfer of Agrobacterium plasmids is controlled by a quorum-sensing system consisting of TraR and its acyl-homoserine lactone (HSL) ligand. The acyl-HSL is essential for the TraR-mediated activation of the Ti plasmid Tra genes. Strains A6 and C58 of Agrobacterium tumefaciens produce a lactonase, BlcC (AttM), that can degrade the quormone, leading some to conclude that the enzyme quenches the quorum-sensing system. We tested this hypothesis by examining the effects of the mutation, induction, or mutational derepression of blcC on the accumulation of acyl-HSL and on the conjugative competence of strain C58. The induction of blc resulted in an 8- to 10-fold decrease in levels of extracellular acyl-HSL but in only a twofold decrease in intracellular quormone levels, a measure of the amount of active intracellular TraR. The induction or mutational derepression of blc as well as a null mutation in blcC had no significant effect on the induction of or continued transfer of pTiC58 from donors in any stage of growth, including stationary phase. In matings performed in developing tumors, wild-type C58 transferred the Ti plasmid to recipients, yielding transconjugants by 14 to 21 days following infection. blcC-null donors yielded transconjugants 1 week earlier, but by the following week, transconjugants were recovered at numbers indistinguishable from those of the wild type. Donors mutationally derepressed for blcC yielded transconjugants in planta at numbers 10-fold lower than those for the wild type at weeks 2 and 3, but by week 4, the two donors showed no difference in recoverable transconjugants. We conclude that BlcC has no biologically significant effect on Ti plasmid transfer or its regulatory system.

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Zhu, J., and S. C. Winans. "Autoinducer binding by the quorum-sensing regulator TraR increases affinity for target promoters in vitro and decreases TraR turnover rates in whole cells." Proceedings of the National Academy of Sciences 96, no. 9 (April 27, 1999): 4832–37. http://dx.doi.org/10.1073/pnas.96.9.4832.

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Schmidt-Eisenlohr, Heike, Natalie Domke, and Christian Baron. "TraC of IncN Plasmid pKM101 Associates with Membranes and Extracellular High-Molecular-Weight Structures inEscherichia coli." Journal of Bacteriology 181, no. 18 (September 15, 1999): 5563–71. http://dx.doi.org/10.1128/jb.181.18.5563-5571.1999.

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ABSTRACT Conjugative transfer of IncN plasmid pKM101 is mediated by the TraI-TraII region-encoded transfer machinery components. Similar to the case for the related Agrobacterium tumefaciens T-complex transfer apparatus, this machinery is needed for assembly of pili to initiate cell-to-cell contact preceding DNA transfer. Biochemical and cell biological experiments presented here show extracellular localization of TraC, as suggested by extracellular complementation of TraC-deficient bacteria by helper cells expressing a functional plasmid transfer machinery (S. C. Winans, and G. C. Walker, J. Bacteriol. 161:402–410, 1985). Overexpression of TraC and its export in large amounts into the periplasm of Escherichia coliallowed purification by periplasmic extraction, ammonium sulfate precipitation, and column chromatography. Whereas TraC was soluble in overexpressing strains, it partly associated with the membranes in pKM101-carrying cells, possibly due to protein-protein interactions with other components of the TraI-TraII region-encoded transfer machinery. Membrane association of TraC was reduced in strains carrying pKM101 derivatives with transposon insertions in genes coding for other essential components of the transfer machinery,traM, traB, traD, andtraE but not eex, coding for an entry exclusion protein not required for DNA transfer. Cross-linking identified protein-protein interactions of TraC in E. coli carrying pKM101 but not derivatives with transposon insertions in essentialtra genes. Interactions with membrane-bound Tra proteins may incorporate TraC into a surface structure, suggested by its removal from the cell by shearing as part of a high-molecular-weight complex. Heterologous expression of TraC in A. tumefaciens partly compensated for the pilus assembly defect in strains deficient for its homolog VirB5, which further supported its role in assembly of conjugative pili. In addition to its association with high-molecular-weight structures, TraC was secreted into the extracellular milieu. Conjugation experiments showed that secreted TraC does not compensate transfer deficiency of TraC-deficient cells, suggesting that extracellular complementation may rely on cell-to-cell transfer of TraC only as part of a bona fide transfer apparatus.

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Costa, Esther D., Yunrong Chai, and Stephen C. Winans. "The quorum-sensing protein TraR of Agrobacterium tumefaciens is susceptible to intrinsic and TraM-mediated proteolytic instability." Molecular Microbiology 84, no. 5 (April 19, 2012): 807–15. http://dx.doi.org/10.1111/j.1365-2958.2012.08037.x.

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Lawley, Trevor D., Matthew W. Gilmour, James E. Gunton, Leah J. Standeven, and Diane E. Taylor. "Functional and Mutational Analysis of Conjugative Transfer Region 1 (Tra1) from the IncHI1 Plasmid R27." Journal of Bacteriology 184, no. 8 (April 15, 2002): 2173–80. http://dx.doi.org/10.1128/jb.184.8.2173-2180.2002.

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ABSTRACT The conjugative transfer region 1 (Tra1) of the IncHI1 plasmid R27 was subjected to DNA sequence analysis, mutagenesis, genetic complementation, and an H-pilus-specific phage assay. Analysis of the nucleotide sequence indicated that the Tra1 region contains genes coding for mating pair formation (Mpf) and DNA transfer replication (Dtr) and a coupling protein. Insertional disruptions of 9 of the 14 open reading frames (ORFs) in the Tra1 region resulted in a transfer-deficient phenotype. Conjugative transfer was restored for each transfer mutant by genetic complementation. An intergenic region between traH and trhR was cloned and mobilized by R27, indicating the presence of an origin of transfer (oriT). The five ORFs immediately downstream of the oriT region are involved in H-pilus production, as determined by an H-pilus-specific phage assay. Three of these ORFs encode proteins homologous to Mpf proteins from IncF plasmids. Upstream of the oriT region are four ORFs required for plasmid transfer but not H-pilus production. TraI contains sequence motifs that are characteristic of relaxases from the IncP lineage but share no overall homology to known relaxases. TraJ contains both an Arc repressor motif and a leucine zipper motif. A putative coupling protein, TraG, shares a low level of homology to the TraG family of coupling proteins and contains motifs that are important for DNA transfer. This analysis indicates that the Mpf components of R27 share a common lineage with those of the IncF transfer system, whereas the relaxase of R27 is ancestrally related to that of the IncP transfer system.

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Lin, Mei-Hui, and Shih-Tung Liu. "Stabilization of pSW100 from Pantoea stewartii by the F Conjugation System." Journal of Bacteriology 190, no. 10 (March 14, 2008): 3681–89. http://dx.doi.org/10.1128/jb.00846-07.

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ABSTRACT Plasmid pSW100 is 1 of the 13 plasmids from Pantoea stewartii subsp. stewartii SW2 which has a replicon that resembles that of ColE1. This work uses a pSW100 derivative, pSW140K, to study how the pSW100 replicon is stably maintained in its hosts. Our results indicate that although pSW140K is stable in Escherichia coli HB101, the plasmid is rapidly lost in another E. coli strain, DH5α, indicating that the genetic background of an E. coli strain affects the stability of pSW140K. Mutagenesis of E. coli HB101 with EZ::TN <DHFR-1> revealed that mutations in traC, traF, traG, traN, and traV, which encode the components of the sex pilus assembly, reduce plasmid stability. Furthermore, this work identified that a 38-bp region located immediately upstream of the RNAII promoter is critical to the maintenance of plasmid stability in E. coli HB101. TraC binds to the region, and in addition, deleting the region destabilizes the plasmid. Furthermore, inserting this 38-bp fragment into a plasmid that contains the minimal replicon from pSW200 stabilizes the plasmid in E. coli HB101. Fluorescence in situ hybridization and immunofluorescence staining also revealed that derivatives of pSW100, pSW128A, and TraC are colocalized in cells, suggesting that pSW100 may use the sex pilus assembly as a partition apparatus to ensure the even distribution of the plasmid during cell division, which may thus maintain the plasmid's stability.

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Hwang, Ingyu, Audra J. Smyth, Zhao-Qing Luo, and Stephen K. Farrand. "Modulating quorum sensing by antiactivation: TraM interacts with TraR to inhibit activation of Ti plasmid conjugal transfer genes." Molecular Microbiology 34, no. 2 (October 1999): 282–94. http://dx.doi.org/10.1046/j.1365-2958.1999.01595.x.

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Wang, Chao, Hai-Bao Zhang, Guozhou Chen, Lingling Chen, and Lian-Hui Zhang. "Dual Control of Quorum Sensing by Two TraM-Type Antiactivators in Agrobacterium tumefaciens Octopine Strain A6." Journal of Bacteriology 188, no. 7 (April 1, 2006): 2435–45. http://dx.doi.org/10.1128/jb.188.7.2435-2445.2006.

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ABSTRACT Agrobacterium tumefaciens wild-type strains have a unique quorum-sensing (QS)-dependent Ti plasmid conjugative transfer phenotype in which QS signaling is activated by corresponding conjugative opine inducers. Strain K588, with a nopaline-type chromosomal background harboring an octopine-type Ti plasmid, however, is a spontaneous mutant displaying a constitutive phenotype in QS. In this study, we show that a single amino acid mutation (L54P) in the QS antiactivator TraM encoded by the traM gene of Ti plasmid is responsible for the constitutive phenotype of strain K588. Introduction of the L54P point mutation to the TraM of wild-type strain A6 by allelic replacement, however, failed to generate the expected constitutive phenotype in this octopine-type strain. Intriguingly, the QS-constitutive phenotype appeared when the pTiA6 carrying the mutated traM was placed in the chromosomal background of the nopaline-type strain C58C1RS, suggesting an unknown inhibitory factor(s) encoded by the chromosomal background of strain A6 but not by C58C1RS. Low-stringency Southern blotting analysis showed that strain A6, but not strain C58 and its derivatives, contains a second traM homologue. The homologue, designated traM2, has 64% and 65% identities with traM at the DNA and peptide levels, respectively. Similar to TraM, TraM2 is a potent antiactivator that functions by blocking TraR, the QS activator, from specific binding to the tra gene promoters. Deletion of traM2 in strain A6 harboring the mutated traM confers a constitutive QS phenotype. The results demonstrate that the QS system in strain A6 is subjected to the dual control of TraM and TraM2.

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Farago, Emica, Draženka Blaži, and Martina Vuković Ogrizek. "Artikulacijsko-fonološke sposobnosti djece s cerebralnom paralizom." Hrvatska revija za rehabilitacijska istraživanja 52, no. 1 (July 8, 2016): 17–29. http://dx.doi.org/10.31299/hrri.52.1.2.

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Cilj ovog istraživanja bio je ispitati i usporediti artikulacijske i fonološke sposobnosti djece s cerebralnom paralizom s njihovim vršnjacima urednoga razvoja. Ispitivanje je provedeno na uzorku 15-ero djece s cerebralnom paralizom te 15- ero djece urednoga razvoja. Skupine su bile izjednačene po spolu i starosnoj dobi. Upotrijebljeni mjerni instrumenti sastojali su se od Testa artikulacije i zadataka za ispitivanje fonoloških vještina. Primijenjeni su relevantni statistički postupci (osnovni statistici, robusna diskriminativna analiza, t-test, hi-kvadrat test, Cochran-Cox metoda). Dobiveni rezultati ukazali su na postojanje statistički značajnih razlika u artikulacijskim i fonološkim sposobnostima između djece s cerebralnom paralizom (CP) i djece urednoga razvoja (UR). Razlikovanju skupina, u manifestnom prostoru, u najvećoj mjeri pridonose varijable brisanje konsonanata (BRISK) te broj artikulacijskih pogrešaka (TESTA). U kreiranju diskriminacijske funkcije značajno sudjeluju i varijable: izdvajanje početnoga glasa (IZPG), fonemska analiza (FONAN) i traženje rime (TRAR). Rezultati potvrđuju i statistički značajne razlike u artikulacijskim i fonološkim sposobnostima prema tipu cerebralne paralize. Skupina djece sa spastičnim tipom cerebralne paralize bila je uspješnija u zadatcima koji su ispitivali fonemsku analizu (FONAN), rastavljanje na slogove, brisanje konsonanata (BRISK), traženje rime (TRAR) te su imali bolje artikulacijske sposobnosti.

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Maneewannakul, K., and K. Ippen-Ihler. "Construction and analysis of F plasmid traR, trbJ, and trbH mutants." Journal of Bacteriology 175, no. 5 (1993): 1528–31. http://dx.doi.org/10.1128/jb.175.5.1528-1531.1993.

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Blankschien, Matthew D., Katarzyna Potrykus, Elicia Grace, Abha Choudhary, Daniel Vinella, Michael Cashel, and Christophe Herman. "TraR, a Homolog of a RNAP Secondary Channel Interactor, Modulates Transcription." PLoS Genetics 5, no. 1 (January 16, 2009): e1000345. http://dx.doi.org/10.1371/journal.pgen.1000345.

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Vreede, Jocelyne, Klaas J. Hellingwerf, and Wim Crielaard. "TraR auto-inducer enhances protein backbone fluctuations in DNA binding domain." FEBS Letters 582, no. 5 (February 12, 2008): 805–9. http://dx.doi.org/10.1016/j.febslet.2008.01.062.

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Fuqua, C., M. Burbea, and S. C. Winans. "Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene." Journal of bacteriology 177, no. 5 (1995): 1367–73. http://dx.doi.org/10.1128/jb.177.5.1367-1373.1995.

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Journal articles on the topic 'TraR'

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