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Stephen K Farrand - One of the best experts on this subject based on the ideXlab platform.
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the agrobacterium tumefaciens rnd homolog is required for Trar mediated quorum dependent activation of ti plasmid Tra Gene expression
Journal of Bacteriology, 2001Co-Authors: Stephen K FarrandAbstract: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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conjugal Transfer but not quorum dependent Tra Gene induction of ptic58 requires a solid surface
Applied and Environmental Microbiology, 1999Co-Authors: Kevin R Piper, Stephen K FarrandAbstract:Donors of Agrobacterium tumefaciens harboring a Transfer-constitutive derivative of the nopaline-type Ti plasmid pTiC58 Transferred this element at frequencies 3 to 4 orders of magnitude higher in matings conducted on solid surfaces than in those conducted in liquid medium. However, as measured with a lacZ reporter fusion, the Tra Genes of the wild-type Ti plasmid were inducible by opines to indistinguishable levels on solid and in liquid medium. Donors induced in liquid Transferred the Ti plasmid at high frequency when mated with recipients on solid medium. We conclude that while formation of stable mating pairs and subsequent Transfer of the Ti plasmid is dependent on a solid sTratum, the regulatory system can activate Tra Gene expression to equivalent levels in liquid and on solid surfaces.
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Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid Transfer by autoinduction
Nature, 1993Co-Authors: Kevin R Piper, Susanne B. Von Bodman, Stephen K FarrandAbstract:CONJUGAL Transfer of Ti plasmids from Agrobacterium donors to bacterial recipients is controlled by two types of diffusible signal molecules. Induction is mediated by novel compounds, called opines, that are secreted by crown gall tumours. These neoplasias result from infection of susceptible plants by virulent agrobacteria. The second diffusible signal, called conjugation factor, is synthesized by the donor bacteria themselves. Production of this factor is induced by the opine. Here we show that conjugation is regulated directly by a Transcriptional activator, TraR, which requires conjugation factor as a coinducer to activate Tra Gene expression. TraR is a homologue of LuxR, the lux Gene activator from Vibrio fischeri which also requires an endogenously synthesized diffusible coinducer. The two regulatory systems are related; the two activator proteins show amino-acid sequence similarities and the lux system cofactor, autoinducer, will substitute for conjugation factor in the TraR-dependent activation of Ti plasmid Tra Genes.
Stanley N Cohen - One of the best experts on this subject based on the ideXlab platform.
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Unraveling the essential role in conjugation of the Tra protein of Streptomyces lividans plasmid pIJ101
Antonie van Leeuwenhoek, 2001Co-Authors: Gregg S Pettis, Stanley N CohenAbstract:Plasmid pIJ101 from Streptomyces lividans encodes a single Gene, Tra , that is essential for both plasmid Transfer and mobilization of chromosomes during mating. The Tra Gene product (Tra) is a membrane protein, a portion of which shows similarity to Transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. This paper reviews past and present work that has focused on elucidating the precise role of the Tra protein of pIJ101 in conjugation in Streptomyces .
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Mutational Analysis of the Tra Locus of the Broad-Host-Range Streptomyces Plasmid pIJ101
Journal of Bacteriology, 2000Co-Authors: Gregg S Pettis, Stanley N CohenAbstract:The Tra Gene of Streptomyces lividans plasmid pIJ101 encodes a 621-amino-acid protein that can mediate both plasmid Transfer and the interbacterial Transfer of chromosomal Genes (i.e., chromosome-mobilizing ability [Cma]) during mating. Here we report the results of in-frame insertional mutaGenesis studies aimed at defining regions of Tra required for these functions. While hexameric linker insertions throughout the Tra Gene affected plasmid and chromosomal Gene Transfer, insertions in a 200-amino-acid region of the Tra protein that contains presumed nucleotide-binding motifs and that is widely conserved among a functionally diverse family of bacterial and plasmid proteins (K. J. Begg, S. J. Dewar, and W. D. Donachie, J. Bacteriol. 177:6211–6222, 1995) had especially prominent effects on both functions. Insertions near the N terminus of Tra reduced Cma for either circular or linear host chromosomes to a much greater extent than pIJ101 plasmid Transfer. Our results suggest that Cma involves Tra functions incremental to those needed for plasmid DNA Transfer.
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plasmid Transfer and expression of the Transfer Tra Gene product of plasmid pij101 are temporally regulated during the streptomyces lividans life cycle
Molecular Microbiology, 1996Co-Authors: Gregg S Pettis, Stanley N CohenAbstract:: We previously have shown that a chromosomally integrated copy of the Tra Gene of plasmid pIJ101 under the control of the KorA repressor protein, which regulates Transcription of Tra as well as its own synthesis, can promote the conjugal Transfer of both chromosomal and plasmid Genes in Streptomyces lividans. Using an antibody Generated against a fusion protein containing the C-terminal portion of Tra, we show here that this essential conjugation protein is present in membrane fractions of both surface-grown S. lividans, which mate readily, and of cells grown in liquid culture, where mating has not been found. Expression of Tra during the S. lividans life cycle was temporally regulated and was reduced late during vegetative growth so that little or no Tra protein was detected in cells as they began to differentiate morphologically and produce secondary metabolites. Comparison of the membrane concenTration of Tra protein with Tra mRNA concenTration during the S. lividans life cycle indicated that the disappearance of Tra is post-Transcriptionally controlled and thus is not mediated by KorA. The results of 'interrupted mating' experiments, together with the time of appearance of Tra in S. lividans membranes, indicate that the intermycelial Transfer of pIJ101 in S. lividans is complete by the onset of cellular differentiation.
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Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis‐acting function dispensable for chromosomal Gene Transfer
Molecular Microbiology, 1994Co-Authors: Gregg S Pettis, Stanley N CohenAbstract:The Tra Gene of Streptomyces lividans plasmid pIJ101 is required for both plasmid DNA Transfer and pIJ101-induced mobilization of chromosomal Genes during mating. We show that a chromosomally inserted copy of Tra mediates Transfer of chromosomal DNA at high frequency but promotes efficient Transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from pIJ101 reduced plasmid Transfer mediated by either plasmid-borne or chromosomally located Tra by at least three orders of magnitude, abolished the Transfer-associated pocking phenomenon, and interfered with the ability of Tra + plasmids to promote Transfer of chromosomal DNA
Clay Fuqua - One of the best experts on this subject based on the ideXlab platform.
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quorum sensing in rhizobium sp sTrain ngr234 regulates conjugal Transfer Tra Gene expression and influences growth rate
Journal of Bacteriology, 2003Co-Authors: Xuesong He, William Chang, Deanne L Pierce, Laura Ort Seib, Jennifer K Wagner, Clay FuquaAbstract: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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Analogs of the Autoinducer 3-Oxooctanoyl-Homoserine Lactone Strongly Inhibit Activity of the TraR Protein of Agrobacterium tumefaciens
Journal of Bacteriology, 1998Co-Authors: John W. Beaber, Margret I. Moré, Clay Fuqua, Anatol Eberhard, Stephen C. WinansAbstract:The TraR and TraI proteins of Agrobacterium tumefaciens mediate cell-density-dependent expression of the Ti plasmid Tra regulon. TraI synthesizes the autoinducer pheromone N-(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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Conserved cis-acting promoter elements are required for density-dependent Transcription of Agrobacterium tumefaciens conjugal Transfer Genes.
Journal of Bacteriology, 1996Co-Authors: Clay Fuqua, Stephen C. WinansAbstract:Ti plasmids of Agrobacterium tumefaciens, in addition to Transferring oncogenic DNA to the nuclei of infected plant cells, can conjugally Transfer between agrobacteria. Conjugation of wide-host-range octopine-type Ti plasmids requires a tumor-released arginine derivative called octopine. Octopine stimulates expression of the TraR Gene, whose product directly activates other Tra Genes in the presence of an acylated homoserine lactone called Agrobacterium autoinducer (AAI). We have localized the Transcription starts of three Tra promoters and find conserved elements (Tra boxes) at virtually identical positions upstream of each promoter. Disruption of these Tra boxes abolished induction of each promoter. Deletion analysis of the TraI promoter indicates that Tra boxes are the only upstream elements required for Transcriptional activation. Since Ti plasmid donor cells both produce and respond to AAI, we tested whether expression of Tra promoters was enhanced by high concenTrations of bacteria. Both Tra Gene expression and conjugation itself were strongly stimulated either by high donor densities or by exogenous AAI.
Gregg S Pettis - One of the best experts on this subject based on the ideXlab platform.
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Unraveling the essential role in conjugation of the Tra protein of Streptomyces lividans plasmid pIJ101
Antonie van Leeuwenhoek, 2001Co-Authors: Gregg S Pettis, Stanley N CohenAbstract:Plasmid pIJ101 from Streptomyces lividans encodes a single Gene, Tra , that is essential for both plasmid Transfer and mobilization of chromosomes during mating. The Tra Gene product (Tra) is a membrane protein, a portion of which shows similarity to Transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. This paper reviews past and present work that has focused on elucidating the precise role of the Tra protein of pIJ101 in conjugation in Streptomyces .
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Minimal and contributing sequence determinants of the cis-acting locus of Transfer (clt) of streptomycete plasmid pIJ101 occur within an intrinsically curved plasmid region.
Journal of Bacteriology, 2000Co-Authors: Matthew J. Ducote, Shubha Prakash, Gregg S PettisAbstract:Efficient interbacterial Transfer of streptomycete plasmid pIJ101 requires the pIJ101 Tra Gene, as well as a cis-acting plasmid function known as clt. Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3′ end of the korB regulatory Gene. Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt Transfer function and demonsTration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 Transfer.
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Mutational Analysis of the Tra Locus of the Broad-Host-Range Streptomyces Plasmid pIJ101
Journal of Bacteriology, 2000Co-Authors: Gregg S Pettis, Stanley N CohenAbstract:The Tra Gene of Streptomyces lividans plasmid pIJ101 encodes a 621-amino-acid protein that can mediate both plasmid Transfer and the interbacterial Transfer of chromosomal Genes (i.e., chromosome-mobilizing ability [Cma]) during mating. Here we report the results of in-frame insertional mutaGenesis studies aimed at defining regions of Tra required for these functions. While hexameric linker insertions throughout the Tra Gene affected plasmid and chromosomal Gene Transfer, insertions in a 200-amino-acid region of the Tra protein that contains presumed nucleotide-binding motifs and that is widely conserved among a functionally diverse family of bacterial and plasmid proteins (K. J. Begg, S. J. Dewar, and W. D. Donachie, J. Bacteriol. 177:6211–6222, 1995) had especially prominent effects on both functions. Insertions near the N terminus of Tra reduced Cma for either circular or linear host chromosomes to a much greater extent than pIJ101 plasmid Transfer. Our results suggest that Cma involves Tra functions incremental to those needed for plasmid DNA Transfer.
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plasmid Transfer and expression of the Transfer Tra Gene product of plasmid pij101 are temporally regulated during the streptomyces lividans life cycle
Molecular Microbiology, 1996Co-Authors: Gregg S Pettis, Stanley N CohenAbstract:: We previously have shown that a chromosomally integrated copy of the Tra Gene of plasmid pIJ101 under the control of the KorA repressor protein, which regulates Transcription of Tra as well as its own synthesis, can promote the conjugal Transfer of both chromosomal and plasmid Genes in Streptomyces lividans. Using an antibody Generated against a fusion protein containing the C-terminal portion of Tra, we show here that this essential conjugation protein is present in membrane fractions of both surface-grown S. lividans, which mate readily, and of cells grown in liquid culture, where mating has not been found. Expression of Tra during the S. lividans life cycle was temporally regulated and was reduced late during vegetative growth so that little or no Tra protein was detected in cells as they began to differentiate morphologically and produce secondary metabolites. Comparison of the membrane concenTration of Tra protein with Tra mRNA concenTration during the S. lividans life cycle indicated that the disappearance of Tra is post-Transcriptionally controlled and thus is not mediated by KorA. The results of 'interrupted mating' experiments, together with the time of appearance of Tra in S. lividans membranes, indicate that the intermycelial Transfer of pIJ101 in S. lividans is complete by the onset of cellular differentiation.
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Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis‐acting function dispensable for chromosomal Gene Transfer
Molecular Microbiology, 1994Co-Authors: Gregg S Pettis, Stanley N CohenAbstract:The Tra Gene of Streptomyces lividans plasmid pIJ101 is required for both plasmid DNA Transfer and pIJ101-induced mobilization of chromosomal Genes during mating. We show that a chromosomally inserted copy of Tra mediates Transfer of chromosomal DNA at high frequency but promotes efficient Transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from pIJ101 reduced plasmid Transfer mediated by either plasmid-borne or chromosomally located Tra by at least three orders of magnitude, abolished the Transfer-associated pocking phenomenon, and interfered with the ability of Tra + plasmids to promote Transfer of chromosomal DNA
Kevin R Piper - One of the best experts on this subject based on the ideXlab platform.
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conjugal Transfer but not quorum dependent Tra Gene induction of ptic58 requires a solid surface
Applied and Environmental Microbiology, 1999Co-Authors: Kevin R Piper, Stephen K FarrandAbstract:Donors of Agrobacterium tumefaciens harboring a Transfer-constitutive derivative of the nopaline-type Ti plasmid pTiC58 Transferred this element at frequencies 3 to 4 orders of magnitude higher in matings conducted on solid surfaces than in those conducted in liquid medium. However, as measured with a lacZ reporter fusion, the Tra Genes of the wild-type Ti plasmid were inducible by opines to indistinguishable levels on solid and in liquid medium. Donors induced in liquid Transferred the Ti plasmid at high frequency when mated with recipients on solid medium. We conclude that while formation of stable mating pairs and subsequent Transfer of the Ti plasmid is dependent on a solid sTratum, the regulatory system can activate Tra Gene expression to equivalent levels in liquid and on solid surfaces.
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Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid Transfer by autoinduction
Nature, 1993Co-Authors: Kevin R Piper, Susanne B. Von Bodman, Stephen K FarrandAbstract:CONJUGAL Transfer of Ti plasmids from Agrobacterium donors to bacterial recipients is controlled by two types of diffusible signal molecules. Induction is mediated by novel compounds, called opines, that are secreted by crown gall tumours. These neoplasias result from infection of susceptible plants by virulent agrobacteria. The second diffusible signal, called conjugation factor, is synthesized by the donor bacteria themselves. Production of this factor is induced by the opine. Here we show that conjugation is regulated directly by a Transcriptional activator, TraR, which requires conjugation factor as a coinducer to activate Tra Gene expression. TraR is a homologue of LuxR, the lux Gene activator from Vibrio fischeri which also requires an endogenously synthesized diffusible coinducer. The two regulatory systems are related; the two activator proteins show amino-acid sequence similarities and the lux system cofactor, autoinducer, will substitute for conjugation factor in the TraR-dependent activation of Ti plasmid Tra Genes.