scholarly journals A new regulatory element modulates homoserine lactone-mediated autoinduction of Ti plasmid conjugal transfer.

1995 ◽  
Vol 177 (2) ◽  
pp. 449-458 ◽  
Author(s):  
I Hwang ◽  
D M Cook ◽  
S K Farrand
Keyword(s):  
Regulatory Element ◽  
Homoserine Lactone ◽  
Ti Plasmid ◽  
Conjugal Transfer

scholarly journals Quorum Sensing but Not Autoinduction of Ti Plasmid Conjugal Transfer Requires Control by the Opine Regulon and the Antiactivator TraM

2000 ◽  
Vol 182 (4) ◽  
pp. 1080-1088 ◽  
Author(s):  
Kevin R. Piper ◽  
Stephen K. Farrand
Keyword(s):  
Quorum Sensing ◽  
Control Process ◽  
Population Level ◽  
Homoserine Lactone ◽  
Ti Plasmid ◽  
Conjugal Transfer ◽  
Population Densities ◽  
Independent Manner ◽  
Ti Plasmids ◽  
Donor Population

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.

scholarly journals Quorum Sensing in Rhizobium sp. Strain NGR234 Regulates Conjugal Transfer (tra) Gene Expression and Influences Growth Rate

2003 ◽  
Vol 185 (3) ◽  
pp. 809-822 ◽  
Author(s):  
Xuesong He ◽  
William Chang ◽  
Deanne L. Pierce ◽  
Laura Ort Seib ◽  
Jennifer Wagner ◽  
...  
Keyword(s):  
Growth Rate ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Ti Plasmid ◽  
Conjugal Transfer ◽  
Signal Molecule ◽  
Sensing Systems ◽  
Ti Plasmids ◽  
Wide Range ◽  
Low Levels

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.

scholarly journals Modulating quorum sensing by antiactivation: TraM interacts with TraR to inhibit activation of Ti plasmid conjugal transfer genes

1999 ◽  
Vol 34 (2) ◽  
pp. 282-294 ◽  
Author(s):  
Ingyu Hwang ◽  
Audra J. Smyth ◽  
Zhao-Qing Luo ◽  
Stephen K. Farrand
Keyword(s):  
Quorum Sensing ◽  
Ti Plasmid ◽  
Conjugal Transfer ◽  
Transfer Genes

scholarly journals TraG from RP4 and TraG and VirD4 from Ti Plasmids Confer Relaxosome Specificity to the Conjugal Transfer System of pTiC58

2000 ◽  
Vol 182 (6) ◽  
pp. 1541-1548 ◽  
Author(s):  
Claire M. Hamilton ◽  
Hyewon Lee ◽  
Pei-Li Li ◽  
David M. Cook ◽  
Kevin R. Piper ◽  
...  
Keyword(s):  
Mating Systems ◽  
Pair Formation ◽  
Ti Plasmid ◽  
Dna Transfer ◽  
Conjugal Transfer ◽  
Transfer System ◽  
Essential Factor ◽  
Ti Plasmids ◽  
Coupling Protein ◽  
Formation System

ABSTRACT Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraGRP4) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraGRP4. A. tumefaciensdonors transferred a chimeric plasmid that contains theoriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraGRP4 was expressed in the donors. Mutations intraG RP4 with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, thetra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraGRP4 nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG RP4 mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraGRP4-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraGRP4 and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.

scholarly journals 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

2000 ◽  
Vol 182 (1) ◽  
pp. 179-188 ◽  
Author(s):  
Pei-Li Li ◽  
Stephen K. Farrand
Keyword(s):  
Quorum Sensing ◽  
Copy Number ◽  
Constitutive Expression ◽  
Plasmid Copy Number ◽  
Ti Plasmid ◽  
Conjugal Transfer ◽  
Plasmid Copy ◽  
Sensing System ◽  
Promoter Recognition ◽  
Quorum Sensing System

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.

scholarly journals Co-Dependent and Interdigitated: Dual Quorum Sensing Systems Regulate Conjugative Transfer of the Ti Plasmid and the At Megaplasmid in Agrobacterium tumefaciens 15955

2020 ◽  
Author(s):  
Ian S Barton ◽  
Justin L Eagan ◽  
Priscila A Nieves-Otero ◽  
Ian P Reynolds ◽  
Thomas G Patt ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Ti Plasmid ◽  
Conjugative Transfer ◽  
Gene Promoters ◽  
Transcription Activator ◽  
Conjugation System ◽  
Direct Production ◽  
Sensing Systems

Members of the Rhizobiaceae, often carry multiple secondary replicons in addition to the primary chromosome with compatible repABC-based replication systems. Unlike secondary chromosomes and chromids, repABC-based megaplasmids and plasmids can undergo copy number fluctuations and are capable of conjugative transfer in response to environmental signals. Several Agrobacterium tumefaciens lineages harbor three secondary repABC-based replicons, including a secondary chromosome (often linear), the Ti (tumor-inducing) plasmid and the At megaplasmid. The Ti plasmid is required for virulence and encodes a conjugative transfer (tra) system that is strictly regulated by a subset of plant-tumor released opines and a well-described acyl-homoserine lactone (AHL)-based quorum-sensing mechanism. At plasmids are generally not required for virulence, but carry genes that enhance rhizosphere survival, and these plasmids are often conjugatively proficient. We report that the At megaplasmid of the octopine-type strain A. tumefaciens 15955 encodes a quorum-controlled conjugation system that directly interacts with the paralogous quorum sensing system on the co-resident Ti plasmid. Both the pAt15955 and pTi15955 plasmids carry homologues of a TraI-type AHL synthase, a TraR-type AHL-responsive transcription activator, and a TraM-type anti-activator. The traI genes from both pTi15955 and pAt15955 can direct production of the inducing AHL (3-octanoyl-L-homoserine lactone) and together contribute to the overall AHL pool. The TraR protein encoded on each plasmid activates AHL-responsive transcription of target tra gene promoters. The pAt15955 TraR can cross-activate tra genes on the Ti plasmid as strongly as its cognate tra genes, whereas the pTi15955 TraR preferentially biased towards its own tra genes. Putative tra box elements are located upstream of target promoters, and comparing between plasmids, they are in similar locations and share an inverted repeat structure, but have distinct consensus sequences. The two AHL quorum sensing systems have a combinatorial effect on conjugative transfer of both plasmids. Overall, the interactions described here have implications for the horizontal transfer and evolutionary stability of both plasmids and, in a broad sense, are consistent with other repABC systems that often have multiple quorum-sensing controlled secondary replicons.

scholarly journals Two Opines Control Conjugal Transfer of an Agrobacterium Plasmid by Regulating Expression of Separate Copies of the Quorum-Sensing Activator Gene traR

2002 ◽  
Vol 184 (4) ◽  
pp. 1121-1131 ◽  
Author(s):  
Philippe Oger ◽  
Stephen K. Farrand
Keyword(s):  
Quorum Sensing ◽  
Regulatory System ◽  
Homoserine Lactone ◽  
Conjugal Transfer ◽  
Environmental Cues ◽  
Upstream Gene ◽  
Catabolic Plasmid ◽  
Ti Plasmids ◽  
Control Transfer ◽  
Crown Gall Tumors

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.

scholarly journals Essential Components of the Ti Plasmidtrb System, a Type IV Macromolecular Transporter

1999 ◽  
Vol 181 (16) ◽  
pp. 5033-5041 ◽  
Author(s):  
Pei-Li Li ◽  
Ingyu Hwang ◽  
Heather Miyagi ◽  
Heather True ◽  
Stephen K. Farrand
Keyword(s):  
Insertion Site ◽  
Ti Plasmid ◽  
Conjugal Transfer ◽  
Insertion Mutation ◽  
Type Iv ◽  
System A ◽  
Catabolic Plasmid ◽  
Ti Plasmids ◽  
Essential Components ◽  
Insertion Mutations

ABSTRACT The trb operon from pTiC58 is one of three loci that are required for conjugal transfer of this Ti plasmid. The operon, which probably codes for the mating bridge responsible for pair formation and DNA transfer, contains 12 genes, 11 of which are related to genes from other members of the type IV secretion system family. The 12th gene, traI, codes for production ofAgrobacterium autoinducer (AAI). Insertion mutations were constructed in each of the 12 genes, contained on a full-length clone of the trb region, using antibiotic resistance cassettes or a newly constructed transposon. This transposon, called mini-Tn5Ptrb, was designed to express genes downstream of the insertion site from a promoter regulated by TraR and AAI. Each mutation could trans complement downstream Tn3HoHo1 insertions in the trb operon of full-sized Ti plasmids. When marker-exchanged into the transfer-constitutive Ti plasmid pTiC58ΔaccR mutations intrbB, -C, -D, -E, -L, -F, -G, and -Habolished conjugal transfer from strain UIA5, which lacks the 450-kb catabolic plasmid pAtC58. However, these mutants retained residual conjugal transfer activity when tested in strain NT1, which contains this large plasmid. The trbJ mutant failed to transfer at a detectable frequency from either strain, while the trbImutant transferred at very low but detectable levels from both donors. Only the trbK mutant was unaffected in conjugal transfer from either donor. Transfer of each of the marker-exchange mutants was restored by a clone expressing only the wild-type allele of the corresponding mutant trb gene. An insertion mutation intraI abolished the production of AAI and also conjugal transfer. This defect was restored by culturing the mutant donor in the presence of AAI. We conclude that all of the trb genes except trbI and trbK are essential for conjugal transfer of pTiC58. We also conclude that mutations in any one of thetrb genes except traI and trbJ can be complemented by functions coded for by pAtC58.

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