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

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.

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The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.

Journal of Bacteriology ◽

10.1128/jb.178.14.4248-4257.1996 ◽

1996 ◽

Vol 178 (14) ◽

pp. 4248-4257 ◽

Cited By ~ 66

Author(s):

J Alt-Mörbe ◽

J L Stryker ◽

C Fuqua ◽

P L Li ◽

S K Farrand ◽

...

Keyword(s):

Agrobacterium Tumefaciens ◽

Ti Plasmid ◽

Conjugal Transfer ◽

Transfer System ◽

Ti Plasmids ◽

Vir Genes

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Functional and Mutational Analysis of Conjugative Transfer Region 1 (Tra1) from the IncHI1 Plasmid R27

Journal of Bacteriology ◽

10.1128/jb.184.8.2173-2180.2002 ◽

2002 ◽

Vol 184 (8) ◽

pp. 2173-2180 ◽

Cited By ~ 42

Author(s):

Trevor D. Lawley ◽

Matthew W. Gilmour ◽

James E. Gunton ◽

Leah J. Standeven ◽

Diane E. Taylor

Keyword(s):

Leucine Zipper ◽

Mutational Analysis ◽

Dna Transfer ◽

Genetic Complementation ◽

Conjugative Transfer ◽

Transfer System ◽

Sequence Motifs ◽

Specific Phage ◽

Transfer Region ◽

Coupling Protein

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

Journal of Bacteriology ◽

10.1128/jb.181.16.5033-5041.1999 ◽

1999 ◽

Vol 181 (16) ◽

pp. 5033-5041 ◽

Cited By ~ 35

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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Genetic and Sequence Analysis of the pTiC58 trb Locus, Encoding a Mating-Pair Formation System Related to Members of the Type IV Secretion Family

Journal of Bacteriology ◽

10.1128/jb.180.23.6164-6172.1998 ◽

1998 ◽

Vol 180 (23) ◽

pp. 6164-6172 ◽

Cited By ~ 40

Author(s):

Pei-Li Li ◽

Dawn M. Everhart ◽

Stephen K. Farrand

Keyword(s):

Sequence Analysis ◽

Pair Formation ◽

Type Iv Secretion ◽

Ti Plasmid ◽

Open Reading Frames ◽

Conjugal Transfer ◽

Mating Pair ◽

Secretion Systems ◽

Type Iv ◽

And Function

ABSTRACT Conjugal transfer of pTiC58 requires two regions, trawhich contains the oriT and several genes involved in DNA processing and a region of undefined size and function that is located at the 2-o’clock position of the plasmid. Using transposon mutagenesis with Tn3HoHo1 and a binary transfer system, we delimited this second region, called trb, to an 11-kb interval between the loci for vegetative replication and nopaline catabolism. DNA sequence analysis of this region identified 13 significant open reading frames (ORFs) spanning 11,003 bp. The first, encodingtraI, already has been described and is responsible for the synthesis of Agrobacterium autoinducer (AAI) (I. Hwang, P.-L. Li, L. Zhang, K. R. Piper, D. M. Cook, M. E. Tate, and S. K. Farrand, Proc. Natl. Acad. Sci. USA 91:4639–4643, 1994). Translation products of the next 11 ORFs showed similarities to those of trbB, -C, -D,-E, -J, -K, -L,-F, -G, -H, and -I of the trb region of the octopine-type Ti plasmid pTi15955 and of the tra2 core region of RP4. In RP4, these genes encode mating-pair formation functions and are essential for the conjugal transfer of the IncP plasmid. Each of the trb gene homologues is oriented counterclockwise on the Ti plasmid. Expression of these genes, as measured by using the lacZ fusions formed by Tn3HoHo1, required the traI promoter and the transcriptional activator TraR along with its coinducer, AAI. While related to that of RP4, the trb system of pTiC58 did not allow propagation of the trb-specific bacteriophages PRD1, PRR1, and Pf3. The products of several trb genes of the Ti plasmid are similar to those of other loci that encode DNA transfer or protein secretion systems, all of which are members of the type IV secretion family.

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

Journal of Bacteriology ◽

10.1128/jb.182.4.1080-1088.2000 ◽

2000 ◽

Vol 182 (4) ◽

pp. 1080-1088 ◽

Cited By ~ 63

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.

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Octopine-type Ti plasmids code for a mannopine-inducible dominant-negative allele of traR, the quorum-sensing activator that regulates Ti plasmid conjugal transfer

Molecular Microbiology ◽

10.1046/j.1365-2958.1998.00671.x ◽

1998 ◽

Vol 27 (2) ◽

pp. 277-288 ◽

Cited By ~ 72

Author(s):

Philippe Oger ◽

Kun-Soo Kim ◽

Rebecca L. Sackett ◽

Kevin R. Piper ◽

Stephen K. Farrand

Keyword(s):

Quorum Sensing ◽

Ti Plasmid ◽

Dominant Negative ◽

Conjugal Transfer ◽

Ti Plasmids ◽

Dominant Negative Allele

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

Journal of Bacteriology ◽

10.1128/jb.185.3.809-822.2003 ◽

2003 ◽

Vol 185 (3) ◽

pp. 809-822 ◽

Cited By ~ 84

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.

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A New Type IV Secretion System Promotes Conjugal Transfer in Agrobacterium tumefaciens

Journal of Bacteriology ◽

10.1128/jb.184.17.4838-4845.2002 ◽

2002 ◽

Vol 184 (17) ◽

pp. 4838-4845 ◽

Cited By ~ 54

Author(s):

Lishan Chen ◽

Yuching Chen ◽

Derek W. Wood ◽

Eugene W. Nester

Keyword(s):

Agrobacterium Tumefaciens ◽

Bartonella Henselae ◽

Secretion System ◽

Type Iv Secretion ◽

Ti Plasmid ◽

Dna Transfer ◽

Conjugal Transfer ◽

Type Iv Secretion System ◽

Plant Host ◽

Type Iv

ABSTRACT Two DNA transfer systems encoded by the tumor-inducing (Ti) plasmid have been previously identified in Agrobacterium tumefaciens. The virB operon is required for the transfer of transferred DNA to the plant host, and the trb system encodes functions required for the conjugal transfer of the Ti plasmid between cells of Agrobacterium. Recent availability of the genome sequence of Agrobacterium allowed us to identify a third system that is most similar to the VirB type IV secretion system of Bartonella henselae. We have designated this system avhB for Agrobacterium virulence homologue virB. The avhB loci reside on pAtC58 and encode at least 10 proteins (AvhB2 through AvhB11), 7 of which display significant similarity to the corresponding virulence-associated VirB proteins of the Ti plasmid. However, the AvhB system is not required for tumor formation; rather, it mediates the conjugal transfer of the pAtC58 cryptic plasmid between cells of Agrobacterium. This transfer occurs in the absence of the Ti plasmid-encoded VirB and Trb systems. Like the VirB system, AvhB products promote the conjugal transfer of the IncQ plasmid RSF1010, suggesting that these products comprise a mating-pair formation system. The presence of plasmid TiC58 or plasmid RSF1010 reduces the conjugal transfer efficiency of pAtC58 10- or 1,000-fold, respectively. These data suggest that complex substrate interactions exist among the three DNA transfer systems of Agrobacterium.

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