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.

Conjugal transfer of the nopaline-type Agrobacterium Ti plasmid pTiC58 is regulated by a trasriptional activator, TraR, and a diffusible signal molecule, conjugation factor (CF). CF is a member of a family of substituted homoserine lactones (HSLs) that act as coinducers for regulating gene expression in diverse Gram-negative bacteria by a mechanism called antoinduction. In Vibrio fischeur HSL production is conferred by the luxI gene. Homologues of this gene are responsible for HSL production by other Gram-negative bacteria. A gene that we call tral, conferring production of material with CF activity, was localized to a 1-kb region at the upstream end of tra of pTiC58. Spectroscopy showed that the activity was authentic CF. Sequence analysis showed that Otn could encode a protein of 211 amino acids, TraI, that is related to the proteins responsible for HSL production by other bacteria. A second, partial open reading frame immiatel downstream of tral could encode a protein related to TrbB of plasmid RP4, which is required for conjugal transer. Transcription of tnal and of the downstream tra3 genes requires TraR and CF and initiates from the tral promoter. The results show that tnd is responsible for CF production, that it is the first gene of the n3 operon, and that expression of this operon is regulated by autoinduction.

Conjugation of the Agrobacterium Ti plasmid pTiC58 is regulated by a hierarchy involving induction by the opines agrocinopines A and B and a quorum‐sensing system. Regulation by the opines is mediated by the repressor AccR, while quorum sensing is effected by the transcriptional activator TraR and its ligand, the acyl‐homoserine lactone signal molecule Agrobacterium autoinducer (AAI). These last two elements combine to activate expression of the tra system at high population densities. Sequence analysis indicated that traR is the fourth gene of an operon, which we named arc, that is transcribed divergently from accR. Complementation analysis of mutations in the genes 5′ to traR showed that the other members of the arc operon are not required for conjugation. Analysis of lacZ reporter fusions demonstrated that traR expression is regulated directly by AccR. Deletion analysis showed that AccR‐regulated expression of traR initiates from a promoter located in the intergenic region between accR and orfA, the first gene of the arc operon. Reverse transcriptase–polymerase chain reaction (RT–PCR) and primer extension analyses indicated that the arc transcript initiates upstream of orfA and proceeds uninterrupted through traR. These results are consistent with a model in which quorum sensing is subordinate to the opine regulon because traR has become associated with an operon controlled by the opine‐responsive transcriptional regulator.

SummaryConjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by two hierarchal signalling systems. Transfer is dependent on a subset of opines produced by the plant tumours induced by the bacterium. Induction also requires an acyl-homoserine lactone signal, called AAI, that is produced by the bacteria themselves. AAI is the co-inducer for TraR, the transcriptional activator required for expression of the tra regulon. Octopine induces conjugation of the octopine-mannityl opine-type Ti plasmids by regulating the expression of traR via OccR, the octopine-dependent activator of the opine regulon. We have discovered a second traR-like gene, trlR, on the octopine-mannityl opine-type Ti plasmids pTi15955 and pTiR10. This gene is located in an operon coding for a mannopine transport system and is expressed as part of the mannityl opine regulon. Sequence analysis indicated that trlR is a frameshift allele of traR, and the resulting protein lacks the carboxy-terminal domain thought to constitute the DNA-binding region of TraR. Expression of trlR inhibited octopine-induced conjugation of pTi15955 and pTiR10 by suppressing the TraRmediated transcription of the tra and trb operons. Although TrlR had no effect on the expression of traR, TraR activated the expression of trlR. Southern hybridizations indicated that several other Ti and opine-catabolic plasmids contain more than one copy of genes homologous to traR. We propose that trlR is a dominant negative allele of traR and that TrlR inhibits conjugation by forming inactive heteromultimers with TraR.

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 (TraG RP4 ) 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 TraG RP4 . A. tumefaciens donors transferred a chimeric plasmid that contains the oriT 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 TraG RP4 was expressed in the donors. Mutations in traG 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, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG RP4 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. TraG RP4 -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 TraG RP4 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.Plasmid conjugation conceptually can be divided into two functions. In the first, the DNA is processed by a complex of proteins, one of which introduces a single-strand nick at the nic site within the oriT recognition sequence. Called the relaxosome, the proteins of this complex are coded for by genes of the Dtr (DNA transfer and replication) component of the transfer system. In the second, the nucleoprotein transfer intermediate comprised of the nicked strand covalently linked at the 5Ј end to the relaxase is secreted from the donor directly into the recipient via a bridge that forms between the mating pair. This translocation apparatus is a complex membraneassociated structure coded for by the Mpf (mating pair formation) genes.The relaxosome of one conjugal plasmid may or may not be transferrable by the Mpf system of another. Specificity is conferred, in part, by a...

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 10 7 cells per cm 2 . 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 tra regulon, as assessed by monitoring a traA::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 lacking traM, 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.Conjugal transfer of the Ti plasmids from Agrobacterium tumefaciens is regulated directly by the transcriptional activator TraR and its acyl-homoserine lactone (acyl-HSL) ligand, Agrobacterium autoinducer [AAI; N-(3-oxo-octanoyl)-L-homoserine lactone] (16, 34, 40; reviewed in reference 12). TraR, in its interaction with AAI, controls conjugation by autoinduction, a process by which the bacteria induce gene sets in response to signals they themselves produce. This regulatory strategy is believed to tie plasmid transfer to the population density of the donor in what has come to be called the quorumsensing effect (reviewed in reference 18). The acyl-HSL autoinducers, which are produced by the bacteria themselves, are released into the environment, w...

We describe a transcriptional analysis platform consisting of a universal micro-array system (UMAS) combined with an enzymatic manipulation step that is capable of generating expression profiles from any organism without requiring a priori species-specific knowledge of transcript sequences. The transcriptome is converted to cDNA and processed with restriction endonucleases to generate low-complexity pools (approximately 80-120) of equal length DNA fragments. The resulting material is amplified and detected with the UMAS system, comprising all possible 4,096 (4(6)) DNA hexamers. Ligation to the arrays yields thousands of 14-mer sequence tags. The compendium of signals from all pools in the array-of-universal arrays comprises a full-transcriptome expression profile. The technology was validated by analysis of the galactose response of Saccharomyces cerevisiae, and the resulting profiles showed excellent agreement with the literature and real-time PCR assays. The technology was also used to demonstrate expression profiling from a hybrid organism in a proof-of-concept experiment where a T-cell receptor gene was expressed in yeast.

Agrobacterium tumefaciens strain C58 can transform plant cells to produce and secrete the sugar-phosphate conjugate opines agrocinopines A and B. The bacterium then moves in response to the opines and utilizes them as exclusive sources of carbon, energy, and phosphate via the functions encoded by the acc operon. These privileged opine-involved activities contribute to the formation of agrobacterial niches in the environment. We found that the expression of the acc operon is induced by agrocinopines and also by limitation of phosphate. The main promoter is present in front of the first gene, accR, which codes for a repressor. This operon structure enables efficient repression when opine levels are low. The promoter contains two putative operators, one overlapping the ؊10 sequence and the other in the further upstream from it; two partly overlapped putative pho boxes between the two operators; and two consecutive transcription start sites. DNA fragments containing either of the operators bound purified repressor AccR in the absence of agrocinopines but not in the presence of the opines, demonstrating the on-off switch of the promoter. Induction of the acc operon can occur under low-phosphate conditions in the absence of agrocinopines and further increases when the opines also are present. Such opine-phosphate dual regulatory system of the operon may ensure maximum utilization of agrocinopines when available and thereby increase the chances of agrobacterial survival in the highly competitive environment with limited general food sources.During agrobacterial infection of susceptible plants, a copy of the T region in a tumor-inducing plasmid (Ti plasmid), called the T-DNA, is transferred from the bacterium to the plant where it becomes integrated into the chromosome (10)(11)(12)25). Expression of the genes on the integrated T-DNA results in the tumorous phenotype and also the production of unusual low-molecular-weight carbon compounds that belong to a large group of plant tumor-specific metabolites collectively called opines (15,20,58,66).Crown gall tumors induced by the classic nopaline-type Agrobacterium tumefaciens strain C58 synthesize and secrete two families of tumor metabolites: agrocinopines A and B and nopaline (20,43). The genes responsible for the biosynthesis of agrocinopines and of nopaline are carried on the T region of pTiC58, harbored by the strain C58 (7,14,29). Strain C58 can catabolize nopaline and agrocinopines by using functions encoded by two different sets of genes located on the nontransferred region of the Ti plasmid (6, 27, 34, 76). These loci, called acc (agrocinopine catabolism) and noc (nopaline catabolism), code for the transport and catabolism of, as well as chemotaxis to, their cognate opines (34,35,51,76).The process by which A. tumefaciens transforms plant cells to produce opines has been termed genetic colonization of the plant (60). The genetic colonization theory leads to the opine concept, proposed by Tempé et al. (68), which states that the opines are the chemical mediators of t...