TrlR, a defective TraR-like protein of Agrobacterium tumefaciens, blocks TraR function in vitro by forming inactive TrlR:TraR dimers.

Octopine-type Ti plasmids of Agrobacterium tumefaciens require the quorum-sensing proteins TraR and TraI and the diffusible pheromone 3-oxooctanoyl homoserine lactone (AAI) to regulate genes required for conjugal transfer. TraR activity is inhibited by a protein called TrlR, which closely resembles amino acids 1-181 of TraR but is truncated as a result of a shift in the reading frame at codon 182. This frameshift does not affect synthesis of the amino-terminal domain, which is thought to bind autoinducer and mediate protein dimerization, but abolishes translation of the carboxyl-terminal, DNA-binding domain. In this study, we show that TrlR, like TraR, requires AAI for solubility when overexpressed in Escherichia coli. TrlR bound one molecule of AAI per protein monomer, supporting the prediction that the amino-terminal domain of TraR contains the AAI binding site. Purified TrlR blocked TraR for both specific DNA binding and transcription of a tra promoter, supporting previous studies performed with whole cells. When TrlR and a TraR fusion protein were co-expressed in E. coli, these proteins readily formed heterodimeric complexes that were inactive in DNA-binding activity. These data support the hypotheses that (i) the amino-terminal half of TraR binds AAI and mediates protein dimerization; (ii) both DNA-binding domains in a TraR dimer are required for stable DNA binding; and (iii) TrlR blocks TraR by direct protein-protein interactions.

The quorum-sensing transcriptional regulator TraR requires its cognate signaling ligand for protein folding, protease resistance, and dimerization.

Complexes between the quorum-sensing regulator TraR and its inducing ligand autoinducer (AAI) are soluble in Escherichia coli, whereas apo-TraR is almost completely insoluble. Here we show that the lack of soluble TraR is due in large part to rapid proteolysis, inasmuch as apo-TraR accumulated to high levels in an E. coli strain deficient in Clp and Lon proteases. In pulse labeling experiments, AAI protected TraR against proteolysis only when it was added before the radiolabel. This observation indicates that TraR proteins can productively bind AAI only during their own synthesis on polysomes, whereas fully synthesized apo-TraR proteins are not functional AAI receptors. Purified apo-TraR was rapidly degraded by trypsin to oligopeptides, whereas TraR-AAI complexes were more resistant to trypsin and were cleaved at discrete interdomain linkers, indicating that TraR requires AAI to attain its mature tertiary structure. TraR-AAI complexes eluted from a gel filtration column as dimers and bound DNA as dimers. In contrast, apo-TraR was monomeric, and incubation with AAI under a variety of conditions did not cause dimerization. We conclude that AAI is critical for the folding of nascent TraR protein into its mature tertiary structure and that full-length apo-TraR cannot productively bind AAI and is consequently targeted for rapid proteolysis.

The right end of the vir region of an octopine-type Ti plasmid contains four new members of the vir regulon that are not essential for pathogenesis.

We sequenced the virD-virE, virE-virF, and virF-T-DNA intergenic regions of an octopine Ti plasmid. Four newly described genes were induced by the vir gene inducer acetosyringone, two of which are conserved in the nopaline-type Ti plasmid pTiC58. One gene resembles a family of phosphatase genes. Each of these genes is dispensable for tumorigenesis.

The phenolic vir gene inducer ferulic acid is O-demethylated by the VirH2 protein of an Agrobacterium tumefaciens Ti plasmid.

Some or possibly all Ti plasmids of Agrobacterium tumefaciens encode a bicistronic operon designated virH, which encodes two proteins, VirH1 and VirH2, that resemble a family of cytochrome P450-type monooxygenases. Expression of this operon is induced by a family of phenolic compounds that induce all other operons within the vir regulon. We hypothesized that either or both of these proteins might metabolize some or all of these phenolic compounds. We therefore tested induction of a vir promoter by a variety of phenolic compounds in isogenic strains that express or lack virH1 and virH2. Although some compounds were equally effective inducers regardless of the virH status, other compounds induced vir expression far more effectively in the virH mutant than in the virH-proficient host. For all tested compounds, VirH2 appeared to be solely responsible for this effect. One such compound, ferulic acid, was chosen for biochemical analysis. Ferulic acid was degraded by a VirH-proficient host but not by a VirH mutant. The wild-type strain released large amounts of a more hydrophilic compound into the cell supernatant. This compound was tested by mass spectroscopy, nuclear magnetic resonance and UV spectroscopy and found to consist of caffeic acid. This indicates that wild-type strains convert virtually all added ferulic acid to caffeic acid, and that VirH2 is essential for this O-demethylation reaction. Ferulic acid was far more toxic than caffeic acid to the wild-type strain, although the wild-type strain was more resistant to ferulic acid than was the virH mutant. Caffeic acid was slowly removed from the broth, suggesting further metabolic reactions.

An Lrp-type transcriptional regulator from Agrobacterium tumefaciens condenses more than 100 nucleotides of DNA into globular nucleoprotein complexes.

The PutR protein of Agrobacterium tumefaciens positively regulates expression of the putA gene in response to exogenous proline, resulting in the utilization of proline as a source of carbon and nitrogen. PutR activity required a region of DNA extending more than 106 nt upstream of the putA transcription start site. Purified PutR bound to this region with high degree of affinity and repressed expression of the putR promoter in vitro. PutR also activated the putA promoter in vitro in the presence of proline, though less strongly than in whole cells. PutR protected a DNA interval extending from nucleotides -30 to -140, but protected only one helical face over most of this interval, suggesting that it may bind only to this face of the DNA. The addition of proline caused a slight decrease in binding affinity and altered DNase I protection patterns along the entire length of the binding site. PutR-DNA complexes were found by atomic force microscopy to be globular rather than elongated. Although the DNA fragment in these complexes was 190 nm in length, the length of the visible DNA was only 150 nm, indicating that 40 nm of DNA (115 nt) must be condensed with protein. PutR caused a net bend of this binding site, and under some conditions, proline shifted the center of this bend by one helical turn.

Autoinducer binding by the quorum-sensing regulator TraR increases affinity for target promoters in vitro and decreases TraR turnover rates in whole cells.

TraR is an Agrobacterium transcriptional regulator whose activity requires the pheromone N-3-oxooctanoyl-L-homoserine lactone. TraR was purified as a complex with the pheromone and contained one pheromone molecule per protein monomer. TraR-pheromone complexes bound to a single DNA site and activated two promoters that flank this site. Promoter expression was elevated 30-fold by using a supercoiled template. Pheromone binding increased the affinity of TraR for this binding site. Pheromone also increased TraR abundance in vivo by causing a 20-fold decrease in TraR turnover rates.

Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1.

The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonuclease cleavage patterns within their tra regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (oriT). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three tra complementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both traK and traI were required for efficient interplasmid site-specific recombination at oriT, while traJ was not required. The leading region of pKM101 contains three genes (stbA, stbB, and stbC), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.

Mannopinic acid and agropinic acid catabolism region of the octopine-type Ti plasmid pTi15955.

Octopine-type Ti plasmids such as pTi15955, pTiA6 and pTiR10 direct the catabolism of at least eight compounds called opines that are released from crown gall tumours. Four of these compounds are denoted mannityl opines, each of which possesses a D-mannityl substituent on the nitrogen atom of either glutamate or glutamine. We have analysed a 20 kb region of the Ti plasmid pTi15955 that is required for the catabolism of two such opines, mannopinic acid and agropinic acid. A total of 12 genes in four operons were identified by DNA sequence analysis. Transposons Tn5lacZ and MudK were used to mutagenize these genes and to create aga-lacZ and moa-lacZ translational fusions. The expression of all fusions was induced by agropinic acid and by mannopinic acid. One of these four operons encodes an agropinic acid permease, whereas a second one encodes a mannopinic acid permease. A third operon contains three genes encoding probable catabolic enzymes, two of which (AgaF and AgaG) are thought to convert agropinic acid to mannopinic acid, while the third (AgaE) probably converts mannopinic acid to mannose and glutamate. AgaE resembles a bacterial amino acid deaminase, whereas AgaF and AgaG resemble two bacterial proteins that together catabolize substituted hydantoins, whose chemical structure resembles that of agropinic acid. The remaining operon encoded the MoaR protein, a negative regulator of itself and of the other three operons.

Analogs of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens.

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.

Wound-released chemical signals may elicit multiple responses from an Agrobacterium tumefaciens strain containing an octopine-type Ti plasmid.

The vir regions of octopine-type and nopaline-type Ti plasmids direct the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to the nuclei of host plant cells. Previous studies indicate that at least two genetic loci at the left ends of these two vir regions are sufficiently conserved to form heteroduplexes visible in the electron microscope. To initiate an investigation of these genetic loci, we determined the DNA sequences of these regions of both Ti plasmids and identified both conserved loci. One of these is the 2.5-kb virH locus, which was previously identified on the octopine-type Ti plasmid but thought to be absent from the nopaline-type Ti plasmid. The virH operon contains two genes that resemble P-450-type monooxygenases. The other locus encodes a 0.5-kb gene designated virK. In addition, we identified other potential genes in this region that are not conserved between these two plasmids. To determine (i) whether these genes are members of the vir regulon and, (ii) whether they are required for tumorigenesis, we used a genetic technique to disrupt each gene and simultaneously fuse its promoter to lacZ. Expression of these genes was also measured by nuclease S1 protection assays. virK and two nonconserved genes, designated virL and virM, were strongly induced by the vir gene inducer acetosyringone. Disruptions of virH, virK, virL, or virM did not affect tumorigenesis of Kalanchöe diagramontiana leaves or carrot disks, suggesting that they may play an entirely different role during pathogenesis.

Activity of the quorum-sensing regulator TraR of Agrobacterium tumefaciens is inhibited by a truncated, dominant defective TraR-like protein.

Horizontal transfer of Agrobacterium tumefaciens tumour-inducing plasmids requires opines, which are released from plant tumours as nutrients for the bacteria. The opine octopine causes synthesis of the quorum-sensing TraR protein, which activates several tra promoters in the presence of a pheromone called Agrobacterium autoinducer (AAI). A gene, traS, was previously found on the same Ti plasmid in an operon that directs the uptake of mannopine, another opine. TraS strongly resembles TraR but lacks a DNA-binding module. TraS did not activate a TraR-dependent promoter and blocked TraR function, probably by forming inactive heteromultimers. Expression of traS was induced by mannopine, although this induction was strongly inhibited by the favoured catabolites succinate, glutamine and tryptone. Mannopine inhibited conjugation in a TraS-dependent fashion, and artificial overexpression of TraS also inhibited conjugation. Favoured catabolites restored tra gene expression in wild-type strains but not in strains that overexpress TraS. Downstream of traS is a gene encoding a truncated, defective chemoreceptor whose expression abolished chemotaxis.

Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria.

We describe several plasmids that are designed to create fusions between chromosomal or plasmid-encoded genes and the lacZ, phoA or gfp reporter genes. These plasmids all contain the vegetative origin of R6K, but lack the R6K pir gene, and therefore fail to replicate in strains lacking pir. Fragments of target genes are introduced into these plasmids, and fusions are created in a single step as a consequence of (Campbell-type) integration of the entire plasmid by homologous recombination. Cloned fragments containing either an intact 5'-end of the target gene including its promoter or an intact 3'-end of the gene preserve a functional copy of that gene, while fragments lacking both 5'- and 3'-ends of the target gene cause a gene disruption. In addition to facilitating measurements of gene expression, some plasmids create translational fusions to beta-galactosidase or alkaline phosphatase and are therefore useful in studying the membrane topology of a target protein. We demonstrate the utility of these plasmids by constructing and testing two operon fusions and two protein fusions between the virG gene of Agrobacterium tumefaciens and lacZ.

The hydrophobic TraM protein of pKM101 is required for conjugal transfer and sensitivity to donor-specific bacteriophage.

pKM101 is a self-transmissible plasmid of the IncN incompatibility group. Analysis of the DNA sequences of the genes required for conjugal transfer suggested the existence of a previously uncharacterized open reading frame, designated traM, that might be required for conjugation. Merodiploid strains containing transposon insertion mutations either in traM or in neighboring tra genes were used to demonstrate that traM constitutes a new complementation group essential for conjugation and donor phage sensitivity. The hydrophobicity profile of TraM suggests that it contains a signal sequence. The remainder of TraM is also composed predominantly of hydrophobic amino acids but contains one possible surface exposed loop. TraM-alkaline phosphatase and TraM-beta-galactosidase fusion proteins supported the hypothesis that TraM has a small cytoplasmic loop. We were unable to detect heterologous complementation between any tra mutation and its homolog from the virB operon of Agrobacterium tumefaciens.

Transcriptional regulation and locations of Agrobacterium tumefaciens genes required for complete catabolism of octopine.

By screening for octopine-inducible gene expression, we previously identified all the genes required for utilization of octopine as a source of carbon, nitrogen, and energy. They are (i) octopine oxidase, which converts octopine to arginine and pyruvate and is encoded by the ooxAB operon, (ii) arginase, which converts arginine to ornithine and urea and is encoded by arcA, (iii) ornithine cyclodeaminase, which converts ornithine to proline and ammonia and is encoded by the homologous arcB and ocd genes, and (iv) proline dehydrogenase, which converts proline to glutamate and is encoded by putA. Here we describe the regulation and localization of each of these genes. The ooxA-ooxB-ocd operon was previously shown to reside on the Ti plasmid and to be directly inducible by octopine. The arcAB operon is directly inducible by arginine, while it is induced by octopine only in strains that can convert octopine to arginine. Ornithine may also be a direct inducer of arcAB. putA is directly inducible by proline, while induction by octopine and by arginine (and probably by ornithine) requires their conversion to proline. Genetic studies indicate that arcAB and putA are localized on a conjugal genetic element. This element can be transferred to other Agrobacterium tumefaciens strains by a mechanism that does not require recA-dependent homologous recombination. Transfer of this genetic element from A. tumefaciens R10 requires at least one tra gene found on its Ti plasmid, indicating that this element is not self-transmissible but is mobilizable by the Ti plasmid. The DNA containing the arcAB and putA genes comigrates with a 243-kb linear molecular weight standard on field inversion electrophoretic gels.

The putA gene of Agrobacterium tumefaciens is transcriptionally activated in response to proline by an Lrp-like protein and is not autoregulated.

The Agrobacterium tumefaciens putA gene, which encodes proline dehydrogenase, is transcriptionally induced by exogenous proline. In contrast to the putA genes of enteric bacteria, the A. tumefaciens putA gene is not regulated by the PutA protein, as the putA promoter remained strongly proline inducible in strains lacking PutA. A putA null mutation increased the expression of the putA promoter under a variety of conditions. However, this mutation is predicted to increase the cytoplasmic concentration of proline, and this alone probably accounts for its effects on putA expression. The putA promoter was also strongly induced by valine, and the putA genotype did not affect expression by this gratuitous inducer. An open reading frame (ORF) encoding an Lrp-like protein was found transcribed divergently from putA. Disruption of this ORF, designated putR, abolished induction of the putA promoter by proline or valine. In addition to activating putA, PutR also repressed its own transcription, and this autorepression was only slightly affected by exogenous proline. The transcription start sites for the putA and putR genes are separated by 64 nucleotides, suggesting that PutR could regulate both promoters by binding to a single operator.

Pleiotropic phenotypes caused by genetic ablation of the receiver module of the Agrobacterium tumefaciens VirA protein.

The VirA protein of Agrobacterium tumefaciens is a transmembrane sensory kinase that phosphorylates the VirG response regulator in response to chemical signals released from plant wound sites. VirA contains both a two-component kinase module and, at its carboxyl terminus, a receiver module. We previously provided evidence that this receiver module inhibited the activity of the kinase module and that inhibition might be neutralized by phosphorylation. In this report, we provide additional evidence for this model by showing that overexpressing the receiver module in trans can restore low-level basal activity to a VirA mutant protein lacking the receiver module. We also show that ablation of the receiver module restores activity to the inactive VirA (delta324-413) mutant, which has a deletion within a region designated the linker module. This indicates that deletion of the linker module does not denature the kinase module, but rather locks the kinase into a phenotypically inactive conformation, and that this inactivity requires the receiver module. These data provide genetic evidence that the kinase and receiver modules of VirA attain their native conformations autonomously. The receiver module also restricts the variety of phenolic compounds that have stimulatory activity, since removal of this module causes otherwise nonstimulatory phenolic compounds such as 4-hydroxyacetophenone to stimulate vir gene expression.

Resection and mutagenesis of the acid pH-inducible P2 promoter of the Agrobacterium tumefaciens virG gene.

Transcription of the virG gene initiates from two tandem promoters, designated P1 and P2, that are located 50 nucleotides apart. Transcription of the P2 promoter is induced by extracellular acidity. cis-acting sites required for P2 activity were identified by constructing and assaying a series of 5' and 3' resections and site-directed nucleotide substitutions. Nucleotides between positions -9 and -37 were sufficient for regulated promoter activity. Within this region, nucleotide substitutions at the predicted -10 and -35 regions strongly reduced P2 expression. In addition, alterations in the region between nucleotides -24 and -32 also eliminated or strongly reduced promoter activity. These data suggest that this promoter may be regulated by a positive transcription factor that binds to nucleotide residues in this interval.

Genes encoding the pKM101 conjugal mating pore are negatively regulated by the plasmid-encoded KorA and KorB proteins.

The IncN plasmid pKM101 contains a group of 11 genes thought to be required for the synthesis of its conjugal pilus and mating pore. Within this region are two genes, kilA and kilB, either of which is conditionally lethal to the cell. kilA was previously shown to be allelic with traL, and we now show that kilB is allelic with traE. In the same region, genetic studies previously defined two loci, korA and korB (kor for kill override), which together prevent lethality mediated by kilA and kilB. We now identify the genes that encode KorA and KorB functions. To determine whether KorA and KorB proteins influence tra gene transcription, we constructed beta-galactosidase fusions to three promoters in this region and measured their expression in the presence of KorA, KorB, and both proteins. KorA and KorB together repressed transcription of all three promoters, while neither protein alone affected transcription. We identified all three transcriptional start sites by primer extension analysis. Two putative binding sites for these proteins, designated kor boxes, contain 26 identical nucleotides in a 29-nucleotide region. The electrophoretic mobilities (of DNA fragments containing kor boxes were retarded by cell extracts containing both KorA and KorB but were not retarded by extracts containing just KorA or just KorB. DNase I footprinting analysis of one of these promoters demonstrates that KorA and/or KorB binds to a region containing a kor box.