The MexE/MexF/AmeC Efflux Pump of Agrobacterium tumefaciens and Its Role in Ti Plasmid Virulence Gene Expression.

Expression of the tumor-inducing (Ti) plasmid virulence genes of Agrobacterium tumefaciens is required for the transfer of DNA from the bacterium into plant cells, ultimately resulting in the initiation of plant tumors. The vir genes are induced as a result of exposure to certain phenol derivatives, monosaccharides, and low pH in the extracellular milieu. The soil, as well as wound sites on a plant-the usual site of the virulence activity of this bacterium-can contain these signals, but vir gene expression in the soil would be a wasteful utilization of energy. This suggests that mechanisms may exist to ensure that vir gene expression occurs only at the higher concentrations of inducers typically found at a plant wound site. In a search for transposon-mediated mutations that affect sensitivity for the virulence gene-inducing activity of the phenol, 3,5-dimethoxy-4-hydroxyacetophenone (acetosyringone [AS]), an RND-type efflux pump homologous to the MexE/MexF/OprN pump of Pseudomonas aeruginosa was identified. Phenotypes of mutants carrying an insertion or deletion of pump components included hypersensitivity to the vir-inducing effects of AS, hypervirulence in the tobacco leaf explant virulence assay, and hypersensitivity to the toxic effects of chloramphenicol. Furthermore, the methoxy substituents on the phenol ring of AS appear to be critical for recognition as a pump substrate. These results support the hypothesis that the regulation of virulence gene expression is integrated with cellular activities that elevate the level of plant-derived inducers required for induction so that this occurs preferentially, if not exclusively, in a plant environment.IMPORTANCE Expression of genes controlling the virulence activities of a bacterial pathogen is expected to occur preferentially at host sites vulnerable to that pathogen. Host-derived molecules that induce such activities in the plant pathogen Agrobacterium tumefaciens are found in the soil, as well as in the plant. Here, we tested the hypothesis that mechanisms exist to suppress the sensitivity of Agrobacterium species to a virulence gene-inducing molecule by selecting for mutant bacteria that are hypersensitive to its inducing activity. The mutant genes identified encode an efflux pump whose proposed activity increases the concentration of the inducer necessary for vir gene expression; this pump is also involved in antibiotic resistance, demonstrating a relationship between cellular defense activities and the control of virulence in Agrobacterium.

An extra repABC locus in the incRh2 Ti plasmid pTiBo542 exerts incompatibility toward an incRh1 plasmid.

Ti/Ri plasmids in pathogenic Agrobacterium species are repABC replicons that are stably maintained by the function of repABC genes. Two Ti plasmids, pTiBo542 and pTiS4, belonging to incRh2 and incRh4 incompatibility groups, respectively, were reported to carry two repABC loci. In the present study, to reveal the roles of the two repABC loci in the two plasmids, we constructed mini-replicons carrying any one or both of the repABC loci (referred to as repABC1 and repABC2 here) and examined their replication and incompatibility properties. The introduction of mini-replicons into A. tumefaciens C58C1 strains suggested that repABC1 functions as replicator genes but repABC2 does not in both the Ti plasmids. Because the components of repABC2 of pTiBo542 have highly similar amino acid and nucleotide sequences to those of the incRh1-type repABC replicon, we introduced repABC2-containing replicons into cells harboring an incRh1 plasmid in order to check their incompatibility traits. As a result, the repABC2-containing replicon expelled the resident incRh1 plasmid, indicating that the extra repABC locus is dispensable for replication and could work as an incompatibility determinant against incRh1 group plasmids. We suggest that the locus contributes to plasmid retention by eliminating the burden of co-existing competitive plasmids in host cells through its incompatibility.

Quorum-quenching limits quorum-sensing exploitation by signal-negative invaders.

Some bacteria produce and perceive quorum-sensing (QS) signals that coordinate several behaviours, including the costly processes that are exoenzyme production and plasmid transfer. In the case of plasmid transfer, the emergence of QS signal-altered invaders and their policing are poorly documented. In Agrobacterium tumefaciens, the virulence Ti-plasmid encodes both synthesis and sensing of QS-signals, which promote its transfer from a donor to a recipient cell. Here, we reported that QS-altered A. tumefaciens mutants arose during experimental evolution. All showed improved growth compared to their ancestor. Genome sequencing revealed that, though some had lost the Ti-plasmid, most were defective for QS-signal synthesis and Ti-plasmid conjugation (traR mutations) and one exhibited a QS-signal exploitation behaviour, using signal produced by other cells to enhance its own Ti-plasmid transfer. We explored mechanisms that can limit this QS-hijacking. We showed that the A. tumefaciens capacity to inactivate QS-signals by expressing QS-degrading enzyme could attenuate dissemination of the QS signal-negative Ti-plasmids. This work shows that enzymatic QS-disruption whether encoded by the QS-producing Ti-plasmid itself, by a companion plasmid in the same donor cells, or by one in the recipient cells, in all cases can serve as a mechanism for controlling QS exploitation by QS signal-negative mutants.

Plant GABA:proline ratio modulates dissemination of the virulence Ti plasmid within the Agrobacterium tumefaciens hosted population.

Accumulation of amino acids is a common plant response to several biotic and abiotic stresses, even if the roles of these accumulations remain often poorly understood. In a recent study we measured the levels of different amino acids in tumors of Arabidopsis thaliana induced by the phytopathogen Agrobacterium tumefaciens and correlated these data with changes of gene expressions in both organisms. This led to the demonstration that the non-protein amino acid GABA plays an important role for the adaptation of the bacteria to the plant tumor environment, and especially in the control of the virulent Ti plasmid dissemination. Here we present a model that describes how different GABA:proline ratios in the A. thaliana host may have different impacts on the conjugation of A. tumefaciens Ti plasmid, and advance the view that the amino acid metabolism of plant hosts could be critical for the propagation of the virulence genes in A. tumefaciens populations.

Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein.

During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

RepC protein of the octopine-type Ti plasmid binds to the probable origin of replication within repC and functions only in cis.

Vegetative replication and partitioning of many plasmids and some chromosomes of alphaproteobacteria are directed by their repABC operons. RepA and RepB proteins direct the partitioning of replicons to daughter cells, while RepC proteins are replication initiators, although they do not resemble any characterized replication initiation protein. Here we show that the replication origin of an Agrobacterium tumefaciens Ti plasmid resides fully within its repC gene. Purified RepC bound to a site within repC with moderate affinity, high specificity and with twofold cooperativity. The binding site was localized to an AT-rich region that contains a large number of GANTC sites, which have been implicated in replication regulation in related organisms. A fragment of RepC containing residues 26-158 was sufficient to bind DNA, although with limited sequence specificity. This portion of RepC is predicted to have structural homology to members of the MarR family of transcription factors. Overexpression of RepC in A. tumefaciens caused large increases in copy number in cis but did not change the copy number of plasmids containing the same oriV sequence in trans, confirming other observations that RepC functions only in cis.

The BlcC (AttM) lactonase of Agrobacterium tumefaciens does not quench the quorum-sensing system that regulates Ti plasmid conjugative transfer.

The conjugative transfer of Agrobacterium plasmids is controlled by a quorum-sensing system consisting of TraR and its acyl-homoserine lactone (HSL) ligand. The acyl-HSL is essential for the TraR-mediated activation of the Ti plasmid Tra genes. Strains A6 and C58 of Agrobacterium tumefaciens produce a lactonase, BlcC (AttM), that can degrade the quormone, leading some to conclude that the enzyme quenches the quorum-sensing system. We tested this hypothesis by examining the effects of the mutation, induction, or mutational derepression of blcC on the accumulation of acyl-HSL and on the conjugative competence of strain C58. The induction of blc resulted in an 8- to 10-fold decrease in levels of extracellular acyl-HSL but in only a twofold decrease in intracellular quormone levels, a measure of the amount of active intracellular TraR. The induction or mutational derepression of blc as well as a null mutation in blcC had no significant effect on the induction of or continued transfer of pTiC58 from donors in any stage of growth, including stationary phase. In matings performed in developing tumors, wild-type C58 transferred the Ti plasmid to recipients, yielding transconjugants by 14 to 21 days following infection. blcC-null donors yielded transconjugants 1 week earlier, but by the following week, transconjugants were recovered at numbers indistinguishable from those of the wild type. Donors mutationally derepressed for blcC yielded transconjugants in planta at numbers 10-fold lower than those for the wild type at weeks 2 and 3, but by week 4, the two donors showed no difference in recoverable transconjugants. We conclude that BlcC has no biologically significant effect on Ti plasmid transfer or its regulatory system.

Identification of pTi-SAKURA DNA region conferring enhancement of plasmid incompatibility and stability.

In Agrobacterium tumefaciens, the stability of Ti plasmids differs depending on the strain. So far, little is known about genes that cause the difference in stability. The repABC operon is responsible for replication and incompatibility of Ti plasmids. We constructed recombinant plasmids carrying the repABC operon and different portions of pTi-SAKURA. Cells having the recombinant plasmids that harbored a 2.6-kbp NheI fragment of pTi-SAKURA were found to be transformed via conjugation 100-fold less frequently with a small incompatible repABC plasmid than cells having the recombinant plasmids lacking the 2.6-kbp NheI fragment. Since the phenomenon occurred only when the resident and incoming plasmids belonged to the same incompatibility group, it was suggested that the 2.6-kbp NheI fragment bears the potential enhancing incompatibility. The fragment contained an operon consisting of two open reading frames, tiorf24 and tiorf25. tiorf24 is an orphan gene, whereas tiorf25 is a homologue of a group of plasmid stability genes. Removal of the 2.6-kbp fragment from the resident pTi-SAKURA increased the resident plasmid ejection ratio by the incoming repABC plasmid, whereas addition of the fragment to pTiC58 decreased the ejection ratio, and the loss ratio during growth at 37 degrees C. These data suggest that tiorf24 and tiorf25 are responsible for the stability of pTi-SAKURA, and reduce, in the host bacterium, the frequency of ejection of the resident plasmid, presumably through an incompatibility mechanism.

Molecular basis of transcriptional antiactivation. TraM disrupts the TraR-DNA complex through stepwise interactions.

Conjugative transfer of Agrobacterium Ti plasmids is regulated by TraR, a quorum-sensing activator. Quorum dependence requires TraM, which binds to and inactivates TraR. In this study, we showed that TraR and TraM form a 151-kDa stable complex composed of two TraR and two TraM dimers both in vitro and in vivo. When interacted with TraR bound to tra box DNA, wild-type TraM formed a nucleoprotein complex of 77 kDa composed of one dimer of each protein and DNA. The complex converted to the 151-kDa species with concomitant release of DNA with a half-life of 1.6 h. TraR in the complex still retained tightly bound autoinducer. From these results, we conclude that TraM interacts in a two-step process with DNA-TraR to form a large, stable antiactivation complex. Mutagenesis identified residues of TraR important for interacting with TraM. These residues form two patches, possibly defining the binding interfaces. Consistent with this interpretation, comparison of the trypsin-digested polypeptides of TraR and of TraM with that of the TraR-TraM complex revealed that a tryptic site at position 177 of TraR around these patches is accessible on free TraR but is blocked by TraM in the complex. From these genetic and structural considerations, we constructed three-dimensional models of the complex that shed light on the mechanism of TraM-mediated inhibition of TraR and on TraM-mediated destabilization of the TraR-DNA complex.

Agrobacterium virulence gene induction.

The ability of Agrobacterium to transform plants and other organisms is under highly regulated genetic control. Two Virulence (Vir) proteins, VirA and VirG, function as a two-component regulatory system to sense particular phenolic compounds synthesized by wounded plant tissues. Induction by these phenolic compounds, in the presence of certain neutral or acid sugars, results in activation of other vir genes, leading to the processing of T-DNA from the Ti-plasmid and transfer of T-DNA to recipient host cells. Many plant, and most nonplant, species do not provide sufficient quantities of the correct phenolic compounds to permit efficient Agrobacterium-mediated genetic transformation to occur. In order to transform these species, phenolic inducing compounds must be added to agrobacteria before and/or during cocultivation of recipient cells with the bacteria. This chapter discusses conditions for efficient induction of Agrobacterium virulence genes by phenolic compounds.

Parameters affecting the efficiency of Agrobacterium tumefaciens-mediated transformation of Colletotrichum graminicola.

We have developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for the plant pathogenic fungus Colletotrichum graminicola, the cause of anthracnose leaf blight and stalk rot of corn. The ATMT results in higher transformation efficiencies than previously available polyethylene glycol-mediated protocols, and falcate spores can be used instead of protoplasts for transformation. Various experimental parameters were tested for their effects on transformation efficiencies. The parameters with the greatest influence were the A. tumefaciens strain used and the Ti-plasmid it carried, the ratio of bacterium to fungus during cocultivation, and the length of cocultivation. Southern analysis demonstrated that most transformants (80%) contained tandem integrations of plasmid sequences, and at least 36% had integrations at multiple sites in the genome. In a majority of cases (70%), the whole Ti-plasmid, and not just the T-DNA, had integrated as a series of tandem repeats. Tandem integrations, especially of the whole plasmid, make it difficult to rescue DNA from both flanks of the integrations with standard PCR-based approaches. Thus, ATMT may be unsuitable for insertional mutagenesis of C. graminicola without further modification.

A small antisense RNA downregulates expression of an essential replicase protein of an Agrobacterium tumefaciens Ti plasmid.

Tumour-inducing (Ti) plasmids of Agrobacterium tumefaciens replicate via the products of the repABC genes, which are highly conserved among plasmids of the alpha-Proteobacteria. RepA and RepB direct stable partitioning of daughter plasmids, while the RepC directs replicative DNA synthesis. We have identified a new gene (repE) within the repB-repC intergenic region of an octopine-type Ti plasmid. This gene encodes a small, non-translated RNA that is transcribed in the direction opposite to the repABC mRNA. Increased expression of repE blocked plasmid replication of a repABC-dependent miniplasmid, while decreased repE expression increased plasmid copy number. Direct RNA measurements and repC-lacZ fusions demonstrated that RepE inhibits the expression of RepC at the transcriptional level and possibly also at the translational level. Based on our experimental results and an RNA folding algorithm, we predict that RepE binding to the repABC mRNA would promote termination of the repABC transcript near the start codon of repC. Sequence analysis suggests that this phenomenon may be widespread among plasmids of this family.

Isolation, purification, and identification of the virulence protein VirE2 from Agrobacterium tumefaciens.

Bacteria of the genus Agrobacterium can transfer a portion of their Ti plasmid (T-DNA) in complex with the VirE2 and VirD2 proteins into the plant-cell nucleus and cause it to be integrated in the host-cell chromosomes. The mechanism of T-DNA transfer across the plant-cell membrane and cytoplasm is unknown. The aim of this study was to isolate the virulence protein VirE2 in order to explore its role in T-DNA transfer across the eukaryotic-cell membrane and cytoplasm. To obtain VirE2, we cloned the virE2 gene into plasmid pQE31 in Escherichia coli cells. VirE2 protein was isolated from E. coli XL-1 blue cells containing a recombinant plasmid, pQE31-virE2. The cells were ultrasonically disrupted, and the protein containing six histidine residues at the N-terminal end was isolated by affinity chromatography on Ni-NTA agarose. The purified preparation was tested by immunodot, by using polyclonal rabbit antibodies and miniantibodies produced toward VirE2. The capacity of the recombinant protein VirE2 for interacting with single-stranded DNA was tested by the formation of complexes, recorded by agarose-gel electrophoresis. In summary, A. tumefaciens virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into the plant cell, was isolated, purified, and partially characterized. Anti-VirE2 miniantibodies were obtained, and direct labeling of VirE2 with colloidal gold was done for the first time.

Dimerization properties of TraM, the antiactivator that modulates TraR-mediated quorum-dependent expression of the Ti plasmid tra genes.

TraM, an 11.2 kDa antiactivator, modulates the acyl-homoserine lactone-mediated autoinduction of Ti plasmid conjugative transfer by interacting directly with TraR, the quorum-sensing transcriptional activator. Most antiactivators and antisigma factors examined to date act in dimer form. However, whether, and if so, how TraM dimerizes is unknown. Analyses based on a genetic assay using fusions of TraM to the lambda cI DNA binding domain, and biochemical assays using chemical crosslinking and gel filtration chromatography showed that TraM forms homodimers. Although SDS-PAGE studies suggested that the lone cysteine residue at position 71 was involved in interprotomer disulfide-bridging in TraM, altering Cys-71 to a serine did not significantly affect dimerization or the antiactivator activity of this mutant protein when expressed at wild-type levels in vivo. Analysis of N-terminal, C-terminal, and internal deletion mutants of TraM identified two regions of the protein involved in dimerization; one located within a segment between residues 20 and 50, and the other located to a segment between residues 67 and 96. Both regions are required for formation of fully stable dimers. Analysis of the activity of these deletion mutants in vivo, and their ability to bind TraR and to disrupt TraR-DNA complexes in vitro, suggests that while the internal segment of the protein is required for dimerization, determinants located at the far C-terminus and beginning at between residues 10 and 20 at the N-terminus play a role in TraR binding and antiactivator function. When co-expressed with lambda cI'::TraR fusions, wild-type TraM mediated quormone-independent dimerization of the transcriptional activator, suggesting that dimers of TraM can multimerize TraR.

The RepA and RepB autorepressors and TraR play opposing roles in the regulation of a Ti plasmid repABC operon.

The replicator regions of the Ti plasmids of Agrobacterium tumefaciens belong to the repABC family of replication and partitioning systems, members of which are widely distributed among alpha proteobacteria. In the region upstream of the octopine-type Ti plasmid repABC operon, three promoters were recently shown to be activated by the LuxR-type regulator TraR. Activation of these promoters by TraR led to enhanced rep gene expression and increased Ti plasmid copy number. Here we describe a fourth promoter, designated P4. This promoter lies directly upstream of repA and is not regulated by TraR. The promoter was localized by subcloning and demonstrated to be strongly autorepressed. RepA is the major cis-acting autorepressor of this promoter, though RepB enhanced repression and was essential for RepA-mediated repression in trans. Purified RepA bound to an approximately 70-nucleotide operator site overlapping the P4 promoter and extending well downstream. Binding affinity was increased by adenosine di- and tri-phosphates and also by purified RepB. Activation of P1, P2, and P3 enhanced the activity of P4, suggesting that P4 somehow communicates with the upstream promoters. These findings demonstrate that both autoinduction and autorepression play critical and opposing roles in regulating repABC expression and hence in the replication, stability and copy number of the Ti plasmid.