Search

We previously described a plasmid of Agrobacterium spp., pAoF64/95, in which the quorum‐sensing system that controls conjugative transfer is induced by the opine mannopine. We also showed that the quorum‐sensing regulators TraR, TraM, and TraI function similarly to their counterparts in other repABC plasmids. However, traR, unlike its counterpart on Ti plasmids, is monocistronic and not located in an operon that is inducible by the conjugative opine. Here, we report that both traR and traM are expressed constitutively and not regulated by growth with mannopine. We report two additional regulatory genes, mrtR and tmsP, that are involved in a novel mechanism of control of TraR activity. Both genes are located in the distantly linked region of pAoF64/95 encoding mannopine utilization. MrtR, in the absence of mannopine, represses the four‐gene mocC operon as well as tmsP, which is the distal gene of the eight‐gene motA operon. As judged by a bacterial two‐hybrid analysis, TmsP, which shows amino acid sequence relatedness with the TraM‐binding domain of TraR, interacts with the antiactivator. We propose a model in which mannopine, acting through the repressor MrtR, induces expression of TmsP which then titrates the levels of TraM thereby freeing TraR to activate the tra regulon.

The production of cellulose fibrils is involved in the attachment of Agrobacterium tumefaciens to its plant host. Consistent with previous studies, we reported recently that a putative diguanylate cyclase, celR , is required for synthesis of this polymer in A. tumefaciens . In this study, the effects of celR and other components of the regulatory pathway of cellulose production were explored. Mutational analysis of celR demonstrated that the cyclase requires the catalytic GGEEF motif, as well as the conserved aspartate residue of a CheY-like receiver domain, for stimulating cellulose production. Moreover, a site-directed mutation within the PilZ domain of CelA, the catalytic subunit of the cellulose synthase complex, greatly reduced cellulose production. In addition, deletion of divK , the first gene of the divK-celR operon, also reduced cellulose production. This requirement for divK was alleviated by expression of a constitutively active form of CelR, suggesting that DivK acts upstream of CelR activation. Based on bacterial two-hybrid assays, CelR homodimerizes but does not interact with DivK. The mutation in divK additionally affected cell morphology, and this effect was complementable by a wild-type copy of the gene, but not by the constitutively active allele of celR . These results support the hypothesis that CelR is a bona fide c-di-GMP synthase and that the nucleotide signal produced by this enzyme activates CelA via the PilZ domain. Our studies also suggest that the DivK/CelR signaling pathway in Agrobacterium regulates cellulose production independent of cell cycle checkpoint systems that are controlled by divK .

Cellulose fibrils play a role in attachment of Agrobacterium tumefaciens to its plant host. While the genes for cellulose biosynthesis in the bacterium have been identified, little is known concerning the regulation of the process. The signal molecule cyclic di-GMP (c-di-GMP) has been linked to the regulation of exopolysaccharide biosynthesis in many bacterial species, including A. tumefaciens . In this study, we identified two putative diguanylate cyclase genes, celR ( atu1297 ) and atu1060 , that influence production of cellulose in A. tumefaciens . Overexpression of either gene resulted in increased cellulose production, while deletion of celR , but not atu1060 , resulted in decreased cellulose biosynthesis. celR overexpression also affected other phenotypes, including biofilm formation, formation of a polar adhesion structure, plant surface attachment, and virulence, suggesting that the gene plays a role in regulating these processes. Analysis of celR and Δ cel mutants allowed differentiation between phenotypes associated with cellulose production, such as biofilm formation, and phenotypes probably resulting from c-di-GMP signaling, which include polar adhesion, attachment to plant tissue, and virulence. Phylogenetic comparisons suggest that species containing both celR and celA , which encodes the catalytic subunit of cellulose synthase, adapted the CelR protein to regulate cellulose production while those that lack celA use CelR, called PleD, to regulate specific processes associated with polar localization and cell division.

Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI q - lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha- and gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZ α. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens , a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.

Summary Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is controlled by a hierarchical system in which opines, substrates produced by crown gall tumours, induce a quorum-sensing system. The cascade results from the control of expression of traR, the quorum-sensing activator, by a regulator responsive to the opine. In the two cases studied to date, the gene arrangements responsible for the cascade differ remarkably, suggesting that considerable diversity exists among the many Ti-like plasmids in the agrobacteria. In this study, we demonstrated that the novel Ti plasmid pTiChry5 is induced to transfer at high frequency by extracts from tumours initiated by strain Chry5. The purified inducer had the chemical and biological properties of agrocinopines C and D, a set of sugar phosphodiester opines known to induce transfer of another Ti plasmid, pTiBo542. The T-region of pTiChry5 contained a gene whose product, called AcsChry5, is virtually identical to the agrocinopine C1D synthase from the T-region of pTiBo542. The two genes are less closely related to acs of pTiC58, which is responsible for the production of agrocinopines A1B, a similar but not identical set of phosphodiester opines by tumours induced by strain C58. Agrocinopines A1B induce transfer of pTiC58 but did not induce transfer of pTiChry5. A single copy of traR was identified at the 11 o’clock region of pTiChry5, where it is part of a two-gene operon called arcChry5. Although altered by deletions, arcChry5 is related to the five-gene arc operon that controls the expression of traR on pTiC58. Expression of traRChry5 was induced by agrocinopines C1D and the opines isolated from Chry5 tumours but not by agrocinopines A1B. A mutation in traRChry5 abolished transfer, and transfer was restored by complementation in trans. We conclude that the agrocinopine opines and the corresponding opine-meditated conjugal regulatory regions of pTiChry5 and pTiC58 share a common origin, but that the opine signals for the two Ti plasmids have evolved divergently through changes in the opine synthase enzymes. The alterations in the opines, in turn, necessitated a coevolutionary change in the opine recognition systems responsible for controlling expression of the traR genes on these two types of Ti plasmids.

Conjugative transfer of the Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of TraR and its signal N -(3-oxo-octanoyl)- l -homoserine lactone. This system is, in turn, controlled by the conjugative opines produced by crown gall tumors induced on plants by the bacteria. Using nonpolar traI mutants, we examined the kinetics of induction of conjugative transfer in response to exogenous acyl-homoserine lactone. In the absence of the antiactivator TraM, onset of induction of transfer requires about 30 min, 15 to 20 min of which is needed for expression and construction of the conjugative apparatus. TraM delays the onset of conjugation by 30 min. While the rate of development of conjugative competence was not significantly affected by levels of TraR, maximum efficiencies of transfer were correlated with amounts of the activator in the donors. Donors harboring Ti plasmids lacking TraM were fully induced by the quormone at concentrations as low as 100 pM. TraM raised the concentration of signal required for maximum activity to 1 nM. Donors grown in batch culture retained conjugative competence following signal removal, even when in stationary phase. However, donors kept in balanced growth rapidly lost transfer ability following signal removal. Loss of transfer was mirrored by a decrease in levels of active TraR. Decreases in TraR activity and conjugative competence could be accounted for by dilution associated with cell division, suggesting that while induction of Ti plasmid conjugation is an active process, the cells lack a mechanism for disassembling the conjugative apparatus when signals become limiting.

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.

Agrobacterium tumefaciens possesses three iron-containing superoxide dismutases (FeSods) encoded by distinct genes with differential expression patterns. SodBI and SodBII are cytoplasmic isozymes, while SodBIII is a periplasmic isozyme. sodBI is expressed at a high levels throughout all growth phases. sodBII expression is highly induced upon exposure to superoxide anions in a SoxR-dependent manner. sodBIII is expressed only during stationary phase. Analysis of the physiological function of sod s reveals that the inactivation of sodBI markedly reduced levels of resistance to a superoxide generator, menadione. A mutant lacking all three Sod enzymes is the most sensitive to menadione treatment, indicating that all sod s contribute at various levels towards the overall menadione resistance level. Sods also have important roles in A. tumefaciens virulence toward a host plant. A sodBI but not a sodBII or sodBIII mutant showed marked reduction in its ability to induce tumors on tobacco leaf discs, while the triple sod null mutant is avirulent.

Phenazine production by Pseudomonas fluorescens 2-79 and P. chlororaphis isolates 30-84 and PCL1391 is regulated by quorum sensing through the activator PhzR and acyl-homoserine lactones (acyl-HSLs) synthesized by PhzI. PhzI from P. fluorescens 2-79 produces five acyl-HSLs that include four 3-hydroxy species. Of these, N -(3-hydroxyhexanoyl)-HSL is the biologically relevant ligand for PhzR. The quorum-sensing systems of P. chlororaphis strains 30-84 and PCL1391 have been reported to produce and respond to N -(hexanoyl)-HSL. These differences were of interest since PhzI and PhzR of strain 2-79 share almost 90% sequence identity with orthologs from strains 30-84 and PCL1391. In this study, as assessed by thin-layer chromatography, the three strains produce almost identical complements of acyl-HSLs. The major species produced by P. chlororaphis 30-84 were identified by mass spectrometry as 3-OH-acyl-HSLs with chain lengths of 6, 8, and 10 carbons. Heterologous bacteria expressing cloned phzI from strain 30-84 produced the four 3-OH acyl-HSLs in amounts similar to those seen for the wild type. Strain 30-84, but not strain 2-79, also produced N -(butanoyl)-HSL. A second acyl-HSL synthase of strain 30-84, CsaI, is responsible for the synthesis of this short-chain signal. Strain 30-84 accumulated N -(3-OH-hexanoyl)-HSL to the highest levels, more than 100-fold greater than that of N -(hexanoyl)-HSL. In titration assays, PhzR 30-84 responded to both N -(3-OH-hexanoyl)- and N -(hexanoyl)-HSL with equal sensitivities. However, only the 3-OH-hexanoyl signal is produced by strain 30-84 at levels high enough to activate PhzR. We conclude that strains 2-79, 30-84, and PCL1391 use N -(3-OH-hexanoyl)-HSL to activate PhzR.

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.

The large repABC plasmids of the order Rhizobiales with Class I quorum-regulated conjugative transfer systems often define the nature of the bacterium that harbors them. These otherwise diverse plasmids contain a core of highly conserved genes for replication and conjugation raising the question of their evolutionary relationships. In an analysis of 18 such plasmids these elements fall into two organizational classes, Group I and Group II, based on the sites at which cargo DNA is located. Cladograms constructed from proteins of the transfer and quorum-sensing components indicated that those of the Group I plasmids, while coevolving, have diverged from those coevolving proteins of the Group II plasmids. Moreover, within these groups the phylogenies of the proteins usually occupy similar, if not identical, tree topologies. Remarkably, such relationships were not seen among proteins of the replication system; although RepA and RepB coevolve, RepC does not. Nor do the replication proteins coevolve with the proteins of the transfer and quorum-sensing systems. Functional analysis was mostly consistent with phylogenies. TraR activated promoters from plasmids within its group, but not between groups and dimerized with TraR proteins from within but not between groups. However, oriT sequences, which are highly conserved, were processed by the transfer system of plasmids regardless of group. We conclude that these plasmids diverged into two classes based on the locations at which cargo DNA is inserted, that the quorum-sensing and transfer functions are coevolving within but not between the two groups, and that this divergent evolution extends to function.

Many pseudomonads, including the generalist Pseudomonas aeruginosa and fluorescent soil forms, such as Pseudomonas fluorescens, Pseudomonas aureofaciens, and Pseudomonas chlororaphis, produce phenazine-1-carboxylic acid (PCA) or its derivatives (7, 11, 49). These secondary metabolites exhibit antimicrobial activity against a wide range of prokaryotic and eukaryotic microbes, and the synthesis and secretion of these compounds is thought to confer an advantage on the producer bacteria when in competition with other microflora in the soil habitat (29). Production of substituted phenazines is also required by these bacteria to fully protect plants against diseases caused by fungal and bacterial pathogens that are susceptible to the antibiotics (2, 7, 9, 33, 49). Such biocontrol properties are of considerable interest, since their application in the field could reduce reliance on the use of environmentally toxic antimicrobial chemicals. However, for such strategies to be successful, the antibiotics must be synthesized reliably in the appropriate environmental sites. Hence, it is important to identify the genetic determinants required for synthesis of the phenazines, as well as the mechanisms by which expression of these genes is regulated. The phz cluster is organized as an operon of seven genes, the products of which catalyze the synthesis of chorismate and its conversion to PCA (9, 27, 28, 35). This parent compound can be substituted at several positions, depending upon the producer strain, and the genes coding for the enzymes involved in such substitutions are restricted to those strains producing the modified compounds. While gene nomenclature differs with the isolate (for an example, see reference 27), the order of the seven genes and the amino acid sequences of their products are conserved in all phz operons examined to date (9, 27, 28, 35). Although regulation is complex and multifactorial, the phz operons of P. aureofaciens 30-84 and P. chlororaphis PCL1391 are controlled at the transcriptional level by a ligand-dependent transcription factor called PhzR (8, 34). This activator is a member of the LuxR family and, as such, requires a low-molecular-weight signal, an acyl-homoserine lactone (acyl-HSL), for activity (8, 56). Signal production in both pseudomonads is catalyzed by PhzI, an acyl-HSL synthase of the LuxI family (8, 56). The acyl-HSL most probably transits out of and back into the cell by diffusion and thus accumulates within the environment as the population size of the dividing bacteria increases. When the concentration of the signal reaches a critical level, which corresponds to a particular population size of the bacteria, it interacts with and activates PhzR, thus initiating expression of the phz operon. In P. aureofaciens 30-84 and P. chlororaphis PCL1391, phzR and phzI are adjacent and convergently oriented, with phzR being located just upstream of and in opposite orientation to the phz operon (8, 56). Similarly, the phz operon of P. fluorescens 2-79 is preceded by phzR and phzI homologs organized in identical manners (Fig. ​(Fig.1).1). Moreover, the three phzR genes and the three phzI genes, as well as their encoded protein products, are all strongly orthologous (8). FIG. 1. Gene structure of the phz locus from P. fluorescens 2-79 and its mutants. (A) The organizations of the phz operon and the two regulatory genes phzR and phzI are shown by arrows. Alterations in the three mutants—strain 2-79IR, in which phzR and ... Strain 2-79 has served as a model for the genetics, biochemistry, and enzymology of phenazine biosynthesis. We previously reported that this isolate produces at least six acyl-HSLs, including the 3-hydroxy forms, N-(3-hydroxy-hexanoyl)-l-homoserine lactone (3-OH-C6-HSL), N-(3-hydroxy-octanoyl)-l-homoserine lactone (3-OH-C8-HSL), and N-(3-hydroxy-decanoyl)-l-homoserine lactone (3-OH-C10-HSL); the alkanoyl forms hexanoyl-homoserine lactone (C6-HSL) and octanoyl-homoserine lactone (C8-HSL); and an active signal compound of unknown structure (45). However, P. aureofaciens 30-84 and P. chlororaphis PCL1391 both have been reported to produce and utilize the alkanoyl signal, C6-HSL (8, 57). Moreover, the last two strains have not been reported to produce 3-hydroxy-acyl-HSLs. Given the very close amino acid sequence relatedness of the PhzI and PhzR proteins from these three strains, it became important to determine what acyl-HSL signals are produced by PhzI of P. fluorescens 2-79 and which of these signals activates PhzR of this strain. In this report, we show that PhzI catalyzes synthesis of all of the 3-OH- and alkanoyl-acyl-HSLs produced by P. fluorescens 2-79. We also show that 3-OH-C6 accumulates to the highest concentration in culture supernatants and that PhzR from strain 2-79 responds with greatest sensitivity to this signal. In addition, we show that activation of the phz operon by PhzR is dependent upon an intact inverted repeat, the phz box, located in the divergent phzA-phzR promoter region and that PhzR weakly autoactivates its own transcription, also in a phz box-dependent manner.

Amadori compounds form spontaneously in decomposing plant material and can be found in the rhizosphere. As such, these compounds could influence microbial populations by serving as sources of carbon, nitrogen and energy to microorganisms expressing suitable catabolic pathways. Two distinct sets of genes for utilization of deoxyfructosyl glutamine (DFG), an Amadori compound, have been identified in isolates of Agrobacterium spp. One, the soc gene set, is encoded by pAtC58, a 543 kb plasmid in A. tumefaciens strain C58. The second, mocD dissimilates DFG formed in the pathway for catabolism of mannopine (MOP) a non-Amadori, imine-type member of the mannityl opine family characteristic of certain Ti and Ri plasmids. To assess the level of dispersal of these two Amadori-utilizing systems, isolates of Agrobacterium spp. and related bacteria in the family Rhizobiaceae were examined by Southern analysis for homologs of socD and mocD. Homologs of mocD were associated only with Ti plasmid-encoded pathways for catabolism of MOP. Homologs of socD were more widely distributed, being detectable in many but not all of the isolates of Agrobacterium, Sinorhizobium, and Rhizobium spp. tested. However, this gene was never associated with the virulence elements, such as the Ti and Ri plasmids, in these strains. Regardless of genus most of the isolates containing socD homologs could utilize DFG as sole source of carbon, nitrogen and energy. Correlation studies suggested that mocD has evolved uniquely as part of the mannityl opine catabolic pathway while socD has evolved for the general utilization of Amadori compounds. Certain isolates of Agrobacterium and Rhizobium that lacked detectable homologs of socD and mocD also could utilize DFG suggesting the existence of additional, unrelated pathways for the catabolism of this Amadori compound. These results suggest that Amadori compounds constitute a source of nutrition that is important to microflora in the rhizosphere.

TraR, a quorum-sensing activator, induces transcription from its binding site, the tra-box, located upstream of Ti plasmid target promoters. TraR activated expression of a lacZ reporter in Escherichia coli only when RpoAAt from Agrobacterium tumefaciens was co-expressed. As assessed by gel retardation assays RpoAAt, but not RpoAEc, formed a ternary complex with TraR and a tra-box probe in vitro. TraR formed similar ternary complexes with alphaCTDAt but not with NTDAt, the C- and N-terminal segments of RpoAAt. As measured by surface plasmon resonance refractometry, TraR interacted directly with RpoAAt with an affinity about five times greater than that observed for its interaction with RpoAEc. The activator interacted with alphaCTDAt with kinetics and affinities similar to those of the full-sized -subunit. Positive control (PC) mutations at Asp-10 and Gly-123 of TraR did not affect DNA binding but greatly decreased the TraR-RpoAAt interaction. These two residues combine to form two patches on the activator, one of which may be involved in interaction with RpoA. When co-expressed, mutants of TraR with substitutions at Asp-10 complementing mutants with substitutions at Gly-123 for gene activation in an allele-specific manner. Co-expression studies with TraR and its PC mutants, and also with complementary PC alleles of TraR, coupled with three-dimensional structure are consistent with a hypothesis that both Asp-10/Gly-123 patches are required for activator function.

Summary 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 λ 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 λ cI′::TraR fusions, wild-type TraM mediated quormone-independent dimerization of the transcriptional activator, suggesting that dimers of TraM can multimerize TraR.

TraR, the quorum-sensing activator of theAgrobacterium tumefaciens Ti plasmid conjugation system, induces gene expression in response to its quormone,N-(3-oxooctanoyl)-l-homoserine lactone. Ligand binding results in dimerization of TraR and is required for its activity. Analysis of N- and C-terminal deletion mutants of TraR localized the quormone-binding domain to a region between residues 39 and 140 and the primary dimerization domain to a region between residues 119 and 156. The dominant-negative properties of these mutants predicted a second dimerization domain at the C terminus of the protein. Analysis of fusions of N-terminal fragments of TraR to λcI′ confirmed the dimerization activity of these two domains. Fifteen single amino acid substitution mutants of TraR defective in dimerization were isolated. According to the analysis of these mutants, Asp-70 and Gly-113 are essential for quormone binding, whereas Ala-38 and Ala-105 are important, but not essential. Additional residues located within the N-terminal half of TraR, including three located in α-helix 9, contribute to dimerization, but are not required for ligand binding. These results and the recently reported crystal structure of TraR are consistent with and complement each other and together define some of the structural and functional relationships of this quorum-sensing activator.

Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N -(3-oxo-octanoyl)- l -homoserine lactone. We isolated four Tn5-induced mutants of A. tumefaciens C58 deficient in TraR-mediated activation of tra genes on pTiC58Δ accR . These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing. In all four mutants Tn 5 was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by rnd of Escherichia coli , an RNase known to be involved in tRNA processing. The wild-type allele of the rnd homolog cloned from C58 restored the two phenotypes to each mutant. Several ORFs, including a homolog of cya2 , surround A. tumefaciens rnd , but none of these genes exerted a detectable effect on the expression of the tra reporter. In the mutant, traR was expressed from the Ti plasmid at a level about twofold lower than that in NT1. The expression of tra , but not the growth rate, was partially restored by increasing the copy number of traR or by disrupting traM , a Ti plasmid gene coding for an antiactivator specific for TraR. The mutation in rnd also slightly reduced expression of two tested vir genes but had no detectable effect on tumor induction by this mutant. Our data suggest that the defect in tra gene induction in the mutants results from lowered levels of TraR. In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D.

Promoter binding by TraR and LuxR, the activators of two bacterial quorum-sensing systems, requires their cognate acyl-homoserine lactone (acyl-HSL) signals, but the role the signal plays in activating these transcription factors is not known. Soluble active TraR, when purified from cells grown with the acyl-HSL, contained bound signal and was solely in dimer form. However, genetic and cross-linking studies showed that TraR is almost exclusively in monomer form in cells grown without signal. Adding signal resulted in dimerization of the protein in a concentration-dependent manner. In the absence of signal, monomer TraR localized to the inner membrane while growth with the acyl-HSL resulted in the appearance of dimer TraR in the cytoplasmic compartment. Affinity chromatography indicated that the N-terminus of TraR from cells grown without signal is hidden. Analysis of heterodimers formed between TraR and its deletion mutants localized the dimerization domain to a region between residues 49 and 156. We conclude that binding signal drives dimerization of TraR and its release from membranes into the cytoplasm.

The nine decades since Smith and Townsend demonstrated that Agrobacterium tumefaciens causes plant tumors (95) have been marked by a series of surprises. Among the most important of these was the report in 1958 that these tumors could be excised and propagated in vitro without exogenous plant hormones (7). Equally important were a series of reports beginning about the same time that tumors released compounds that agrobacteria could use as nutrients (24). Perhaps the most exciting discoveries, reported in the 1970s and 1980s, were that tumorigenesis required the transfer of fragments of oncogenic DNA to infected plant cells (10), that this process evolved from a conjugal transfer system (99), and that the genes that direct this process are expressed in response to host-released chemical signals (47). This DNA transfer process has become a cornerstone of plant molecular genetics. The genus Agrobacterium also has provided excellent models for several aspects of host-pathogen interactions, including intercellular transport of macromolecules (11), bacterial detection of host organisms (47), targeting of proteins to plant cell nuclei (3), and interbacterial chemical signaling via autoinducer-type pheromones (120).

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.

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and beta-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54-95% of the cotyledon explants on MXB selective medium containing 200 microg ml(-1) kanamycin and 500 microg ml(-1) carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4-5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode.

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.

Agrobacterium tumefaciens C58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency. We observed that a mutation affecting a tRNA processing function significantly affected the emergence of such mutants, suggesting that C58 contained a positively acting gene conferring resistance to tetracycline. A cosmid clone conferring resistance to tetracycline in Escherichia coli and Agrobacterium was isolated from a genomic bank of one such mutant. Subcloning, transposon mutagenesis, and DNA sequence analysis revealed that this DNA fragment contained two divergently transcribed genes, tetA and tetR , encoding products that were very similar to proteins of the Tet(A) class of tetracycline resistance systems. In the clone from this mutant, tetR was disrupted by an IS 426 . The homologous region from wild-type NT1 contained an intact tetR gene and did not confer resistance to tetracycline. Hybridization analysis showed that of 22 members of the genus Agrobacterium surveyed, only strains C58 and T37 contained the tet determinant. Moreover, only these two strains mutated to resistance to this antibiotic. Unlike other Tet(A) systems, neither tetracycline nor a series of its derivatives induced the expression of this tet gene unit. Other polycyclic compounds, including many of plant origin, also did not induce this tet gene system. The divergent promoter region of this tet system contained a single inverted repeat element identical to one such operator repeat in the promoter region of the tet determinant from the IncP1α R plasmid RP4. TetR repressor proteins from the Agrobacterium tet system and from RP4 interacted with the heterologous operators. While the repressive effect of the TetR protein from strain C58 (TetR C58 ) on the tetA gene from strain RP4 ( tetA RP4 ) was not relieved by tetracycline, repression of tetA C58 by TetR RP4 was lifted by this antibiotic.

Opines are carbon compounds produced by crown galls and hairy roots induced by Agrobacterium tumefaciens and A. rhizogenes, respectively. These novel condensation products of plant metabolic intermediates are utilized as nutritional sources by the Agrobacterium strains that induced the growths. Thus, opines are thought to favor the propagation of agrobacteria in the tumorsphere. Certain Agrobacterium strains were chemoattracted to opines. The chemotactic activities to octopine, to nopaline, to manno-pine, and to agrocinopines A+B were dependent on the type of the Ti plasmid present in the bacterium. The determinants for chemotaxis to these opines were localized to the regions of the octopine- and nopaline-type Ti plasmids coding for transport and catabolism of that opine. An insertion in accA, which encodes the periplasmic binding protein for agrocinopines A+B, abolished chemotaxis while an insertion in accC, which encodes a component of the transport system, and an insertion in accF, which encodes a function required for agrocinopine catabolism, did not affect chemotaxis to this opine. Thus, transport and catabolism of these opines are not required for the chemo-tactic activity. Analyses of subclones of the acc region confirmed that accA is the only gene required from the Ti plasmid for chemotaxis to agrocinopines A+B.

The mechanisms that ensure that Ti plasmid T-DNA genes encoding proteins involved in the biosynthesis of opines in crown gall tumors are always matched by Ti plasmid genes conferring the ability to catabolize that set of opines on the inducing Agrobacterium strains are unknown. The pathway for the biosynthesis of the opine agropine is thought to require an enzyme, mannopine cyclase, coded for by the ags gene located in the T(R) region of octopine-type Ti plasmids. Extracts prepared from agropine-type tumors contained an activity that cyclized mannopine to agropine. Tumor cells containing a T region in which ags was mutated lacked this activity and did not contain agropine. Expression of ags from the lac promoter conferred mannopine-lactonizing activity on Escherichia coli. Agrobacterium tumefaciens strains harboring an octopine-type Ti plasmid exhibit a similar activity which is not coded for by ags. Analysis of the DNA sequence of the gene encoding this activity, called agcA, showed it to be about 60% identical to T-DNA ags genes. Relatedness decreased abruptly in the 5' and 3' untranslated regions of the genes. ags is preceded by a promoter that functions only in the plant. Expression analysis showed that agcA also is preceded by its own promoter, which is active in the bacterium. Translation of agcA yielded a protein of about 45 kDa, consistent with the size predicted from the DNA sequence. Antibodies raised against the agcA product cross-reacted with the anabolic enzyme. These results indicate that the agropine system arose by a duplication of a progenitor gene, one copy of which became associated with the T-DNA and the other copy of which remained associated with the bacterium.

Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. With the exception of N -butanoyl- l -homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains of all lengths tested. The intensity of the response was proportional to the amount of the signal molecule chromatographed. Each of the 3-oxo- and the 3-unsubstituted derivatives migrated with a unique mobility. Using the assay, we showed that some bacteria produce as many as five detectable signal molecules. Structures could be assigned tentatively on the basis of mobility and spot shape. The dominant species produced by Pseudomonas syringae pv. tabaci chromatographed with the properties of N -(3-oxohexanoyl)- l -homoserine lactone, a structure that was confirmed by mass spectrometry. An isolate of Pseudomonas fluorescens produced five detectable species, three of which had novel chromatographic properties. These were identified as the 3-hydroxy- forms of N -hexanoyl-, N -octanoyl-, and N -decanoyl- l -homoserine lactone. The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules.

Two regions of the nopaline-type Ti plasmid pTiC58 are important for conjugal transfer of this element to recipient bacteria. These two regions were cloned into two independent replicons to produce a binary transfer system. For one region, oriT/tra, we constructed two derivatives, pFRtra and pDCtra-5. Each contains the oriT site and the two flanking, divergently transcribed tra operons that encode the DNA processing functions associated with the relaxosome. These two plasmids also carry traR, which encodes the transcriptional activator necessary for expression of transfer genes. The two plasmids differ by the amounts of traB sequence or sequence downstream of traG present in the construct. The second replicon, pPLE2, carries the traI/trb region. The traI gene confers production of the Agrobacterium tumefaciens N-acyl homoserine lactone autoinducer, while the remaining genes in the trb operon encode components of the mating bridge. Donors harboring the two plasmids mobilized the transfer of the plasmid carrying the oriT/tra region to an A. tumefaciens recipient at frequencies similar to that at which the intact Ti plasmid transferred. Plasmid pFRtra, which encodes most of traB, was mobilized at a frequency almost 10-fold higher than was pDCtra-5, which lacks most of the gene. A. tumefaciens donors also mobilized pFRtra to Escherichia coli and Pseudomonas fluorescens recipients at frequencies similar to those observed with A. tumefaciens recipients. Rhizobium meliloti harboring the binary system also transferred the oriT/tra component to these recipients. However, E. coli or P. fluorescens donors harboring the binary system did not transfer pFRtra to any of the recipients. Furthermore, while the A. tumefaciens and R. meliloti donors produced high levels of the autoinducer, the P. fluorescens and E. coli donors produced only trace amounts of this signal molecule. These results indicate that the tra system of pTiC58 is fully contained within the characterized tra and trb regions of the Ti plasmid, that conjugation does not require functions encoded by the vir system for maximal activity, and that while the Ti plasmid tra system recognizes diverse gram-negative bacteria as recipients, of the hosts tested, it functions only in members of the family Rhizobiaceae.

The Ti plasmids of Agrobacterium tumefaciens encode two transfer systems. One mediates the translocation of the T-DNA from the bacterium to a plant cell, while the other is responsible for the conjugal transfer of the entire Ti plasmid from one bacterium to another. The determinants responsible for conjugal transfer map to two regions, tra and trb, of the nopaline-type Ti plasmid pTiC58. By using transposon mutagenesis with Tn3HoHo1, we localized the tra determinants to an 8.5-kb region that also contains the oriT region. Fusions to lacZ formed by transposon insertions indicated that this region is expressed as two divergently transcribed units. We determined the complete nucleotide sequence of an 8,755-bp region of the Ti plasmid encompassing the transposon insertions defining tra. The region contains six identifiable genes organized as two units divergently transcribable from a 258-bp inter-genic region that contains the oriT site. One unit encodes traA, traF, and traB, while the second encodes traC, traD, and traG. Reporter insertions located downstream of both sets of genes did not affect conjugation but were expressed, suggesting that the two units encode additional genes that are not involved in transfer under the conditions tested. Proteins of the predicted sizes were expressible from traA, traC, traD, and traG. The products of several Ti plasmid tra genes are related to those of other conjugation systems. The 127-kDa protein expressed from traA contains domains related to MobA of RSF1O1O and to the helicase domain of TraI of plasmid F. The translation product of traF is related to TraF of RP4, and that of traG is related to TraG of RP4 and to VirD4 of the Ti plasmid T-DNA transfer system. Genetic analysis indicated that at least traG and traF are essential for conjugal transfer, while sequence analysis predicts that traA also encodes an essential function. traB, while not essential, is required for maximum frequency of transfer. Patterns of sequence relatedness indicate that the oriT and the predicted cognate site-specific endonuclease encoded by traA share lineage with those of the transfer systems of RSF1010 and plasmid F, while genes of the Ti plasmid encoding other essential tra functions share common ancestry with genes of the RP4 conjugation system.

The mocC gene encoded by the octopine/mannityl opine-type Ti plasmid pTi15955 is related at the nucleotide sequence level to mas1' encoded by the T region of this plasmid. While Mas1 is required for the synthesis of mannopine (MOP) by crown gall tumor cells, MocC is essential for the utilization of MOP by Agrobacterium spp. A cosmid clone of pTi15955, pYDH208, encodes mocC and confers the utilization of MOP on strain NT1 and on strain UIA5, a derivative of NT1 lacking the 450-kb cryptic plasmid pAtC58. NT1 or UIA5 harboring pYDH208 with an insertion mutation in mocC failed to utilize MOP as the sole carbon source. Plasmid pSa-C, which encodes only mocC, complemented this mutation in both strains. This plasmid also was sufficient to confer utilization of MOP on NT1 but not on UIA5. Computer analysis showed that MocC is related at the amino acid sequence level to members of the short-chain alcohol dehydrogenase family of oxidoreductases. Lysates prepared from Escherichia coli cells expressing mocC contained an enzymatic activity that oxidizes MOP to deoxyfructosyl glutamine (santhopine [SOP]) in the presence of NAD+. The reaction catalyzed by the MOP oxidoreductase is reversible; in the presence of NADH, the enzyme reduced SOP to MOP. The apparent Km values of the enzyme for MOP and SOP were 6.3 and 1.2 mM, respectively. Among analogs of MOP tested, only N-1-(1-deoxy-D-lyxityl)-L-glutamine and N-1-(1-deoxy-D-mannityl)-L-asparagine served as substrates for MOP oxidoreductase. These results indicate that mocC encodes an oxidoreductase that, as an oxidase, is essential for the catabolism of MOP. The reductase activity of this enzyme is precisely the reaction ascribed to its T-region-encoded homolog, Mas1, which is responsible for biosynthesis of mannopine in crown gall tumors.

Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.

Catabolic mannopine (MOP) cyclase encoded by certain Agrobacterium Ti and Ri plasmids lactonizes MOP to agropine (AGR). The enzyme, purified to homogeneity from a recombinant clone, has a molecular mass of 45 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography. The enzyme catalyzed the lactonization of MOP to AGR without the need for any cofactors. The enzyme also converted AGR to MOP with the lactonizing activity being predominant over the reverse reaction. MOP cyclase is specific for imine conjugates of D-hexose and L-glutamine and was not inhibited by sugars or amino acids. The enzyme lactonized deoxyfructosyl glutamine, a natural intermediate of MOP synthesis and catabolism, to a product indistinguishable from chrysopine, a newly discovered crown gall opine. The enzyme also lactonized N-l-(1,2-dideoxy-D-mannityl)-L-glutamine, indicating that a hydroxyl group at carbon atom 2 of the sugar moiety is not required for the enzymatic reaction.

Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium .

The plasmid pYDH208, which confers the ability to catabolize the mannityl opines mannopine and agropine, was mobilized into the nonpathogenic Pseudomonas syringae strain Cit7. The growth of the mannityl opine-catabolizing strain Cit7(pYDH208) was compared with that of the near-isogenic non-opine-catabolizing strain Cit7xylE on leaves of wild-type tobacco (Nicotiana tabacum cv. Xanthi) and transgenic mannityl opine-producing tobacco plants (N. tabacum cv. Xanthi, line 2-26). The population size of Cit7(pYDH208) was significantly greater on the lower leaves of transgenic plants than on middle or upper leaves of those plants. The population size of Cit7(pYDH208) on lower leaves of transgenic plants was also significantly greater than the population size of Cit7xylE on similar leaves of wild-type plants. High-voltage paper electrophoresis demonstrated higher levels of mannityl opines in washings from lower- and mid-level leaves than in washings from upper-level leaves. The ability of Cit7(pYDH208) to catabolize mannityl opines in the carbon-limited phyllosphere increased the carrying capacity of the lower leaves of transgenic plants for Cit7(pYDH208). In coinoculations, the increase in the ratio of population sizes of Cit7(pYDH208) to Cit7xylE on transgenic plants was apparently due to a subtle difference in the growth rates of the two strains and to the difference in final population sizes. An ability to utilize additional carbon sources on the transgenic plants also enabled Cit7(pYDH208) to achieve a higher degree of coexistence with Cit7xylE on transgenic plants than on wild-type plants. This supports the hypothesis that the level of coexistence between epiphytic bacterial populations can be altered through nutritional resource partitioning.

We engineered an expression unit composed of three eukaryotic genes driven by a single plant-active promoter and demonstrated functional expression in planta. The individual genes were linked as translational fusions to produce a polyprotein using spacer sequences encoding specific heptapeptide cleavage recognition sites for NIa protease of tobacco vein mottling virus (TVMV). The NIa gene itself was included as the second gene of the multi-gene unit. The first and third genes, obtained from the TR region of pTi15955, encoded enzymatic functions associated with the mannityl opine biosynthetic pathway. The mannityl opine conjugase gene (mas2) was the first unit of the construct and provided the native plant-active promoter and 5' untranslated regulatory sequence. The third gene (mas1), encoding the mannityl opine reductase, furnished the native 3' untranslated region. Cis-processing of the polyprotein by the NIa protease domain was demonstrated in vitro using rabbit reticulocyte lysate and wheat germ cell-free translation systems. Tobacco plant cells transformed with the multi-gene unit produced detectable levels of mannopine, mannopinic acid, and their biosynthetic intermediates, deoxyfructosyl-glutamate and deoxyfructosyl-glutamine. This indicates that the polygene construct results in a set of functional enzymatic activities that constitute a complete metabolic pathway.

Conjugal transfer of the Agrobacterium tumefaciens nopaline-type Ti plasmid pTiC58 is induced by agrocinopines A and B, opines secreted by crown gall tumors induced by the bacterium. This regulation functions through the transcriptional repressor, AccR. However, actual transcription of the tra genes is regulated by autoinduction through the activator TraR and the substituted homoserine lactone second messenger, Agrobacterium autoinducer (AAI). We have identified a new regulatory element that modulates the response of TraR to AAI. The gene, called traM, suppresses TraR-AAI activation of transcription of tra genes carried on recombinant clones. The suppression could be relieved by increasing the expression of TraR but not by increasing AAI levels. traM is located between traR and traAF on pTiC58 and is transcribed in the clockwise direction. The 306-bp gene encodes an 11.2-kDa protein showing no significant relatedness to other proteins in the databases. Mutations in traM in pTiC58 conferred a transfer-constitutive phenotype, and strains harboring the Ti plasmid produced easily detectable amounts of AAI. These same mutations engineered into the transfer-constitutive Ti plasmid pTiC58 delta accR conferred a hyperconjugal phenotype and very high levels of AAI production. Expression of traM required TraR, indicating that transcription of the gene is regulated by the autoinduction system. TraM had no effect on the expression of traR, demonstrating that the suppressive effect is not due to repression of the gene encoding the activator. These results suggest that TraM is not a direct transcriptional regulator. Since the suppressive effect is demonstrable only when traM is overexpressed with respect to traR, we suggest that TraM functions to sequester TraR from the very small amounts of AAI produced under conditions when the agrocinopines are not present.

Catabolic mannopine (MOP) cyclase encoded by Ti or Ri plasmids lactonizes MOP to agropine (AGR). The gene of the octopine-type Ti plasmid pTi15955 encoding the catabolic MOP cyclase enzyme previously was localized to a 1.6-kb segment within a cosmid clone, pYDH208. A subclone containing only this region complemented the AGR catabolism-negative phenotype conferred by a derivative of the octopine-type plasmid pTiB6S3 containing a Tn7 insertion in the region encoding the MOP cyclase enzyme. Uptake assays of strains harboring pRiA4 or pArA4a, along with complementation analyses, indicate that MOP cyclase is not sufficient for catabolism of AGR but that the strains must also express an AGR transport system. To determine the requirement for MOP cyclase in opine catabolism unequivocally, a site-specific, nonpolar deletion mutation abolishing only MOP cyclase activity was introduced into pYDH208, a cosmid clone that confers utilization of MOP, AGR, and mannopinic acid (MOA). Strains harboring this MOP cyclase-negative mutant clone, pYDPH208, did not utilize AGR but continued to utilize MOP. Growth on AGR was restored in this strain upon introduction of clones encoding the pTi15955-derived catabolic or anabolic MOP cyclase genes. The induction pattern of MOA catabolism shown by strain NT1 harboring the MOP cyclase-deficient pYDPH208 suggests that AGR is converted into MOP by MOP cyclase and that MOP, but not AGR, induces catabolism of MOA. Genetic and biochemical analyses of MOP and AGR metabolism suggest that only the conversion of AGR to MOP is directly involved in catabolism of AGR, even though the reaction catalyzed by MOP cyclase predominantly lies in the lactonization of MOP to AGR.

Conjugal transfer of the nopaline-type Agrobacterium Ti plasmid pTiC58 is regulated by a transcriptional 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 autoinduction. In Vibrio fischeri 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 traI, conferring production of material with CF activity, was localized to a 1-kb region at the upstream end of tra3 of pTiC58. Spectroscopy showed that the activity was authentic CF. Sequence analysis showed that traI 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 immediately downstream of traI could encode a protein related to TrbB of plasmid RP4, which is required for conjugal transfer. Transcription of traI and of the downstream tra3 genes requires TraR and CF and initiates from the traI promoter. The results show that traI is responsible for CF production, that it is the first gene of the tra3 operon, and that expression of this operon is regulated by autoinduction.

The 47.7-kb plasmid pAgK84, present in Agrobacterium radiobacter strain K84, confers production of a novel, highly specific, antiagrobacterial antibiotic called agrocin 84. Strain K84 is used commercially to biocontrol crown gall caused by agrocin 84-susceptible strains of Agrobacterium tumefaciens. Efficient biocontrol is dependent upon production of agrocin 84 by strain K84. Starting with a derivative of pAgK84 containing a Tn5 insertion, a series of deletion derivatives of the plasmid were isolated. The smallest of these, pJS500, contains about 8 kb of the original agrocin plasmid and localized the replication functions to between 4 and 6 o'clock on the physical map. A smaller derivative, produced by clonal rescue of a Tn5 insertion in the 4 o'clock region, further localized the minimal replication functions to a 1.5-kb region mapping between coordinates 18.1 and 19.6. Analysis of plasmid stability indicated that functions required for maintenance of the plasmid under nonselective conditions are tightly linked to the minimal replication region. This region also encodes incompatibility functions; the deletion derivatives were all incompatible with the wild-type pAgK84. The stability/replication locus of pAgK84 maps just anticlockwise from the Tra region. This region is retained fully in pAgK1026, the directed Tra- derivative of pAgK84 which is now in use as the primary crown gall biocontrol agent in Australia. One of the deletion derivatives, the 15-kb pJS400, was used as a vector to clone the KpnI fragments of an octopine-type Ti plasmid. Traits known to be encoded on these fragments were expressed and properly regulated in Agrobacterium hosts. One clone, encoding the Ti plasmid replication/incompatibility region, was used to cure IncRh1 Ti plasmids from their hosts. This clone also was found to be incompatible with pAtK84b, a large plasmid encoding opine catabolism present in A. radiobacter strain K84. This indicates that the opine catabolic plasmid is closely related to the IncRh1 Ti plasmids.

Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 × 106 electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 × 10−4. More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.

Three genes from the T(R) region of pTi15955 were introduced into tobacco (Nicotiana tabacum L.) to direct the synthesis of the mannityl opines from hexose sugars and glutamine or glutamate. Opines were present in all tissue types tested and accumulated to levels of 100 to 150 micrograms per milligram dry weight in root, stem, and leaf tissues. Opine-producing plants appeared normal with respect to morphology and development. Transgenic plants grown for 60 days under sterile autotrophic conditions produced up to 540 micrograms of the mannityl opines per milligrams dry weight of tissue as root exudates. Opines were also detected in leaf and seed washes from soil-grown plants.

The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed. Transfer is induced by the conjugal opines, a group of unique carbon compounds synthesized in crown gall tumors. The opines also induce Ti plasmid-encoded genes required by the bacteria for opine catabolism. We have cloned and sequenced a gene from the Ti plasmid pTiC58, whose product mediates the opine-dependent regulation of conjugal transfer and catabolism of the conjugal opines, agrocinopines A and B. The gene, accR, is closely linked to the agrocinopine catabolic locus. A spontaneous mutant Ti plasmid, pTiC58Trac, which constitutively expresses conjugal transfer and opine catabolism, was complemented in trans by a clone of wild-type accR. Comparative sequence analysis identified a 5-base-pair deletion close to the 5' end of the mutant accR allele from pTiC58Trac. Analysis of lacZ fusions in conjugal transfer and opine catabolic structural genes demonstrated that the accR-encoded function is a transcriptional repressor. accR can encode a 28-kDa protein. This protein is related to a class of repressor proteins that includes LacR, GutR, DeoR, FucR, and GlpR that regulate sugar catabolic systems in several bacterial genera.

Positive control (PC) mutants defining 20 residues of the quorum-sensing activator TraR were isolated that bind DNA but show defects in activating transcription from class I, class II or both types of promoters. These PC residues, located in both the N- and C-terminal regions, combine to form three patches, one on the top (II) and two near the DNA binding domain on both lateral faces of the dimer (I and III). Patches I and II, but not patch III, involve residues from both protomers and are essential for activation. TraR-mediated activation in Escherichia coli requires expression of the alpha-subunit of Agrobacterium (alpha(At)). We report that TraR also activates a class II promoter in E. coli when coexpressed with sigma(70)(At). Analyses in E. coli expressing alpha(At), sigma(70)(At) or both subunits indicate that most of the PC residues are important for interactions with alpha(At) and that these interactions are predominant for activation of class II promoters. Using the E. coli system we identified nine residues in the C-terminal domain of alpha(At) that are required for stimulating TraR-mediated activation. We conclude that N- and C-terminal residues of TraR from both protomers cooperate to define regions of the protein important for interactions with RNAP.

Agrobacterium radiobacter K84 is a commercial agent used worldwide to control crown gall disease caused by pathogenic isolates of A. tumefaciens. More than 2,000 transposon insertion derivatives of strain K84 were screened by a standardized greenhouse bioassay to identify mutants defective in biocontrol. Three mutants affected in biocontrol properties were identified. All three mutants displayed normal levels of attachment to tomato seed and root colonization. One of these mutants, M19-164, exhibited partial biocontrol and did not produce detectable levels of agrocin 84. In this mutant, the transposon is located in the agn locus of pAgK84, which codes for agrocin 84 biosynthesis. The second mutant, M19-158, also exhibited partial biocontrol and produced reduced amounts of agrocin 84 as a result of a mutation in a chromosomal gene of unknown function. The third mutant, M9-22, failed to biocontrol, was impaired in both growth in minimal medium and siderophore production, and failed to produce detectable levels of agrocin 84. The chromosomal gene ahcY, which encodes S-adenosyl-l-homocysteine hydrolase, was disrupted in this mutant. Expression of a functional copy of ahcY in M9-22 restored all of the altered phenotypes. The fact that all identified biocontrol mutants exhibited a partial or total defect in production of agrocin 84 indicates that this antibiotic is required for optimum biocontrol. This study also identified two chromosomally encoded genes required for agrocin 84 production. That a mutation in ahcY abolishes biocontrol suggests that the intracellular ratio of S-adenosyl-l-methionine to S-adenosyl-l-homocysteine is an important factor for agrocin 84 biosynthesis. Finally, we demonstrate that the ahcY gene in strain K84 is also required for optimal growth as well as for antibiotic production and biocontrol of crown gall disease.