An 8× draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10-13) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Many bacteria sense an appropriate growth condition or a critical population density for gene expression by producing acylhomoserine lactones (acyl‐HSLs) that act as intercellular autoinduction signals. We recently showed that, in Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium, acyl‐HSL production requires solI, which encodes a putative acyl‐HSL synthase, and that its expression is positively regulated by the acyl‐HSL‐responsive SolR transcriptional regulator. This acyl‐HSL‐dependent autoinduction system is noteworthy because (i) it is regulated by a ‘higher level’ autoinducer system (responsive to 3‐hydroxypalmitic acid methyl ester) via PhcA, a LysR‐type transcriptional regulator and (ii) acyl‐HSL production requires two additional unlinked loci. As reported here, cloning and sequencing of one of these other loci revealed that it encodes a homologue of RpoS, an alternative sigma factor (σS) that in other bacteria activates gene expression during stationary phase or in response to stress conditions. R. solanacearum RpoS (RpoSRso) was demonstrated to function as a σ factor because when introduced in trans into an Escherichia coli rpoS mutant it largely restored expression of the RpoS‐dependent bolAp1 gene. Mutation of rpoSRso in R. solanacearum reduced survival during starvation and low pH conditions, but did not affect survival during exposure to hydrogen peroxide, high osmolarity or high temperature. This mutant was also altered in its production of several virulence factors and wilted tomato plants several days more slowly than the wild‐type parent. Transcription of solR and solI were decreased in an rpoSRso background (thereby reducing acyl‐HSL production), but neither mutations in solR, solI or phcA nor addition of acyl‐HSLs affected rpoSRso expression. Therefore, in R. solanacearum the acyl‐HSL‐dependent autoinduction system is controlled both by a second autoinduction system and by the RpoSRso sigma factor.

Susceptible plants infected by Pseudomonas solanacearum usually will, largely due to extracellular proteins (EXPs) and the high-molecular-mass extracellular polysaccharide (EPS I) this pathogen produces. Circumstantial evidence suggested that a 28-kDa protein, the single most abundant EXP made by P. solanacearum in culture, is associated with production of EPS I, and thus might have a role in pathogenesis. The 28-kDa EXP was purified and, based on its N-terminal amino acid sequence, an oligonucleotide mixture was made and used as a hybridization probe to clone the gene encoding it. DNA sequence analysis suggested that the coding sequence for the 28-kDa EXP is within a gene, designated tek, that encodes a 58-kDa membrane-associated precursor protein that is processed by signal peptidase II during export. Analysis of radiolabeled polypeptides expressed from tek confirmed that it encodes a 58-kDa precursor protein, which is exported out of the cells as a 55-kDa preprotein and processed extracellularly to release the very basic 28-kDa EXP from its C terminus. The position, transcriptional direction, and regulated expression of tek suggest that it is cotranscribed with xpsR, a gene essential for regulating biosynthesis of EPS I, and reinforces the association of the 28-kDa EXP with virulence. However, since P. solanacearum mutants lacking only the 28-kDa EXP produced wild-type amounts of EPS I and were fully virulent, the function of this protein remains unclear.

We have discovered an unusual and complex regulatory network used by the phytopathogen Pseudomonas solanacearum to control transcription of eps, which encodes for production of its primary virulence factor, the exopolysaccharide EPS I. The major modules of this network were shown to be three separate signal transduction systems: PhcA, a LysR-type transcriptional regulator, an dual two-component regulatory systems, VsrA/VsrD and VsrB/VsrC. Using lacZ fusions and RNA analysis, we found that both PhcA and VsrA/VsrD control transcription of another network component, xpsR, which in turn acts in conjunction with vsrB/vsrC to increase transcription of the eps promoter by > 25-fold. Moreover, gel shift DNA binding assays showed that PhcA specifically binds to the xpsR promoter region. Thus, the unique XpsR protein interconnects the three signal transduction systems, forming a network for convergent control of EPS I in simultaneous response to multiple environmental inputs. In addition, we demonstrate that each individual signaling system of the network also acts independently to divergently regulate other unique sets of virulence factors. The purpose of this complex network may be to allow this phytopathogen to both coordinately or independently regulate diverse virulence factors in order to cope with the dynamic situations and conditions encountered during interactions with plants.

SummaryThe wilt‐inducing phytopathogen Pseudomonas solanacearum produces several extracellular virulence factors, both polysaccharides (EPS I) and proteins (EXPs), which are independently regulated by a LysR‐type transcriptional regulator, PhcA, and a histidine kinase sensor, VsrB. Here we characterize a third locus, vsrA, which is also required for normal production of EPS I, some EXPs and wilt disease. Analysis of eps::lacZ reporters in vsrA mutants showed that, like vsrB and phcA, vsrA is required for maximal expression (transcription) of eps, which contains some of the genes necessary for production of EPS I. Unlike vsrB and phcA mutants, however, eps transcription (and EPS I production) by vsrA mutants varies from 3 to 17% of wild‐type levels, depending on growth conditions. Inactivation of vsrA also causes a dramatic reduction in production of three species of EXPs (28kDa, 48kDa, and 66kDa), and an apparent increase in production of a few other EXPs. Unlike most other EPS‐deficient P. solanacearum strains, vsrA mutants caused almost no disease symptoms when 104 cells were stem‐inoculated into tomato plants. This correlated with a greater than 10‐fold reduction in their ability to grow in plants. vsrA was cloned from a P. solanacearum genomic library by complementation of the vsrA mutant and was further subcloned on a 2.3kb DNA fragment. PhoA fusion analysis and subcellular localization of the vsrA gene product in Escherichia coli maxicells suggest that it is a 53 kDa membrane‐associated protein. Analysis of the nucleotide sequence of vsrA revealed a 502 residue open reading frame with homology to the histidine kinase domain of sensors in the two‐component regulator family. This discovery shows that EPS I production by P. solanacearum is simultaneously controlled by dual two‐component sensors.

A complex regulatory network controls virulence genes of Pseudomonas solanacearum. Analysis of the transposon-generated mutant AW1-83 suggests that a new locus, designated phcB, may play a role in this network. AW1-83 (phcB83) produced at least 30-fold less than the wild type of extracellular polysaccharide (EPS I, encoded in part by eps) and at least seven extracellular proteins, but these traits were fully restored in response to one or more extracellular factors (EF) released by wild-type P. solanacearum. Presence of EF increased transcription of a genomic eps::lacZ fusion in a phcB83 background more than 50-fold, restoring wild-type expression. The EF made by P. solanacearum was present in both the aqueous and the vapor phases. Millimolar levels of methanol (but not larger alcohols) and micromolar levels of C14- to C18-fatty acid methyl esters (but not larger or smaller methyl esters) also restored nearly wild-type expression of eps::lacZ in a phcB83 background. The methoxy group was essential for this increase, since neither free fatty acids nor the ethyl or propyl esters were active. Growth with the C16-methyl ester restored normal production of EPS I and extracellular proteins by AW1-83. The wild-type phcB locus was subcloned on a 4-kb fragment and delimited to less than 2 kb by transposon inactivation and complementation studies. Genomic phcB::Tn3HoHo1 mutations appeared to eliminate EF production but did not uniformly reduce production of EPS I and extracellular proteins. Site-specific recombination of the phcB83 allele into the genome of five other P. solanacearum strains revealed that they have a structurally and functionally conserved phcB locus. Although all 80 wild-type strains of P. solanacearum tested made some EF, out of seven genera of bacteria tested, only Agrobacterium produced an EF-like activity that stimulated visible EPS production by AW1-83. Our results suggest that the EF may be an extracellular signal molecule in P. solanacearum that is different from the acyl-homoserine lactone signal compounds produced by Vibrio fischeri and other Gram-negative bacteria.

A complex regulatory network controls production of some extracellular macromolecules that Pseudomonas solanacearum strain AWl needs for full virulence on tomato. Expression of genes for extracellular polysaccharide (EPS) and other virulence factors is coordinately controlled by phcA, since inactivation of this gene results in a pleiotropic change called phenotype conversion (PC). PhcA is likely to be a trans-acting DNA-binding protein because it has strong similarity at the amino acid level to the LysR family of transcriptional activators. At least three different insertions were detected within phcA after spontaneous PC, suggesting that random inactivation of this gene is responsible for this phenomenon. Another locus, phcB, is necessary for production by wild-type P. solanacearum strains of a volatile inducer compound that appears to be required for full activity of PhcA. Cell density, and thus levels ofthe endogenous inducer, appear to mediate expression of a Phc.4-regulated eps::lacZ fusion. Expression of eps genes also depends on three additional loci (xpsR, vsrA. and vsrB), two of which encode membraneassociated proteins and thus may serve as environmental sensors. These findings suggest that current concepts regarding the physiology, ecology, and possible strategies for controlling P. solanacearum should be re-evaluated. IN the last ten years there has been substantial progress in elucidating the genetic and biochemical bases of pathogenesis of P. solanacearum. For example, the genes encoding several of the putative virulence factors identified in the 1950s were cloned and subsequently inactivated to test their contribution to the typical wilt symptoms. It is now clear that, in planta, the extracellular polysaccharide (EPS) is the primary virulence factor (Denny and Baek 1991; Kao and Sequeira 1992). whereas the extracellular endoglucanase (EG) and en do-polygalacturonase (endo-PG) enzymes. which may act on plant cell walls, are relatively minor virulence factors (Denny et aL 1990). In contrast to these factors that enhance virulence but are dispensable, the hrp genes are essential for pathogenesis on compatible hosts and the hypersensitive response on incompatible hosts (Boucher et al. 1992). Unlike virulence mutants, strains with one or more hrp genes inactivated grow very poorly in planta (Macol 1989; ·Departments of Plant Pathology and Microbiology. 2105 Plant Science Building, University of Georgia, Athens GA 30602,USA. Trigalet and Demery 1986) and cause no disease symptoms. Despite these and other significant advances, our understanding of the pathogenic processes of P. solanacearum is still incomplete. Not all of the putative virulence factors have been examined (Denny and Schell 1992) and even those mentioned above have not been tested for their role in other aspects of pathogenesis or saprophytic survival. Even less is known about regulation of genes encoding pathogenesisspecific molecules. This paper will discuss some of our recent research on phenotype conversion in P. solanacearum and the associated regulatory net work that controls virulence. Phenotype Conversion Nearly four decades ago Kelman (1954) reported that when P. solanacearum spontaneously changes from a mucoid to a nonmucoid colony morphology there is a concomitant loss of its capacity to wilt plants. These non-mucoid strains are not hrp mutants, because they stilI grow in planta (Denny and Baek 1991) and cause

Phenotype conversion (PC) in Pseudomonas solanacearum is the coordinated change in production of extracellular polysaccharide and a variety of extracellular proteins, some of which contribute to virulence. Although PC is normally spontaneous, it is mimicked by transposon inactivation of the phcA locus (S. M. Brumbley and T. P. Denny, J. Bacteriol. 172:5677-5685, 1990). The DNA sequence of a 1.8-kb region from strain AW1 that contains phcA revealed one open reading frame that should encode a polypeptide of 38.6 kDa. The PhcA protein produced in Escherichia coli by using a T7 RNA polymerase expression system was of the predicted size. The deduced amino acid sequence of PhcA is similar to that of some members of the LysR transcriptional activator gene family, especially in the amino terminus, where a putative helix-turn-helix DNA-binding motif was identified. An analogous allele (phcA1) was cloned from the spontaneous PC mutant strain AW1-PC and found to be nonfunctional in complementation studies. When phcA1 was expressed in E. coli, the PhcA1 protein was 35.5 kDa, 3 kDa smaller than PhcA. Sequence analysis of phcA1 and chimeric constructs of phcA and phcA1 confirmed that PhcA1 is truncated by a 2-bp insertion 147 nucleotides upstream of the carboxyl terminus of PhcA. Southern blot analysis of 10 additional independently isolated PC mutants of strain AW1 revealed that two strains have larger insertions (0.2 and 1.0 kb) within phcA. These results suggest that phcA encodes a DNA-binding protein that regulates the transcription of one or more of the genes involved in P. solanacearum virulence and that spontaneous PC can be attributed to one of several different insertions within this locus.

Pseudomonas solanacearum, an important wilt pathogen of many plants, produces several extracellular proteins (EXPs) and extracellular polysaccharides (EPSs) that contribute to its virulence. Using TnphoA mutagenesis, we discovered a new gene, vsrB, that when inactivated causes a major reduction in the virulence and production of an EPS. Analysis of eps::lacZ reporters showed that vsrB is required for maximal expression (transcription) of eps, whose products are required for production of EPS I, a major virulence determinant. Analysis of EXPs in culture supernatants revealed that inactivation of vsrB also causes reduced production of two major EXPs, with molecular masses of 28 and 97 kDa, and a simultaneous 15-fold increase in levels of another EXP, PglA endopolygalacturonase. The vsrB gene was cloned from a P. solanacearum genomic library by complementation of the nonmucoid phenotype of the vsrB::TnphoA mutant and then subcloned on a 2.4-kb DNA fragment. TnphoA fusion analysis and subcellular localization of the vsrB gene product in Escherichia coli maxicells suggest that it is a ca. 60-kDa transmembrane protein. The nucleotide sequence of the 2.4-kb DNA fragment was determined, and a 638-amino-acid open reading frame was found for VsrB. A search of the GenBank data base found that the central part of VsrB has homology with the histidine kinase domain of sensors in the two-component regulator family, while the C terminus has homology with the phosphate receiver domain of response regulators in the same family. Genetic analysis suggests that the receiver domain is not required for vsrB function.

Pseudomonas solanacearum produces an acidic, nitrogen-rich, extracellular polysaccharide (EPS) that is required for wilting and killing of infected plants Biosynthesis of EPS is partially encoded by the 18-kb eps locus; additional loci near eps are also required for EPS production, but only under specific growth conditions. All these loci are transcriptionally controlled by an interacting regulatory network involving the products of at least five distinct regulatory loci: phcA, phcB, vsrA,vsrB, and xpsR. This network, which also regulates other virulence genes, may control transcription in response to various environmental signals, such as nutritional status and cell density.

Pseudomonas solanacearum undergoes a spontaneous mutation that pleiotropically reduces extracellular polysaccharide (EPS) production, endoglucanase activity, and virulence and increases motility. We refer to the process that coordinately affects these traits as phenotype conversion (PC) and the resulting mutants as PC types. Previous research with the wild-type strain AW1 suggested that inactivation of a single locus could mimic phenotype conversion (T. P. Denny, F. W. Makini, and S. M. Brumbley, Mol. Plant-Microbe Interact. 1:215-223, 1988). Additional Tn5 mutagenesis of AW1 generated three more mutants (AW1-81, AW1-82, and AW1-84) that were indistinguishable from the PC type and one slightly leaky mutant (AW1-87); all four had single insertions in the same 4.0-kilobase (kb) EcoRI fragment that were responsible for the PC-like phenotype. Another insertion mutant, AW1-83, which lacks an insertion in this 4.0-kb fragment, resembled the PC type except that it was reversibly induced to produce wild-type levels of EPS when cultured adjacent to AW1. The wild-type region containing the gene that controls traits affected by phenotype conversion in AW1, designated phcA, was cloned on a 2.2-kb DNA fragment that restored all the phcA::Tn5 mutants and 11 independent spontaneous PC-type derivatives of AW1 to wild-type status. Homology with the phcA region was found in diverse wild-type strains of P. solanacearum, although restriction fragment length polymorphisms were seen. No major DNA alterations were observed in the phcA homologous region of PC types from strain AW1 or 82N. PC types from 7 of 11 conjugal strains of P. solanacearum were restored to EPS+ by phcA from AW1; however, only some PC types of strain K60 were restored, whereas others were not. We believe that a functional phcA gene is required to maintain the wild-type phenotype in P. solanacearum, and for most strains phenotype conversion results from a loss of phcA gene expression or the function of its gene product.

The egl gene of Pseudomonas solanacearum was cloned on a cosmid and expressed in Escherichia coli. Restriction endonuclease mapping, transposon mutagenesis, and subclone analysis showed that the egl gene was located on a 2.7-kilobase XhoI-SalI P. solanacearum DNA fragment. Immunoabsorption experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that the egl gene encodes the 43-kilodalton endoglucanase that is the major excreted endoglucanase of P. solanacearum. In E. coli, the egl gene appeared to be expressed from its own promoter, but its product was restricted to the cytoplasm. The cloned egl gene was mutagenized with Tn5 and used to specifically mutate the chromosomal egl gene of P. solanacearum by site-directed mutagenesis. The resultant mutant was identical to the wild-type strain in production of extracellular polysaccharide and extracellular polygalacturonase as well as several other excreted proteins but produced at least 200-fold less endoglucanase. This mutant strain was significantly less virulent on tomato than the wild-type strain in plant bioassay experiments. Virulence of the endoglucanase-deficient strain was restored to near wild-type levels by complementation in trans with the cloned egl gene, indicating that the egl gene is important but not absolutely required for pathogenesis.

A major endopolygalacturonase excreted by Pseudomonas solanacearum was purified to greater than 95% homogeneity and shown to have an isoelectric point of 9.0 and a subunit molecular mass of 52 kilodaltons (kDa). The gene encoding this enzyme (pglA) was isolated from a genomic library of P. solanacearum DNA based on its expression in Escherichia coli and shown to be contained on a 1.8-kilobase DNA fragment. The identity of the pglA gene product and the 52-kDa polygalacturonase was demonstrated by immunoadsorption and isoelectric focusing experiments. The cloned pglA gene was apparently expressed from its own promoter in E. coli and its product was partially secreted into the periplasm. The pglA gene was insertionally inactivated in vitro and used to mutate the chromosomal pglA gene of P. solanacearum by marker exchange mutagenesis. The resulting mutant strain was deficient in production of the 52-kDa polygalacturonase and took twice as long to wilt and kill tomato plants as the wild-type parent in plant bioassay experiments. Complementation in trans with the wild-type cloned pglA gene restored virulence to near wild-type levels. The data indicate that the pglA gene is important, but not absolutely necessary, for pathogenesis.