Ecological genomics of marine Roseobacters.

Bacterioplankton of the marine Roseobacter clade have genomes that reflect a dynamic environment and diverse interactions with marine plankton. Comparative genome sequence analysis of three cultured representatives suggests that cellular requirements for nitrogen are largely provided by regenerated ammonium and organic compounds (polyamines, allophanate, and urea), while typical sources of carbon include amino acids, glyoxylate, and aromatic metabolites. An unexpectedly large number of genes are predicted to encode proteins involved in the production, degradation, and efflux of toxins and metabolites. A mechanism likely involved in cell-to-cell DNA or protein transfer was also discovered: vir-related genes encoding a type IV secretion system typical of bacterial pathogens. These suggest a potential for interacting with neighboring cells and impacting the routing of organic matter into the microbial loop. Genes shared among the three roseobacters and also common in nine draft Roseobacter genomes include those for carbon monoxide oxidation, dimethylsulfoniopropionate demethylation, and aromatic compound degradation. Genes shared with other cultured marine bacteria include those for utilizing sodium gradients, transport and metabolism of sulfate, and osmoregulation.

Twitching motility of Ralstonia solanacearum requires a type IV pilus system.

Twitching motility is a form of bacterial translocation over firm surfaces that requires retractile type IV pili. Microscopic colonies of Ralstonia solanacearum strains AW1, K60 and GMI1000 growing on the surface of a rich medium solidified with 1.6% agar appeared to exhibit twitching motility, because early on they divided into motile 'rafts' of cells and later developed protruding 'spearheads' at their margins. Individual motile bacteria were observed only when they were embedded within masses of other cells. Varying degrees of motility were observed for 33 of 35 strains of R. solanacearum in a selected, diverse collection. Timing was more important than culture conditions for observing motility, because by the time wild-type colonies were easily visible by eye (about 48 h) this activity ceased and the spearheads were obscured by continued bacterial multiplication. In contrast, inactivation of PhcA, a transcriptional regulator that is essential for R. solanacearum to cause plant disease, resulted in colonies that continued to expand for at least several additional days. Multiple strains with mutations in regulatory genes important for virulence were tested, but all exhibited wild-type motility. Many of the genes required for production of functional type IV pili, and hence for twitching motility, are conserved among unrelated bacteria, and pilD, pilQ and pilT orthologues were identified in R. solanacearum. Colonies of R. solanacearum pilQ and pilT mutants did not develop spearheads or rafts, confirming that the movement of cells that had been observed was due to twitching motility. Compared to the wild-type parents, both pilQ and pilT mutants caused slower and less severe wilting on susceptible tomato plants. This is the first report of twitching motility by a phytopathogenic bacterium, and the first example where type IV pili appear to contribute significantly to plant pathogenesis.

Multicomponent transcriptional regulation at the complex promoter of the exopolysaccharide I biosynthetic operon of Ralstonia solanacearum.

High-level transcription of eps, an operon encoding biosynthesis of an exopolysaccharide virulence factor of the phytopathogen Ralstonia (Pseudomonas) solanacearum, requires the products of at least seven regulatory genes (phcA, phcB, xpsR, vsrA-vsrD, and vsrB-vsrC), which are organized in three converging signal transduction cascades. Because xpsR and the vsrB-vsrC two-component system are the most downstream cascade components required for activation of eps, we explored how these components control transcription from the eps promoter (P(eps)). Deletion and PCR mutagenesis identified an upstream region of P(eps) (nucleotides -82 to -62) that is critical for transcription activation by VsrB-VsrC and XpsR and also is required for negative control of P(eps) by the putative eps regulator EpsR. Using PCR mutagenesis we generated the vsrC1 allele that encodes a response regulator that constitutively activates P(eps) in the absence of its cognate sensor, VsrB. However, activation of P(eps) by vsrC1 still required xpsR. Unexpectedly, the amino acid substitution conferring the constitutive phenotype on VsrC1 is 12 residues from its C terminus, outside the known functional domains of response regulators. Finally, a modified DNase I footprinting method was used to demonstrate specific binding of both VsrC1 and VsrC to the -72 to -62 upstream region of P(eps).

Evidence that Ralstonia eutropha (Alcaligenes eutrophus) contains a functional homologue of the Ralstonia solanacearum Phc cell density sensing system.

In the phytopathogen Ralstonia (Pseudomonas) solanacearum, control of many virulence genes is partly mediated by the Phc cell density sensing system. Phc uses a novel self-produced signal molecule [3-hydroxypalmitic acid methyl ester (3-OH PAME)], an atypical two-component system (PhcS/PhcR), and a LysR-type activator (PhcA) to regulate a reversible switching between two different physiological states. While Phc is present in most R. solanacearum strains, it is apparently absent from other pseudomonad plant pathogens and prokaryotic genomes that have been sequenced. Here, we report discovery of a phcA orthologue in the non-pathogenic, facultative chemolithoautotroph Ralstonia eutropha (Alcaligenes eutrophus) that fully complements R. solanacearum phcA mutants. We also demonstrate that some R. eutropha produce an extracellular factor that complements R. solanacearum mutants deficient in production of the 3-OH PAME signal molecule that controls phcA. Additionally, Southern blot hybridization analysis suggested that R. eutropha harbours other Phc components, such as PhcB (a biosynthetic enzyme for 3-OH PAME) and PhcS (a 3-OH PAME-responsive sensor kinase). Analysis of a phcA-null mutant of R. eutropha showed that phcA (and probably Phc) positively activates motility, in contrast to R. solanacearum where it represses motility. Similarly, the R. eutropha phcA mutant was unaffected in siderophore production, whereas inactivation of phcA in R. solanacearum increases siderophore production. Although our data strongly suggest that R. eutropha has a functional Phc-like system and support the phylogeny of Ralstonia, it implies that Phc may have a different physiological and ecological function in R. eutropha.

Footprinting with an automated capillary DNA sequencer.

Footprinting is a valuable tool for studying DNA-protein contacts. However, it usually involves expensive, tedious and hazardous steps such as radioactive labeling and analyses on polyacrylamide sequencing gels. We have developed an easy four-step footprinting method involving (i) the generation and purification of a PCR fragment that is fluorescently labeled at one end with 6-carboxyfluorescein; (ii) brief exposure of the fragment to a DNA-binding protein and then DNase I; (iii) spin-column purification; and (iv) analysis of partial digestion products on the ABI Prism 310 capillary DNA sequencer/genetic analyzer. Very detailed and sensitive footprints of large (> 400 bp) DNA fragments can be easily obtained, as illustrated by our use of this method to characterize binding of PhcA, a LysR-type activator, to two sites greater than 100 bp apart in the 5' untranslated region of xpsR, one of its regulated target genes. The advantages of this new method are that it (i) uses long-lived, safe and easy-to-make fluorescently labeled target fragments; (ii) uses sensitive, robust and highly reproducible fragment analysis using an automated DNA sequencer, instead of gel electrophoresis and autoradiography; and (iii) is cost effective.

Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum.

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10(7) cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of beta-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.

Spatial-Temporal and Quantitative Analysis of Growth and EPS I Production by Ralstonia solanacearum in Resistant and Susceptible Tomato Cultivars.

ABSTRACT One susceptible and two resistant cultivars of tomato were tested for differences in infection by Ralstonia solanacearum and for the subsequent multiplication, colonization, and production of the wilt-inducing virulence factor, exopolysaccharide I (EPS I). Bacterial ingress into the taproot was fastest in the susceptible cv. Marion, followed by the resistant cvs. L285 (fivefold slower) and Hawaii 7996 (15-fold slower). Once inside the taproot, R. solanacearum colonized, to some extent, almost all regions of the resistant and susceptible plants. However, colonization occurred sooner in the susceptible than in the resistant cultivars, as measured by viablecell counts of bacteria in the midstems. Rates of multiplication and maximum bacterial cell densities were also greater in the susceptible than in the resistant cultivars. Growth experiments utilizing xylem fluid from infected and uninfected plants indicated that neither antimicrobial activities nor reduced levels of growth-supporting nutrients in the xylem fluids were responsible for the reduced bacterial multiplication in the resistant cultivars. Quantification of EPS I in the infected plants, using an enzyme-linked immunosorbent assay, revealed that the bacterial populations in the susceptible cultivar produced greater amounts of EPS I per plant than those in the resistant cultivars. Immunofluorescence microscopy using antibodies against either EPS I or R. solanacearum cells revealed that bacteria and EPS I were distributed throughout the vascular bundles and intercellular spaces of the pith in the susceptible cultivar, whereas in the resistant cultivars, bacteria and EPS I were restricted to the vascular tissues.

Characterization of genes involved in biosynthesis of a novel antibiotic from Burkholderia cepacia BC11 and their role in biological control of Rhizoctonia solani.

Genetic manipulation of fluorescent pseudomonads has provided major insight into their production of antifungal molecules and their role in biological control of plant disease. Burkholderia cepacia also produces antifungal activities, but its biological control activity is much less well characterized, in part due to difficulties in applying genetic tools. Here we report genetic and biochemical characterization of a soil isolate of B. cepacia relating to its production of an unusual antibiotic that is very active against a variety of soil fungi. Purification and preliminary structural analyses suggest that this antibiotic (called AFC-BC11) is a novel lipopeptide associated largely with the cell membrane. Analysis of conditions for optimal production of AFC-BC11 indicated stringent environmental regulation of its synthesis. Furthermore, we show that production of AFC-BC11 is largely responsible for the ability of B. cepacia BC11 to effectively control the damping-off of cotton caused by the fungal pathogen Rhizoctonia solani in a gnotobiotic system. Using Tn5 mutagenesis, we identified, cloned, and characterized a region of the genome of strain BC11 that is required for production of this antifungal metabolite. DNA sequence analysis suggested that this region encodes proteins directly involved in the production of a nonribosomally synthesized lipopeptide.

Joint transcriptional control of xpsR, the unusual signal integrator of the Ralstonia solanacearum virulence gene regulatory network, by a response regulator and a LysR-type transcriptional activator.

Ralstonia (Pseudomonas) solanacearum is a soil-borne phytopathogen that causes a wilting disease of many important crops. It makes large amounts of the exopolysaccharide EPS I, which it requires for efficient colonization, wilting, and killing of plants. Transcription of the eps operon, encoding biosynthetic enzymes for EPS I, is controlled by a unique and complex sensory network that responds to multiple environmental signals. This network is comprised of the novel transcriptional activator XpsR, three distinct two-component regulatory systems (VsrAD, VsrBC, and PhcSR), and the LysR-type regulator PhcA, which is under the control of PhcSR. Here we show that the xpsR promoter (PxpsR) is simultaneously controlled by PhcA and VsrD, permitting XpsR to act like a signal integrator, simultaneously coordinating signal input into the eps promoter from both VsrAD and PhcSR. Additionally, we used in vivo expression analysis and in vitro DNA binding assays with substitution and deletion mutants of PxpsR to show the following. (i) PhcA primarily interacts with a typical 14-bp LysR-type consensus sequence around position -77, causing a sixfold activation of PxpsR; a weaker, less-defined binding site between -183 and -239 likely enhances PhcA binding and activation via the -77 site another twofold. (ii) Full 70-fold activation of PxpsR requires the additional interaction of the VsrD response regulator (or its surrogate) with a 14-bp dyadic sequence centered around -315 where it enhances activation (and possibly binding) by PhcA; however, VsrD alone cannot activate PxpsR. (iii) Increasing the distance between the putative VsrD binding site from that of PhcA by up to 232 bp did not dramatically affect PxpsR activation or regulation.

An RpoS (sigmaS) homologue regulates acylhomoserine lactone-dependent autoinduction in Ralstonia solanacearum.

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 soll, 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 (sigmaS) that in other bacteria activates gene expression during stationary phase or in response to stress conditions. R. solanacearum RpoS (RpoS(Rso)) was demonstrated to function as a sigma factor because when introduced in trans into an Escherichia coli rpoS mutant it largely restored expression of the RpoS-dependent bolAp1 gene. Mutation of rpoS(Rso) 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 soll were decreased in an rpoS(Rso) background (thereby reducing acyl-HSL production), but neither mutations in solR, soll or phcA nor addition of acyl-HSLs affected rpoS(Rso) expression. Therefore, in R. solanacearum the acyl-HSL-dependent autoinduction system is controlled both by a second autoinduction system and by the RpoS(Rso) sigma factor.

Hierarchical autoinduction in Ralstonia solanacearum: control of acyl-homoserine lactone production by a novel autoregulatory system responsive to 3-hydroxypalmitic acid methyl ester.

Bacteria employ autoinduction systems to sense the onset of appropriate cell density for expression of developmental genes. In many gram-negative bacteria, autoinduction involves the production of and response to diffusible acylated-homoserine lactones (acyl-HSLs) and is mediated by members of the LuxR and LuxI families. Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium that appears to autoregulate its virulence genes, produces compounds that promote expression of several heterologous acyl-HSL-responsive reporter gene constructs. High-pressure liquid chromatography of highly concentrated ethyl acetate extracts revealed that culture supernatants of strain AW1 contained two compounds with retention times similar to N-hexanoyl- and N-octanoyl-HSL. To investigate the role of these acyl-HSLs in R. solanacearum virulence gene expression, transposon mutants that were deficient for inducing an acyl-HSL-responsive reporter in Agrobacterium tumefaciens were generated. Three loci involved in normal acyl-HSL production were identified, one of which was shown to contain the divergently transcribed solR and solI genes, the luxR and luxI homologs, respectively. A 4.1-kb fragment containing solR and solI enabled all of the mutants (regardless of the locus inactivated) and a naturally acyl-HSL-defective strain of R. solanacearum to produce acyl-HSLs. Inactivation of solI abolished production of all detectable acyl-HSLs but affected neither the expression of virulence genes in culture nor the ability to wilt tomato plants. AW1 has a functional autoinduction system, because (i) expression of solI required SolR and acyl-HSL and (ii) expression of a gene linked to solR and solI, designated aidA, was acyl-HSL dependent. Because AidA has no homologs in the protein databases, its discovery provided no clues as to the role of acyl-HSLs in R. solanacearum gene regulation. However, expression of solR and solI required the global LysR-type virulence regulator PhcA, and both solR and solI exhibited a cell density-associated pattern of expression similar to other PhcA-regulated genes. The acyl-HSL-dependent autoinduction system in R. solanacearum is part of a more complex autoregulatory hierarchy, since the transcriptional activity of PhcA is itself controlled by a novel autoregulatory system that responds to 3-hydroxypalmitic acid methyl ester.

A two-component system in Ralstonia (Pseudomonas) solanacearum modulates production of PhcA-regulated virulence factors in response to 3-hydroxypalmitic acid methyl ester.

Expression of virulence factors in Ralstonia solanacearum is controlled by a complex regulatory network, at the center of which is PhcA, a LysR family transcriptional regulator. We report here that expression of phcA and production of PhcA-regulated virulence factors are affected by products of the putative operon phcBSR(Q). phcB is required for production of an extracellular factor (EF), tentatively identified as the fatty acid derivative 3-hydroxypalmitic acid methyl ester (3-OH PAME), but a biochemical function for PhcB could not be deduced from DNA sequence analysis. The other genes in the putative operon are predicted to encode proteins homologous to members of two-component signal transduction systems: PhcS has amino acid similarity to histidine kinase sensors, whereas PhcR and OrfQ are similar to response regulators. PhcR is quite unusual because its putative output domain strongly resembles the histidine kinase domain of a sensor protein. Production of the PhcA-regulated factors exopolysaccharide I, endoglucanase, and pectin methyl esterase was reduced 10- to 100-fold only in mutants with a nonpolar insertion in phcB [which express phcSR(Q) in the absence of the EF]; simultaneously, expression of phcA was reduced fivefold. Both a wild-type phenotype and phcA expression were restored by addition of 3-OH PAME to growing cultures. Mutants with polar insertions in phcB or lacking the entire phcBSR(Q) region produced wild-type levels of PhcA-regulated virulence factors. The genetic data suggest that PhcS and PhcR function together to regulate expression of phcA, but the biochemical mechanism for this is unclear. At low levels of the EF, it is likely that PhcS phosphorylates PhcR, and then PhcR interacts either with PhcA (which is required for full expression of phcA) or an unknown component of the signal cascade to inhibit expression of phcA. When the EF reaches a threshold concentration, we suggest that it reduces the ability of PhcS to phosphorylate PhcR, resulting in increased expression of phcA and production of PhcA-regulated factors.

Differential Expression of Virulence Genes and Motility in Ralstonia (Pseudomonas) solanacearum during Exponential Growth.

A complex network regulates virulence in Ralstonia solanacearum (formerly Pseudomonas solanacearum); central to this system is PhcA, a LysR-type transcriptional regulator. We report here that two PhcA-regulated virulence factors, endoglucanase (Egl) and acidic exopolysaccharide I (EPS I), and motility are expressed differentially during exponential growth in batch cultures. Tests with strains carrying lacZ fusions in a wild-type genetic background revealed that expression (on a per-cell basis) of phcA was constant but expression of egl and epsB increased 20- to 50-fold during multiplication from 1 x 10(sup7) to 5 x 10(sup8) CFU/ml. Expression of xpsR, an intermediate regulator downstream of PhcA in the regulatory cascade for eps expression, was similar to that of epsB and egl. Motility track photography revealed that all strains were essentially nonmotile at 10(sup6) CFU/ml. As cell density increased, 30 to 50% of wild-type cells were motile between 10(sup7) and 10(sup8) CFU/ml, but this population was again nonmotile at 10(sup9) CFU/ml. In contrast, about 60% of the cells of phcB and phcA mutants remained motile at 10(sup9) CFU/ml. Expression of phcB, which is not positively regulated by PhcA, was the inverse of epsB, egl, and xpsR (i.e., it decreased 20-fold at high cell density). PhcB is essential for production of an extracellular factor, tentatively identified as 3-hydroxypalmitic acid methyl ester (3-OH PAME), that might act as an exponential-phase signal to activate motility or expression of virulence genes. However, growth of the lacZ fusion strains in medium containing excess 3-OH PAME did not result in motility or expression of virulence genes at dramatically lower cell densities, suggesting that 3-OH PAME is not the only factor controlling these traits.

Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum.

Expression of virulence genes in Ralstonia solanacearum, a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs. Production of several virulence determinants is coordinately reduced by inactivation of phcB, but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wild-type strains of R. solanacearum. The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography. Gas chromatography and mass spectroscopy identified 3-hydroxypalmitic acid methyl ester (3-OH PAME) as the major component in the single peak of VEF activity. Authentic 3-OH PAME and the purified VEF were active at < or =1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant. Authentic 3-OH PAME also increased the production of three virulence factors by a phcB mutant over 20-fold to wild-type levels, restored normal cell density-associated expression of eps and increased expression of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small-molecule S-adenosylmethionine-dependent methyltransferase, which might catalyse synthesis of 3-OH PAME from a naturally occurring fatty acid. Biologically active concentrations of extracellular 3-OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild-type R. solanacearum. Other than acyl-homoserine lactones, 3-OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long-distance intercellular communication.

Role of Extracellular Polysaccharide and Endoglucanase in Root Invasion and Colonization of Tomato Plants by Ralstonia solanacearum.

ABSTRACT Ralstonia solanacearum is a soilborne plant pathogen that normally invades hosts through their roots and then systemically colonizes aerial tissues. Previous research using wounded stem infection found that the major factor in causing wilt symptoms was the high-molecular-mass acidic extracellular polysaccharide (EPS I), but the beta-1,4-endoglucanase (EG) also contributes to virulence. We investigated the importance of EPS I and EG for invasion and colonization of tomato by infesting soil of 4-week-old potted plants with either a wild-type derivative or genetically well-defined mutants lacking EPS I, EG, or EPS I and EG. Bacteria of all strains were recovered from surface-disinfested roots and hypocotyls as soon as 4 h after inoculation; that bacteria were present internally was confirmed using immunofluorescence microscopy. However, the EPS-minus mutants did not colonize stems as rapidly as the wild type and the EG-minus mutant. Inoculations of wounded petioles also showed that, even though the mutants multiplied as well as the wild type in planta, EPS-minus strains did not spread as well throughout the plant stem. We conclude that poor colonization of stems by EPS-minus strains after petiole inoculation or soil infestation is due to reduced bacterial movement within plant stem tissues.

Cloning and characterization of tek, the gene encoding the major extracellular protein of Pseudomonas solanacearum.

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.

Impact of a genetically engineered bacterium with enhanced alkaline phosphatase activity on marine phytoplankton communities.

An indigenous marine Achromobacter sp. was isolated from coastal Georgia seawater and modified in the laboratory by introduction of a plasmid with a phoA hybrid gene that directed constitutive overproduction of alkaline phosphatase. The effects of this "indigenous" genetically engineered microorganism (GEM) on phosphorus cycling were determined in seawater microcosms following the addition of a model dissolved organic phosphorus compound, glycerol 3-phosphate, at a concentration of 1 or 10 (mu)M. Within 48 h, a 2- to 10-fold increase in the concentration of inorganic phosphate occurred in microcosms containing the GEM (added at an initial density equivalent to 8% of the total bacterial population) relative to controls containing only natural microbial populations, natural populations with the unmodified Achromobacter sp., or natural populations with the Achromobacter sp. containing the plasmid but not the phoA gene. Secondary effects of the GEM on the phytoplankton community were observed after several days, evident as sustained increases in phytoplankton biomass (up to 14-fold) over that in controls. Even in the absence of added glycerol 3-phosphate, a numerically stable GEM population (averaging 3 to 5% of culturable bacteria) was established within 2 to 3 weeks of introduction into seawater. Moreover, alkaline phosphatase activity in microcosms with the GEM was substantially higher than that in controls for up to 25 days, and microcosms containing the GEM maintained the potential for net phosphate accumulation above control levels for longer than 1 month.

X-ray analysis of crystals of polygalacturonase A from Pseudomonas solanacearum.

Crystals of the pectolytic protein, polygalacturonase A, have been obtained from polyethylene glycol 8000 using vapor diffusion methods. The 52.4 kDa protein is secreted by the plant pathogenic bacteria Pseudomonas solanacearum, and is important in the virulence of this plant pathogen. The protein crystallizes in space group P2(1) and has unit-cell parameters of a = 101.9, b = 124.6, c = 48.1 A, and beta= 105 degrees 50'. The crystal has two molecules in the asymmetric unit, and diffracts maximally to a resolution of 2.1 A.

catM encodes a LysR-type transcriptional activator regulating catechol degradation in Acinetobacter calcoaceticus.

On the basis of the constitutive phenotypes of two catM mutants of Acinetobacter calcoaceticus, the CatM protein was proposed to repress expression of two different loci involved in catechol degradation, catA and catBCIJFD (E. Neidle, C. Hartnett, and L. N. Ornston, J. Bacteriol. 171:5410-5421, 1989). In spite of its proposed negative role as a repressor, CatM is similar in amino acid sequence to positive transcriptional activators of the LysR family. Investigating this anomaly, we found that insertional inactivation of catM did not cause the phenotype expected for the disruption of a repressor-encoding gene: in an interposon-generated catM mutant, no cat genes were expressed constitutively, but rather catA and catB were still inducible by muconate. Moreover, this catM mutant grew poorly on benzoate, a process requiring the expression of all cat genes. The inducibility of the cat genes in this catM mutant was completely eliminated by a 3.5-kbp deletion 10 kbp upstream of catM. In this double mutant, catM in trans restored muconate inducibility to both catA and catB. These results suggested the presence of an additional regulatory locus controlling cat gene expression. The ability of CatM to function as an activator was also suggested by these results. In support of this hypothesis, in vivo methylation protection assays showed that CatM protects two guanines in a dyad 65 nucleotides upstream of the catB transcriptional start site, in a location and pattern typical of LysR-type transcriptional activators. Gel mobility shift assays indicated that CatM also binds to a region upstream of catA. DNA sequence analysis revealed a nucleotide near the 3' end of catM not present in the published sequence. Translation of the corrected sequence resulted in the deduced CatM protein being 52 residues longer than previously reported. The size, amino acid sequence, and mode of action of CatM now appear similar to, and typical of, what has been found for transcriptional activators in the LysR family. Analysis of one of the constitutive alleles of catM previously thought to encode a dysfunctional repressor indicated instead that it encodes an inducer-independent transcriptional activator.

A complex network regulates expression of eps and other virulence genes of Pseudomonas solanacearum.

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.