Timothy P. Denny

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 co‐ordinately 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 ≤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.

Pathogenomics of the Ralstonia solanacearum species complex.

Ralstonia solanacearum is a major phytopathogen that attacks many crops and other plants over a broad geographical range. The extensive genetic diversity of strains responsible for the various bacterial wilt diseases has in recent years led to the concept of an R. solanacearum species complex. Genome sequencing of more than 10 strains representative of the main phylogenetic groups has broadened our knowledge of the evolution and speciation of this pathogen and led to the identification of novel virulence-associated functions. Comparative genomic analyses are now opening the way for refined functional studies. The many molecular determinants involved in pathogenicity and host-range specificity are described, and we also summarize current understanding of their roles in pathogenesis and how their expression is tightly controlled by an intricate virulence regulatory network.

Expression of a cecropin B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas solanacearum

Abstract Cecropin B is a naturally-occurring lytic peptide found in Hyalophora cecropia, the Giant Silk Moth. It is thought to comprise part of an inducible humoral defense system that combats infection in the insect. Two 38 amino acid peptides, SB-37 and Shiva-1, were produced as substitution analogs of Cecropin B. SB-37 is 95% homologous to Cecropin B while Shiva-1 retains only 46% homology to the natural molecule. However, hydrophobic properties and charge density of the native structure were conserved at 100% in the synthetic peptides. The genes for both peptides were chemically synthesized and cloned into the binary vector pBI121 under the control of a constitutive or wound-inducible plant promoter. Transgenic tobacco plants (RO) were subsequently obtained via Agrobacterium transformation. Bioassays to test disease resistance of R1 progeny indicate that, compared to transgenic control and SB-37 plants, Shiva-1 seedlings exhibited delayed wilt symptoms and reduced disease severity and mortality after infection with a highly virulent strain of Pseudomonas solanacearum.

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.

Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence

As reported previously for Ralstonia solanacearum strain GMI1000, wild‐type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching‐positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43‐amino‐acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10‐fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10‐fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities.

Identification of Open Reading Frames Unique to a Select Agent: Ralstonia solanacearum Race 3 Biovar 2

An 8x 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.

Genetic Diversity and Relationships of Two Pathovars of Pseudomonas syringae

To determine genetic relationships within and between two pathovars of Pseudomonas syringae, strains typical of P. syringae pv. tomato (P. s. tomato) and selected strains of P. syringae pv. syringae (P. s. syringae) were characterized by three methods. DNA-DNA hybridization experiments showed that strains of P. s. tomato and P. s. syringae were, respectively, 86-100% and 37-47% homologous to DNA from a P. s. tomato reference strain when tested under stringent conditions. An analysis of electrophoretic variation in enzymes encoded by 26 loci placed 17 P. s. tomato strains studied in a group of four electrophoretic types, and these strains had a mean genetic diversity per locus of 0.076. Six P. s. syringae strains formed a second group of six electrophoretic types, which had a higher mean genetic diversity per locus of 0.479. The mean genetic distance separating P. s. tomato from P. s. syringae (D = 0.94) was unexpectedly large for strains of a single species. An analysis of restriction fragment length polymorphisms (RFLPs) with three cloned hybridization probes demonstrated that each of the P. s. tomato and P. s. syringae strains was unique. A method was developed to quantify the RFLP difference between pairs of strains, and cluster analysis revealed relationships among P. s. tomato, but not among P. s. syringae, that were similar to those based on enzyme polymorphisms. Implications of these findings for bacterial systematics and epidemiology are discussed.

Control of the Ralstonia solanacearum Type III secretion system (Hrp) genes by the global virulence regulator PhcA

Expression of several virulence factors in the plant pathogen bacterium Ralstonia solanacearum is controlled by a complex regulatory network, at the center of which is PhcA. We provide genetic evidence that PhcA also represses the expression of hrp genes that code for the Type III protein secretion system, a major pathogenicity determinant in this bacterium. The repression of hrp genes in complete medium is relieved in a phcA mutant and two distinct signals, a quorum‐sensing signal and complex nitrogen sources, appear to trigger this PhcA‐dependent repression. This control of hrp gene expression by PhcA is realized at the level of the HrpG regulatory protein.

Inactivation of multiple virulence genes reduces the ability of Pseudomonas solanacearum to cause wilt symptoms

A logical extension of our earlier work was to determine what effect inactivating multiple virulence genes would have on the ability of P. solanacearum to wilt tomato plants. A double mutant that is EPS i and EG − was of special interest, since it should resemble the spontaneous mutant strain AW1-PC. We also wished to know whether the observed virulence of the mutants would be altered if the bacteria were required to infect plants via the roots

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 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.