Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments.
(1/152)
Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60 available at GenBank, the Ral_fliC PCR primer system was designed; this system yielded a single 724-bp product with the DNAs of all of the R. solanacearum strains tested. However, R. pickettii and four environmental Ralstonia isolates also yielded amplicons. The Ral_fliC PCR products obtained with 12 strains (R. solanacearum, R. pickettii, and environmental isolates) were sequenced. By sequence alignment, Rsol_fliC primers specific for R. solanacearum were designed. With this primer system, a specific 400-bp PCR product was obtained from all 82 strains of R. solanacearum tested. Six strains of R. pickettii and several closely related environmental isolates yielded no PCR product; however, a product was obtained with one Pseudomonas syzygii strain. A GC-clamped 400-bp fliC product could be separated in denaturing gradient gels and allowed us to distinguish P. syzygii from R. solanacearum. The Rsol_fliC PCR system was applied to detect R. solanacearum in soil. PCR amplification, followed by Southern blot hybridization, allowed us to detect about one target DNA molecule per PCR, which is equivalent to 10(3) CFU g of bulk soil(-1). The system was applied to survey soils from different geographic origins for the presence of R. solanacearum. (+info)
Propionyl-coenzyme A synthetases of Ralstonia solanacearum and Salmonella choleraesuis display atypical kinetics.
(2/152)
Propionyl-coenzyme A synthetases (PrpE) of Salmonella choleraesuis and Ralstonia solanacearum sharing 62% identity in amino acid sequence to each other were cloned, expressed in Escherichia coli and purified. Both enzymes catalyzed acetyl-, propionyl-, butyryl- and acrylyl-coenzyme A formation with the highest k(cat)/K(m) values for propionate. They displayed sigmoidal homotrophic autoactivation kinetics for propionate but not for the other acyl substrates tested. Besides, substrate inhibition kinetics was observed for co-substrates, i.e. ATP and CoA. Based on the kinetic data reported herein, the reaction mechanisms of the enzyme are discussed. (+info)
Characterization of the cis-acting regulatory element controlling HrpB-mediated activation of the type III secretion system and effector genes in Ralstonia solanacearum.
(3/152)
The ability of Ralstonia solanacearum to cause disease on plants depends on its type III secretion system (TTSS) encoded by hrp genes. The expression of hrp genes and known TTSS substrates is coordinately regulated by HrpB, a member of the AraC family of transcriptional regulators. Two HrpB-regulated promoters (hrpY and popABC) were characterized by deletion analysis, and the HrpB-dependent activation of these promoters was found to be conferred by a 25-nucleotide DNA element, the hrp(II) box (TTCGn16TTCG), which is present in other hrp promoters. The hrp(II) box element is an imperfect plant inducible promoter box, an element which was originally found in hrp promoters of Xanthomonas campestris (S. Fenselau and U. Bonas, Mol. Plant-Microbe Interact. 8:845-854, 1995) but which was not characterized at the molecular level. Site-directed mutagenesis showed that the hrp(II) box is essential for hrpY promoter activation in vivo. Functional analysis of the hrp(II) box element identified critical parameters that are required for HrpB-dependent activity. Further mapping analyses of several other hrpB-dependent promoters also indicated that the position of the hrp(II) box is conserved, at -70 to -47 bp from the transcriptional start. As a first step toward identifying novel TTSS effectors, we used the hrp(II) box consensus sequence to search for potential HrpB-regulated promoters in the complete genome sequence of R. solanacearum strain GMI1000. Among the 114 genes identified, a subset of promoters was found to have a structural relationship with hrp promoters, thus providing a pool of candidate genes encoding TTSS effectors. (+info)
Swimming motility, a virulence trait of Ralstonia solanacearum, is regulated by FlhDC and the plant host environment.
(4/152)
Swimming motility allows the bacterial wilt pathogen Ralstonia solanacearum to efficiently invade and colonize host plants. However, the bacteria are essentially nonmotile once inside plant xylem vessels. To determine how and when motility genes are expressed, we cloned and mutated flhDC, which encodes a major regulator of flagellar biosynthesis and bacterial motility. An flhDC mutant was nonmotile and less virulent than its wild-type parent on both tomato and Arabidopsis; on Arabidopsis, the flhDC mutant also was less virulent than a nonmotile fliC flagellin mutant. Genes in the R. solanacearum motility regulon had strikingly different expression patterns in culture and in the plant. In culture, as expected, flhDC expression depended on PehSR, a regulator of early virulence factors; and, in turn, FlhDC was required for fliC (flagellin) expression. However, when bacteria grew in tomato plants, flhDC was expressed in both wild-type and pehR mutant backgrounds, although PehSR is necessary for motility both in culture and in planta. Both flhDC and pehSR were significantly induced in planta relative to expression levels in culture. Unexpectedly, the fliC gene was expressed in planta at cell densities where motile bacteria were not observed, as well as in a nonmotile flhDC mutant. Thus, expression of flhDC and flagellin itself are uncoupled from bacterial motility in the host environment, indicating that additional signals and regulatory circuits repress motility during plant pathogenesis. (+info)
Flagellin is not a major defense elicitor in Ralstonia solanacearum cells or extracts applied to Arabidopsis thaliana.
(5/152)
The phytopathogenic bacterium Ralstonia solanacearum requires motility for full virulence, and its flagellin is a candidate pathogen-associated molecular pattern that may elicit plant defenses. Boiled extracts from R. solanacearum contained a strong elicitor of defense-associated responses. However, R. solanacearum flagellin is not this elicitor, because extracts from wild-type bacteria and fliC or flhDC mutants defective in flagellin production all elicited similar plant responses. Equally important, live R. solanacearum caused similar disease on Arabidopsis ecotype Col-0, regardless of the presence of flagellin in the bacterium or the FLS2-mediated flagellin recognition system in the plant. Unlike the previously studied flg22 flagellin peptide, a peptide based on the corresponding conserved N-terminal segment of R. solanacearum, flagellin did not elicit any response from Arabidopsis seedlings. Thus recognition of flagellin plays no readily apparent role in this pathosystem. Flagellin also was not the primary elicitor of responses in tobacco. The primary eliciting activity in boiled R. solanacearum extracts applied to Arabidopsis was attributable to one or more proteins other than flagellin, including species purifying at approximately 5 to 10 kDa and also at larger molecular masses, possibly due to aggregation. Production of this eliciting activity did not require hrpB (positive regulator of type III secretion), pehR (positive regulator of polygalacturonase production and motility), gspM (general secretion pathway), or phcA (LysR-type global virulence regulator). Wild-type R. solanacearum was virulent on Arabidopsis despite the presence of this elicitor in pathogen extracts. (+info)
Distribution and sequence analysis of a family of type ill-dependent effectors correlate with the phylogeny of Ralstonia solanacearum strains.
(6/152)
In Ralstonia solanacearum, we previously have reported on the characterization of popP1 and popP2 genes. These genes encode type III-dependent pathogenicity effectors related to the large family of AvrRxv/YopJ cysteine proteases that are shared among pathogens of plants and animals. In this study, we identify a third gene, named popP3, that is inactivated in the genome sequence of strain GMI1000 by insertion of a copy of the insertion sequence ISRso13. The three popP genes are localized on two large chromosomal pathogenicity islands, with popP1 and popP2 being present on the same island. Phylogenic analysis demonstrated that the PopP2 and PopP3 proteins are clearly distinct from other effectors of this family previously characterized in plant and animal pathogens. Analysis of the distribution and allelic variations of the three genes in 30 strains representative of the biodiversity of R. solanacearum established that popP genes are distributed widely among strains from two of the three phyla previously defined on the basis of the structure of the core genome. Sequencing of the popP genes from the different strains revealed limited allelic variations at the three loci but did not show evidence of recombination between the popP genes. Limited allelic variation together with occurrence of insertion sequences within or in the close vicinity of popP genes and the presence of gene duplications in these pathogenicity islands suggest that genomic rearrangements might be a major evolutionary driving force controlling evolution of the genes encoded in these regions. The implications of these observations in terms of bacterial evolution, gene acquisition, and horizontal gene transfers are discussed. (+info)
Ralstonia solanacearum iron scavenging by the siderophore staphyloferrin B is controlled by PhcA, the global virulence regulator.
(7/152)
PhcA is a transcriptional regulator that activates expression of multiple virulence genes in the plant pathogen Ralstonia solanacearum. Relative to their wild-type parents, phcA mutants overproduced iron-scavenging activity detected with chrome azurol S siderophore detection medium. Transposon mutagenesis of strain AW1-PC (phcA1) generated strain GB6, which was siderophore negative but retained weak iron-scavenging activity. The ssd gene inactivated in GB6 encodes a protein similar to group IV amino acid decarboxylases, and its transcription was repressed by iron(III) and PhcA. ssd is the terminal gene in a putative operon that also appears to encode three siderophore synthetase subunits, a integral membrane exporter, and three genes with no obvious role in siderophore production. A homologous operon was found in the genomes of Ralstonia metallidurans and Staphylococcus aureus, both of which produce the polycarboxylate siderophore staphyloferrin B. Comparison of the siderophores present in culture supernatants of R. solanacearum, R. metallidurans, and Bacillus megaterium using chemical tests, a siderophore utilization bioassay, thin-layer chromatography, and mass spectroscopy indicated that R. solanacearum produces staphyloferrin B rather than schizokinen as was reported previously. Inactivation of ssd in a wild-type AW1 background resulted in a mutant almost incapable of scavenging iron but normally virulent on tomato plants. AW1 did not produce siderophore activity when cultured in tomato xylem sap, suggesting that the main location in tomato for R. solanacearum during pathogenesis is iron replete. (+info)
Relationship between avirulence gene (avrA) diversity in Ralstonia solanacearum and bacterial wilt incidence.
(8/152)
Bacterial wilt, caused by Ralstonia solanacearum, is a serious disease of tobacco in North and South Carolina. In contrast, the disease rarely occurs on tobacco in Georgia and Florida, although bacterial wilt is a common problem on tomato. We investigated whether this difference in disease incidence could be explained by qualitative characteristics of avirulence gene avrA in the R. solanacearum population in the southeastern United States. Sequence analysis established that wild-type avrA has a 792-bp open reading frame. Polymerase chain reaction (PCR) amplification of avrA from 139 R. solanacearum strains generated either 792-bp or approximately 960-bp DNA fragments. Strains that elicited a hypersensitive reaction (HR) on tobacco contained the 792-bp allele, and were pathogenic on tomato and avirulent on tobacco. All HR-negative strains generated a approximately 960-bp DNA fragment, and wilted both tomato and tobacco. The DNA sequence of avrA in six HR-negative strains revealed the presence of one of two putative miniature inverted-repeat transposable elements (MITEs): a 152-bp MITE between nucleotides 542 and 543, or a 170-bp MITE between nucleotides 461 and 462 or 574 and 575. Southern analysis suggested that the 170-bp MITE is unique to strains from the southeastern United States and the Caribbean. Mutated avrA alleles were present in strains from 96 and 75% of North and South Carolina sites, respectively, and only in 13 and 0% of the sites in Georgia and Florida, respectively. Introduction of the wildtype allele on a plasmid into four HR-negative strains reduced their virulence on both tobacco and tomato. Inactivation of avrA in an HR-positive, avirulent strain, resulted in a mutant that was weakly virulent on tobacco. Thus, the incidence of bacterial wilt of tobacco in the southeastern United States is partially explained by which avrA allele dominates the local R. solanacearum population. (+info)