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Sample records for ralstonia eutropha class

  1. Polyhydroxyalkanoate (PHA) synthesis by class IV PHA synthases employing Ralstonia eutropha PHB(-)4 as host strain.

    PubMed

    Hyakutake, Manami; Saito, Yuta; Tomizawa, Satoshi; Mizuno, Kouhei; Tsuge, Takeharu

    2011-01-01

    Class IV polyhydroxyalkanoate (PHA) synthase from Bacillus cereus YB-4 (PhaRC(YB4)) or B. megaterium NBRC15308(T) (PhaRC(Bm)) was expressed in Ralstonia eutropha PHB(-)4 to compare the ability to produce PHA and the substrate specificity of PhaRCs. PhaRC(YB4) produced significant amounts of PHA and had broader substrate specificity than PhaRC(Bm).

  2. Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism.

    PubMed

    Jia, Y; Yuan, W; Wodzinska, J; Park, C; Sinskey, A J; Stubbe, J

    2001-01-30

    The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium vinosum, respectively, catalyze the polymerization of beta-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. vinosum synthase suggested that it is structurally homologous to bacterial lipases and allowed identification of active site residues important for catalysis [Jia, Y., Kappock, T. J., Frick, T., Sinskey, A. J., and Stubbe, J. (2000) Biochemistry 39, 3927-3936]. Sequence alignments between the Class I and III synthases revealed similar residues in the R. eutropha synthase. Site-directed mutants of these residues were prepared and examined using HBCoA and a terminally saturated trimer of HBCoA (sT-CoA) as probes. These studies reveal that the R. eutropha synthase possesses an essential catalytic dyad (C319-H508) in which the C319 is involved in covalent catalysis. A conserved Asp, D480, was shown not to be required for acylation of C319 by sT-CoA and is proposed to function as a general base catalyst to activate the hydroxyl of HBCoA for ester formation. Studies of the [(3)H]sT-CoA with wild-type and mutant synthases reveal that 0.5 equiv of radiolabel is covalently bound per monomer of synthase, suggesting that a dimeric form of the enzyme is involved in elongation. These studies, in conjunction with search algorithms for secondary structure, suggest that the Class I and III synthases are mechanistically similar and structurally homologous, despite their physical and kinetic differences.

  3. Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from Ralstonia eutropha.

    PubMed

    Han, Xuerong; Satoh, Yasuharu; Satoh, Toshifumi; Matsumoto, Ken'ichiro; Kakuchi, Toyoji; Taguchi, Seiichi; Dairi, Tohru; Munekata, Masanobu; Tajima, Kenji

    2011-11-01

    A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 × 10(5), and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T (g), T (m), and T (d) (10%) were observed at -1.1°C, 158.8°C, and 252.7°C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.

  4. Catabolism of 3-Nitrophenol by Ralstonia eutropha JMP 134

    PubMed Central

    Schenzle, A.; Lenke, H.; Fischer, P.; Williams, P. A.; Knackmuss, H.

    1997-01-01

    Ralstonia eutropha JMP 134 utilizes 3-nitrophenol as the sole source of nitrogen, carbon, and energy. The entire catabolic pathway of 3-nitrophenol is chromosomally encoded. An initial NADPH-dependent reduction of 3-nitrophenol was found in cell extracts of strain JMP 134. By use of a partially purified 3-nitrophenol nitroreductase from 3-nitrophenol-grown cells, 3-hydroxylaminophenol was identified as the initial reduction product. Resting cells of R. eutropha JMP 134 metabolized 3-nitrophenol to N-acetylaminohydroquinone under anaerobic conditions. With cell extracts, 3-hydroxylaminophenol was converted into aminohydroquinone. This enzyme-mediated transformation corresponds to the acid-catalyzed Bamberger rearrangement. Enzymatic conversion of the analogous hydroxylaminobenzene yields a mixture of 2- and 4-aminophenol. PMID:16535572

  5. CDC Group IV c-2: a New Ralstonia Species Close to Ralstonia eutropha

    PubMed Central

    Moissenet, Didier; Goujon, Christophe P.; Garbarg-Chenon, Antoine; Vu-Thien, Hoang

    1999-01-01

    CDC group IV c-2, an environmental gram-negative bacillus recently proposed for inclusion in the genus Ralstonia, has been isolated in several human infections. Biochemical characterization and 16S ribosomal DNA (rDNA) sequencing with phylogenetic analysis were used to characterize eight clinical isolates and four type strains. Other typing tools, such as pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) analysis, were also used. PFGE typing of clinical isolates was unsuccessful because the DNA was degraded, and RAPD analysis was poorly discriminatory. In contrast, the type strains were clearly distinguished with both PFGE and RAPD analysis. All of the 16S rDNA sequences were identical. Comparison of the 16S rDNA sequences to the GenBank sequences showed that they were consistent with CDC group IV c-2 belonging to the genus Ralstonia. The closest matches were obtained with Ralstonia eutropha. However, four differences in 32 biochemical tests separated R. eutropha from CDC group IV c-2, which suggests that CDC group IV c-2 is a new species of the genus Ralstonia. PMID:10325323

  6. Wautersia gen. nov., a novel genus accommodating the phylogenetic lineage including Ralstonia eutropha and related species, and proposal of Ralstonia [Pseudomonas] syzygii (Roberts et al. 1990) comb. nov.

    PubMed

    Vaneechoutte, Mario; Kämpfer, Peter; De Baere, Thierry; Falsen, Enevold; Verschraegen, Gerda

    2004-03-01

    Comparative 16S rDNA sequence analysis indicates that two distinct sublineages, with a sequence dissimilarity of >4 % (bootstrap value, 100 %), exist within the genus RALSTONIA: the Ralstonia eutropha lineage, which comprises Ralstonia basilensis, Ralstonia campinensis, R. eutropha, Ralstonia gilardii, Ralstonia metallidurans, Ralstonia oxalatica, Ralstonia paucula, Ralstonia respiraculi and Ralstonia taiwanensis; and the Ralstonia pickettii lineage, which comprises Ralstonia insidiosa, Ralstonia mannitolilytica, R. pickettii, Ralstonia solanacearum and Ralstonia syzygii comb. nov. (previously Pseudomonas syzygii). This phylogenetic discrimination is supported by phenotypic differences. Members of the R. eutropha lineage have peritrichous flagella, do not produce acids from glucose and are susceptible to colistin, in contrast to members of the R. pickettii lineage, which have one or more polar flagella, produce acid from several carbohydrates and are colistin-resistant. Members of the R. pickettii lineage are viable for up to 6 days on tryptic soy agar at 25 degrees C, whereas members of the R. eutropha lineage are viable for longer than 9 days. It is proposed that species of the R. eutropha lineage should be classified in a novel genus, Wautersia gen. nov. Finally, based on the literature and new DNA-DNA hybridization data, it is proposed that Pseudomonas syzygii should be renamed Ralstonia syzygii comb. nov.

  7. Metabolic engineering of Ralstonia eutropha for the biosynthesis of 2-hydroxyacid-containing polyhydroxyalkanoates.

    PubMed

    Park, Si Jae; Jang, Young-Ah; Lee, Hyuk; Park, A-Reum; Yang, Jung Eun; Shin, Jihoon; Oh, Young Hoon; Song, Bong Keun; Jegal, Jonggeon; Lee, Seung Hwan; Lee, Sang Yup

    2013-11-01

    Polyhydroxyalkanoates (PHAs) are bio-based and biodegradable polyesters synthesized by numerous microorganisms. PHAs containing 2-hydroxyacids as monomer units have attracted much attention, but their production has not been efficient. Here, we metabolically engineered Ralstonia eutropha strains for the in vivo synthesis of PHAs containing 2-hydroxyacids as monomers. This was accomplished by replacing the R. eutropha phaC gene in the chromosome with either the R. eutropha phaC S506G A510K gene, which contains two point mutations, or the Pseudomonas sp. MBEL 6-19 phaC1437 gene. In addition, the R. eutropha phaAB genes in the chromosome were replaced with the Clostridium propionicum pct540 gene. All of the engineered R. eutropha strains produced PHAs containing 2-hydroxyacid monomers, including lactate and 2-hydroxybutyrate (2HB), along with 3-hydroxybutyrate (3HB) and/or 3-hydroxyvalerate (3HV), when they were cultured in nitrogen-free medium containing 5 g/L lactate or 4 g/L 2HB and 20 g/L glucose as carbon sources. Expression of the Escherichia coli ldhA gene in engineered R. eutropha strains allowed production of poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from glucose as the sole carbon source. This is the first report on the production of 2-hydroxyacid-containing PHAs by metabolically engineered R. eutropha.

  8. Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

    PubMed Central

    Burgdorf, Tanja; De Lacey, Antonio L.; Friedrich, Bärbel

    2002-01-01

    The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD+ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H2-oxidizing activity. In one of these isolates (HoxH I64A), H2 binding was impaired. Class II mutants revealed a high D2/H+ exchange rate relative to a low H2-oxidizing activity. A representative (HoxH H16L) displayed D2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O2-sensitive growth on hydrogen due to an O2-sensitive SH protein. PMID:12399498

  9. Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16.

    PubMed

    Pohlmann, Anne; Fricke, Wolfgang Florian; Reinecke, Frank; Kusian, Bernhard; Liesegang, Heiko; Cramm, Rainer; Eitinger, Thomas; Ewering, Christian; Pötter, Markus; Schwartz, Edward; Strittmatter, Axel; Voss, Ingo; Gottschalk, Gerhard; Steinbüchel, Alexander; Friedrich, Bärbel; Bowien, Botho

    2006-10-01

    The H(2)-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H(2) and CO(2) as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism's ability to produce and store large amounts of poly[R-(-)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.

  10. A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha.

    PubMed

    Solaiman, Daniel K Y; Swingle, Bryan M; Ashby, Richard D

    2010-08-01

    Ralstonia eutropha (formerly Alcaligenes eutrophus) is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and molecular engineering of this organism. We report here a versatile gene expression method for the genetic engineering of R. eutropha. This method, based on a simplified electroporation protocol, uses a recombinant plasmid, pBS29-P2, containing a Pseudomonas syringae promoter (P2) and two antibiotic-resistance markers (i.e., genes coding for kanamycin (Km)- and tetracycline (Tc)-resistance). Using this method, we successfully achieved transformation of wild-type R. eutropha and its poly(hydroxyalkanoate)-negative mutant, R. eutropha PHB(-)4, with various pBS29-P2-based recombinants. A transformation frequency as high as 4x10(3) Km-resistance colonies/mug DNA was obtained per electroporation experiment. We further demonstrated the successful expression of a heterologous gene coding for green-fluorescent-protein by fluorescence measurement. In addition, our results indicated the expression of a truncated but active Streptomyces coelicolor alpha-galactosidase in R. eutropha.

  11. Metabolic engineering of Ralstonia eutropha for the production of polyhydroxyalkanoates from sucrose.

    PubMed

    Park, Si Jae; Jang, Young-Ah; Noh, Won; Oh, Young Hoon; Lee, Hyuk; David, Yokimiko; Baylon, Mary Grace; Shin, Jihoon; Yang, Jung Eun; Choi, So Young; Lee, Seung Hwan; Lee, Sang Yup

    2015-03-01

    A sucrose utilization pathway was established in Ralstonia eutropha NCIMB11599 and R. eutropha 437-540 by introducing the Mannheimia succiniciproducens MBEL55E sacC gene that encodes β-fructofuranosidase. These engineered strains were examined for the production of poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)], respectively, from sucrose as a carbon source. It was found that β-fructofuranosidase excreted into the culture medium could hydrolyze sucrose to glucose and fructose, which were efficiently used as carbon sources by recombinant R. eutropha strains. When R. eutropha NCIMB11599 expressing the sacC gene was cultured in nitrogen-free chemically defined medium containing 20 g/L of sucrose, a high P(3HB) content of 73.2 wt% could be obtained. In addition, R. eutropha 437-540 expressing the Pseudomonas sp. MBEL 6-19 phaC1437 gene and the Clostridium propionicum pct540 gene accumulated P(3HB-co-21.5 mol% LA) to a polymer content of 19.5 wt% from sucrose by the expression of the sacC gene and the Escherichia coli ldhA gene. The molecular weights of P(3HB) and P(3HB-co-21.5 mol%LA) synthesized in R. eutropha using sucrose as a carbon source were 3.52 × 10(5) (Mn ) and 2.19 × 10(4) (Mn ), respectively. The engineered R. eutropha strains reported here will be useful for the production of polyhydroxyalkanoates (PHAs) from sucrose, one of the most abundant and relatively inexpensive carbon sources.

  12. Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha.

    PubMed

    Lu, Jingnan; Brigham, Christopher J; Gai, Claudia S; Sinskey, Anthony J

    2012-10-01

    Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.

  13. Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

    SciTech Connect

    Lu, JN; Brigham, CJ; Gai, CS; Sinskey, AJ

    2012-08-04

    Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.

  14. Genomic Plasticity in Ralstonia eutropha and Ralstonia pickettii: Evidence for Rapid Genomic Change and Adaptation

    SciTech Connect

    Terence L. MArsh

    2007-06-27

    The proposed foci of our investigations were on Ralstonia eutropha and Rasltonia pickettii. We have 18 derived lineages of the former as well as their progenitor and eleven isolates of the latter. Our goal was to measure the level of plasticity in these strains and attempt to derive a mechanistic understanding of how genomic plasticity formed. Extensive attempts to reproducibly induce conformational changes in the genome of R. eutropha were unsuccessful. We thought that we had a reasonable lead on this inasmuch as we had shown that the ancestral strain along with many of the derivative lineages exhibited “temperature induced mutation and mortality akin to R. metallodurans. However we were unable to get subtractive hybridization working to the degree that it revealed differences between the lineages. During this time the R. pickettii analysis was proving quite fruitful and so we concentrated our efforts on our analyses of R. pickettii. These strains were isolated from a copper-contaminated lake sediment and were resistant to copper at 800 µg/ml (CuSO4). Our results in the investigation of R. pickettii permitted a view into the adaptation of a beta-proteobacteria to an extreme environment. Our worked revealed that within the same ecosystem two genomovars with structurally different genomes and genome sizes were present and apparently filling similar if not identical niches. The genomovars were detected with REP & BOX-PCR, pulse field gel electrophoresis, and DNA:DNA hybridizations. Moreover there were different metal resistance patterns associated with the different genomovars, one showing resistance to Zn and Cd while the other had resistance to Ni. Five of the isolates had a high-copy number extrachromosomal element that was identified as the replicative form of a filamentous phage. Mature virions were isolated from culture broth using PEG precipitation and CsCl density centrifugation. The DNA associated with the filamentous particles was single stranded and had

  15. Engineering of Ralstonia eutropha H16 for Autotrophic and Heterotrophic Production of Methyl Ketones

    PubMed Central

    Müller, Jana; MacEachran, Daniel; Burd, Helcio; Sathitsuksanoh, Noppadon; Bi, Changhao; Yeh, Yi-Chun; Lee, Taek Soon; Hillson, Nathan J.; Chhabra, Swapnil R.; Singer, Steven W.

    2013-01-01

    Ralstonia eutropha is a facultatively chemolithoautotrophic bacterium able to grow with organic substrates or H2 and CO2 under aerobic conditions. Under conditions of nutrient imbalance, R. eutropha produces copious amounts of poly[(R)-3-hydroxybutyrate] (PHB). Its ability to utilize CO2 as a sole carbon source renders it an interesting new candidate host for the production of renewable liquid transportation fuels. We engineered R. eutropha for the production of fatty acid-derived, diesel-range methyl ketones. Modifications engineered in R. eutropha included overexpression of a cytoplasmic version of the TesA thioesterase, which led to a substantial (>150-fold) increase in fatty acid titer under certain conditions. In addition, deletion of two putative β-oxidation operons and heterologous expression of three genes (the acyl coenzyme A oxidase gene from Micrococcus luteus and fadB and fadM from Escherichia coli) led to the production of 50 to 65 mg/liter of diesel-range methyl ketones under heterotrophic growth conditions and 50 to 180 mg/liter under chemolithoautotrophic growth conditions (with CO2 and H2 as the sole carbon source and electron donor, respectively). Induction of the methyl ketone pathway diverted substantial carbon flux away from PHB biosynthesis and appeared to enhance carbon flux through the pathway for biosynthesis of fatty acids, which are the precursors of methyl ketones. PMID:23686271

  16. Engineering the heterotrophic carbon sources utilization range of Ralstonia eutropha H16 for applications in biotechnology.

    PubMed

    Volodina, Elena; Raberg, Matthias; Steinbüchel, Alexander

    2016-12-01

    Ralstonia eutropha H16 is an interesting candidate for the biotechnological production of polyesters consisting of hydroxy- and mercaptoalkanoates, and other compounds. It provides all the necessary characteristics, which are required for a biotechnological production strain. Due to its metabolic versatility, it can convert a broad range of renewable heterotrophic resources into diverse valuable compounds. High cell density fermentations of the non-pathogenic R. eutropha can be easily performed. Furthermore, this bacterium is accessible to engineering of its metabolism by genetic approaches having available a large repertoire of genetic tools. Since the complete genome sequence of R. eutropha H16 has become available, a variety of transcriptome, proteome and metabolome studies provided valuable data elucidating its complex metabolism and allowing a systematic biology approach. However, high production costs for bacterial large-scale production of biomass and biotechnologically valuable products are still an economic challenge. The application of inexpensive raw materials could significantly reduce the expenses. Therefore, the conversion of diverse substrates to polyhydroxyalkanoates by R. eutropha was steadily improved by optimization of cultivation conditions, mutagenesis and metabolic engineering. Industrial by-products and residual compounds like glycerol, and substrates containing high carbon content per weight like palm, soybean, corn oils as well as raw sugar-rich materials like molasses, starch and lignocellulose, are the most promising renewable substrates and were intensively studied.

  17. Cloning and Expression of a Ralstonia eutropha HF39 Gene Mediating Indigo Formation in Escherichia coli

    PubMed Central

    Drewlo, Sascha; Brämer, Christian O.; Madkour, Mohamed; Mayer, Frank; Steinbüchel, Alexander

    2001-01-01

    On complex medium Escherichia coli strains carrying hybrid plasmid pBEC/EE:11.0, pSKBEC/BE:9.0, pSKBEC/PP:3.3, or pSKBEC/PP:2.4 harboring genomic DNA of Ralstonia eutropha HF39 produced a blue pigment characterized as indigo by several chemical and spectroscopic methods. A 1,251-bp open reading frame (bec) was cloned and sequenced. The deduced amino acid sequence of bec showed only weak similarities to short-chain acyl-coenzyme A dehydrogenases, and the gene product catalyzed formation of indoxyl, a reactive preliminary stage for production of indigo. PMID:11282658

  18. Development of a broad-host synthetic biology toolbox for ralstonia eutropha and its application to engineering hydrocarbon biofuel production

    PubMed Central

    2013-01-01

    Background The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. Results We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5’ mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters PBAD, T7, Pxyls/PM, PlacUV5, and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. Conclusion We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well. PMID:24219429

  19. Development of a broad-host synthetic biology toolbox for Ralstonia eutropha and its application to engineering hydrocarbon biofuel production.

    PubMed

    Bi, Changhao; Su, Peter; Müller, Jana; Yeh, Yi-Chun; Chhabra, Swapnil R; Beller, Harry R; Singer, Steven W; Hillson, Nathan J

    2013-11-13

    The chemoautotrophic bacterium Ralstonia eutropha can utilize H2/CO2 for growth under aerobic conditions. While this microbial host has great potential to be engineered to produce desired compounds (beyond polyhydroxybutyrate) directly from CO2, little work has been done to develop genetic part libraries to enable such endeavors. We report the development of a toolbox for the metabolic engineering of Ralstonia eutropha H16. We have constructed a set of broad-host-range plasmids bearing a variety of origins of replication, promoters, 5' mRNA stem-loop structures, and ribosomal binding sites. Specifically, we analyzed the origins of replication pCM62 (IncP), pBBR1, pKT (IncQ), and their variants. We tested the promoters P(BAD), T7, P(xyls/PM), P(lacUV5), and variants thereof for inducible expression. We also evaluated a T7 mRNA stem-loop structure sequence and compared a set of ribosomal binding site (RBS) sequences derived from Escherichia coli, R. eutropha, and a computational RBS design tool. Finally, we employed the toolbox to optimize hydrocarbon production in R. eutropha and demonstrated a 6-fold titer improvement using the appropriate combination of parts. We constructed and evaluated a versatile synthetic biology toolbox for Ralstonia eutropha metabolic engineering that could apply to other microbial hosts as well.

  20. Polyhydroxyalkanoates production with Ralstonia eutropha from low quality waste animal fats.

    PubMed

    Riedel, Sebastian L; Jahns, Stefan; Koenig, Steven; Bock, Martina C E; Brigham, Christopher J; Bader, Johannes; Stahl, Ulf

    2015-11-20

    Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polyesters considered as alternatives to petroleum-based plastics. Ralstonia eutropha is a model organism for PHA production. Utilizing industrially rendered waste animal fats as inexpensive carbon feedstocks for PHA production is demonstrated here. An emulsification strategy, without any mechanical or chemical pre-treatment, was developed to increase the bioavailability of solid, poorly-consumable fats. Wild type R. eutropha strain H16 produced 79-82% (w/w) polyhydroxybutyrate (PHB) per cell dry weight (CDW) when cultivated on various fats. A productivity of 0.3g PHB/(L × h) with a total PHB production of 24 g/L was achieved using tallow as carbon source. Using a recombinant strain of R. eutropha that produces poly(hydroxybutyrate-co-hydroxyhexanoate) [P(HB-co-HHx)], 49-72% (w/w) of PHA per CDW with a HHx content of 16-27 mol% were produced in shaking flask experiments. The recombinant strain was grown on waste animal fat of the lowest quality available at lab fermenter scale, resulting in 45 g/L CDW with 60% (w/w) PHA per CDW and a productivity of 0.4 g PHA/(L × h). The final HHx content of the polymer was 19 mol%. The use of low quality waste animal fats as an inexpensive carbon feedstock exhibits a high potential to accelerate the commercialization of PHAs. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Production of Poly (3-Hydroxybutyric Acid) by Ralstonia eutropha in a Biocalorimeter and its Thermokinetic Studies.

    PubMed

    Anusha, Subramanian Mohanakrishnan; Leelaram, Santharam; Surianarayanan, Mahadevan

    2016-07-01

    Bioplastic production from microbial sources is an emerging area which provides opportunities even to convert the wastes into bioplastics. Poly (3-hydroxybutyric acid), commonly called as PHB, is a bioplastic, which is stored as intracellular cytoplasmic inclusions in microorganisms. The objectives of this study are to calorimetrically monitor the PHB production and evaluate the thermokinetic data in a bioreaction calorimeter (BioRC1e). Thus, a well-known PHB-producing bacteria Ralstonia eutropha was selected for batch process in a bioreaction calorimeter. The metabolic heat generated was found to be correlated with the biomass, substrate consumption, oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) and PHB production. The OUR pattern explained the oxidative metabolism of the strain R. eutropha. The heat yields due to biomass and glucose consumption during PHB production were found to be 12.56 and 13.56 kJ/g, respectively. The oxycalorific value obtained for the PHB production was 443.80 kJ/mol of O2. The concentration of PHB obtained in BioRC1e was 4.33 g/L with a production rate of 0.09 g/L/h. The chemical structure of the extracted PHB by R. eutropha was confirmed using fourier transform infrared spectroscopy (FT-IR) and (1)H and (13)C nuclear magnetic resonance (NMR) analysis.

  2. Enhancement of glycerol utilization ability of Ralstonia eutropha H16 for production of polyhydroxyalkanoates.

    PubMed

    Fukui, Toshiaki; Mukoyama, Masaharu; Orita, Izumi; Nakamura, Satoshi

    2014-09-01

    Ralstonia eutropha H16 is a well-studied bacterium with respect to biosynthesis of polyhydroxyalkanoates (PHAs), which has attracted attentions as biodegradable bio-based plastics. However, this strain shows quite poor growth on glycerol of which bulk supply has been increasing as a major by-product of biodiesel industries. This study examined enhancement of glycerol assimilation ability of R. eutropha H16 by introduction of the genes of aquaglyceroporin (glpF) and glycerol kinase (glpK) from Escherichia coli. Although introduction of glpFK Ec into the strain H16 using a multi-copy vector was not successful, a recombinant strain possessing glpFK Ec within the chromosome showed much faster growth on glycerol than H16. Further analyses clarified that weak expression of glpK Ec alone allowed to establish efficient glycerol assimilation pathway, indicating that the poor growth of H16 on glycerol was caused by insufficient kination activity to glycerol, as well as this strain had a potential ability for uptake of extracellular glycerol. The engineered strains expressing glpFK Ec or glpK Ec produced large amounts of poly[(R)-3-hydroxybutyrate] [P(3HB)] from glycerol with much higher productivity than H16. Unlike other glycerol-utilizable wild strains of R. eutropha, the H16-derived engineered strains accumulated P(3HB) with no significant decrease in molecular weights on glycerol, and the polydispersity index of the glycerol-based P(3HB) synthesized by the strains expressing glpFK Ec was lower than those by the parent strains. The present study demonstrated possibility of R. eutropha H16-based platform for production of useful compounds from inexpensive glycerol.

  3. Low temperature-induced viable but not culturable state of Ralstonia eutropha and its relationship to accumulated polyhydroxybutyrate

    PubMed Central

    Nowroth, Verena; Marquart, Lisa; Jendrossek, Dieter

    2016-01-01

    The culturability of Escherichia coli, Ralstonia eutropha and Bacillus subtilis after incubation in phosphate-buffered saline at either 5°C or 30°C was determined. The culturability of B. subtilis showed little dependence on temperature. The culturability of E. coli rapidly decreased at 30°C but remained almost constant at 5°C. In contrast, the culturability of R. eutropha decreased by three orders of magnitude at 5°C within 24 h but only moderately decreased (one order of magnitude) at 30°C. Remarkably, prolonged incubation of R. eutropha at 30°C resulted in a full recovery of colony forming units in contrast to only a partial recovery at 5°C. Ralstonia eutropha cells at 30°C remained culturable for 3 weeks while culturability at 5°C constantly decreased. The effect of temperature was significantly stronger in a polyhydroxybutyrate-negative mutant. Our data show that accumulated polyhydroxybutyrate has a cold-protective function and can prevent R. eutropha entering the viable but not culturable state. PMID:27810883

  4. Large scale extraction of poly(3-hydroxybutyrate) from Ralstonia eutropha H16 using sodium hypochlorite

    PubMed Central

    2012-01-01

    Isolation of polyhydroxyalkanoates (PHAs) from bacterial cell matter is a critical step in order to achieve a profitable production of the polymer. Therefore, an extraction method must lead to a high recovery of a pure product at low costs. This study presents a simplified method for large scale poly(3-hydroxybutyrate), poly(3HB), extraction using sodium hypochlorite. Poly(3HB) was extracted from cells of Ralstonia eutropha H16 at almost 96% purity. At different extraction volumes, a maximum recovery rate of 91.32% was obtained. At the largest extraction volume of 50 L, poly(3HB) with an average purity of 93.32% ± 4.62% was extracted with a maximum recovery of 87.03% of the initial poly(3HB) content. This process is easy to handle and requires less efforts than previously described processes. PMID:23164136

  5. Synthesis of microbial poly(beta-hydroxybutyrate) modified with oligo(pentaerythritol ethoxylate) by Ralstonia eutropha.

    PubMed

    Jenzsch, M; Volk, N; Kressler, J; Scholz, C

    2001-01-01

    Poly(beta-hydroxybutyrate) (PHB) modified with different amounts of pentaerythritol ethoxylate (PEE) has been synthesized using Ralstonia eutropha. The growth kinetics and the synthesis of PHB in the presence of PEE were modeled using appropriate differential equations for the mass balance of the two-stage process. The influence of PEE addition on the morphology of PHB was studied by various microscopic and scattering techniques. Light microscopic and wide-angle X-ray measurements indicated that the addition of PEE had a nucleating effect on the crystallization of PHB. The spherulite growth rate was widely independent of the PEE addition. The lamellae of PHB became more disordered when PEE was added as demonstrated by atomic force microscopy. Furthermore, small-angle X-ray data indicated a decrease in the long period with increasing PEE content of the modified PHB.

  6. Large scale extraction of poly(3-hydroxybutyrate) from Ralstonia eutropha H16 using sodium hypochlorite.

    PubMed

    Heinrich, Daniel; Madkour, Mohamed H; Al-Ghamdi, Mansour A; Shabbaj, Ibraheem I; Steinbüchel, Alexander

    2012-11-19

    Isolation of polyhydroxyalkanoates (PHAs) from bacterial cell matter is a critical step in order to achieve a profitable production of the polymer. Therefore, an extraction method must lead to a high recovery of a pure product at low costs. This study presents a simplified method for large scale poly(3-hydroxybutyrate), poly(3HB), extraction using sodium hypochlorite. Poly(3HB) was extracted from cells of Ralstonia eutropha H16 at almost 96% purity. At different extraction volumes, a maximum recovery rate of 91.32% was obtained. At the largest extraction volume of 50 L, poly(3HB) with an average purity of 93.32% ± 4.62% was extracted with a maximum recovery of 87.03% of the initial poly(3HB) content. This process is easy to handle and requires less efforts than previously described processes.

  7. A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha

    PubMed Central

    Srinivasan, Sriram; Barnard, Gavin C.; Gerngross, Tillman U.

    2002-01-01

    We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (>1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli. PMID:12450812

  8. Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

    PubMed Central

    Wenning, Leonie; Stöveken, Nadine; Wübbeler, Jan Hendrik

    2015-01-01

    Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs. PMID:26590284

  9. Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16

    SciTech Connect

    Lu, JN; Brigham, CJ; Plassmeier, JK; Sinskey, AJ

    2014-08-01

    2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by l-valine (IC50 = 1.2 mM), l-isoleucine (IC50 = 2.3 mM), and l-leucine (IC50 = 5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (K-M = 10.5 mu M) and is highly selective towards 2-ketobutyrate (R = 140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2

  10. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16.

    PubMed

    Pötter, Markus; Madkour, Mohamed H; Mayer, Frank; Steinbüchel, Alexander

    2002-08-01

    Regulation of expression of the phasin PhaP, which is the major protein at the surface of polyhydroxyalkanoate (PHA) granules in Ralstonia eutropha H16, was studied and analysed at the molecular level. The regulation of PhaP expression is achieved by an autoregulated repressor, which is encoded by phaR in R. eutropha. The occurrence of PhaR homologues and the organization of phaR genes was analysed in detail in 29 different bacteria. Three kinds of molecule to which PhaR binds were identified in cells of R. eutropha, as revealed by gel-mobility-shift assays, DNaseI footprinting, cell fractionation, immunoelectron microscopy studies employing anti-PhaR antibodies raised against purified N-terminal hexahistidine-tagged PhaR and in vitro binding studies employing artificial PHA granules. PhaR binds upstream of phaP at two sites comprising the transcriptional start site plus the -10 region and a region immediately upstream of the -35 region of the sigma(70) promoter of phaP, where two imperfect 12 bp repeat sequences (GCAMMAAWTMMD) were identified on the sense and anti-sense strands. PhaR also binds 86 bp upstream of the phaR translational start codon, where the sigma(54)-dependent promoter was identified. PhaR also binds to the surface of PHA granules. In the cytoplasm of a phaROmegaKm mutant of R. eutropha H16, increased quantities of PhaP were detected and the cells formed by this strain were much smaller and had many more PHA granules present than the wild-type. These data support the following model for the regulation of phaP expression. Under cultivation conditions not permissive for PHA biosynthesis or in mutants defective in PHA biosynthesis, PhaR binds to the phaP promoter region and represses transcription of this gene. After the onset of PHA biosynthesis, under conditions that are permissive for the formation of nascent granules, PhaR binds to PHA granules and phaP is transcribed. At the later stages of PHA accumulation, PhaR no longer binds to the granules

  11. Biosynthesis and thermal properties of PHBV produced from levulinic acid by Ralstonia eutropha.

    PubMed

    Wang, Yuanpeng; Chen, Ronghui; Cai, JiYuan; Liu, Zhenggui; Zheng, Yanmei; Wang, Haitao; Li, Qingbiao; He, Ning

    2013-01-01

    Levulinic acid (LA) can be cost-effectively produced from a vast array of renewable carbohydrate-containing biomaterials. LA could facilitate the commercialization of the polymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and PHBV-based products as carbon substrates. Therefore, this paper focused on the production of PHBV by Ralstonia eutropha with LA for hydroxyvalerate (HV) production, which plays an important role in enhancing the thermal properties of PHBV. Accordingly, the HV content of PHBV varied from 0-40.9% at different concentrations of LA. Stimulation of cell growth and PHBV accumulation were observed when 2-6 g L(-1) LA was supplied to the culture. The optimal nitrogen sources were determined to be 0.5 g L(-1) ammonium chloride and 2 g L(-1) casein peptone. It was determined that the optimal pH for cell growth and PHBV accumulation was 7.0. When the cultivation was performed in large scale (2 L fermenter) with a low DO concentration of 30% and a pH of 7.0, a high maximum dry cell weight of 15.53 g L(-1) with a PHBV concentration of 12.61 g L(-1) (53.9% HV), up to 81.2% of the dry cell weight, was obtained. The melting point of PHBV found to be decreased as the fraction of HV present in the polymer increased, which resulted in an improvement in the ductility and flexibility of the polymer. The results of this study will improve the understanding of the PHBV accumulation and production by R. eutropha and will be valuable for the industrial production of biosynthesized polymers.

  12. Production of branched-chain alcohols by recombinant Ralstonia eutropha in fed-batch cultivation

    SciTech Connect

    Fei, Q; Brigham, CJ; Lu, JN; Fu, RZ; Sinskey, AJ

    2013-09-01

    Branched-chain alcohols are considered promising green energy sources due to their compatibility with existing infrastructure and their high energy density. We utilized a strain of Ralstonia eutropha capable of producing branched-chain alcohols and examined its production in flask cultures. In order to increase isobutanol and 3-methyl-1-butanol (isoamyl alcohol) productivity in the engineered strain, batch, fed-batch, and two-stage fed-batch cultures were carried out in this work. The effects of nitrogen source concentration on branched-chain alcohol production were investigated under four different initial concentrations in fermenters. A maximum 380 g m(-3) of branched-chain alcohol production was observed with 2 kg m(-3) initial NH4Cl concentration in batch cultures. A pH-stat control strategy was utilized to investigate the optimum carbon source amount fed during fed-batch cultures for higher cell density. In cultures of R. eutropha strains that did not produce polyhydroxyalkanoate or branched-chain alcohols, a maximum cell dry weight of 36 kg m(-3) was observed using a fed-batch strategy, when 10 kg m(-3) carbon source was fed into culture medium. Finally, a total branched-chain alcohol titer of 790 g m(-3), the highest branched-chain alcohol yield of 0.03 g g(-1), and the maximum branched-chain alcohol productivity of 8.23 g m(-3) h(-1) were obtained from the engineered strain Re2410/pJL26 in a two-stage fed-batch culture system with pH-stat control. Isobutanol made up over 95% (mass fraction) of the total branched-chain alcohols titer produced in this study. (C) 2013 Published by Elsevier Ltd.

  13. Novel, Oxygen-Insensitive Group 5 [NiFe]-Hydrogenase in Ralstonia eutropha

    PubMed Central

    Schäfer, Caspar; Friedrich, Bärbel

    2013-01-01

    Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H2 per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2 of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low Km of group 5 hydrogenases and makes atmospheric H2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2. PMID:23793632

  14. Dynamic mathematical models for biodegradation of formaldehyde by Ralstonia eutropha in a batch bioreactor.

    PubMed

    Habibi, Alireza; Vahabzadeh, Farzaneh; Zaiat, Marcelo

    2013-11-15

    Degradation of formaldehyde by Ralstonia eutropha was studied in a batch bioreactor operated in recycling mode (30 °C, initial pH of 6.5, aeration rate 0.5 vvm, and a recycling flow rate of 6 mL min(-1)). Growth kinetics equations were described using four substrate inhibition models, and the initial formaldehyde concentration ranged from 54.5 to 993.0 mg L(-1). In each case, model parameters were estimated interactively using nonlinear regression analysis and on the basis of the goodness of fit, the fitness of the model to the experimental data was obtained (i.e., the coefficient of determination and the percent of standard deviation). The estimated parameters according to the Luong equation were μmax = 0.101 h(-1), KS = 54.1 mg L(-1), Sm = 1329 mg L(-1), and n = 2.07. According to the maintenance energies explained by Pirt, cell maintenance was quantified with q = Aμ + B; where A and B are the associated and non-associated growth parts of substrate consumption, respectively. The importance of these terms was verified using the developed models, which would efficiently describe the dynamic nature of growth and formaldehyde biodegradation.

  15. Phosphotransferase protein EIIANtr interacts with SpoT, a key enzyme of the stringent response, in Ralstonia eutropha H16.

    PubMed

    Karstens, Katja; Zschiedrich, Christopher P; Bowien, Botho; Stülke, Jörg; Görke, Boris

    2014-04-01

    EIIA(Ntr) is a member of a truncated phosphotransferase (PTS) system that serves regulatory functions and exists in many Proteobacteria in addition to the sugar transport PTS. In Escherichia coli, EIIA(Ntr) regulates K(+) homeostasis through interaction with the K(+) transporter TrkA and sensor kinase KdpD. In the β-Proteobacterium Ralstonia eutropha H16, EIIA(Ntr) influences formation of the industrially important bioplastic poly(3-hydroxybutyrate) (PHB). PHB accumulation is controlled by the stringent response and induced under conditions of nitrogen deprivation. Knockout of EIIA(Ntr) increases the PHB content. In contrast, absence of enzyme I or HPr, which deliver phosphoryl groups to EIIA(Ntr), has the opposite effect. To clarify the role of EIIA(Ntr) in PHB formation, we screened for interacting proteins that co-purify with Strep-tagged EIIA(Ntr) from R. eutropha cells. This approach identified the bifunctional ppGpp synthase/hydrolase SpoT1, a key enzyme of the stringent response. Two-hybrid and far-Western analyses confirmed the interaction and indicated that only non-phosphorylated EIIA(Ntr) interacts with SpoT1. Interestingly, this interaction does not occur between the corresponding proteins of E. coli. Vice versa, interaction of EIIA(Ntr) with KdpD appears to be absent in R. eutropha, although R. eutropha EIIA(Ntr) can perfectly substitute its homologue in E. coli in regulation of KdpD activity. Thus, interaction with KdpD might be an evolutionary 'ancient' task of EIIA(Ntr) that was subsequently replaced by interaction with SpoT1 in R. eutropha. In conclusion, EIIA(Ntr) might integrate information about nutritional status, as reflected by its phosphorylation state, into the stringent response, thereby controlling cellular PHB content in R. eutropha.

  16. Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products.

    PubMed

    Riedel, Sebastian L; Lu, Jingnan; Stahl, Ulf; Brigham, Christopher J

    2014-02-01

    Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.

  17. Chemoselective Nitro Group Reduction and Reductive Dechlorination Initiate Degradation of 2-Chloro-5-Nitrophenol by Ralstonia eutropha JMP134

    PubMed Central

    Schenzle, Andreas; Lenke, Hiltrud; Spain, Jim C.; Knackmuss, Hans-Joachim

    1999-01-01

    Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134. 2-Chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene. PMID:10347008

  18. Chemoselective nitro group reduction and reductive dechlorination initiate degradation of 2-chloro-5-nitrophenol by Ralstonia eutropha JMP134

    SciTech Connect

    Schenzle, A.; Lenke, H.; Knackmuss, H.J.; Spain, J.C.

    1999-06-01

    Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134, 2-chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene.

  19. Production and purification of a soluble hydrogenase from Ralstonia eutropha H16 for potential hydrogen fuel cell applications.

    PubMed

    Jugder, Bat-Erdene; Lebhar, Helene; Aguey-Zinsou, Kondo-Francois; Marquis, Christopher P

    2016-01-01

    The soluble hydrogenase (SH) from Ralstonia eutropha H16 is a promising candidate enzyme for H2-based biofuel application as it favours H2 oxidation and is relatively oxygen-tolerant. In this report, bioprocess development studies undertaken to produce and purify an active SH are described, based on the methods previously reported [1], [2], [3], [4]. Our modifications are: •Upstream method optimizations were undertaken on heterotrophic growth media and cell lysis involving ultrasonication.•Two anion exchangers (Q Sepharose and RESOURCE Q) and size exclusion chromatographic (Superdex 200) matrices were successfully employed for purification of a hexameric SH from R. eutropha.•The H2 oxidizing activity of the SH was demonstrated spectrophotometrically in solution and also immobilized on an EPG electrode using cyclic voltammetry.

  20. Engineering of Ralstonia eutropha for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose.

    PubMed

    Zhang, Ying-Zi; Liu, Gui-Ming; Weng, Wei-Qi; Ding, Jiu-Yuan; Liu, Shuang-Jiang

    2015-02-10

    Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with Ralstonia eutropha relies on the addition of propionate during fermentation, and propionate consumption is one of the major factors affecting the cost of PHBV production. In this study, 7 strains were obtained by genetic manipulating the methylcitric acid cycle and the methylmalonyl-CoA pathway in R. eutropha. Disruption of prpC1 and prpC2 genes did not affect cell growth and PHBV accumulation. All 7 strains were able to accumulation high amounts of PHBVs with 3HV fractions of 0.41-29.1 mol% during cultivation in flasks. Fermentation in 7.5-L fermenter showed that genetically engineered Rem-8 was able to yield biomass of 132.8 CDWg/L, of which 68.6% were PHBV with 3HV fraction of 26.0 mol% in the biopolymer, indicating promising potentials of commercialization in the future.

  1. Production of fatty acids in Ralstonia eutropha H16 by engineering β-oxidation and carbon storage

    PubMed Central

    Chen, Janice S.; Colón, Brendan; Dusel, Brendon; Ziesack, Marika; Torella, Joseph P.

    2015-01-01

    Ralstonia eutropha H16 is a facultatively autotrophic hydrogen-oxidizing bacterium capable of producing polyhydroxybutyrate (PHB)-based bioplastics. As PHB’s physical properties may be improved by incorporation of medium-chain-length fatty acids (MCFAs), and MCFAs are valuable on their own as fuel and chemical intermediates, we engineered R. eutropha for MCFA production. Expression of UcFatB2, a medium-chain-length-specific acyl-ACP thioesterase, resulted in production of 14 mg/L laurate in wild-type R. eutropha. Total fatty acid production (22 mg/L) could be increased up to 2.5-fold by knocking out PHB synthesis, a major sink for acetyl-CoA, or by knocking out the acyl-CoA ligase fadD3, an entry point for fatty acids into β-oxidation. As ΔfadD3 mutants still consumed laurate, and because the R. eutropha genome is predicted to encode over 50 acyl-CoA ligases, we employed RNA-Seq to identify acyl-CoA ligases upregulated during growth on laurate. Knockouts of the three most highly upregulated acyl-CoA ligases increased fatty acid yield significantly, with one strain (ΔA2794) producing up to 62 mg/L free fatty acid. This study demonstrates that homologous β-oxidation systems can be rationally engineered to enhance fatty acid production, a strategy that may be employed to increase yield for a range of fuels, chemicals, and PHB derivatives in R. eutropha. PMID:26664804

  2. "Intelligent" descriptions of microbial kinetics in finitely dispersed bioreactors: neural and cybernetic models for PHB biosynthesis by Ralstonia eutropha.

    PubMed

    Patnaik, Pratap R

    2007-08-08

    For many microbial processes, the complexity of the metabolisms and the responses to transient and realistic conditions are difficult to capture in mechanistic models. The cells seem to have an innate intelligence that enables them to respond optimally to environmental changes. Some "intelligent" models have therefore been proposed and compared with a mechanistic model for fed-batch cultures of Ralstonia eutropha. Two kinds of models have been proposed to describe such cellular behavior. Cybernetic models are derived through postulates of cellular intelligence and memory, and neural models use artificial intelligence through neural networks. Some competing models of both kinds have been compared for their ability to portray and optimize the synthesis of poly-beta-hydroxybutyrate by Ralstonia eutropha in fed-batch cultures with finite dispersion. Neural models enabled the formation of more of the polymer than cybernetic models, with lesser utilization of the carbon and nitrogen substrates. Both types of models were decidedly superior to a mechanistic model used as a reference, thus supporting the value of intelligent descriptions of microbial kinetics in incompletely dispersed bioreactors. Neural and cybernetic models describe and optimize unsteady state fed-batch microbial reactors with finite dispersion more effectively than mechanistic models. However, these "intelligent" models too have weaknesses, and hence a hybrid approach combining such models with some mechanistic features is suggested.

  3. "Intelligent" descriptions of microbial kinetics in finitely dispersed bioreactors: neural and cybernetic models for PHB biosynthesis by Ralstonia eutropha

    PubMed Central

    Patnaik, Pratap R

    2007-01-01

    Background For many microbial processes, the complexity of the metabolisms and the responses to transient and realistic conditions are difficult to capture in mechanistic models. The cells seem to have an innate intelligence that enables them to respond optimally to environmental changes. Some "intelligent" models have therefore been proposed and compared with a mechanistic model for fed-batch cultures of Ralstonia eutropha. Results Two kinds of models have been proposed to describe such cellular behavior. Cybernetic models are derived through postulates of cellular intelligence and memory, and neural models use artificial intelligence through neural networks. Some competing models of both kinds have been compared for their ability to portray and optimize the synthesis of poly-β-hydroxybutyrate by Ralstonia eutropha in fed-batch cultures with finite dispersion. Neural models enabled the formation of more of the polymer than cybernetic models, with lesser utilization of the carbon and nitrogen substrates. Both types of models were decidedly superior to a mechanistic model used as a reference, thus supporting the value of intelligent descriptions of microbial kinetics in incompletely dispersed bioreactors. Conclusion Neural and cybernetic models describe and optimize unsteady state fed-batch microbial reactors with finite dispersion more effectively than mechanistic models. However, these "intelligent" models too have weaknesses, and hence a hybrid approach combining such models with some mechanistic features is suggested. PMID:17686143

  4. Liquid Fuel From Bacteria: Engineering Ralstonia eutropha for Production of Isobutanol (IBT) Motor Fuel from CO2, Hydrogen, and Oxygen

    SciTech Connect

    2010-07-15

    Electrofuels Project: MIT is using solar-derived hydrogen and common soil bacteria called Ralstonia eutropha to turn carbon dioxide (CO2) directly into biofuel. This bacteria already has the natural ability to use hydrogen and CO2 for growth. MIT is engineering the bacteria to use hydrogen to convert CO2 directly into liquid transportation fuels. Hydrogen is a flammable gas, so the MIT team is building an innovative reactor system that will safely house the bacteria and gas mixture during the fuel-creation process. The system will pump in precise mixtures of hydrogen, oxygen, and CO2, and the online fuel-recovery system will continuously capture and remove the biofuel product.

  5. Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

    PubMed Central

    Ushimaru, Kazunori; Mizuno, Shoji

    2015-01-01

    Recombinant Ralstonia eutropha strain PHB−4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB−4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB−4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone. PMID:25645560

  6. Metabolic flux modeling of detoxification of acetic acid by Ralstonia eutropha at slightly alkaline pH levels.

    PubMed

    Yu, J; Wang, J

    2001-06-20

    Ralstonia eutropha grows on and produces polyhydroxyalkanoates (PHAs) from fermentation acids. Acetic acid, one major organic acid from acidogenesis of organic wastes, has an inhibitory effect on the bacterium at slightly alkaline pH (6 g HAc/L at pH 8). The tolerance of R. eutropha to acetate, however, was increased significantly up to 15 g/L at the slightly alkaline pH level with high cell mass concentration. A metabolic cell model with five fluxes is proposed to depict the detoxification mechanism including mass transfer and acetyl-CoA formation of acetic acid and the formation of three final metabolic products, polyhydroxybutyrate (PHB), active biomass, and CO(2). The fluxes were measured under different conditions such as cell mass concentration, acetic acid concentration, and medium composition. The experimental results indicate that the acetate detoxification by high cell mass concentration is attributed to the increased fluxes at high extracellular acetate concentrations. The fluxes could be doubled to reduce and hence detoxify the accumulated intracellular acetate anions.

  7. Impact of Ralstonia eutropha's poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB storage in recombinant Escherichia coli.

    PubMed

    Eggers, Jessica; Steinbüchel, Alexander

    2014-12-01

    The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay

  8. Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model.

    PubMed

    Rehm, Bernd H A; Antonio, Regina V; Spiekermann, Patricia; Amara, Amro A; Steinbüchel, Alexander

    2002-01-31

    A threading model of the Ralstonia eutropha polyhydroxyalkanoate (PHA) synthase was developed based on the homology to the Burkholderia glumae lipase, whose structure has been resolved by X-ray analysis. The lid-like structure in the model was discussed. In this study, various R. eutropha PHA synthase mutants were generated employing random as well as site-specific mutagenesis. Four permissive mutants (double and triple mutations) were obtained from single gene shuffling, which showed reduced activity and whose mutation sites mapped at variable surface-exposed positions. Six site-specific mutations were generated in order to identify amino acid residues which might be involved in substrate specificity. Replacement of residues T323 (I/S) and C438 (G), respectively, which are located in the core structure of the PHA synthase model, abolished PHA synthase activity. Replacement of the two amino acid residues Y445 (F) and L446 (K), respectively, which are located at the surface of the protein model and adjacent to W425, resulted in reduced activity without changing substrate specificity and indicating a functional role of these residues. The E267K mutant exhibited only slightly reduced activity with a surface-exposed mutation site. Four site-specific deletions were generated to evaluate the role of the C-terminus and variant amino acid sequence regions, which link highly conserved regions. Deleted regions were D281-D290, A372-C382, E578-A589 and V585-A589 and the respective PHA synthases showed no detectable activity, indicating an essential role of the variable C-terminus and the linking regions between conserved blocks 2 and 3 as well as 3 and 4. Moreover, the N-terminal part of the class II PHA synthase (PhaC(Pa)) from Pseudomonas aeruginosa and the C-terminal part of the class I PHA synthase (PhaC(Re)) from R. eutropha were fused, respectively, resulting in three fusion proteins with no detectable in vivo activity. However, the fusion protein F1 (PhaC(Pa)-1-265-Pha

  9. PhaM is the physiological activator of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha.

    PubMed

    Pfeiffer, Daniel; Jendrossek, Dieter

    2014-01-01

    Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.

  10. Increasing the metabolic capacity of Escherichia coli for hydrogen production through heterologous expression of the Ralstonia eutropha SH operon.

    PubMed

    Ghosh, Dipankar; Bisaillon, Ariane; Hallenbeck, Patrick C

    2013-08-26

    Fermentative hydrogen production is an attractive means for the sustainable production of this future energy carrier but is hampered by low yields. One possible solution is to create, using metabolic engineering, strains which can bypass the normal metabolic limits to substrate conversion to hydrogen. Escherichia coli can degrade a variety of sugars to hydrogen but can only convert electrons available at the pyruvate node to hydrogen, and is unable to use the electrons available in NADH generated during glycolysis. Here, the heterologous expression of the soluble [NiFe] hydrogenase from Ralstonia eutropha H16 (the SH hydrogenase) was used to demonstrate the introduction of a pathway capable of deriving substantial hydrogen from the NADH generated by fermentation. Successful expression was demonstrated by in vitro assay of enzyme activity. Moreover, expression of SH restored anaerobic growth on glucose to adhE strains, normally blocked for growth due to the inability to re-oxidize NADH. Measurement of in vivo hydrogen production showed that several metabolically engineered strains were capable of using the SH hydrogenase to derive 2 mol H2 per mol of glucose consumed, close to the theoretical maximum. Previous introduction of heterologous [NiFe] hydrogenase in E. coli led to NAD(P)H dependent activity, but hydrogen production levels were very low. Here we have shown for the first time substantial in vivo hydrogen production by a heterologously expressed [NiFe] hydrogenase, the soluble NAD-dependent H2ase of R. eutropha (SH hydrogenase). This hydrogenase was able to couple metabolically generated NADH to hydrogen production, thus rescuing an alcohol dehydrogenase (adhE) mutant. This enlarges the range of metabolism available for hydrogen production, thus potentially opening the door to the creation of greatly improved hydrogen production. Strategies for further increasing yields should revolve around making additional NADH available.

  11. Engineering Ralstonia eutropha for Production of Isobutanol (IBT) Motor Fuel from Carbon Dioxide, Hydrogen, and Oxygen Project Final Report

    SciTech Connect

    Sinskey, Anthony J.; Worden, Robert Mark; Brigham, Christopher; Lu, Jingnan; Quimby, John Westlake; Gai, Claudia; Speth, Daan; Elliott, Sean; Fei, John Qiang; Bernardi, Amanda; Li, Sophia; Grunwald, Stephan; Grousseau, Estelle; Maiti, Soumen; Liu, Chole

    2013-12-16

    This research project is a collaboration between the Sinskey laboratory at MIT and the Worden laboratory at Michigan State University. The goal of the project is to produce Isobutanol (IBT), a branched-chain alcohol that can serve as a drop-in transportation fuel, through the engineered microbial biosynthesis of Carbon Dioxide, Hydrogen, and Oxygen using a novel bioreactor. This final technical report presents the findings of both the biological engineering work at MIT that extended the native branched-chain amino acid pathway of the wild type Ralstonia eutropha H16 to perform this biosynthesis, as well as the unique design, modeling, and construction of a bioreactor for incompatible gasses at Michigan State that enabled the operational testing of the complete system. This 105 page technical report summarizing the three years of research includes 72 figures and 11 tables of findings. Ralstonia eutropha (also known as Cupriavidus necator) is a Gram-negative, facultatively chemolithoautotrophic bacteria. It has been the principle organism used for the study of polyhydroxybutyrate (PHB) polymer biosynthesis. The wild-type Ralstonia eutropha H16 produces PHB as an intracellular carbon storage material while under nutrient stress in the presence of excess carbon. Under this stress, it can accumulate approximately 80 % of its cell dry weight (CDW) as this intracellular polymer. With the restoration of the required nutrients, the cells are then able to catabolize this polymer. If extracted from the cell, this PHB polymer can be processed into biodegradable and biocompatible plastics, however for this research, it is the efficient metabolic pathway channeling the captured carbon that is of interest. R. eutropha is further unique in that it contains two carbon-fixation Calvin–Benson–Bassham cycle operons, two oxygen-tolerant hydrogenases, and several formate dehydrogenases. It has also been much studied for its ability in the presence of oxygen, to fix carbon dioxide

  12. Crystal structure and biochemical characterization of beta-keto thiolase B from polyhydroxyalkanoate-producing bacterium Ralstonia eutropha H16

    SciTech Connect

    Kim, Eun-Jung; Son, Hyeoncheol Francis; Kim, Sangwoo; Ahn, Jae-Woo; Kim, Kyung-Jin

    2014-02-14

    Highlights: • We determined a crystal structure of β-keto thiolase from Ralstonia eutropha H16 (ReBktB). • Distinct substrate binding mode ReBktB was elucidated. • Enzymatic kinetic parameters of ReBktB were revealed. - Abstract: ReBktB is a β-keto thiolase from Ralstonia eutropha H16 that catalyzes condensation reactions between acetyl-CoA with acyl-CoA molecules that contains different numbers of carbon atoms, such as acetyl-CoA, propionyl-CoA, and butyryl-CoA, to produce valuable bioproducts, such as polyhydroxybutyrate, polyhydroxybutyrate-hydroxyvalerate, and hexanoate. We solved a crystal structure of ReBktB at 2.3 Å, and the overall structure has a similar fold to that of type II biosynthetic thiolases, such as PhbA from Zoogloea ramigera (ZrPhbA). The superposition of this structure with that of ZrPhbA complexed with CoA revealed the residues that comprise the catalytic and substrate binding sites of ReBktB. The catalytic site of ReBktB contains three conserved residues, Cys90, His350, and Cys380, which may function as a covalent nucleophile, a general base, and second nucleophile, respectively. For substrate binding, ReBktB stabilized the ADP moiety of CoA in a distinct way compared to ZrPhbA with His219, Arg221, and Asp228 residues, whereas the stabilization of β-mercaptoethyamine and pantothenic acid moieties of CoA was quite similar between these two enzymes. Kinetic study of ReBktB revealed that K{sub m}, V{sub max}, and K{sub cat} values of 11.58 μM, 1.5 μmol/min, and 102.18 s{sup −1}, respectively, and the catalytic and substrate binding sites of ReBktB were further confirmed by site-directed mutagenesis experiments.

  13. Characterization of poly-3-hydroxybutyrate (PHB) produced from Ralstonia eutropha using an alkali-pretreated biomass feedstock.

    PubMed

    Saratale, Ganesh D; Oh, Min-Kyu

    2015-09-01

    Alkaline pretreatment using NaOH, KOH, or NaOCl has been applied to various types of waste biomass to enhance enzymatic digestibility. Pretreatment (2% NaOH, 121 °C, 30 min) of rice paddy straw (PS) resulted in a maximum yield of 703 mg of reducing sugar per gram of PS with 84.19% hydrolysis yield after a two-step enzymatic hydrolysis process. Ralstonia eutropha ATCC 17699 was tested for its ability to synthesize poly-3-hydroxybutyrate (PHB) using PS hydrolysates as its sole carbon source. It is noteworthy that dry cell weight, polyhydroxyalkanoate (PHA) accumulation and PHB yield with the use of laboratory-grade sugars were similar to those achieved with PS-derived sugars. Under optimized conditions, we observed maximal PHA accumulation (75.45%) and PHB production (11.42 g/L) within 48 h of fermentation. After PHB recovery, the physicochemical properties of PHB were determined by various analytical techniques, showed the results were consistent with the characteristics of a standard polymer of PHB. Thus, the PS hydrolysate proved to be an excellent cheap carbon substrate for PHB production.

  14. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of β-ketothiolase B from Ralstonia eutropha H16.

    PubMed

    Kim, Eun-Jung; Son, Hyeoncheol Francis; Chang, Jeong Ho; Kim, Kyung-Jin

    2014-03-01

    Polyhydroxyalkanoates are linear polyesters that are produced by bacterial fermentation and are used as biodegradable bioplastics. β-Ketothiolase B (BktB) from Ralstonia eutropha (ReBktB) is a key enzyme for the production of various types of copolymers by catalyzing the condensation reactions of acetyl-CoA with propionyl-CoA and butyryl-CoA. The ReBktB protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 25% polyethylene glycol 3350, 0.1 M bis-tris pH 6.5, 0.2 M lithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space group C2221, with unit-cell parameters a = 106.95, b = 107.24, c = 144.14 Å. With two molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 2.54 Å(3) Da(-1), which corresponds to a solvent content of approximately 51.5%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.

  15. Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha.

    PubMed

    Niks, Dimitri; Duvvuru, Jayant; Escalona, Miguel; Hille, Russ

    2016-01-15

    We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD(+)-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo(V) state and demonstrated the direct transfer of the substrate Cα hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase from Ralstonia eutropha*

    PubMed Central

    Niks, Dimitri; Duvvuru, Jayant; Escalona, Miguel; Hille, Russ

    2016-01-01

    We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD+-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its MoV state and demonstrated the direct transfer of the substrate Cα hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center. PMID:26553877

  17. A trimeric supercomplex of the oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha H16.

    PubMed

    Frielingsdorf, Stefan; Schubert, Torsten; Pohlmann, Anne; Lenz, Oliver; Friedrich, Bärbel

    2011-12-20

    The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit.

  18. Expression and activity of the Calvin-Benson-Bassham cycle transcriptional regulator CbbR from Acidithiobacillus ferrooxidans in Ralstonia eutropha.

    PubMed

    Esparza, Mario; Jedlicki, Eugenia; Dopson, Mark; Holmes, David S

    2015-08-01

    Autotrophic fixation of carbon dioxide into cellular carbon occurs via several pathways but quantitatively, the Calvin-Benson-Bassham cycle is the most important. CbbR regulates the expression of the cbb genes involved in CO2 fixation via the Calvin-Benson-Bassham cycle in a number of autotrophic bacteria. A gene potentially encoding CbbR (cbbR(AF)) has been predicted in the genome of the chemolithoautotrophic, extreme acidophile Acidithiobacillus ferrooxidans. However, this microorganism is recalcitrant to genetic manipulation impeding the experimental validation of bioinformatic predictions. Two novel functional assays were devised to advance our understanding of cbbR(AF) function using the mutated facultative autotroph Ralstonia eutropha H14 ΔcbbR as a surrogate host to test gene function: (i) cbbR(AF) was expressed in R. eutropha and was able to complement ΔcbbR; and (ii) CbbR(AF) was able to regulate the in vivo activity of four A. ferrooxidans cbb operon promoters in R. eutropha. These results open up the use of R. eutropha as a surrogate host to explore cbbR(AF) activity. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. A closer look on the polyhydroxybutyrate- (PHB-) negative phenotype of Ralstonia eutropha PHB-4.

    PubMed

    Raberg, Matthias; Voigt, Birgit; Hecker, Michael; Steinbüchel, Alexander

    2014-01-01

    The undefined poly(3-hydroxybutyrate)- (PHB-) negative mutant R. eutropha PHB-4 was generated in 1970 by 1-nitroso-3-nitro-1-methylguanidine (NMG) treatment. Although being scientific relevant, its genotype remained unknown since its isolation except a recent first investigation. In this study, the mutation causing the PHA-negative phenotype of R. eutropha PHB-4 was confirmed independently: sequence analysis of the phaCAB operon identified a G320A mutation in phaC yielding a stop codon, leading to a massively truncated PhaC protein of 106 amino acids (AS) in R. eutropha PHB-4 instead of 589 AS in the wild type. No other mutations were observed within the phaCAB operon. As further mutations probably occurred in the genome of mutant PHB-4 potentially causing secondary effects on the cells' metabolism, the main focus of the study was to perform a 2D PAGE-based proteome analysis in order to identify differences in the proteomes of the wild type and mutant PHB-4. A total of 20 differentially expressed proteins were identified which provide valuable insights in the metabolomic changes of mutant PHB-4. Besides excretion of pyruvate, mutant PHB-4 encounters the accumulation of intermediates such as pyruvate and acetyl-CoA by enhanced expression of the observed protein species: (i) ThiJ supports biosynthesis of cofactor TPP and thereby reinforces the 2-oxoacid dehydrogenase complexes as PDHC, ADHC and OGDHC in order to convert pyruvate at a higher rate and the (ii) 3-isopropylmalate dehydrogenase LeuB3 apparently directs pyruvate to synthesis of several amino acids. Different (iii) acylCoA-transferases enable transfer reactions between organic acid intermediates, and (iv) citrate lyase CitE4 regenerates oxaloacetate from citrate for conversion with acetyl-CoA in the TCC in an anaplerotic reaction. Substantial amounts of reduction equivalents generated in the TCC are countered by (v) synthesis of more ubiquinones due to enhanced synthesis of MenG2 and MenG3, thereby

  20. Analysis of 4-Phosphopantetheinylation of Polyhydroxybutyrate Synthase from Ralstonia eutropha: Generation of β-Alanine Auxotrophic Tn5 Mutants and Cloning of the panD Gene Region

    PubMed Central

    Hoppensack, Astrid; Rehm, Bernd H. A.; Steinbüchel, Alexander

    1999-01-01

    The postulated posttranslational modification of the polyhydroxybutyrate (PHA) synthase from Ralstonia eutropha by 4-phosphopantetheine was investigated. Four β-alanine auxotrophic Tn5-induced mutants of R. eutropha HF39 were isolated, and two insertions were mapped in an open reading frame with strong similarity to the panD gene from Escherichia coli, encoding l-aspartate-1-decarboxylase (EC 4.1.1.15), whereas two other insertions were mapped in an open reading frame (ORF) with strong similarity to the NAD(P)+ transhydrogenase (EC 1.6.1.1) alpha 1 subunit, encoded by the pntAA gene from Escherichia coli. The panD gene was cloned by complementation of the panD mutant of R. eutropha Q20. DNA sequencing of the panD gene region (3,312 bp) revealed an ORF of 365 bp, encoding a protein with 63 and 67% amino acid sequence similarity to PanD from E. coli and Bacillus subtilis, respectively. Subcloning of only this ORF into vectors pBBR1MCS-3 and pBluescript KS− led to complementation of the panD mutants of R. eutropha and E. coli SJ16, respectively. panD-encoded l-aspartate-1-decarboxylase was further confirmed by an enzymatic assay. Upstream of panD, an ORF with strong similarity to pntAA from E. coli, encoding NAD(P)+ transhydrogenase subunit alpha 1 was found; downstream of panD, two ORFs with strong similarity to pntAB and pntB, encoding subunits alpha 2 and beta of the NAD(P)+ transhydrogenase, respectively, were identified. Thus, a hitherto undetermined organization of pan and pnt genes was found in R. eutropha. Labeling experiments using one of the R. eutropha panD mutants and [2-14C]β-alanine provided no evidence that R. eutropha PHA synthase is covalently modified by posttranslational attachment of 4-phosphopantetheine, nor did the E. coli panD mutant exhibit detectable labeling of functional PHA synthase from R. eutropha. PMID:10049372

  1. Genome-scale reconstruction and in silico analysis of the Ralstonia eutropha H16 for polyhydroxyalkanoate synthesis, lithoautotrophic growth, and 2-methyl citric acid production

    PubMed Central

    2011-01-01

    Background Ralstonia eutropha H16, found in both soil and water, is a Gram-negative lithoautotrophic bacterium that can utillize CO2 and H2 as its sources of carbon and energy in the absence of organic substrates. R. eutropha H16 can reach high cell densities either under lithoautotrophic or heterotrophic conditions, which makes it suitable for a number of biotechnological applications. It is the best known and most promising producer of polyhydroxyalkanoates (PHAs) from various carbon substrates and is an environmentally important bacterium that can degrade aromatic compounds. In order to make R. eutropha H16 a more efficient and robust biofactory, system-wide metabolic engineering to improve its metabolic performance is essential. Thus, it is necessary to analyze its metabolic characteristics systematically and optimize the entire metabolic network at systems level. Results We present the lithoautotrophic genome-scale metabolic model of R. eutropha H16 based on the annotated genome with biochemical and physiological information. The stoichiometic model, RehMBEL1391, is composed of 1391 reactions including 229 transport reactions and 1171 metabolites. Constraints-based flux analyses were performed to refine and validate the genome-scale metabolic model under environmental and genetic perturbations. First, the lithoautotrophic growth characteristics of R. eutropha H16 were investigated under varying feeding ratios of gas mixture. Second, the genome-scale metabolic model was used to design the strategies for the production of poly[R-(-)-3hydroxybutyrate] (PHB) under different pH values and carbon/nitrogen source uptake ratios. It was also used to analyze the metabolic characteristics of R. eutropha when the phosphofructokinase gene was expressed. Finally, in silico gene knockout simulations were performed to identify targets for metabolic engineering essential for the production of 2-methylcitric acid in R. eutropha H16. Conclusion The genome-scale metabolic model

  2. New Insights in PhaM-PhaC-mediated Localization of PHB Granules in Ralstonia eutropha H16.

    PubMed

    Bresan, Stephanie; Jendrossek, Dieter

    2017-04-07

    Formation and localization of polyhydroxybutyrate (PHB) granules in Ralstonia eutropha is controlled by PhaM that interacts both with the PHB synthase (PhaC) and with the bacterial nucleoid. Here, we studied the importance of proline and lysine residues of two C-terminal PAKKA motifs in PhaM for their importance to attach PHB granules to DNA by in vitro and in vivo methods. Substitution of the lysine residues but not of the proline residues resulted in a detachment of formed PHB granules from the nucleoid. Instead, a formation of PHB granule clusters at polar regions of the rod-shaped cells and an unequal distribution of PHB granules to daughter cells was observed. The formation of PHB granules was studied by the expression of chromosomally anchored gene fusions of fluorescent proteins with PhaM and PhaC in different backgrounds. PhaM and PhaC fusions showed a distinct co-localization at formed PHB granules in the nucleoid region of the wild type. In a ΔphaC background, PhaM and the catalytically inactive PhaC(C319A) proteins were not able to form fluorescent foci indicating that correct positioning requires the formation of PHB. Furthermore, time-lapse experiments revealed that PhaC and PhaM proteins detach at later stages from formed PHB granules resulting in an inhomogeneous population of PHB granules. This could explain why growth of individual PHB granules stops under PHB permissive conditions at a certain size.Importance PHB granules are storage compounds for carbon and energy in many prokaryotes. Equal distribution of accumulated PHB granules during cell division is therefore important for optimal fitness of the daughter cells. In R. eutropha, PhaM is responsible for maximal activity of PHB synthase, for initiation of PHB granule formation at discrete regions in the cells and for association of formed PHB granules to the nucleoid. Here we found out that four lysine residues of C-terminal PhaM sequence motifs are essential for association of PHB granules to

  3. Stable Carbon Isotope Discrimination by Form IC Rubisco Enzymes of the Extremely Metabolically Versatile Rhodobacter sphaeroides and Ralstonia eutropha}

    NASA Astrophysics Data System (ADS)

    Thomas, P. J.; Boller, A. J.; Zhao, Z.; Tabita, F. R.; Cavanaugh, C. M.; Scott, K. M.

    2006-12-01

    Variations in the relative amounts of 12C and 13C in microbial biomass can be used to infer the pathway(s) autotrophs use to fix and assimilate dissolved inorganic carbon. Discrimination against 13C by the enzymes catalyzing autotrophic carbon fixation is a major factor dictating biomass stable carbon isotopic compositions (δ13C = {[13C/12Csample/13C/12Cstandard] - 1} × 1000). Five different forms of RubisCO (IA, IB, IC, ID, and II) are utilized by algae and autotrophic bacteria reliant on the Calvin-Benson cycle for carbon fixation. To date, isotope discrimination has been measured for form IA, IB, and II RubisCOs, and their ɛ values (={[12k/13k] - 1} × 1000; 12k and 13k = rates of 12C and 13C fixation) range from 18 to 29‰, explaining the variation in biomass δ13C values of autotrophs utilizing these enzymes. Isotope discrimination by form IC RubisCO has not been measured, despite the presence of this enzyme in many proteobacteria of ecological interest, including marine manganese-oxidizing bacteria, some nitrifying and nitrogen-fixing bacteria, and extremely metabolically versatile organisms such as Rhodobacter sphaeroides and Ralstonia eutropha. The purpose of this work was to determine the ɛ values for form IC RubisCO enzymes from R. sphaeroides and R. eutropha. Recombinant form IC RubisCOs were purified by conventional column chromatography procedures. Assay conditions (pH, dissolved inorganic carbon concentration) were tested to determine which parameters were conducive to the high rates of carbon fixation necessary for ɛ determination. Under standard conditions (pH 8.5 and 5 mM DIC), form IC RubisCO activities were sufficient for ɛ determination. Experiments are currently being conducted to measure the ɛ values of these enzymes. Sampling the full phylogenetic breadth of RubisCO enzymes for isotopic discrimination makes it possible to constrain the range of δ13C values of organisms fixing carbon via the Calvin-Benson cycle. These results are

  4. Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16.

    PubMed

    Sznajder, Anna; Pfeiffer, Daniel; Jendrossek, Dieter

    2015-03-01

    Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.

  5. Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16

    PubMed Central

    Tumlirsch, Tony; Sznajder, Anna

    2015-01-01

    A protein (PhaX) that interacted with poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 and with PHB granule-associated phasin protein PhaP2 was identified by two-hybrid analysis. Deletion of phaX resulted in an increase in the level of polyphosphate (polyP) granule formation and in impairment of PHB utilization in nutrient broth-gluconate cultures. A procedure for enrichment of polyP granules from cell extracts was developed. Twenty-seven proteins that were absent in other cell fractions were identified in the polyP granule fraction by proteome analysis. One protein (A2437) harbored motifs characteristic of type 1 polyphosphate kinases (PPK1s), and two proteins (A1212, A1271) had PPK2 motifs. In vivo colocalization with polyP granules was confirmed by expression of C- and N-terminal fusions of enhanced yellow fluorescent protein (eYFP) with the three polyphosphate kinases (PPKs). Screening of the genome DNA sequence for additional proteins with PPK motifs revealed one protein with PPK1 motifs and three proteins with PPK2 motifs. Construction and subsequent expression of C- and N-terminal fusions of the four new PPK candidates with eYFP showed that only A1979 (PPK2 motif) colocalized with polyP granules. The other three proteins formed fluorescent foci near the cell pole (apart from polyP) (A0997, B1019) or were soluble (A0226). Expression of the Ralstonia eutropha ppk (ppkReu) genes in an Escherichia coli Δppk background and construction of a set of single and multiple chromosomal deletions revealed that both A2437 (PPK1a) and A1212 (PPK2c) contributed to polyP granule formation. Mutants with deletion of both genes were unable to produce polyP granules. The formation and utilization of PHB and polyP granules were investigated in different chromosomal backgrounds. PMID:26407880

  6. Catalytic Properties of the Isolated Diaphorase Fragment of the NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha

    PubMed Central

    Lauterbach, Lars; Idris, Zulkifli; Vincent, Kylie A.; Lenz, Oliver

    2011-01-01

    The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H2-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O2. It comprises six subunits, HoxHYFUI2, and incorporates a [NiFe] H+/H2 cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with KI values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O2, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis. PMID:22016788

  7. Rapid selection of glucose-utilizing variants of the polyhydroxyalkanoate producer Ralstonia eutropha H16 by incubation with high substrate levels.

    PubMed

    Franz, A; Rehner, R; Kienle, A; Grammel, H

    2012-01-01

    The application of Ralstonia eutropha H16 for producing polyhydroxyalkanoates as bioplastics is limited by the incapability of the bacterium to utilize glucose as a growth substrate. This study aims in characterizing glucose-utilizing strains that arose after incubation with high glucose levels, in comparison with previously published mutants, generated either by mutagenesis or by metabolic engineering. Cultivations on solid and liquid media showed that the application of high substrate concentrations rapidly induced a glucose-positive phenotype. The time span until the onset of growth and the frequency of glucose-utilizing colonies were correlated to the initial glucose concentration. All mutants exhibited elevated activities of glucose-6-phosphate dehydrogenase. The glucose-positive phenotype was abolished after deleting genes for the N-acetylglucosamine phosphotransferase system. A procedure is provided for selecting glucose-utilizing R. eutropha H16 in an unprecedented short time period and without any mutagenic treatment. An altered N-acetylglucosamine phosphotransferase system appears to be a common motif in all glucose-utilizing mutants examined so far. The correlation of the applied glucose concentration and the appearance of glucose-utilizing mutants poses questions about the randomness or the specificity of adaptive mutations in general. Furthermore, glucose-adapted strains of R. eutropha H16 could be useful for the production of bioplastics. © 2011 The Authors. Letters in Applied Microbiology ©2011 The Society for Applied Microbiology.

  8. Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16

    SciTech Connect

    Brigham, CJ; Speth, DR; Rha, C; Sinskey, AJ

    2012-10-22

    Poly(3-hydroxybutyrate) (PHB) production and mobilization in Ralstonia eutropha are well studied, but in only a few instances has PHB production been explored in relation to other cellular processes. We examined the global gene expression of wild-type R. eutropha throughout the PHB cycle: growth on fructose, PHB production using fructose following ammonium depletion, and PHB utilization in the absence of exogenous carbon after ammonium was resupplied. Our results confirm or lend support to previously reported results regarding the expression of PHB-related genes and enzymes. Additionally, genes for many different cellular processes, such as DNA replication, cell division, and translation, are selectively repressed during PHB production. In contrast, the expression levels of genes under the control of the alternative sigma factor sigma(54) increase sharply during PHB production and are repressed again during PHB utilization. Global gene regulation during PHB production is strongly reminiscent of the gene expression pattern observed during the stringent response in other species. Furthermore, a ppGpp synthase deletion mutant did not show an accumulation of PHB, and the chemical induction of the stringent response with DL-norvaline caused an increased accumulation of PHB in the presence of ammonium. These results indicate that the stringent response is required for PHB accumulation in R. eutropha, helping to elucidate a thus-far-unknown physiological basis for this process.

  9. Efficient biological conversion of carbon monoxide (CO) to carbon dioxide (CO2) and for utilization in bioplastic production by Ralstonia eutropha through the display of an enzyme complex on the cell surface.

    PubMed

    Hyeon, Jeong Eun; Kim, Seung Wook; Park, Chulhwan; Han, Sung Ok

    2015-06-25

    An enzyme complex for biological conversion of CO to CO2 was anchored on the cell surface of the CO2-utilizing Ralstonia eutropha and successfully resulted in a 3.3-fold increase in conversion efficiency. These results suggest that this complexed system may be a promising strategy for CO2 utilization as a biological tool for the production of bioplastics.

  10. Extension of the Substrate Utilization Range of Ralstonia eutropha Strain H16 by Metabolic Engineering To Include Mannose and Glucose ▿

    PubMed Central

    Sichwart , Shanna; Hetzler, Stephan; Bröker, Daniel; Steinbüchel, Alexander

    2011-01-01

    The Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha strain H16 is known for its narrow carbohydrate utilization range, which limits its use for biotechnological production of polyhydroxyalkanoates and possibly other products from renewable resources. To broaden its substrate utilization range, which is for carbohydrates and related compounds limited to fructose, N-acetylglucosamine, and gluconate, strain H16 was engineered to use mannose and glucose as sole carbon sources for growth. The genes for a facilitated diffusion protein (glf) from Zymomonas mobilis and for a glucokinase (glk), mannofructokinase (mak), and phosphomannose isomerase (pmi) from Escherichia coli were alone or in combination constitutively expressed in R. eutropha strain H16 under the control of the neokanamycin or lac promoter, respectively, using an episomal broad-host-range vector. Recombinant strains harboring pBBR1MCS-3::glf::mak::pmi or pBBR1MCS-3::glf::pmi grew on mannose, whereas pBBR1MCS-3::glf::mak and pBBR1MCS-3::glf did not confer the ability to utilize mannose as a carbon source to R. eutropha. The recombinant strain harboring pBBR1MCS-3::glf::pmi exhibited slower growth on mannose than the recombinant strain harboring pBBR1MCS-3::glf::mak::pmi. These data indicated that phosphomannose isomerase is required to convert mannose-6-phosphate into fructose-6-phosphate for subsequent catabolism via the Entner-Doudoroff pathway. In addition, all plasmids also conferred to R. eutropha the ability to grow in the presence of glucose. The best growth was observed with a recombinant R. eutropha strain harboring plasmid pBBR1MCS-2::Pnk::glk::glf. In addition, expression of the respective enzymes was demonstrated at the transcriptional and protein levels and by measuring the activities of mannofructokinase (0.622 ± 0.063 U mg−1), phosphomannose isomerase (0.251 ± 0.017 U mg−1), and glucokinase (0.518 ± 0.040 U mg−1). Cells of recombinant strains of R

  11. Versatile plasmid-based expression systems for Gram-negative bacteria--General essentials exemplified with the bacterium Ralstonia eutropha H16.

    PubMed

    Gruber, Steffen; Schwab, Helmut; Koefinger, Petra

    2015-12-25

    The Gram-negative bacterium Escherichia coli is currently the most efficient and widely used prokaryotic host for recombinant protein and metabolite production. However, due to some limitations and to various interesting features of other Gram-negative bacteria efficient vector systems applicable to a broad range are desired. Basic building blocks for plasmid-based vectors include besides the need for a suitable selection marker in the first line a proper replication and maintenance system. In addition to these basic requirements, further elements are needed for Gram-negative bacteria beyond E. coli, such as Pseudomonas pudita, Ralstonia eutropha, Burkholderia glumae or Acinetobacter sp.. Established building blocks have to be adapted and new building blocks providing the desired functions need to be identified and exploited. This minireview addresses so far described and used genetic elements for broad host range replication, efficient plasmid maintenance, and conjugative plasmid transfer as well as expression elements and protein secretion signals. The industrially important bacterium R. eutropha H16 was chosen as a model organism to provide specific data on the effectivity and utility of building blocks based on such genetic elements.

  12. Modification of β-oxidation pathway in Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from soybean oil.

    PubMed

    Insomphun, Chayatip; Mifune, Jun; Orita, Izumi; Numata, Keiji; Nakamura, Satoshi; Fukui, Toshiaki

    2014-02-01

    Ralstonia eutropha H16 is a useful platform for metabolic engineering aiming at efficient production of polyhydroxyalkanaotes being attracted as practical bioplastics. This study focused on bifunctional (S)-specific 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase encoded by fadB to obtain information regarding β-oxidation in this bacterium and to achieve compositional regulation of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] synthesized from soybean oil. In addition to two FadB homologs (FadB1 and FadB') encoded within the previously identified β-oxidation gene clusters on the chromosome 1, a gene of third homolog (FadB2) was found on chromosome 2 of R. eutropha. The fadB homologs were disrupted in R. eutropha strain NSDG expressing a mutant gene of PHA synthase from Aeromonas caviae. The gene disruptions affected neither growth nor PHA production on fructose. On soybean oil, fadB' deletion led to reduction of PHA quantity attributed to decrease of 3HB unit, while fadB1 deletion slightly increased 3HHx composition without serious negative impact on both cell growth and PHA biosynthesis. Double deletion of fadB1 and fadB' significantly impaired the cell growth and PHA biosynthesis, indicating the major roles of fadB1 and fadB' in β-oxidation. When fadB1 was deleted in several engineered strains of R. eutropha possessing additional (R)-enoyl-CoA hydratase gene(s), the net amounts of 3HHx unit in the PHA fractions showed 6-21% increase probably due to slightly enhanced supply of medium-chain-length 2-enoyl-CoAs through the partially impaired β-oxidation. These results demonstrated that modification of β-oxidation by fadB1 deletion was effective for increasing 3HHx composition in the copolyesters produced from soybean oil.

  13. PHA productivity and yield of Ralstonia eutropha when intermittently or continuously fed a mixture of short chain fatty acids.

    PubMed

    Chakraborty, Panchali; Muthukumarappan, Kasiviswanathan; Gibbons, William R

    2012-01-01

    The research described in this present study was part of a larger effort focused on developing a dual substrate, dual fermentation process to produce Polyhydroxyalkanoate (PHA). The focus of this study was developing and optimizing a strategy for feeding a mixture of SCFAs (simulated ARF) and maximizing PHA production in a cost-effective way. Three different feeding strategies were examined in this study. The substrate evaluated in this study for the growth phase of R. eutropha was condensed corn solubles, a low-value byproduct of the dry-mill, corn ethanol industry. The culture was grown to high cell densities in nitrogen-supplemented condensed corn solubles media in 5 L bioreactors. The overall growth rate of R. eutropha was 0.2 h(-1). The 20 mL ARF feeding every 3 h from 48 to 109 h strategy gave the best results in terms of PHA production. PHA productivity (0.0697 g L(-1) h(-1)), PHA concentration (8.37 g L(-1)), and PHA content (39.52%) were the highest when ARF was fed every 3 h for 61 h. This study proved that condensed corn solubles can be potentially used as a growth medium to boost PHA production by R. eutropha thus reducing the overall cost of biopolymer production.

  14. Reduction of unusual iron-sulfur clusters in the H2-sensing regulatory Ni-Fe hydrogenase from Ralstonia eutropha H16.

    PubMed

    Buhrke, Thorsten; Löscher, Simone; Lenz, Oliver; Schlodder, Eberhard; Zebger, Ingo; Andersen, Lars K; Hildebrandt, Peter; Meyer-Klaucke, Wolfram; Dau, Holger; Friedrich, Bärbel; Haumann, Michael

    2005-05-20

    The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H(2) at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RH(WT) [HoxBC](2) and in a monodimeric derivative designated RH(stop) lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RH(WT) seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RH(stop). Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC](2) and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha.

  15. Phenol degradation by Ralstonia eutropha: Colorimetric determination of 2-hydroxymuconate semialdehyde accumulation to control feed strategy in fed-batch fermentations

    SciTech Connect

    Leonard, D; Destruhaut, C; Lindley, N D; Youssef, C B; Queinnec, I

    1999-11-20

    Phenol biodegradation by Ralstonia eutropha was modeled in different culture modes to assess phenol feeding in biotechnological depollution processes. The substrate-inhibited growth of R. eutropha was described by the Haldane equation with a K[sub s] of 2 mg/L, a K[sub i] of 350 mg/L and a [micro][sub max] of 0.41 h[sup [minus]1]. Furthermore, growth in several culture modes was characterized by the appearance of a yellow color, due to production of a metabolic intermediate of the phenol catabolic pathway, 2-hydroxymuconic semialdehyde (2phms) which was directly correlated to the growth rate and/or the phenol-degradation rate, because these two parameters are coupled. This correlation between color appearance and metabolic activity was used to develop a control procedure for optimal phenol degradation. A mass-balance equation modeling approach combined with a filtering step using an extended Kalman filter enabled state variables of the biological system to be simulated. A PI controller, using the estimation of the phenol concentration provided by the modeling step, was then built to maintain the phenol concentration at a constant set-point of 0.1 g/L which corresponded to a constant specific growth rate of 0.3 h[sup [minus]1], close to the maximal specific growth value of the strain. This monitoring strategy, validated for two fed-batch cultures, could lead, in self-cycling fermentation systems, to a productivity of more than 19 kg of phenol consumed/m[sup 3]/d which is the highest value reported to date in the literature. This system of monitoring metabolic activity also protected the bacterial culture against toxicity problems due to the transient accumulation of phenol.

  16. Implications of various phosphoenolpyruvate-carbohydrate phosphotransferase system mutations on glycerol utilization and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16

    PubMed Central

    2011-01-01

    The enhanced global biodiesel production is also yielding increased quantities of glycerol as main coproduct. An effective application of glycerol, for example, as low-cost substrate for microbial growth in industrial fermentation processes to specific products will reduce the production costs for biodiesel. Our study focuses on the utilization of glycerol as a cheap carbon source during cultivation of the thermoplastic producing bacterium Ralstonia eutropha H16, and on the investigation of carbohydrate transport proteins involved herein. Seven open reading frames were identified in the genome of strain H16 to encode for putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS). Although the core components of PEP-PTS, enzyme I (ptsI) and histidine phosphocarrier protein (ptsH), are available in strain H16, a complete PTS-mediated carbohydrate transport is lacking. Growth experiments employing several PEP-PTS mutants indicate that the putative ptsMHI operon, comprising ptsM (a fructose-specific EIIA component of PTS), ptsH, and ptsI, is responsible for limited cell growth and reduced PHB accumulation (53%, w/w, less PHB than the wild type) of this strain in media containing glycerol as a sole carbon source. Otherwise, the deletion of gene H16_A0384 (ptsN, nitrogen regulatory EIIA component of PTS) seemed to largely compensate the effect of the deleted ptsMHI operon (49%, w/w, PHB). The involvement of the PTS homologous proteins on the utilization of the non-PTS sugar alcohol glycerol and its effect on cell growth as well as PHB and carbon metabolism of R. eutropha will be discussed. PMID:21906371

  17. Effects of Homologous Phosphoenolpyruvate-Carbohydrate Phosphotransferase System Proteins on Carbohydrate Uptake and Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha H16▿†

    PubMed Central

    Kaddor, Chlud; Steinbüchel, Alexander

    2011-01-01

    Seven gene loci encoding putative proteins of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PEP-PTS) were identified in the genome of Ralstonia eutropha H16 by in silico analysis. Except the N-acetylglucosamine-specific PEP-PTS, an additional complete PEP-PTS is lacking in strain H16. Based on these findings, we generated single and multiple deletion mutants defective mainly in the PEP-PTS genes to investigate their influence on carbon source utilization, growth behavior, and poly(3-hydroxybutyrate) (PHB) accumulation. As supposed, the H16 ΔfrcACB and H16 ΔnagFEC mutants exhibited no growth when cultivated on fructose and N-acetylglucosamine, respectively. Furthermore, a transposon mutant with a ptsM-ptsH insertion site did not grow on both carbon sources. The observed phenotype was not complemented, suggesting that it results from an interaction of genes or a polar effect caused by the Tn5::mob insertion. ptsM, ptsH, and ptsI single, double, and triple mutants stored much less PHB than the wild type (about 10 to 39% [wt/wt] of cell dry weight) and caused reduced PHB production in mutants lacking the H16_A2203, H16_A0384, frcACB, or nagFEC genes. In contrast, mutant H16 ΔH16_A0384 accumulated 11.5% (wt/wt) more PHB than the wild type when grown on gluconate and suppressed partially the negative effect of the ptsMHI deletion on PHB synthesis. Based on our experimental data, we discussed whether the PEP-PTS homologous proteins in R. eutropha H16 are exclusively involved in the complex sugar transport system or whether they are also involved in cellular regulatory functions of carbon and PHB metabolism. PMID:21478317

  18. Genetic organization of the catabolic plasmid pJP4 from Ralstonia eutropha JMP134 (pJP4) reveals mechanisms of adaptation to chloroaromatic pollutants and evolution of specialized chloroaromatic degradation pathways.

    PubMed

    Trefault, N; De la Iglesia, R; Molina, A M; Manzano, M; Ledger, T; Pérez-Pantoja, D; Sánchez, M A; Stuardo, M; González, B

    2004-07-01

    Ralstonia eutropha JMP134 (pJP4) is a useful model for the study of bacterial degradation of substituted aromatic pollutants. Several key degrading capabilities, encoded by tfd genes, are located in the 88 kb, self-transmissible, IncP-1 beta plasmid pJP4. The complete sequence of the 87,688 nucleotides of pJP4, encoding 83 open reading frames (ORFs), is reported. Most of the coding sequence corresponds to a well-conserved IncP-1 beta backbone and the previously reported tfd genes. In addition, we found hypothetical proteins putatively involved in the transport of aromatic compounds and short-chain fatty acid oxidation. ORFs related to mobile elements, including the Tn501-encoded mercury resistance determinants, an IS1071-based composite transposon and a cryptic class II transposon, are also present in pJP4. These mobile elements are inefficient in transposition and are located in two regions of pJP4 that are rich in remnants of lateral gene transfer events. pJP4 plasmid was able to capture chromosomal genes and form hybrid plasmids with the IncP-1 alpha plasmid RP4. These observations are integrated into a model for the evolution of pJP4, which reveals mechanisms of bacterial adaptation to degrade pollutants.

  19. Cloning, expression, purification, crystallization and X-ray crystallographic analysis of the (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha H16

    PubMed Central

    Kim, Jieun; Kim, Kyung-Jin

    2014-01-01

    The (S)-3-hydroxybutyryl-CoA dehydrogenase PaaH1 from Ralstonia eutropha (RePaaH1) is an enzyme used in the biosynthesis of n-butanol from acetyl-CoA by the reduction of acetoacetyl-CoA to (S)-3-hydroxybutyryl-CoA. The RePaaH1 protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 1.4 M ammonium sulfate, 0.1 M sodium cacodylate pH 6.0, 0.2 M sodium chloride at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.6 Å on a synchrotron beamline. The crystal belonged to space group P3221, with unit-cell parameters a = b = 135.4, c = 97.2 Å. With three molecules per asymmetric unit, the crystal volume per unit protein weight (V M) is 2.68 Å3 Da−1, which corresponds to a solvent content of approximately 54.1%. The structure was solved by the single-wavelength anomalous dispersion method and refinement of the structure is in progress. PMID:25005097

  20. Directed evolution and structural analysis of NADPH-dependent Acetoacetyl Coenzyme A (Acetoacetyl-CoA) reductase from Ralstonia eutropha reveals two mutations responsible for enhanced kinetics.

    PubMed

    Matsumoto, Ken'ichiro; Tanaka, Yoshikazu; Watanabe, Tsuyoshi; Motohashi, Ren; Ikeda, Koji; Tobitani, Kota; Yao, Min; Tanaka, Isao; Taguchi, Seiichi

    2013-10-01

    NADPH-dependent acetoacetyl-coenzyme A (acetoacetyl-CoA) reductase (PhaB) is a key enzyme in the synthesis of poly(3-hydroxybutyrate) [P(3HB)], along with β-ketothiolase (PhaA) and polyhydroxyalkanoate synthase (PhaC). In this study, PhaB from Ralstonia eutropha was engineered by means of directed evolution consisting of an error-prone PCR-mediated mutagenesis and a P(3HB) accumulation-based in vivo screening system using Escherichia coli. From approximately 20,000 mutants, we obtained two mutant candidates bearing Gln47Leu (Q47L) and Thr173Ser (T173S) substitutions. The mutants exhibited kcat values that were 2.4-fold and 3.5-fold higher than that of the wild-type enzyme, respectively. In fact, the PhaB mutants did exhibit enhanced activity and P(3HB) accumulation when expressed in recombinant Corynebacterium glutamicum. Comparative three-dimensional structural analysis of wild-type PhaB and highly active PhaB mutants revealed that the beneficial mutations affected the flexibility around the active site, which in turn played an important role in substrate recognition. Furthermore, both the kinetic analysis and crystal structure data supported the conclusion that PhaB forms a ternary complex with NADPH and acetoacetyl-CoA. These results suggest that the mutations affected the interaction with substrates, resulting in the acquirement of enhanced activity.

  1. The soluble NAD+-Reducing [NiFe]-hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH.

    PubMed

    Burgdorf, Tanja; van der Linden, Eddy; Bernhard, Michael; Yin, Qing Yuan; Back, Jaap W; Hartog, Aloysius F; Muijsers, Anton O; de Koster, Chris G; Albracht, Simon P J; Friedrich, Bärbel

    2005-05-01

    The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).

  2. The Soluble NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha H16 Consists of Six Subunits and Can Be Specifically Activated by NADPH

    PubMed Central

    Burgdorf, Tanja; van der Linden, Eddy; Bernhard, Michael; Yuan Yin, Qing; Back, Jaap W.; Hartog, Aloysius F.; Muijsers, Anton O.; de Koster, Chris G.; Albracht, Simon P. J.; Friedrich, Bärbel

    2005-01-01

    The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD+ at the expense of H2. We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI2. Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I). PMID:15838039

  3. Kinetic and stoichiometric characterization of organoautotrophic growth of Ralstonia eutropha on formic acid in fed-batch and continuous cultures.

    PubMed

    Grunwald, Stephan; Mottet, Alexis; Grousseau, Estelle; Plassmeier, Jens K; Popović, Milan K; Uribelarrea, Jean-Louis; Gorret, Nathalie; Guillouet, Stéphane E; Sinskey, Anthony

    2015-01-01

    Formic acid, acting as both carbon and energy source, is a safe alternative to a carbon dioxide, hydrogen and dioxygen mix for studying the conversion of carbon through the Calvin-Benson-Bassham (CBB) cycle into value-added chemical compounds by non-photosynthetic microorganisms. In this work, organoautotrophic growth of Ralstonia eutropha on formic acid was studied using an approach combining stoichiometric modeling and controlled cultures in bioreactors. A strain deleted of its polyhydroxyalkanoate production pathway was used in order to carry out a physiological characterization. The maximal growth yield was determined at 0.16 Cmole Cmole(-1) in a formate-limited continuous culture. The measured yield corresponded to 76% to 85% of the theoretical yield (later confirmed in pH-controlled fed-batch cultures). The stoichiometric study highlighted the imbalance between carbon and energy provided by formic acid and explained the low growth yields measured. Fed-batch cultures were also used to determine the maximum specific growth rate (μmax  = 0.18 h(-1) ) and to study the impact of increasing formic acid concentrations on growth yields. High formic acid sensitivity was found in R eutropha since a linear decrease in the biomass yield with increasing residual formic acid concentrations was observed between 0 and 1.5 g l(-1) .

  4. Kinetic and stoichiometric characterization of organoautotrophic growth of Ralstonia eutropha on formic acid in fed-batch and continuous cultures

    PubMed Central

    Grunwald, Stephan; Mottet, Alexis; Grousseau, Estelle; Plassmeier, Jens K; Popović, Milan K; Uribelarrea, Jean-Louis; Gorret, Nathalie; Guillouet, Stéphane E; Sinskey, Anthony

    2015-01-01

    Formic acid, acting as both carbon and energy source, is a safe alternative to a carbon dioxide, hydrogen and dioxygen mix for studying the conversion of carbon through the Calvin–Benson–Bassham (CBB) cycle into value-added chemical compounds by non-photosynthetic microorganisms. In this work, organoautotrophic growth of Ralstonia eutropha on formic acid was studied using an approach combining stoichiometric modeling and controlled cultures in bioreactors. A strain deleted of its polyhydroxyalkanoate production pathway was used in order to carry out a physiological characterization. The maximal growth yield was determined at 0.16 Cmole Cmole−1 in a formate-limited continuous culture. The measured yield corresponded to 76% to 85% of the theoretical yield (later confirmed in pH-controlled fed-batch cultures). The stoichiometric study highlighted the imbalance between carbon and energy provided by formic acid and explained the low growth yields measured. Fed-batch cultures were also used to determine the maximum specific growth rate (μmax = 0.18 h−1) and to study the impact of increasing formic acid concentrations on growth yields. High formic acid sensitivity was found in R eutropha since a linear decrease in the biomass yield with increasing residual formic acid concentrations was observed between 0 and 1.5 g l−1. PMID:25123319

  5. Detection of phase-dependent transcriptomic changes and Rubisco-mediated CO2 fixation into poly (3-hydroxybutyrate) under heterotrophic condition in Ralstonia eutropha H16 based on RNA-seq and gene deletion analyses

    PubMed Central

    2013-01-01

    Background Ralstonia eutropha H16 is well known to produce polyhydroxyalkanoates (PHAs), which are potential bio-based biodegradable plastics, in an efficient manner as an energy storage material under unbalanced growth conditions. To obtain further knowledge of PHA biosynthesis, this study performed a quantitative transcriptome analysis based on deep sequencing of the complementary DNA generated from the RNA (RNA-seq) of R. eutropha H16. Results Total RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined using an Illumina high-throughput sequencer. The RNA-seq results indicated the induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in the growth phase; and the repression trends of genes involved in central metabolisms in the PHA production phase. Interestingly, the transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several genes for β-oxidation were significantly induced in the PHA production phase even when the cells were grown on fructose. Moreover, incorporation of 13C was observed in poly(3-hydroxybutyrate) synthesized by R. eutropha H16 from fructose in the presence of NaH13CO3, and further gene deletion analyses revealed that both of the two ribulose 1,5-bisphosphate carboxylase (Rubiscos) in CBB cycle were actually functional in CO2 fixation under the heterotrophic condition. Conclusions The results revealed the phase-dependent transcriptomic changes and a CO2 fixation capability under heterotrophic conditions by PHA-producing R. eutropha. PMID:23879744

  6. Cometabolic degradation of ethyl mercaptan by phenol-utilizing Ralstonia eutropha in suspended growth and gas-recycling trickle-bed reactor.

    PubMed

    Sedighi, Mahsa; Zamir, Seyed Morteza; Vahabzadeh, Farzaneh

    2016-01-01

    The degradability of ethyl mercaptan (EM), by phenol-utilizing cells of Ralstonia eutropha, in both suspended and immobilized culture systems, was investigated in the present study. Free-cells experiments conducted at EM concentrations ranging from 1.25 to 14.42 mg/l, showed almost complete removal of EM at concentrations below 10.08 mg/l, which is much higher than the maximum biodegradable EM concentration obtained in experiments that did not utilize phenol as the primary substrate, i.e. 2.5 mg/l. The first-order kinetic rate constant (kSKS) for EM biodegradation by the phenol-utilizing cells (1.7 l/g biomass/h) was about 10 times higher than by cells without phenol utilization. Immobilized-cells experiments performed in a gas recycling trickle-bed reactor packed with kissiris particles at EM concentrations ranging from 1.6 to 36.9 mg/l, showed complete removal at all tested concentrations in a much shorter time, compared with free cells. The first-order kinetic rate constant (rmaxKs) for EM utilization was 0.04 l/h for the immobilized system compared to 0.06 for the suspended-growth culture, due to external mass transfer diffusion. Diffusion limitation was decreased by increasing the recycling-liquid flow rate from 25 to 65 ml/min. The removed EM was almost completely mineralized according to TOC and sulfate measurements. Shut down and starvation experiments revealed that the reactor could effectively handle the starving conditions and was reliable for full-scale application.

  7. Physiological conditions conducive to high cell density and high cyanophycin content in Ralstonia eutropha strain H16 possessing a KDPG aldolase gene-dependent addiction system.

    PubMed

    Lin, Kaichien; Elbahloul, Yasser; Steinbüchel, Alexander

    2012-03-01

    The recombinant strain of Ralstonia eutropha H16-PHB(-)4-∆eda (pBBR1MCS-2::cphA (6308)/eda (H16)) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH(3), K(2)SO(4), MgSO(4)[Symbol: see text]7H(2)O, Fe(Ш)NH(4)-citrate, CaCl(2)[Symbol: see text]2H(2)O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31 kDa and from 15 to 21 kDa, respectively. High cell densities of up to 82.8 g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.

  8. Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)

    PubMed Central

    Moissenet, Didier; Bidet, Philippe; Garbarg-Chenon, Antoine; Arlet, Guillaume; Vu-Thien, Hoang

    2001-01-01

    Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNAIle and tRNAAla genes, which are identical to genes described for R. pickettii and R. solanacearum. PMID:11136807

  9. Ralstonia paucula (Formerly CDC group IV c-2): unsuccessful strain differentiation with PCR-based methods, study of the 16S-23S spacer of the rRNA operon, and comparison with other Ralstonia species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum).

    PubMed

    Moissenet, D; Bidet, P; Garbarg-Chenon, A; Arlet, G; Vu-Thien, H

    2001-01-01

    Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNA(Ile) and tRNA(Ala) genes, which are identical to genes described for R. pickettii and R. solanacearum.

  10. Altered Composition of Ralstonia eutropha Poly(hydroxyalkanoate) through Expression of PHA Synthase from Allochromatium vinosum ATCC 35206

    USDA-ARS?s Scientific Manuscript database

    The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. We employed a PCR technique coupled with a chromosomal gene-walking method to clone and subsequen...

  11. Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16.

    PubMed

    Pfeiffer, Daniel; Wahl, Andreas; Jendrossek, Dieter

    2011-11-01

    A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1.

  12. Localization of poly(3-hydroxybutyrate) (PHB) granule-associated proteins during PHB granule formation and identification of two new phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16.

    PubMed

    Pfeiffer, Daniel; Jendrossek, Dieter

    2012-11-01

    Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins

  13. The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound [NiFe] Hydrogenase in Ralstonia eutropha H16 ▿ †

    PubMed Central

    Fritsch, Johannes; Lenz, Oliver; Friedrich, Bärbel

    2011-01-01

    The membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha H16 undergoes a complex maturation process comprising cofactor assembly and incorporation, subunit oligomerization, and finally twin-arginine-dependent membrane translocation. Due to its outstanding O2 and CO tolerance, the MBH is of biotechnological interest and serves as a molecular model for a robust hydrogen catalyst. Adaptation of the enzyme to oxygen exposure has to take into account not only the catalytic reaction but also biosynthesis of the intricate redox cofactors. Here, we report on the role of the MBH-specific accessory proteins HoxR and HoxT, which are key components in MBH maturation at ambient O2 levels. MBH-driven growth on H2 is inhibited or retarded at high O2 partial pressure (pO2) in mutants inactivated in the hoxR and hoxT genes. The ratio of mature and nonmature forms of the MBH small subunit is shifted toward the precursor form in extracts derived from the mutant cells grown at high pO2. Lack of hoxR and hoxT can phenotypically be restored by providing O2-limited growth conditions. Analysis of copurified maturation intermediates leads to the conclusion that the HoxR protein is a constituent of a large transient protein complex, whereas the HoxT protein appears to function at a final stage of MBH maturation. UV-visible spectroscopy of heterodimeric MBH purified from hoxR mutant cells points to alterations of the Fe-S cluster composition. Thus, HoxR may play a role in establishing a specific Fe-S cluster profile, whereas the HoxT protein seems to be beneficial for cofactor stability under aerobic conditions. PMID:21441514

  14. Amino acid substitutions in the transcriptional regulator CbbR lead to constitutively active CbbR proteins that elevate expression of the cbb CO2 fixation operons in Ralstonia eutropha (Cupriavidus necator) and identify regions of CbbR necessary for gene activation.

    PubMed

    Dangel, Andrew W; Tabita, F Robert

    2015-09-01

    CbbR is a LysR-type transcriptional regulator that activates expression of the operons containing (cbb) genes that encode the CO2 fixation pathway enzymes in Ralstonia eutropha (Cupriavidus necator) under autotrophic growth conditions. The cbb operons are stringently downregulated during chemoheterotrophic growth on organic acids such as malate. CbbR constitutive proteins (CbbR*s), typically with single amino acid substitutions, were selected and isolated that activate expression of the cbb operons under chemoheterotrophic growth conditions. A large set of CbbR*s from all major domains of the CbbR molecule were identified, except for the DNA-binding domain. The level of gene expression conferred for many of these CbbR*s under autotrophic growth was greater than that conferred by wild-type CbbR. Several of these CbbR*s increase transcription two- to threefold more than wild-type CbbR. One particular CbbR*, a truncated protein, was useful in identifying the regions of CbbR that are necessary for transcriptional activation and, by logical extension, necessary for interaction with RNA polymerase. The reductive assimilation of carbon via CO2 fixation is an important step in the cost-effective production of useful biological compounds. Enhancing CO2 fixation in Ralstonia eutropha through greater transcriptional activation of the cbb operons could prove advantageous, and the use of CbbR*s is one way to enhance product formation.

  15. To be or not to be a poly(3-hydroxybutyrate) (PHB) depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, highly active PHB depolymerases with no detectable role in mobilization of accumulated PHB.

    PubMed

    Sznajder, Anna; Jendrossek, Dieter

    2014-08-01

    The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1 or ΔphaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure.

  16. NUTRITIONAL REQUIREMENTS FOR HYDROGENOMONAS EUTROPHA

    PubMed Central

    Repaske, Roy

    1962-01-01

    Repaske, Roy (National Institute of Allergy and Infectious Diseases, Bethesda, Md.). Nutritional requirements for Hydrogenomonas eutropha. J. Bacteriol. 83: 418–422. 1962.—A simple apparatus for the autotrophic cultivation of Hydrogenomonas eutropha in 100-ml shake cultures is described. Nitrogen, in the form of ammonium, nitrate, or urea, was used for growth; nitrite could not be utilized. Optimal growth occurred at pH 6.4 to 6.8 at 30 C. H. eutropha grew best in an atmosphere containing 15 to 25% oxygen and 10% carbon dioxide. Below these concentrations each of the gases was limiting. Growth was shown to be dependent on iron, and the rate of growth was a function of iron concentration and its state of oxidation. PMID:14491520

  17. Phylogenomic analysis of the genus Ralstonia based on 686 single-copy genes.

    PubMed

    Zhang, Yucheng; Qiu, Sai

    2016-01-01

    The genus Ralstonia contains species that are devastating plant pathogens, opportunistic human pathogens, and/or important degraders of xenobiotic and recalcitrant compounds. However, significant nomenclature problems exist, especially for the Ralstonia solanacearum species complex which consists of four phylotypes. Phylogenomics of the Ralstonia genus was investigated via a comprehensive analysis of 39 Ralstonia genomes as well as four genomes of Cupriavidus necator (more commonly known by its previous name Ralstonia eutropha). These data revealed 686 single-copy orthologs that could be extracted from the Ralstonia core-genome and used to reconstruct the phylogeny of the genus Ralstonia. The generated tree has strong bootstrap support for almost all branches. We also estimated the in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) values between each genome. Our data confirmed that whole genome sequence data provides a powerful tool to resolve the complex taxonomic questions of the genus Ralstonia, e.g. strains of Ralstonia solanacearum phylotype IIA and IIB may represent two subspecies of R. solanacearum, and strains of R. solanacearum phylotype I and III may be classified into two subspecies of Ralstonia pseudosolanacearum. Recently, strains of R. solanacearum phylotype IV were proposed to be reclassified into different subspecies of Ralstonia syzygii; our study, however, showed that phylotype IV strains had high isDDH values (83.8-96.1 %), indicating it may be not appropriate to classify these closely related strains into different subspecies. We also evaluated the performance of six chromosomal housekeeping genes (gdhA, mutS, adk, leuS, rplB and gyrB) used in Ralstonia phylogenetic inference. The multilocus sequence analysis of these six marker genes was able to reliably infer the phylogenetic relationships of the genus Ralstonia.

  18. Properties of a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase with High Specific Activity (PhaZd) in Wautersia eutropha H16

    PubMed Central

    Abe, Tomoko; Kobayashi, Teruyuki; Saito, Terumi

    2005-01-01

    A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases. PMID:16199568

  19. Properties of a novel intracellular poly(3-hydroxybutyrate) depolymerase with high specific activity (PhaZd) in Wautersia eutropha H16.

    PubMed

    Abe, Tomoko; Kobayashi, Teruyuki; Saito, Terumi

    2005-10-01

    A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad (Ser190-Asp266-His330) and oxyanion hole (His108) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T1, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was cloned and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of W. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate oligomers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracellular PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.

  20. Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas eutropha C91: implications for niche adaptation

    SciTech Connect

    Stein, Lisa Y; Arp, D J; Berube, PM; Chain, Patrick S. G.; Hauser, Loren John; Jetten, MSM; Klotz, Martin G; Larimer, Frank W; Norton, Jeanette M.; Op den Camp, HJM; Shin, M; Wei, Xueming

    2007-12-01

    Analysis of the structure and inventory of the genome of Nitrosomonas eutropha C91 revealed distinctive features that may explain the adaptation of N. eutropha-like bacteria to N-saturated ecosystems. Multiple gene-shuffling events are apparent, including mobilized and replicated transposition, as well as plasmid or phage integration events into the 2.66 Mbp chromosome and two plasmids (65 and 56 kbp) of N. eutropha C91. A 117 kbp genomic island encodes multiple genes for heavy metal resistance, including clusters for copper and mercury transport, which are absent from the genomes of other ammonia-oxidizing bacteria (AOB). Whereas the sequences of the two ammonia monooxygenase and three hydroxylamine oxidoreductase gene clusters in N. eutropha C91 are highly similar to those of Nitrosomonas europaea ATCC 19718, a break of synteny in the regions flanking these clusters in each genome is evident. Nitrosomonas eutropha C91 encodes four gene clusters for distinct classes of haem-copper oxidases, two of which are not found in other aerobic AOB. This diversity of terminal oxidases may explain the adaptation of N. eutropha to environments with variable O2 concentrations and/or high concentrations of nitrogen oxides. As with N. europaea, the N. eutropha genome lacks genes for urease metabolism, likely disadvantaging nitrosomonads in low-nitrogen or acidic ecosystems. Taken together, this analysis revealed significant genomic variation between N. eutropha C91 and other AOB, even the closely related N. europaea, and several distinctive properties of the N. eutropha genome that are supportive of niche specialization.

  1. Whole-genome analysis of the ammonia-oxidizing bacterium, Nitrosomonas eutropha C91: implications for niche adaptation.

    PubMed

    Stein, Lisa Y; Arp, Daniel J; Berube, Paul M; Chain, Patrick S G; Hauser, Loren; Jetten, Mike S M; Klotz, Martin G; Larimer, Frank W; Norton, Jeanette M; Op den Camp, Huub J M; Shin, Maria; Wei, Xueming

    2007-12-01

    Analysis of the structure and inventory of the genome of Nitrosomonas eutropha C91 revealed distinctive features that may explain the adaptation of N. eutropha-like bacteria to N-saturated ecosystems. Multiple gene-shuffling events are apparent, including mobilized and replicated transposition, as well as plasmid or phage integration events into the 2.66 Mbp chromosome and two plasmids (65 and 56 kbp) of N. eutropha C91. A 117 kbp genomic island encodes multiple genes for heavy metal resistance, including clusters for copper and mercury transport, which are absent from the genomes of other ammonia-oxidizing bacteria (AOB). Whereas the sequences of the two ammonia monooxygenase and three hydroxylamine oxidoreductase gene clusters in N. eutropha C91 are highly similar to those of Nitrosomonas europaea ATCC 19718, a break of synteny in the regions flanking these clusters in each genome is evident. Nitrosomonas eutropha C91 encodes four gene clusters for distinct classes of haem-copper oxidases, two of which are not found in other aerobic AOB. This diversity of terminal oxidases may explain the adaptation of N. eutropha to environments with variable O(2) concentrations and/or high concentrations of nitrogen oxides. As with N. europaea, the N. eutropha genome lacks genes for urease metabolism, likely disadvantaging nitrosomonads in low-nitrogen or acidic ecosystems. Taken together, this analysis revealed significant genomic variation between N. eutropha C91 and other AOB, even the closely related N. europaea, and several distinctive properties of the N. eutropha genome that are supportive of niche specialization.

  2. Microbial degradation of microcystin-LR by Ralstonia solanacearum.

    PubMed

    Zhang, M L; Yan, H; Pan, G

    2011-12-01

    A bacterial strain was isolated from Lake Dianchi (China) and its degradability and degradative pathways of the cyanobacterial toxin microcystin-LR (MC-LR) were studied. On the basis of morphological, physiological and biochemical tests, the strain was identified as Ralstonia solanacearum. The acute oral toxicity tests showed that Ralstonia solanacearum belongs to a non-toxic class. This bacterium degraded MC-LR at the rate of 9.4 mg/L per day, which was higher than those of the other bacterial strains reported in the literature. As for the degradative pathways, the results showed that the Adda-Arg peptide bond of MC-LR was initially hydrolysed by Ralstonia solanacearum to form a linear molecule as an intermediate. The intermediate product subsequently underwent a cyclisation reaction via dehydration to form a final product with a small peptide ring at one end of the molecule. These biodegradative pathways were different from those reported with other bacterial strains, suggesting that MC-LR may undergo different transformations, and different products were formed due to different compositions of bacteria present in natural lakes and reservoirs. These results suggest that there is a significant potential for Ralstonia solanacearum as a degrader for MC-LR removal from wastewater.

  3. Targeted Enhancement of H2 and CO2 Uptake for Autotrophic Production of Biodiesel in the Lithoautotrophic Bacterium Ralsonia Eutropha

    SciTech Connect

    Eckert, C. A.; Sullivan, R.; Johnson, C.; Yu, J.; Maness, P. C.

    2013-01-01

    CO2 and H2 are promising feedstocks for production of valuable biocompounds. Ralstonia eutropha utilizes these feedstocks to generate energy (ATP) and reductant (NAD(P)H) via oxidation of H2 by a membrane-bound (MBH) and a soluble hydrogenase (SH) for CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle. Increased expression of the enzyme that fixes CO2 (RubisCO) resulted in 6-fold activity improvement in vitro, while increased expression of the MBH operon or the SH operon plus MBH operon maturation factors necessary for activity resulted in a 10-fold enhancement. Current research involves genetic manipulation of two endogenous cbb operons for increased expression, analysis of expression and activity of CBB/MBH/SH, cofactor ratios, and downstream products during autotrophic growth in control versus enhanced strains, and development of strategies for long-term, optimal overexpression. These studies will improve our understanding of autotrophic metabolism and provide a chassis strain for autotrophic production of biodiesel and other valuable carbon biocompounds.

  4. Chemoorganoheterotrophic growth of Nitrosomonas europaea and Nitrosomonas eutropha.

    PubMed

    Schmidt, Ingo

    2009-08-01

    The ammonia oxidizers Nitrosomonas europaea and Nitrosomonas eutropha are able to grow chemoorganotrophically under anoxic conditions with pyruvate, lactate, acetate, serine, succinate, alpha-ketoglutarate, or fructose as substrate and nitrite as terminal electron acceptor. The growth yield of both bacteria is about 3.5 mg protein (mmol pyruvate)(-1) and the maximum growth rates of N. europaea and N. eutropha are 0.094 d(-1) and 0.175 d(-1), respectively. In the presence of pyruvate and CO2 about 80% of the incorporated carbon derives from pyruvate and about 20% from CO2. Pyruvate is used as energy and only carbon source in the absence of CO2 (chemoorganoheterotrophic growth). CO2 stimulates the chemoorganotrophic growth of both ammonia oxidizers and the expression of ribulose bisphosphate carboxylase/oxygenase is down-regulated at increasing CO2 concentration. Ammonium, although required as nitrogen source, is inhibitory for the chemoorganotrophic metabolism of N. europaea and N. eutropha. In the presence of ammonium pyruvate consumption and the expression of the genes aceE, ppc, gltA, odhA, and ppsA (energy conservation) as well as nirK, norB, and nsc (denitrification) are reduced.

  5. Draft Genome Sequence for Ralstonia sp. Strain OR214, a Bacterium with Potential for Bioremediation

    PubMed Central

    Utturkar, Sagar M.; Brzoska, Ryann M.; Klingeman, Dawn M.; Epstein, Slava E.; Palumbo, Anthony V.

    2013-01-01

    Ralstonia sp. strain OR214 belongs to the class Betaproteobacteria and was isolated from subsurface sediments in Oak Ridge, TN. A member of this genus has been described as a potential bioremediation agent. Strain OR214 is tolerant to various heavy metals, such as uranium, nickel, cobalt, and cadmium. We present its draft genome sequence here. PMID:23792748

  6. Ralstonia picketti neonatal sepsis: a case report.

    PubMed

    Sharma, Deepak; Sharma, Pradeep; Soni, Priyanka; Gupta, Basudev

    2017-01-07

    Ralstonia genus are gram negative bacillus and includes four bacteria namely Ralstonia picketti, Ralstonia Solanacearum, Ralstonia insidiosa and Ralstonia mannitolilytica. These are opportunistic pathogens and cause infections in immunocompromised host. The sources of infection are usually contaminated solutions and water. The majority of the reported cases are caused by R. picketti. It is very rare cause of neonatal sepsis with less than twenty cases reported in literature till date. A late preterm male infant, Indian race was admitted to the neonatal intensive care unit for respiratory distress developing soon after birth. The infant was managed with respiratory support and gradually infant improved and diagnosis of transient tachypnea of newborn was made. At age of 84 h of postnatal life, the infant developed features of neonatal sepsis and investigations were suggestive of sepsis. The infant was started on intravenous antibiotic, multiple vasopressors and steroids. The blood culture showed growth of multi-drug resistant R. picketti. The antibiotics were changed as per sensitivity pattern and infant was discharged in good condition and was accepting breast feeding at the time of discharge. There was also no other case of R. picketti in the nursery during the same time period. Ralstonia picketti is an uncommon cause of neonatal sepsis and usually source of infection are contaminated solutions and medical products. The management involves early detection, treatment with appropriate antibiotics and doing surveillance culture to identify the possible source of infection.

  7. Production of polyhydroxyalkanoate (PHA) by Ralstonia eutropha JMP 134 with volatile fatty acids from palm oil mill effluent as precursors.

    PubMed

    Setiadi, Tjandra; Aznury, Martha; Trianto, Azis; Pancoro, Adi

    2015-01-01

    The highest volatile fatty acids (VFAs) concentration from palm oil mill effluent (POME) treated by anaerobic fermentation was achieved for a 1-day process when the main acids used were acetic, propionic and butyric acids. Polyhydroxyalkanoate (PHA) production with VFAs from POME as precursors in the fed-batch mode has advantages over batch mode, both in terms of its productivity and 3HV (3-hydroxyvalerate) composition in the produced polymer. With the fed batch, the productivity increased to 343% and contained more 3HV than those of the batch. The structures of the PHA were identified by different methods and they supported each other; the resulting products consisted of functional groups of 3HB (3-hydroxybutyrate) and 3HV.

  8. Structure of the Catalytic Domain of the Class I Polyhydroxybutyrate Synthase from Cupriavidus necator.

    PubMed

    Wittenborn, Elizabeth C; Jost, Marco; Wei, Yifeng; Stubbe, JoAnne; Drennan, Catherine L

    2016-11-25

    Polyhydroxybutyrate synthase (PhaC) catalyzes the polymerization of 3-(R)-hydroxybutyryl-coenzyme A as a means of carbon storage in many bacteria. The resulting polymers can be used to make biodegradable materials with properties similar to those of thermoplastics and are an environmentally friendly alternative to traditional petroleum-based plastics. A full biochemical and mechanistic understanding of this process has been hindered in part by a lack of structural information on PhaC. Here we present the first structure of the catalytic domain (residues 201-589) of the class I PhaC from Cupriavidus necator (formerly Ralstonia eutropha) to 1.80 Å resolution. We observe a symmetrical dimeric architecture in which the active site of each monomer is separated from the other by ∼33 Å across an extensive dimer interface, suggesting a mechanism in which polyhydroxybutyrate biosynthesis occurs at a single active site. The structure additionally highlights key side chain interactions within the active site that play likely roles in facilitating catalysis, leading to the proposal of a modified mechanistic scheme involving two distinct roles for the active site histidine. We also identify putative substrate entrance and product egress routes within the enzyme, which are discussed in the context of previously reported biochemical observations. Our structure lays a foundation for further biochemical and structural characterization of PhaC, which could assist in engineering efforts for the production of eco-friendly materials. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Improved biovar test for Ralstonia solanacearum

    USDA-ARS?s Scientific Manuscript database

    Race 3, biovar 2 strains of Ralstonia solanacearum are quarantined pathogens in Europe and Canada and Select Agent pathogens in the United States. The biovar classification of R. solanacearum strains is based on their biochemical abilities to utilize a carbohydrate panel. The standard biovar test us...

  10. [Phytotoxic properties of Ralstonia solanacearum lipopolysaccharides].

    PubMed

    Hrytsaĭ, R V; Iakovleva, L M; Varbanets', L D

    2014-01-01

    The study is dedicated to research of phytotoxic properties of Ralstonia solanacearum lipopolysaccharides. This causative agent is one of the most dangerous among potato bacterial diseases. It is revealed that the inhibitory effect of LPS solution on seedlings germination is more noticeable on crops susceptible to brown rot. Maximal total phytotoxic properties have been shown by LPS from strains 35, 52b, TX1 and TS3, which were characterized by relatively low rhamnose content. Relative to the control plants LPS may diminish and some ones--increase the root length, height and weight of seedlings, subject to particular strain. But the stimulation revealed is minor.

  11. Breeding for resistances to Ralstonia solanacearum

    PubMed Central

    Huet, Gaëlle

    2014-01-01

    Ralstonia solanacearum is one of the most devastating bacterial plant pathogens due to its large host range, worldwide geographic distribution and persistence in fields. This soilborne pathogen is the causal agent of bacterial wilt and it can infect major agricultural crops thereby reducing significantly their yield. To favor infection, the bacterium delivers, through the type three secretion system, effectors that manipulate plant immunity. In this review, the relative efficiency of control strategies and existing resistances to R. solanacearum will be presented. Then, the genetic and molecular insights gained from the study of bacterial wilt in model plants will be described. Finally, I will explore how the knowledge gathered from unraveling avirulence and virulence mechanisms of R. solanacearum effectors could help to develop more durable resistances in crop plants toward this destructive pathogen. PMID:25566289

  12. Comparative Analysis of Ralstonia solanacearum Methylomes.

    PubMed

    Erill, Ivan; Puigvert, Marina; Legrand, Ludovic; Guarischi-Sousa, Rodrigo; Vandecasteele, Céline; Setubal, João C; Genin, Stephane; Guidot, Alice; Valls, Marc

    2017-01-01

    Ralstonia solanacearum is an important soil-borne plant pathogen with broad geographical distribution and the ability to cause wilt disease in many agriculturally important crops. Genome sequencing of multiple R. solanacearum strains has identified both unique and shared genetic traits influencing their evolution and ability to colonize plant hosts. Previous research has shown that DNA methylation can drive speciation and modulate virulence in bacteria, but the impact of epigenetic modifications on the diversification and pathogenesis of R. solanacearum is unknown. Sequencing of R. solanacearum strains GMI1000 and UY031 using Single Molecule Real-Time technology allowed us to perform a comparative analysis of R. solanacearum methylomes. Our analysis identified a novel methylation motif associated with a DNA methylase that is conserved in all complete Ralstonia spp. genomes and across the Burkholderiaceae, as well as a methylation motif associated to a phage-borne methylase unique to R. solanacearum UY031. Comparative analysis of the conserved methylation motif revealed that it is most prevalent in gene promoter regions, where it displays a high degree of conservation detectable through phylogenetic footprinting. Analysis of hyper- and hypo-methylated loci identified several genes involved in global and virulence regulatory functions whose expression may be modulated by DNA methylation. Analysis of genome-wide modification patterns identified a significant correlation between DNA modification and transposase genes in R. solanacearum UY031, driven by the presence of a high copy number of ISrso3 insertion sequences in this genome and pointing to a novel mechanism for regulation of transposition. These results set a firm foundation for experimental investigations into the role of DNA methylation in R. solanacearum evolution and its adaptation to different plants.

  13. Comparative Analysis of Ralstonia solanacearum Methylomes

    PubMed Central

    Erill, Ivan; Puigvert, Marina; Legrand, Ludovic; Guarischi-Sousa, Rodrigo; Vandecasteele, Céline; Setubal, João C.; Genin, Stephane; Guidot, Alice; Valls, Marc

    2017-01-01

    Ralstonia solanacearum is an important soil-borne plant pathogen with broad geographical distribution and the ability to cause wilt disease in many agriculturally important crops. Genome sequencing of multiple R. solanacearum strains has identified both unique and shared genetic traits influencing their evolution and ability to colonize plant hosts. Previous research has shown that DNA methylation can drive speciation and modulate virulence in bacteria, but the impact of epigenetic modifications on the diversification and pathogenesis of R. solanacearum is unknown. Sequencing of R. solanacearum strains GMI1000 and UY031 using Single Molecule Real-Time technology allowed us to perform a comparative analysis of R. solanacearum methylomes. Our analysis identified a novel methylation motif associated with a DNA methylase that is conserved in all complete Ralstonia spp. genomes and across the Burkholderiaceae, as well as a methylation motif associated to a phage-borne methylase unique to R. solanacearum UY031. Comparative analysis of the conserved methylation motif revealed that it is most prevalent in gene promoter regions, where it displays a high degree of conservation detectable through phylogenetic footprinting. Analysis of hyper- and hypo-methylated loci identified several genes involved in global and virulence regulatory functions whose expression may be modulated by DNA methylation. Analysis of genome-wide modification patterns identified a significant correlation between DNA modification and transposase genes in R. solanacearum UY031, driven by the presence of a high copy number of ISrso3 insertion sequences in this genome and pointing to a novel mechanism for regulation of transposition. These results set a firm foundation for experimental investigations into the role of DNA methylation in R. solanacearum evolution and its adaptation to different plants. PMID:28450872

  14. Arsenite Oxidase from Ralstonia sp. 22

    PubMed Central

    Lieutaud, Aurélie; van Lis, Robert; Duval, Simon; Capowiez, Line; Muller, Daniel; Lebrun, Régine; Lignon, Sabrina; Fardeau, Marie-Laure; Lett, Marie-Claire; Nitschke, Wolfgang; Schoepp-Cothenet, Barbara

    2010-01-01

    We characterized the aro arsenite oxidation system in the novel strain Ralstonia sp. 22, a β-proteobacterium isolated from soil samples of the Salsigne mine in southern France. The inducible aro system consists of a heterodimeric membrane-associated enzyme reacting with a dedicated soluble cytochrome c554. Our biochemical results suggest that the weak association of the enzyme to the membrane probably arises from a still unknown interaction partner. Analysis of the phylogeny of the aro gene cluster revealed that it results from a lateral gene transfer from a species closely related to Achromobacter sp. SY8. This constitutes the first clear cut case of such a transfer in the Aro phylogeny. The biochemical study of the enzyme demonstrates that it can accommodate in vitro various cytochromes, two of which, c552 and c554, are from the parent species. Cytochrome c552 belongs to the sox and not the aro system. Kinetic studies furthermore established that sulfite and sulfide, substrates of the sox system, are both inhibitors of Aro activity. These results reinforce the idea that sulfur and arsenic metabolism are linked. PMID:20421652

  15. An analysis of the changes in soluble hydrogenase and global gene expression in Cupriavidus necator (Ralstonia eutropha) H16 grown in heterotrophic diauxic batch culture.

    PubMed

    Jugder, Bat-Erdene; Chen, Zhiliang; Ping, Darren Tan Tek; Lebhar, Helene; Welch, Jeffrey; Marquis, Christopher P

    2015-03-25

    Soluble hydrogenases (SH) are enzymes that catalyse the oxidation of molecular hydrogen. The SH enzyme from Cupriavidus necator H16 is relatively oxygen tolerant and makes an attractive target for potential application in biochemical hydrogen fuel cells. Expression of the enzyme can be mediated by derepression of the hox promoter system under heterotrophic conditions. However, the overall impact of hox derepression, from a transcriptomic perspective, has never been previously reported. Derepression of hydrogenase gene expression upon fructose depletion was confirmed in replicate experiments. Using qRT-PCR, hoxF was 4.6-fold up-regulated, hypF2 was up-regulated in the cells grown 2.2-fold and the regulatory gene hoxA was up-regulated by a mean factor of 4.5. A full transcriptomic evaluation revealed a substantial shift in the global pattern of gene expression. In addition to up-regulation of genes associated with hydrogenase expression, significant changes were observed in genes associated with energy transduction, amino acid metabolism, transcription and translation (and regulation thereof), genes associated with cell stress, lipid and cell wall biogenesis and other functions, including cell motility. We report the first full transcriptome analysis of C. necator H16 grown heterotrophically on fructose and glycerol in diauxic batch culture, which permits expression of soluble hydrogenase under heterotrophic conditions. The data presented deepens our understanding of the changes in global gene expression patterns that occur during the switch to growth on glycerol and suggests that energy deficit is a key driver for induction of hydrogenase expression in this organism.

  16. Polyphenol Oxidase Activity Expression in Ralstonia solanacearum

    PubMed Central

    Hernández-Romero, Diana; Solano, Francisco; Sanchez-Amat, Antonio

    2005-01-01

    Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to l-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds. PMID:16269713

  17. Susceptibility of Geranium Cultivars (Pelargonium spp.) to Ralstonia solanacearum

    USDA-ARS?s Scientific Manuscript database

    Sixty-one cultivars of geraniums including zonal, regal, ivy, and scented were tested for susceptibility to three strains of Ralstonia solanacearum: a Race 1 Biovar 1 (R1B1) strain P597 isolated from tomato in Florida, a R1B1 strain P673 obtained from pothos originating in Costa Rica, and a Race 3 B...

  18. Genotypic and phenotypic diversity of Ralstonia pickettii and Ralstonia insidiosa isolates from clinical and environmental sources including High-purity Water. Diversity in Ralstonia pickettii.

    PubMed

    Ryan, Michael P; Pembroke, J Tony; Adley, Catherine C

    2011-08-30

    Ralstonia pickettii is a nosocomial infectious agent and a significant industrial contaminant. It has been found in many different environments including clinical situations, soil and industrial High Purity Water. This study compares the phenotypic and genotypic diversity of a selection of strains of Ralstonia collected from a variety of sources. Ralstonia isolates (fifty-nine) from clinical, industrial and environmental origins were compared genotypically using i) Species-specific-PCR, ii) PCR and sequencing of the 16S-23S rRNA Interspatial region (ISR) iii) the fliC gene genes, iv) RAPD and BOX-PCR and v) phenotypically using biochemical testing. The species specific-PCR identified fifteen out of fifty-nine designated R. pickettii isolates as actually being the closely related species R. insidiosa. PCR-ribotyping of the 16S-23S rRNA ISR indicated few major differences between the isolates. Analysis of all isolates demonstrated different banding patterns for both the RAPD and BOX primers however these were found not to vary significantly. R. pickettii species isolated from wide geographic and environmental sources appear to be reasonably homogenous based on genotypic and phenotypic characteristics. R. insidiosa can at present only be distinguished from R. pickettii using species specific PCR. R. pickettii and R. insidiosa isolates do not differ significantly phenotypically or genotypically based on environmental or geographical origin.

  19. Polyphasic taxonomic revision of the Ralstonia solanacearum species complex: proposal to emend the descriptions of Ralstonia solanacearum and Ralstonia syzygii and reclassify current R. syzygii strains as Ralstonia syzygii subsp. syzygii subsp. nov., R. solanacearum phylotype IV strains as Ralstonia syzygii subsp. indonesiensis subsp. nov., banana blood disease bacterium strains as Ralstonia syzygii subsp. celebesensis subsp. nov. and R. solanacearum phylotype I and III strains as Ralstonia pseudosolanacearum sp. nov.

    PubMed

    Safni, Irda; Cleenwerck, Ilse; De Vos, Paul; Fegan, Mark; Sly, Lindsay; Kappler, Ulrike

    2014-09-01

    The Ralstonia solanacearum species complex has long been recognized as a group of phenotypically diverse strains that can be subdivided into four phylotypes. Using a polyphasic taxonomic approach on an extensive set of strains, this study provides evidence for a taxonomic and nomenclatural revision of members of this complex. Data obtained from phylogenetic analysis of 16S-23S rRNA ITS gene sequences, 16S-23S rRNA intergenic spacer (ITS) region sequences and partial endoglucanase (egl) gene sequences and DNA-DNA hybridizations demonstrate that the R. solanacearum species complex comprises three genospecies. One of these includes the type strain of Ralstonia solanacearum and consists of strains of R. solanacearum phylotype II only. The second genospecies includes the type strain of Ralstonia syzygii and contains only phylotype IV strains. This genospecies is subdivided into three distinct groups, namely R. syzygii, the causal agent of Sumatra disease on clove trees in Indonesia, R. solanacearum phylotype IV strains isolated from different host plants mostly from Indonesia, and strains of the blood disease bacterium (BDB), the causal agent of the banana blood disease, a bacterial wilt disease in Indonesia that affects bananas and plantains. The last genospecies is composed of R. solanacearum strains that belong to phylotypes I and III. As these genospecies are also supported by phenotypic data that allow the differentiation of the three genospecies, the following taxonomic proposals are made: emendation of the descriptions of Ralstonia solanacearum and Ralstonia syzygii and descriptions of Ralstonia syzygii subsp. nov. (type strain R 001(T) = LMG 10661(T) = DSM 7385(T)) for the current R. syzygii strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (type strain UQRS 464(T) = LMG 27703(T) = DSM 27478(T)) for the current R. solanacearum phylotype IV strains, Ralstonia syzygii subsp. celebesensis subsp. nov. (type strain UQRS 627(T

  20. Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum.

    PubMed

    Kai, Kenji; Ohnishi, Hideyuki; Kiba, Akinori; Ohnishi, Kouhei; Hikichi, Yasufumi

    2016-01-01

    Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners.

  1. Comparative study of promoters for the production of polyhydroxyalkanoates in recombinant strains of Wautersia eutropha.

    PubMed

    Delamarre, Soazig C; Batt, Carl A

    2006-08-01

    Recombinant strains of Wautersia eutropha expressing an artificial polyhydroxyalkanoate (PHA) biosynthesis operon under the control of different native promoters linked to polyhydroxybutyrate (PHB) (P(phb)), acetoin (P(acoE), P(acoD), and P(acoX)) or pyruvate (P(pdhE)) metabolism were constructed and tested. The promoters were representative either of the enterobacterial sigma70 (P(phb), P(acoE), and P(pdhE))- or sigma54 (P(acoD) and P(acoX))-dependent promoters. To obtain polymers consisting of C4-C12 monomer units, an artificial operon consisting of the PHA synthase gene from Pseudomonas sp. 61-3 (phaC1 (Ps)) tandemly linked to the W. eutropha genes encoding beta-ketothiolase (phbA (We)) and nicotinamide adenine dinucleotide phosphate dependent acetoacetyl-coenzyme A (CoA) reductase (phbB (We)) was constructed. All recombinant strains produced PHA, indicating that the PHA biosynthesis genes were expressed under the control of the different promoters. Cell growth and PHA synthesis on MS medium complemented with gluconate or octanoate, and different concentrations of acetoin (0, 0.15, and 0.3%) clearly differed among the recombinant strains. While the P(acoD) and P(acoX) promoters mediated only low PHA yields (<1%) in the presence of the inducer acetoin, the remaining promoters-independent of the addition of acetoin-resulted in the production of PHA polymers with high 3HB fractions (90-100 mol%) and with high 3HO contents (70-86 mol%) from gluconate and octanoate, respectively. Interestingly, on octanoate-MS medium with 0.15% acetoin, the P(acoE) promoter mediated the synthesis of PHA with a relatively high 3HB fraction (48 mol%). While PHAs with high 3HB contents were obtained, the overall PHA product yields were low (<10%); thus, their potential application for further commercial exploitation appears limited.

  2. Modeling the low pH limit of Nitrosomonas eutropha in high-strength nitrogen wastewaters.

    PubMed

    Fumasoli, Alexandra; Morgenroth, Eberhard; Udert, Kai M

    2015-10-15

    In wastewater treatment, the rate of ammonia oxidation decreases with pH and stops very often slightly below a pH of 6. Free ammonia (NH3) limitation, inhibition by nitrous acid (HNO2), limitation by inorganic carbon or direct effect of high proton concentrations have been proposed to cause the rate decrease with pH as well as the cessation of ammonia oxidation. In this study, we compare an exponential pH term common for food microbiology with conventionally applied rate laws based on Monod-type kinetics for NH3 limitation and non-competitive HNO2 inhibition as well as sigmoidal pH functions to model the low pH limit of ammonia oxidizing bacteria (AOB). For this purpose we conducted well controlled batch experiments which were then simulated with a computer model. The results showed that kinetics based on NH3 limitation and HNO2 inhibition can explain the rate decrease of ammonia oxidation between pH 7 and 6, but fail in predicting the pH limit of Nitrosomonas eutropha at pH 5.4 and rates close to that limit. This is where the exponential pH term becomes important: this term decreases the rate of ammonia oxidation to zero, as the pH limit approaches. Previously proposed sigmoidal pH functions that affect large pH regions, however, led to an overestimation of the pH effect and could therefore not be applied successfully. We show that the proposed exponential pH term can be explained quantitatively with thermodynamic principles: at low pH values, the energy available from the proton motive force is too small for the NADH production in Nitrosomonas eutropha and related AOB causing an energy limited state of the bacterial cell. Hence, energy limitation and not inhibition or limitation of enzymes is responsible for the cessation of the AOB activity at low pH values.

  3. Native Valve Endocarditis due to Ralstonia pickettii: A Case Report and Literature Review

    PubMed Central

    Orme, Joseph; Rivera-Bonilla, Tomas; Loli, Akil; Blattman, Negin N.

    2015-01-01

    Ralstonia pickettii is a rare pathogen and even more rare in healthy individuals. Here we report a case of R. pickettii bacteremia leading to aortic valve abscess and complete heart block. To our knowledge this is the first case report of Ralstonia species causing infective endocarditis with perivalvular abscess. PMID:25648998

  4. Native Valve Endocarditis due to Ralstonia pickettii: A Case Report and Literature Review.

    PubMed

    Orme, Joseph; Rivera-Bonilla, Tomas; Loli, Akil; Blattman, Negin N

    2015-01-01

    Ralstonia pickettii is a rare pathogen and even more rare in healthy individuals. Here we report a case of R. pickettii bacteremia leading to aortic valve abscess and complete heart block. To our knowledge this is the first case report of Ralstonia species causing infective endocarditis with perivalvular abscess.

  5. prhKLM genes of Ralstonia solanacearum encode novel activators of hrp regulon and are required for pathogenesis in tomato.

    PubMed

    Zhang, Yong; Kiba, Akinori; Hikichi, Yasufumi; Ohnishi, Kouhei

    2011-04-01

    The genes in the hrp regulon encode the proteins composing type III secretion system in Ralstonia solanacearum. The hrp regulon is positively controlled by HrpB, and hrpB expression is activated by both HrpG and PrhG. We have identified three genes, prhK, prhL, and prhM, which positively control the hrp regulon in strain OE1-1. These genes are likely to form an operon, and this operon is well conserved in the genera Ralstonia and Burkholderia. This indicates that the operon is not specific to the plant pathogens. Mutations in each of these three genes abolished hrpB and prhG expression. prhK, prhL, and prhM mutant strains lost pathogenicity toward tomato completely, and they were less virulent toward tobacco. PrhK and PrhL share sequence similarity with allophanate hydrolase and PrhM with LamB. This suggests that the three gene products are not transcriptional regulators in the strict sense, but regulate hrp regulon indirectly. This novel class of virulence-related genes will mark the beginning of new findings regarding the overall infection mode of R. solanacearum.

  6. Ralstonia pickettii-Induced Ataxia in Immunodeficient Mice

    PubMed Central

    Berard, Marion; Medaille, Christine; Simon, Meredith; Serre, Stéphanie; Pritchett-Corning, Kathleen; Dangles-Marie, Virginie

    2009-01-01

    We report here the characterization of an asymmetric ataxia syndrome (head tilt and circling, with death in the most severe cases) demonstrated by profoundly immunodeficient mice housed at the Institut Curie SPF facility. The immune system of the affected mice had been genetically modified so that they were deficient in both B and T cells. Extensive bacteriologic, parasitic, serologic, and histopathologic analysis of the affected animals and their healthy controls led us to identify Ralstonia pickettii as the causative agent of the ataxic syndrome. The outbreak was managed through a test-and-cull process. Even though they also carried Ralstonia pickettii, immunocompetent mice that were kept in the same facility, did not show any of the signs that were expressed by their immunodeficient counterparts. This case highlights the difficulty of maintaining immunocompetent and immunodeficient mice in the same microbiologic unit and the importance of enlarging the spectrum of health monitoring to opportunistic agents when investigating clinical cases in populations of immunocompromised rodents. PMID:19389312

  7. Antibacterial activity against Ralstonia solanacearum of phenolic constituents isolated from dragon's blood.

    PubMed

    Wang, Hui; Luo, Ying; Dai, Haofu; Mei, Wenli

    2013-03-01

    Ralstonia solanacearum is a worldwide and devastating plant pathogen infesting over 200 host species. Synthetic bactericides against the pathogen have only achieved limited success and always cause both crop contamination and environmental pollution. However, natural bactericides are effective for protecting cultivated crops from destruction by disease, without the adverse effects of chemical bactericides. In this paper, fifteen phenolic constituents from dragon's blood were screened for their antimicrobial activity against Ralstonia solanacearum, and all exhibited inhibitory activity. These compounds are potential leading compounds for the development of bactericides against wilt diseases caused by Ralstonia solanacearum.

  8. Ralstonia solanacearum: secrets of a major pathogen unveiled by analysis of its genome.

    PubMed

    Genin, Stéphane; Boucher, Christian

    2002-05-01

    Summary Ralstonia solanacearum Taxonomy: Bacteria; Proteobacteria; beta subdivision; Ralstonia group; genus Ralstonia Microbiological properties: Gram-negative, aerobic, motile rod. Disease symptoms: Agent of bacterial wilt of solanaceous plants, which appears as a sudden wilt. Typically, stem cross-sections ooze a whitish bacterial exudate. R. solanacearum is also the agent of the Moko disease of banana and brown rot of potato. Disease control: Pathogen-free seed and transplants. Few resistant and tolerant plant lines. Sanitation and cultural rotations. Useful web sites: http://ibws.nexenservices.com/;http://sequence.toulouse.inra.fr/R.solanacearum.html.

  9. TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity

    PubMed Central

    Schandry, Niklas; de Lange, Orlando; Prior, Philippe; Lahaye, Thomas

    2016-01-01

    Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47–91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other’s EBEs. Investigation of sequence divergence

  10. TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity.

    PubMed

    Schandry, Niklas; de Lange, Orlando; Prior, Philippe; Lahaye, Thomas

    2016-01-01

    Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47-91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other's EBEs. Investigation of sequence divergence between

  11. Natural products from environmental DNA hosted in Ralstonia metallidurans.

    PubMed

    Craig, Jeffrey W; Chang, Fang-Yuan; Brady, Sean F

    2009-01-16

    Metagenomic studies designed to access new small molecules from the heterologous expression of environmental DNA have focused on the use of two model systems, Escherichia coli and Streptomyces spp., as heterologous hosts. Accessing the biosynthetic potential of DNA extracted from the bacteria present in environmental samples will require the development of a more diverse collection of model bacterial hosts that can be used for screening environmental DNA libraries. In this study the bacterium Ralstonia metallidurans was explored as a heterologous host. Here we report the isolation and characterization of both novel and known metabolites from pigmented and antibacterially active clones found in R. metallidurans based environmental DNA libraries. The clones found in this study do not confer the production of clone-specific metabolites to E. coli, validating R. metallidurans as an orthogonal expression host that can be used to expand the number of metabolites found in future metagenomic discovery efforts.

  12. Ralfuranone thioether production by the plant pathogen Ralstonia solanacearum.

    PubMed

    Pauly, Julia; Spiteller, Dieter; Linz, Jeanine; Jacobs, Jonathan; Allen, Caitilyn; Nett, Markus; Hoffmeister, Dirk

    2013-11-04

    Ralfuranones are aryl-substituted furanone secondary metabolites of the Gram-negative plant pathogen Ralstonia solanacearum. New sulfur-containing ralfuranone derivatives were identified, including the methyl thioether-containing ralfuranone D. Isotopic labeling in vivo, as well as headspace analyses of volatiles from R. solanacearum liquid cultures, established a mechanism for the transfer of an intact methylthio group from L-methionine or α-keto-γ-methylthiobutyric acid. The methylthio acceptor molecule ralfuranone I, a previously postulated biosynthetic intermediate in ralfuranone biosynthesis, was isolated and characterized by NMR. The highly reactive Michael acceptor system of this intermediate readily reacts with various thiols, including glutathione. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Synergistic interaction in dual-species biofilms formation by Escherichia coli O157:H7 and Ralstonia spp

    USDA-ARS?s Scientific Manuscript database

    Introduction: Ralstonia spp., a heterotrophic bacterium that are isolated from produce processing environments as part of the native microflora, have strong potentials for formaing biofilms on various surfaces. When co-cultured, Escherichia coli O157:H7 (EcO157) and Ralstonia spp. displayed a synerg...

  14. Roles of different forms of lipopolysaccharides in Ralstonia solanacearum pathogenesis.

    PubMed

    Li, Chien-Hui; Wang, Kuan-Chung; Hong, Yu-Hau; Chu, Tai-Hsiang; Chu, Yu-Ju; Chou, I-Chun; Lu, Der-Kang; Chen, Chiao-Yen; Yang, Wen-Chieh; Lin, Yu-Mei; Cheng, Chiu-Ping

    2014-05-01

    Lipopolysaccharides (LPS) are critical components for the fitness of most gram-negative bacteria. Ralstonia solanacearum causes a deadly wilting disease in many crops; however, the pathogenic roles of different forms of LPS and their pathways of biogenesis remain unknown. By screening for phage-resistant mutants of R. solanacearum Pss4, whose genome sequence is unavailable, mutants with various types of structural defects in LPS were isolated. Pathogenesis assays of the mutants revealed that production of rough LPS (R-LPS), which does not contain O-polysaccharides, was sufficient to cause necrosis on Nicotiana benthamiana and induce the hypersensitive response on N. tabacum. However, biosynthesis of smooth LPS (S-LPS), which contains O-polysaccharides, was required for bacterial proliferation at infection sites on N. benthamiana leaves and for proliferation and causing wilt on tomato. Complementation tests confirmed the involvement of the previously unidentified cluster RSc2201 to RSc2204 in the formation of R. solanacearum S-LPS. With these data and the availability of the annotated genomic sequence of strain GMI1000, certain loci involved in key steps of R. solanacearum LPS biosynthesis were identified. The strategy of this work could be useful for similar studies in other bacteria without available genome sequences.

  15. Deciphering phenotypic diversity of Ralstonia solanacearum strains pathogenic to potato.

    PubMed

    Cellier, G; Prior, P

    2010-11-01

    Based on the phylotype classification, we questioned how genetically and phenotypically diverse strains of Ralstonia solanacearum pathogenic to potato may be. We studied 129 European and Mediterranean strains along with 57 reference strains known to cover genetic diversity in this species. Phylogeny analysis was done on endoglucanase gene sequences. Pathogenicity to potato, tomato, and eggplant was established at 24 to 30°C and 15 to 24°C, whereas tests on banana were conducted at 24 to 30°C. The ability to cause wilt on species of Solanaceae was shared by strains in all four phylotypes. Brown rot phylotypes IIB-1 and IIB-2 and phylotype IIB-27 established latent infections in banana, and Moko disease-causing phylotypes IIA-6, IIB-3, and IIB-4 were virulent to susceptible potato and tomato, addressing the question of host adaptation mechanisms, which may have undergone a similar bottleneck evolution. Cold-tolerance ability is only shared on species of Solanaceae among brown rot phylotype IIB-1, which gathered the majority of European and Mediterranean strains. We surveyed strain LNPV24.25 as the first report of an emerging phylotype IIB-4NPB strain in France. These findings showed that pathogenicity traits of genetically identified strains still need to be understood, especially in the perspective of post-genomics comparative analysis, to understand bacterial speciation in the R. solanacearum species complex.

  16. Molecular Diversity of Ralstonia solanacearum Isolated from Ginger in Hawaii.

    PubMed

    Yu, Q; Alvarez, A M; Moore, P H; Zee, F; Kim, M S; de Silva, A; Hepperly, P R; Ming, R

    2003-09-01

    ABSTRACT The genetic diversity of Ralstonia solanacearum strains isolated from ginger (Zingiber officinale) growing on the island of Hawaii was determined by analysis of amplified fragment length polymorphisms (AFLPs). Initially 28 strains of R. solanacearum collected from five host plant species worldwide were analyzed by AFLP. A second analysis was conducted on 55 R. solanacearum strains collected from three ginger farms along the Hamakua Coast of Hawaii, the principle area of ginger cultivation in the state. From the initial analysis, R. solanacearum strains from ginger in Hawaii showed a high degree of similarity at 0.853. In contrast, the average genetic similarity between R. solanacearum strains from heliconia and ginger was only 0.165, and strains from ginger showed little similarity with strains from all other hosts. The second analysis of 55 strains from ginger on different Hawaiian farms confirmed that they were distinct from race 1 strains from tomato. Strains from ginger also showed greater diversity among themselves in the second analysis, and the greatest diversity occurred among strains from a farm where ginger is frequently imported and maintained. Our results provide evidence that R. solanacearum strains from ginger in Hawaii are genetically distinct from local strains from tomato (race 1) and heliconia (race 2).

  17. Genomic characterization of Ralstonia solanacearum phage ϕRS138 of the family Siphoviridae.

    PubMed

    Van Truong Thi, Bich; Pham Khanh, Nguyen Huan; Namikawa, Ryuta; Miki, Kaito; Kondo, Akihiro; Dang Thi, Phuong Thao; Kamei, Kaeko

    2016-02-01

    ϕRS138, a bacteriophage of the family Siphoviridae that lyses Ralstonia solanacearum, was isolated. The genomic DNA of ϕRS138 was 41,941 bp long with a GC content of 65.1 % and contained 56 putative open reading frames. The ϕRS138 genome could be divided into three regions based on similarities to other genomes: (1) a region containing genes encoding a putative transcriptional regulator and an integrase, similar to the prophage genes in Ralstonia solanacearum K60-1; (2) a region encoding proteins related to structural modules and virion morphogenesis, similar to genes in the Pseudomonas phages of the family Siphoviridae; and (3) a region highly similar to the genomes of other Ralstonia solanacearum strains.

  18. Novel Tn4371-ICE like element in Ralstonia pickettii and Genome mining for comparative elements

    PubMed Central

    2009-01-01

    Background Integrative Conjugative Elements (ICEs) are important factors in the plasticity of microbial genomes. An element related to the ICE Tn4371 was discovered during a bioinformatic search of the Ralstonia pickettii 12J genome. This element was analysed and further searches carried out for additional elements. A PCR method was designed to detect and characterise new elements of this type based on this scaffold and a culture collection of fifty-eight Ralstonia pickettii and Ralstonia insidiosa strains were analysed for the presence of the element. Results Comparative sequence analysis of bacterial genomes has revealed the presence of a number of uncharacterised Tn4371-like ICEs in the genomes of several β and γ- Proteobacteria. These elements vary in size, GC content, putative function and have a mosaic-like structure of plasmid- and phage-like sequences which is typical of Tn4371-like ICEs. These elements were found after a through search of the GenBank database. The elements, which are found in Ralstonia, Delftia, Acidovorax, Bordetella, Comamonas, Acidovorax, Congregibacter, Shewanella, Pseudomonas Stenotrophomonas, Thioalkalivibrio sp. HL-EbGR7, Polaromonas, Burkholderia and Diaphorobacter sp. share a common scaffold. A PCR method was designed (based on the Tn4371- like element detected in the Ralstonia pickettii 12J genome) to detect and characterise new elements of this type. Conclusion All elements found in this study possess a common scaffold of core genes but contain different accessory genes. A new uniform nomenclature is suggested for ICEs of the Tn4371 family. Two novel Tn4371-like ICE were discovered and characterised, using the novel PCR method described in two different isolates of Ralstonia pickettii from laboratory purified water. PMID:19941653

  19. Novel Tn4371-ICE like element in Ralstonia pickettii and genome mining for comparative elements.

    PubMed

    Ryan, Michael P; Pembroke, J Tony; Adley, Catherine C

    2009-11-26

    Integrative Conjugative Elements (ICEs) are important factors in the plasticity of microbial genomes. An element related to the ICE Tn4371 was discovered during a bioinformatic search of the Ralstonia pickettii 12J genome. This element was analysed and further searches carried out for additional elements.A PCR method was designed to detect and characterise new elements of this type based on this scaffold and a culture collection of fifty-eight Ralstonia pickettii and Ralstonia insidiosa strains were analysed for the presence of the element. Comparative sequence analysis of bacterial genomes has revealed the presence of a number of uncharacterised Tn4371-like ICEs in the genomes of several beta and gamma- Proteobacteria. These elements vary in size, GC content, putative function and have a mosaic-like structure of plasmid- and phage-like sequences which is typical of Tn4371-like ICEs. These elements were found after a through search of the GenBank database. The elements, which are found in Ralstonia, Delftia, Acidovorax, Bordetella, Comamonas, Acidovorax, Congregibacter, Shewanella, Pseudomonas Stenotrophomonas, Thioalkalivibrio sp. HL-EbGR7, Polaromonas, Burkholderia and Diaphorobacter sp. share a common scaffold. A PCR method was designed (based on the Tn4371- like element detected in the Ralstonia pickettii 12J genome) to detect and characterise new elements of this type. All elements found in this study possess a common scaffold of core genes but contain different accessory genes. A new uniform nomenclature is suggested for ICEs of the Tn4371 family. Two novel Tn4371-like ICE were discovered and characterised, using the novel PCR method described in two different isolates of Ralstonia pickettii from laboratory purified water.

  20. Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease.

    PubMed

    de Lange, Orlando; Schreiber, Tom; Schandry, Niklas; Radeck, Jara; Braun, Karl Heinz; Koszinowski, Julia; Heuer, Holger; Strauß, Annett; Lahaye, Thomas

    2013-08-01

    Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs. Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants. Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5' terminal guanine, contrasting with the TALE preference for a 5' thymine. Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  1. Transcriptomes of Ralstonia solanacearum during Root Colonization of Solanum commersonii

    PubMed Central

    Puigvert, Marina; Guarischi-Sousa, Rodrigo; Zuluaga, Paola; Coll, Núria S.; Macho, Alberto P.; Setubal, João C.; Valls, Marc

    2017-01-01

    Bacterial wilt of potatoes—also called brown rot—is a devastating disease caused by the vascular pathogen Ralstonia solanacearum that leads to significant yield loss. As in other plant-pathogen interactions, the first contacts established between the bacterium and the plant largely condition the disease outcome. Here, we studied the transcriptome of R. solanacearum UY031 early after infection in two accessions of the wild potato Solanum commersonii showing contrasting resistance to bacterial wilt. Total RNAs obtained from asymptomatic infected roots were deep sequenced and for 4,609 out of the 4,778 annotated genes in strain UY031 were recovered. Only 2 genes were differentially-expressed between the resistant and the susceptible plant accessions, suggesting that the bacterial component plays a minor role in the establishment of disease. On the contrary, 422 genes were differentially expressed (DE) in planta compared to growth on a synthetic rich medium. Only 73 of these genes had been previously identified as DE in a transcriptome of R. solanacearum extracted from infected tomato xylem vessels. Virulence determinants such as the Type Three Secretion System (T3SS) and its effector proteins, motility structures, and reactive oxygen species (ROS) detoxifying enzymes were induced during infection of S. commersonii. On the contrary, metabolic activities were mostly repressed during early root colonization, with the notable exception of nitrogen metabolism, sulfate reduction and phosphate uptake. Several of the R. solanacearum genes identified as significantly up-regulated during infection had not been previously described as virulence factors. This is the first report describing the R. solanacearum transcriptome directly obtained from infected tissue and also the first to analyze bacterial gene expression in the roots, where plant infection takes place. We also demonstrate that the bacterial transcriptome in planta can be studied when pathogen numbers are low by

  2. Transcriptomes of Ralstonia solanacearum during Root Colonization of Solanum commersonii.

    PubMed

    Puigvert, Marina; Guarischi-Sousa, Rodrigo; Zuluaga, Paola; Coll, Núria S; Macho, Alberto P; Setubal, João C; Valls, Marc

    2017-01-01

    Bacterial wilt of potatoes-also called brown rot-is a devastating disease caused by the vascular pathogen Ralstonia solanacearum that leads to significant yield loss. As in other plant-pathogen interactions, the first contacts established between the bacterium and the plant largely condition the disease outcome. Here, we studied the transcriptome of R. solanacearum UY031 early after infection in two accessions of the wild potato Solanum commersonii showing contrasting resistance to bacterial wilt. Total RNAs obtained from asymptomatic infected roots were deep sequenced and for 4,609 out of the 4,778 annotated genes in strain UY031 were recovered. Only 2 genes were differentially-expressed between the resistant and the susceptible plant accessions, suggesting that the bacterial component plays a minor role in the establishment of disease. On the contrary, 422 genes were differentially expressed (DE) in planta compared to growth on a synthetic rich medium. Only 73 of these genes had been previously identified as DE in a transcriptome of R. solanacearum extracted from infected tomato xylem vessels. Virulence determinants such as the Type Three Secretion System (T3SS) and its effector proteins, motility structures, and reactive oxygen species (ROS) detoxifying enzymes were induced during infection of S. commersonii. On the contrary, metabolic activities were mostly repressed during early root colonization, with the notable exception of nitrogen metabolism, sulfate reduction and phosphate uptake. Several of the R. solanacearum genes identified as significantly up-regulated during infection had not been previously described as virulence factors. This is the first report describing the R. solanacearum transcriptome directly obtained from infected tissue and also the first to analyze bacterial gene expression in the roots, where plant infection takes place. We also demonstrate that the bacterial transcriptome in planta can be studied when pathogen numbers are low by

  3. Effects of Nitrogen Dioxide and Anoxia on Global Gene and Protein Expression in Long-Term Continuous Cultures of Nitrosomonas eutropha C91

    PubMed Central

    Kartal, Boran; Wessels, Hans J. C. T.; van der Biezen, Erwin; Francoijs, Kees-Jan; Jetten, Mike S. M.; Klotz, Martin G.

    2012-01-01

    Nitrosomonas eutropha is an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO2 on gene and protein expression under oxic and anoxic conditions were determined by maintaining N. eutropha strain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO2, and anoxic-plus-NO2 culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure of N. eutropha C91 to NO2 under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO2 resulted in increased representation of proteins and transcripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO2 and anoxic-plus-NO2 conditions. The results indicate that exposure to NO2 results in an energy-deprived state of N. eutropha C91 and that anaerobic growth could not be supported with NO2 as an oxidant. PMID:22562996

  4. Highly sensitive and selective gold(I) recognition by a metalloregulator in Ralstonia metallidurans.

    PubMed

    Jian, Xing; Wasinger, Erik C; Lockard, Jenny V; Chen, Lin X; He, Chuan

    2009-08-12

    A MerR family metalloregulatory protein CupR selectively responds to gold stress in Ralstonia metallidurans. A distorted trigonal geometry appears to be used by CupR to achieve the highly sensitive (K(d) approximately 10(-35) M) and selective recognition of gold(I).

  5. Draft Genome Sequences of Nine Strains of Ralstonia solanacearum Differing in Virulence to Eggplant (Solanum melongena).

    PubMed

    Guinard, Jérémy; Vinatzer, Boris A; Poussier, Stéphane; Lefeuvre, Pierre; Wicker, Emmanuel

    2016-01-28

    Ralstonia solanacearum displays variability in its virulence to solanaceous crops. We report here the draft genome sequences of eight phylotype I strains and one phylotype III strain differing in virulence to the resistant eggplant genotype AG91-25. These data will allow the identification of virulence- and avirulence-related genes. Copyright © 2016 Guinard et al.

  6. Effect of plant essential oils on Ralstonia solanacearum race 4 causing bacterial wilt of edible ginger

    USDA-ARS?s Scientific Manuscript database

    Palmarosa (Cymbopogon martini), lemongrass (C. citratus) and eucalyptus (Eucalyptus globulus) oils were investigated for their effects on Ralstonia solanacearum race 4, and their potential use as bio-fumigants for treating pathogen- infested edible ginger (Zingiber officinale R.) fields. Three conce...

  7. A multiplex PCR assay to detect and differentiate select agent strains of Ralstonia solanacearum

    USDA-ARS?s Scientific Manuscript database

    Ralstonia solanacearum causes bacterial wilt in a variety of cash crops. R. solanacearum race 3 biovar 2 strains are considered select agents by the U.S. Government because they are not endemic to the U.S. and have the potential to cause brown rot disease in our potato production fields. Simple and...

  8. Antagonistic activity and mechanisms of Bacillus subtilis SB1 against Ralstonia solanacearum

    USDA-ARS?s Scientific Manuscript database

    A potential biocontrol agent of bacterial wilt, Bacillus subtilis SB1, showed a broad-spectrum of antimicrobial activity in vitro experiments. In addition to Ralstonia solanacearum, strain SB1 inhibited the growth of many other plant pathogens, including Fusarium oxysporum, Botrytis cinerea, Phytoph...

  9. Genetic diversity and host range variation of Ralstonia solanacearum strains entering the United States

    USDA-ARS?s Scientific Manuscript database

    Each year, large volumes of plant propagative stock are imported into the United States; occasionally, Ralstonia solanacearum is found systemically infecting this plant material. In this study, 106 R. solanacearum strains were collected over a 10-year period from imported ornamental propagative sto...

  10. Draft Genome Sequence of Ralstonia sp. Strain GA3-3, Isolated from Australian Suburban Soil.

    PubMed

    Pearce, Stephen L; Pushiri, Hafizah; Oakeshott, John G; Russell, Robyn J; Pandey, Gunjan

    2013-07-05

    Ralstonia sp. strain GA3-3 is a hexachlorocyclohexane (HCH)-degrading bacterial strain isolated from suburban soil in Canberra, Australia. The genome of strain GA3-3 was sequenced to investigate its ability to degrade α-HCH. Here, we report the annotated genome sequence of this strain.

  11. Complete genome sequence of the plant pathogen Ralstonia solanacearum strain Po82.

    PubMed

    Xu, Jin; Zheng, Hua-jun; Liu, Lei; Pan, Zhe-chao; Prior, Philippe; Tang, Biao; Xu, Jing-sheng; Zhang, Hao; Tian, Qian; Zhang, Li-qing; Feng, Jie

    2011-08-01

    Ralstonia solanacearum strain Po82, a phylotype IIB/sequevar 4 strain, was found to be pathogenic to both solanaceous plants and banana. Here, we report the complete genome sequence of Po82 and its comparison with seven published R. solanacearum genomes.

  12. Complete Genome Sequence of Ralstonia solanacearum FJAT-1458, a Potential Biocontrol Agent for Tomato Wilt

    PubMed Central

    Chen, Deju; Zhu, Yujing; Wang, Jieping; Chen, Zheng; Che, Jiamei; Zheng, Xuefang; Chen, Xiaoqiang

    2017-01-01

    ABSTRACT An avirulent strain of Ralstonia solanacearum FJAT-1458 was isolated from a living tomato. Here, we report the complete R. solanacearum FJAT-1458 genome sequence of 6,059,899 bp and 5,241 genes. This bacterial strain is a potential candidate as a biocontrol agent in the form of a plant vaccine for bacterial wilt. PMID:28385834

  13. Ralstonia solanacearum and R. pseudosolanacearum on Eucalyptus: Opportunists or Primary Pathogens?

    PubMed

    Coutinho, Teresa A; Wingfield, Michael J

    2017-01-01

    Ralstonia solanacearum and R. pseudosolanacearum are well known primary pathogens of herbaceous crops. Reports of wilt caused by these pathogens in tree species are limited other than on Eucalyptus species. Despite the widespread occurrence of so-called bacterial wilt on eucalypts in tropical and sub-tropical parts of Africa, Asia, and the Americas, there remain many contradictions relating to the disease. Our field observations over many years in most regions where the disease occurs on Eucalyptus show that it is always associated with trees that have been subjected to severe stress. The disease is typically diagnosed by immersing cut stems in water and observing bacterial streaming, but the identity of the bacteria within this suspension is seldom considered. To add to the confusion, pathogenicity tests on susceptible species or clones are rarely successful. When they do work, they are on small plants in greenhouse trials. It has become all to easy to attribute Eucalyptus death exclusively to Ralstonia infection. Our data strongly suggest that Ralstonia species and probably other bacteria are latent colonists commonly occurring in healthy and particularly clonally propagated eucalypts. The onset of stress factors provide the bacteria with an opportunity to develop. We believe that the resulting stress weakens the defense systems of the trees allowing Ralstonia and bacterial endophytes to proliferate. Overall our research suggests that R. solanacearum and R. pseudosolanacearum are not primary pathogens of Eucalyptus. Short of clear evidence that they are primary pathogens of Eucalyptus it is inappropriate to attribute this disease solely to infection by Ralstonia species.

  14. Ralstonia solanacearum and R. pseudosolanacearum on Eucalyptus: Opportunists or Primary Pathogens?

    PubMed Central

    Coutinho, Teresa A.; Wingfield, Michael J.

    2017-01-01

    Ralstonia solanacearum and R. pseudosolanacearum are well known primary pathogens of herbaceous crops. Reports of wilt caused by these pathogens in tree species are limited other than on Eucalyptus species. Despite the widespread occurrence of so-called bacterial wilt on eucalypts in tropical and sub-tropical parts of Africa, Asia, and the Americas, there remain many contradictions relating to the disease. Our field observations over many years in most regions where the disease occurs on Eucalyptus show that it is always associated with trees that have been subjected to severe stress. The disease is typically diagnosed by immersing cut stems in water and observing bacterial streaming, but the identity of the bacteria within this suspension is seldom considered. To add to the confusion, pathogenicity tests on susceptible species or clones are rarely successful. When they do work, they are on small plants in greenhouse trials. It has become all to easy to attribute Eucalyptus death exclusively to Ralstonia infection. Our data strongly suggest that Ralstonia species and probably other bacteria are latent colonists commonly occurring in healthy and particularly clonally propagated eucalypts. The onset of stress factors provide the bacteria with an opportunity to develop. We believe that the resulting stress weakens the defense systems of the trees allowing Ralstonia and bacterial endophytes to proliferate. Overall our research suggests that R. solanacearum and R. pseudosolanacearum are not primary pathogens of Eucalyptus. Short of clear evidence that they are primary pathogens of Eucalyptus it is inappropriate to attribute this disease solely to infection by Ralstonia species. PMID:28553301

  15. Ralstonia solanacearum, a widespread bacterial plant pathogen in the post-genomic era.

    PubMed

    Peeters, Nemo; Guidot, Alice; Vailleau, Fabienne; Valls, Marc

    2013-09-01

    Ralstonia solanacearum is a soil-borne bacterium causing the widespread disease known as bacterial wilt. Ralstonia solanacearum is also the causal agent of Moko disease of banana and brown rot of potato. Since the last R. solanacearum pathogen profile was published 10 years ago, studies concerning this plant pathogen have taken a genomic and post-genomic direction. This was pioneered by the first sequenced and annotated genome for a major plant bacterial pathogen and followed by many more genomes in subsequent years. All molecular features studied now have a genomic flavour. In the future, this will help in connecting the classical field of pathology and diversity studies with the gene content of specific strains. In this review, we summarize the recent research on this bacterial pathogen, including strain classification, host range, pathogenicity determinants, regulation of virulence genes, type III effector repertoire, effector-triggered immunity, plant signalling in response to R. solanacearum, as well as a review of different new pathosystems. Bacteria; Proteobacteria; β subdivision; Ralstonia group; genus Ralstonia. Ralstonia solanacearum is the agent of bacterial wilt of plants, characterized by a sudden wilt of the whole plant. Typically, stem cross-sections will ooze a slimy bacterial exudate. In the case of Moko disease of banana and brown rot of potato, there is also visible bacterial colonization of banana fruit and potato tuber. As a soil-borne pathogen, infected fields can rarely be reused, even after rotation with nonhost plants. The disease is controlled by the use of resistant and tolerant plant cultivars. The prevention of spread of the disease has been achieved, in some instances, by the application of strict prophylactic sanitation practices. Stock centre: International Centre for Microbial Resources-French Collection for Plant-associated Bacteria CIRM-CFBP, IRHS UMR 1345 INRA-ACO-UA, 42 rue Georges Morel, 49070 Beaucouzé Cedex, France

  16. Draft Genome Sequences of Two Ralstonia pickettii Strains with Different Aminoglycoside Resistance Phenotypes

    PubMed Central

    Vaz-Moreira, Ivone; Martínez, José Luis

    2016-01-01

    The genomes of two Ralstonia pickettii strains (H2Cu2 and H2Cu5), isolated from hospital effluent in a selective medium containing CuSO4, were sequenced. They presented MICs of >256 and 6 µg/ml for the aminoglycoside gentamicin, respectively. The 5.2-Mb draft genomes have 40 contigs for strain H2Cu2 and 113 for H2Cu5. PMID:27834709

  17. Root-associated bacterial endophytes from Ralstonia solanacearum resistant and susceptible tomato cultivars and their pathogen antagonistic effects

    PubMed Central

    Upreti, Reshmi; Thomas, Pious

    2015-01-01

    This study was undertaken to assess if the root-associated native bacterial endophytes in tomato have any bearing in governing the host resistance to the wilt pathogen Ralstonia solanacearum. Internal colonization of roots by bacterial endophytes was confirmed through confocal imaging after SYTO-9 staining. Endophytes were isolated from surface-sterilized roots of 4-weeks-old seedlings of known wilt resistant (R) tomato cultivar Arka Abha and susceptible (S) cv. Arka Vikas on nutrient agar after plating the tissue homogenate. Arka Abha displayed more diversity with nine distinct organisms while Arka Vikas showed five species with two common organisms (Pseudomonas oleovorans and Agrobacterium tumefaciens). Screening for general indicators of biocontrol potential showed more isolates from Arka Abha positive for siderophore, HCN and antibiotic biosynthesis than from Arka Vikas. Direct challenge against the pathogen indicated strong antagonism by three Arka Abha isolates (P. oleovorans, Pantoea ananatis, and Enterobacter cloacae) and moderate activity by three others, while just one isolate from Arka Vikas (P. oleovorans) showed strong antagonism. Validation for the presence of bacterial endophytes on three R cultivars (Arka Alok, Arka Ananya, Arka Samrat) showed 8–9 antagonistic bacteria in them in comparison with four species in the three S cultivars (Arka Ashish, Arka Meghali, Arka Saurabhav). Altogether 34 isolates belonging to five classes, 16 genera and 27 species with 23 of them exhibiting pathogen antagonism were isolated from the four R cultivars against 17 isolates under three classes, seven genera and 13 species from the four S cultivars with eight isolates displaying antagonistic effects. The prevalence of higher endophytic bacterial diversity and more antagonistic organisms associated with the seedling roots of resistant cultivars over susceptible genotypes suggest a possible role by the root-associated endophytes in natural defense against the pathogen

  18. Genome sequences of Ralstonia insidiosa type strain ATCC 49129 and strain FC1138, a strong biofilm producer isolated from a fresh-cut produce-processing plant

    USDA-ARS?s Scientific Manuscript database

    Ralstonia insidiosa FC1138 is a strong biofilm producer, isolated from a local fresh-cut produce processing plant. Here, we present the complete genome sequence of Ralstonia insidiosa FC1138 which includes two circular chromosomes and a plasmid. To our knowledge, this is the first reported complete ...

  19. Genomic characterization of ϕRS603, a filamentous bacteriophage that is infectious to the phytopathogen Ralstonia solanacearum.

    PubMed

    Van, Truong Thi Bich; Yoshida, Shohei; Miki, Kaito; Kondo, Akihiro; Kamei, Kaeko

    2014-12-01

    A filamentous bacteriophage (ϕ), ϕRS603, which is infectious to the phytopathogen Ralstonia solanacearum was isolated. ϕRS603 was found to have a circular single-stranded DNA genome composed of 7679 nucleotides and to contain 13 putative open reading frames (ORFs). The ϕRS603 genome showed strong similarity with those of Ralstonia phages ϕRSM1 and ϕRSM3, as reported by Askora et al. The ϕRS603 genome had no ORFs corresponding to ORFs 2, 3, 13 and 14 (integrase) of ϕRSM3. ϕRS603 had an ORF that was homologous to other Ralstonia phages ϕRSS0 and ϕRSS1; however, ϕRSM1 and ϕRSM3 did not. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

  20. Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov.

    PubMed

    Coenye, T; Falsen, E; Vancanneyt, M; Hoste, B; Govan, J R; Kersters, K; Vandamme, P

    1999-04-01

    A polyphasic taxonomic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analysis, whole-cell protein and fatty acid analyses, AFLP (amplified fragment length polymorphism) fingerprinting and an extensive biochemical characterization was performed on 10 strains provisionally identified as Alcaligenes faecalis-like bacteria. The six environmental and four human isolates belonged to the genus Ralstonia and were assigned to a new species for which the name Ralstonia gilardii sp. nov. is proposed. The type strain is LMG 5886T.

  1. Improved antibiotic resistance gene cassette for marker exchange mutagenesis in Ralstonia solanacearum and Burkholderia species.

    PubMed

    Um, Hae Young; Chung, Eunsook; Lee, Jai-Heon; Lee, Seon-Woo

    2011-04-01

    Marker exchange mutagenesis is a fundamental approach to understanding gene function at a molecular level in bacteria. New plasmids carrying a kanamycin resistance gene or a trimethoprim resistance gene were constructed to provide antibiotic resistance cassettes for marker exchange mutagenesis in Ralstonia solanacearum and many antibiotic-resistant Burkholderia spp. Insertion sequences present in the flanking sequences of the antibiotic resistance cassette were removed to prevent aberrant gene replacement and polar mutation during mutagenesis in wild-type bacteria. Plasmids provided in this study would be convenient for use in gene cassettes for gene replacement in other Gram-negative bacteria.

  2. Ralstonia pickettii and Burkholderia cepacia complex bloodstream infections related to infusion of contaminated water for injection.

    PubMed

    Moreira, B M; Leobons, M B G P; Pellegrino, F L P C; Santos, M; Teixeira, L M; de Andrade Marques, E; Sampaio, J L M; Pessoa-Silva, C L

    2005-05-01

    Ralstonia pickettii and Burkholderia cepacia complex isolates are causes of healthcare-associated infection related to contamination of intravenously administered products. Based on microbiological and epidemiological data and molecular typing by pulsed-field gel electrophoresis, we report the occurrence of two outbreaks of R. pickettii and B. cepacia complex bloodstream infections. The first outbreak occurred from August 1995 to September 1996, and the second outbreak occurred from 28 March to 8 April 1998, affecting adults and neonates, respectively. Infusion of contaminated water for injection was the source of infection.

  3. [Advances in studies of the type III secretion system in Ralstonia solanacearum--A review].

    PubMed

    Zhang, Yong; Li, Muyuan; Luo, Feng

    2015-06-04

    Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating plant diseases worldwide. The syringe-like type III secretion system (T3SS) plays a crucial role in its pathogenicity. R. solanacearum uses the T3SS to inject effector proteins (Type III effectors) into the cytoplasm of host cells, causing diseases in susceptible plants or triggering the hypersensitive response in resistant plants. In this article we review recent advances in studies of R. solanacearum T3SS and highlight their unique features.

  4. Antibacterial activity of the flavonoids from Dalbergia odorifera on Ralstonia solanacearum.

    PubMed

    Zhao, Xiabo; Mei, Wenli; Gong, Mingfu; Zuo, Wenjian; Bai, Hongjin; Dai, Haofu

    2011-11-25

    Phytohemical investigation on the heartwood of Dalbergia odorifera resulted in the isolation of nine flavonoids. Their structures were elucidated as sativanone (1), (3R)-vestitone (2), (3R)-2',3',7-trihydroxy-4'-methoxyisoflavanone (3), (3R)-4'-methoxy-2',3,7-trihydroxyisoflavanone (4), carthamidin (5), liquiritigenin (6), isoliquiritigenin (7), (3R)-vestitol (8), and sulfuretin (9) based on their spectral data. All compounds were evaluated for their inhibitory activity against Ralstonia solanacearum. This is the first report about anti-R. solanacearum activity of the compounds from D. odorifera.

  5. Suppression of DS1 phosphatidic acid phosphatase confirms resistance to Ralstonia solanacearum in Nicotiana benthamiana.

    PubMed

    Nakano, Masahito; Nishihara, Masahiro; Yoshioka, Hirofumi; Takahashi, Hirotaka; Sawasaki, Tatsuya; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2013-01-01

    Nicotianabenthamiana is susceptible to Ralstonia solanacearum. To analyze molecular mechanisms for disease susceptibility, we screened a gene-silenced plant showing resistance to R. solanacearum, designated as DS1 (Disease suppression 1). The deduced amino acid sequence of DS1 cDNA encoded a phosphatidic acid phosphatase (PAP) 2. DS1 expression was induced by infection with a virulent strain of R. solanacearum in an hrp-gene-dependent manner. DS1 rescued growth defects of the temperature-sensitive ∆lpp1∆dpp1∆pah1 mutant yeast. Recombinant DS1 protein showed Mg(2+)-independent PAP activity. DS1 plants showed reduced PAP activity and increased phosphatidic acid (PA) content. After inoculation with R. solanacearum, DS1 plants showed accelerated cell death, over-accumulation of reactive oxygen species (ROS), and hyper-induction of PR-4 expression. In contrast, DS1-overexpressing tobacco plants showed reduced PA content, greater susceptibility to R. solanacearum, and reduced ROS production and PR-4 expression. The DS1 phenotype was partially compromised in the plants in which both DS1 and NbCoi1 or DS1 and NbrbohB were silenced. These results show that DS1 PAP may affect plant immune responses related to ROS and JA cascades via regulation of PA levels. Suppression of DS1 function or DS1 expression could rapidly activate plant defenses to achieve effective resistance against Ralstonia solanacearum.

  6. Evaluation of antibacterial activity of Stenotrophomonas maltophilia against Ralstonia solanacearum under different application conditions.

    PubMed

    Elhalag, K M; Messiha, N A S; Emara, H M; Abdallah, S A

    2016-06-01

    The aim of this study was the monitoring of different mechanisms involved in the antibacterial activity of the biocontrol agent, Stenotrophomonas maltophilia (PD4560), against Ralstonia solanacearum in vitro and in vivo. Optimization of conditions that favour these mechanisms was the second target of this study. Proteolytic activity of Sten. maltophilia (PD 4560), was tested on skimmed milk medium. The biocontrol agent was able to produce an alkaline serine protease enzyme with a molecular weight of 40 KDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. Spraying of salicylic acid (SA) led to an increase in the efficacy of Sten. maltophilia in controlling the Ralstonia potato wilt while spraying of ammonium sulphate (AmS) did not affect the biocontrol efficacy. The efficacy was correlated with the expression of protease enzyme genes; Prt genes (mainly PrtP and Prt4) and PR genes (mainly PR-1 and PRQ) as evaluated using real-time polymerase chain reaction analysis. The biocontrol activity of Sten. maltophilia can be attributed to the direct mechanism alkaline serine proteolytic enzyme production and through induction of host systemic acquired resistance as indirect mechanism. Tuber bulking was the most suitable physiological growth stage to apply either SA or the biocontrol agent. Both SA and peat-moss as an organic carrier enhanced the antibacterial efficiency of the biocontrol agent. Application of Sten. maltophilia is more suitable under alkaline soil conditions. © 2016 The Society for Applied Microbiology.

  7. Ralstonia mannitolilytica-Induced Septicemia and Homology Analysis in Infected Patients: 3 Case Reports

    PubMed Central

    Liu, Cai-Xia; Yan, Chun; Zhang, Pan; Li, Fang-Qu; Yang, Jing-Hong; Li, Xiang-Yang

    2016-01-01

    Background Ralstonia mannitolilytica is an emerging opportunistic pathogen. Hospital outbreaks of Ralstonia spp. are mainly associated with contaminated treatment water or auxiliary instruments. Objectives In this report, we summarize the clinical infection characteristics of R. mannitolilytica, the drug-susceptibility testing of the bacterial strains, and the results of related infection investigations. Patients and Methods We retrospectively analyzed the clinical information of 3 patients with R. mannitolilytica. Results The patients’ primary-onset symptoms were chills and fever. The disease progressed rapidly and septic shock symptoms developed. Laboratory tests indicated progressively decreased white blood cells and platelets, as well as significant increases in certain inflammation indicators. The effect of treatment with Tazocin was good. The growth period of R. mannitolilytica in sterile distilled water was > 6 months. The pulsed-field gel electrophoresis (PFGE) results revealed that the infectious strains from these 3 patients were not the same clonal strain. This bacterium was not detected in the nosocomial infection samples. Conclusions Our results suggest that R. mannitolilytica-induced septicemia had an acute disease onset and rapid progression. The preferred empirical antibiotic was Tazocin. In these 3 cases, the R. mannitolilytica-induced septicemia was not due to clonal transmission. PMID:27679705

  8. Suppression of DS1 Phosphatidic Acid Phosphatase Confirms Resistance to Ralstonia solanacearum in Nicotiana benthamiana

    PubMed Central

    Nakano, Masahito; Nishihara, Masahiro; Yoshioka, Hirofumi; Takahashi, Hirotaka; Sawasaki, Tatsuya; Ohnishi, Kouhei; Hikichi, Yasufumi; Kiba, Akinori

    2013-01-01

    Nicotianabenthamiana is susceptible to Ralstonia solanacearum. To analyze molecular mechanisms for disease susceptibility, we screened a gene-silenced plant showing resistance to R. solanacearum, designated as DS1 (Disease suppression 1). The deduced amino acid sequence of DS1 cDNA encoded a phosphatidic acid phosphatase (PAP) 2. DS1 expression was induced by infection with a virulent strain of R. solanacearum in an hrp-gene-dependent manner. DS1 rescued growth defects of the temperature-sensitive ∆lpp1∆dpp1∆pah1 mutant yeast. Recombinant DS1 protein showed Mg2+-independent PAP activity. DS1 plants showed reduced PAP activity and increased phosphatidic acid (PA) content. After inoculation with R. solanacearum, DS1 plants showed accelerated cell death, over-accumulation of reactive oxygen species (ROS), and hyper-induction of PR-4 expression. In contrast, DS1-overexpressing tobacco plants showed reduced PA content, greater susceptibility to R. solanacearum, and reduced ROS production and PR-4 expression. The DS1 phenotype was partially compromised in the plants in which both DS1 and NbCoi1 or DS1 and NbrbohB were silenced. These results show that DS1 PAP may affect plant immune responses related to ROS and JA cascades via regulation of PA levels. Suppression of DS1 function or DS1 expression could rapidly activate plant defenses to achieve effective resistance against Ralstonia solanacearum. PMID:24073238

  9. Extracellular DNases of Ralstonia solanacearum modulate biofilms and facilitate bacterial wilt virulence.

    PubMed

    Minh Tran, Tuan; MacIntyre, April; Khokhani, Devanshi; Hawes, Martha; Allen, Caitilyn

    2016-11-01

    Ralstonia solanacearum is a soil-borne vascular pathogen that colonizes plant xylem vessels, a flowing, low-nutrient habitat where biofilms could be adaptive. Ralstonia solanacearum forms biofilm in vitro, but it was not known if the pathogen benefits from biofilms during infection. Scanning electron microscopy revealed that during tomato infection, R. solanacearum forms biofilm-like masses in xylem vessels. These aggregates contain bacteria embedded in a matrix including chromatin-like fibres commonly observed in other bacterial biofilms. Chemical and enzymatic assays demonstrated that the bacterium releases extracellular DNA in culture and that DNA is an integral component of the biofilm matrix. An R. solanacearum mutant lacking the pathogen's two extracellular nucleases (exDNases) formed non-spreading colonies and abnormally thick biofilms in vitro. The biofilms formed by the exDNase mutant in planta contained more and thicker fibres. This mutant was also reduced in virulence on tomato plants and did not spread in tomato stems as well as the wild-type strain, suggesting that these exDNases facilitate biofilm maturation and bacterial dispersal. To our knowledge, this is the first demonstration that R. solanacearum forms biofilms in plant xylem vessels, and the first documentation that plant pathogens use DNases to modulate their biofilm structure for systemic spread and virulence. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  10. Ralstonia insidiosa serves as bridges in biofilm formation by foodborne pathogens Listeria monocytogenes, Salmonella enterica, and enterohemorrhagic Escherichia coli

    USDA-ARS?s Scientific Manuscript database

    Biofilm formation on abiotic surfaces in fresh produce processing facilities might play a role in foodborne outbreaks by providing protective microniches for pathogenic bacteria. Our previous study showed that a strain of Ralstonia insidiosa isolated from a fresh produce processing plant could enhan...

  11. Detection of plant quarantine pathogen Ralstonia solanacearum r3b2 with portable POCKIT™ and BLItz® systems

    USDA-ARS?s Scientific Manuscript database

    Ralstonia solanacearum (Rs) race 3 biovar 2 (r3b2) is designated as a quarantine pathogen in many countries and additionally as a Select Agent in the United States. Rapid, sensitive and accurate detection methods are urgently needed. We report here the development of two portable platforms for r3b...

  12. Draft Genome Sequence of Ralstonia sp. MD27, a Poly(3-Hydroxybutyrate)-Degrading Bacterium, Isolated from Compost

    PubMed Central

    Zhu, Morgan; McCully, Lucy M.; Silby, Mark W.; Charles-Ogan, Tamunonengiyeofori I.

    2015-01-01

    Ralstonia sp. strain MD27, a novel biopolymer-degrading betaproteobacterium, was isolated from compost samples. This organism has been shown to utilize the biopolymer poly(3-hydroxybutyrate) [P(3HB)] as a carbon source for growth. We report the draft genome sequence of MD27 with an estimated total sequence length of 5.9 Mb. PMID:26450738

  13. Evaluation of Chloropicrin as a Soil Fumigant against Ralstonia solanacarum in Ginger (Zingiber officinale Rosc.) Production in China

    PubMed Central

    Ma, Taotao; Liu, Pengfei; Shen, Jin; Li, Yuan; Ouyang, Canbin; Guo, Meixia; Cao, Aocheng

    2014-01-01

    Background Chloropicrin (Pic) offers a potential alternative to methyl bromide (MB) against Ralstonia solanacarum in ginger (Zingiber officinale Rosc.) production. MB is scheduled to be withdrawn from routine use by 2015 in developing countries. Methods Pic treatments were evaluated in a laboratory study and in three commercial ginger fields. Results Laboratory studies showed that the EC50 value and EC80 value of Pic were 2.7 and 3.7 mg a.i. kg−1 soil, respectively. Field trials in highly infested soil revealed that treatments of Pic at the dose of 50 g m−2 covered with totally impermeable film (TIF) or polyethylene film (PE) sharply reduced Ralstonia solanacarum and maintained high ginger yields. Both of the Pic treatments provided results similar to, or in some cases slightly lower than, MB with respect to Ralstonia solanacarum control, plant survival, plant growth and yield. All of the fumigant treatments were significantly better than the non-treated control. Conclusions The present study confirms that the Pic is a promising alternative with good efficacy against Ralstonia solanacarum for ginger production and could be used in integrated pest management programs in China. PMID:24618853

  14. Ectopic expression of an EAR motif deletion mutant of SlERF3 enhances tolerance to salt stress and Ralstonia solanacearum in tomato.

    PubMed

    Pan, I-Chun; Li, Chia-Wen; Su, Ruey-Chih; Cheng, Chiu-Ping; Lin, Choun-Sea; Chan, Ming-Tsair

    2010-10-01

    Ethylene-responsive transcription factors (ERFs) bind specifically to cis-acting DNA regulatory elements such as GCC boxes and play an important role in the regulation of defense- and stress-related genes in plants. In contrast to other ERFs, class II ERFs contain an ERF-associated amphiphilic repression (EAR) domain and act as GCC-mediated transcriptional repressors. In this study, SlERF3, a class II ERF was isolated from tomato and characterized. To examine whether the EAR motif of class II ERF proteins participates in ERF-mediated functions in plants, the EAR domain was deleted to generate SlERF3ΔRD. We show that SlERF3ΔRD protein retains the character of a transcription factor and becomes a GCC-mediated transcriptional activator. Constitutive expression of full-length SlERF3 in tomato severely suppressed growth and, as a result, no transgenic plants were obtained. However, no apparent effects on growth and development of SlERF3ΔRD transgenic plants were observed. Overexpression of SlERF3ΔRD in transgenic tomato induced expression of pathogenesis-related protein genes such as PR1, PR2 and PR5, and enhanced tolerance to Ralstonia solanacearum. Furthermore, transgenic Arabidopsis and tomatoes constitutively expressing SlERF3ΔRD exhibited reduced levels of membrane lipid peroxidation and enhanced tolerance to salt stress. In comparison with wild-type plants grown under stress conditions, transgenic SlERF3ΔRD tomatoes produced more flowers, fruits, and seeds. This study illustrates a gene-enhancing tolerance to both biotic and abiotic stresses in transgenic plants with the deletion of a repressor domain. Our findings suggest that class II ERF proteins may find important use in crop improvement or genetic engineering to increase stress tolerance in plants.

  15. Characterization of bacteria degrading 3-hydroxy palmitic acid methyl ester (3OH-PAME), a quorum sensing molecule of Ralstonia solanacearum.

    PubMed

    Achari, G A; Ramesh, R

    2015-05-01

    Bacterial wilt pathogen Ralstonia solanacearum causes severe crop loss of eggplant, which is of economic importance in India. 3-hydroxy palmitic acid methyl ester (3OH-PAME) is the main quorum sensing molecule governing the expression of virulence factors in R. solanacearum. Ability of 164 bacterial isolates from the xylem of eggplant (Solanum melongena L.), chilli pepper (Capsicum annuum L.) and wild eggplant (Solanum torvum Sw.) to degrade 3OH-PAME was tested by disc diffusion assay. Enzymatic degradation of 3OH-PAME by five bacteria was confirmed by High-Performance Liquid Chromatography-Mass Spectrometry analysis. 3OH-PAME degrading bacteria were identified as Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Rhodococcus corynebacterioides. 3OH-PAME degrading bacteria reduced the expression of virulence factors (exopolysaccharides and endoglucanase) of R. solanacearum in vitro and reduced wilt incidence in eggplant seedlings under greenhouse conditions. Isolates with quorum quenching activity successfully re-colonized eggplant seedlings. Quorum quenching bacteria produced antagonistic compounds, which may act synergistically with quorum quenching in reducing bacterial wilt in eggplant. This is the first report on endophytic bacteria of class Gammaproteobacteria and phylum Actinobacteria having 3OH-PAME degrading activity. This study demonstrates the potential use of endophytic bacteria as quorum quenching biocontrol agents for management of bacterial wilt in eggplant. © 2015 The Society for Applied Microbiology.

  16. Opening the Ralstonia solanacearum type III effector tool box: insights into host cell subversion mechanisms.

    PubMed

    Deslandes, Laurent; Genin, Stephane

    2014-08-01

    Effectors delivered to host cells by the Type III secretion system are essential to Ralstonia solanacearum pathogenicity, as in several other plant pathogenic bacteria. The establishment of exhaustive effector repertoires in multiple R. solanacearum strains drew a first picture of the evolutionary dynamics of the pathogen effector suites. Effector repertoires are diversified, with a core of 20-30 effectors present in most of the strains and the obtention of mutants lacking one or more effector genes revealed the functional overlap among this effector network. Recent functional studies have provided insights into the ability of single effectors to manipulate the host proteasome, elicit cell death, trigger the expression of plant genes, and/or display biochemical activities on plant protein targets. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Extract of Syringa oblata: A new biocontrol agent against tobacco bacterial wilt caused by Ralstonia solanacearum.

    PubMed

    Bai, Wanming; Kong, Fanyu; Lin, Yong; Zhang, Chengsheng

    2016-11-01

    Ralstonia solanacearum causes serious wilt disease in tobacco. To effectively control this disease, the antibacterial activity of 95% ethanol extracts from the flower buds of Syringa oblata was examined. Based on GC-MS analysis and an inhibition experiment against R. solanacearum, the main antibacterial component is eugenol. We further determined the effect of eugenol on the physiology, biochemistry, and cellular morphology of R. solanacearum. The results showed that eugenol can destroy wilt bacteria, leading to the disappearance of flagella, the leakage of contents, and the appearance of a cavity. SDS-PAGE showed that eugenol decreased protein content in R. solanacearum, reduced medium carbohydrate utilization, and inhibited CAT and SDH activity. The above results showed that eugenol had a significant inhibitory effect on R. solanacearum and this component has the potential to prevent tobacco bacterial wilt. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Evaluation of antibacterial effects of carbon nanomaterials against copper-resistant Ralstonia solanacearum.

    PubMed

    Wang, Xiuping; Liu, Xueqin; Han, Heyou

    2013-03-01

    In this paper, we investigated the antibacterial activity and the action mode of carbon nanomaterials (CNMs) against the copper-resistant plant pathogenic bacterium Ralstonia solanacearum (R. solanacearum). Single-walled carbon nanotubes (SWCNTs) dispersion was found to show the strongest antibacterial activity, sequentially followed by graphene oxide (GO), multi-walled carbon nanotubes (MWCNTs), reduced graphene oxide (rGO) and fullerene (C(60)). Our investigation of the antibacterial mechanism of SWCNTs and GO indicated that the damage to the cell membrane leads to the release of cytoplasm materials from the bacterium, which is the causative factor for the inactivation of R. solanacearum bacterial cells. The superior antibacterial effect, and the novel antibacterial mode of SWCNTs and GO suggest that those carbon nanomaterials may have important applications in the control of plant bacterial diseases. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  19. [Regulation of rsc1285 gene in type III secretion system in Ralstonia solanacearum].

    PubMed

    Li, Muyuan; Xu, Pengxia; Zhang, Weiqi; Zhang, Yong

    2015-08-04

    Rsc1285 is one of the putative T3SS-regulated factors in Ralstonia solanacearum, and the regulation of Rsc1285 on T3SS and pathogenicity was characterized. The rsc1285 deletion mutants were constructed by homologous recombination and characterized by complementation. The rsc1285 mutant was significantly less virulent than the wild-type strain to infect tomato plants. Rsc1285 controls the expression of hrpB and HrpB-regulating genes, but it is dispensable for the expression of hrpG and prhG. R. solanacearum uses Rsc1285 to control the T3SS and pathogenicity via a novel pathway, and this finding provides insights into overall infection mode of R. solanacearum.

  20. Comparison between Solanum torvum Sw. and S. melongena L. after Ralstonia solanacearum inoculation.

    PubMed

    Aribaud, M; Noirot, M; Fock-Bastide, I; Vaniet, S; Kodja, H

    2014-09-01

    Bacterial wilt, caused by Ralstonia solanacearum, is one of the most devastating plant diseases, affecting some economically important Solanaceae crops. In contrast, Solanum torvum, also known as wild eggplant, does not wilt when infested with R. solanacearum. In order to describe the mechanism underlying the response of S. torvum, it was compared with the cultivated eggplant, S. melongena, when both were infected with the same R. solanacearum strain. No wilting occurred in S. torvum, although the bacteria colonised roots and stems in both species within the first 24 h. There were marked differences beyond 24 h, consisting of high bacterial mortality in S. torvum. Using the calli model, our investigations revealed an increase in cell wall monoamine oxidase activity in S. torvum after R. solanacearum inoculation, which did not occur in S. melongena. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.

  1. Class Schedules Need Class.

    ERIC Educational Resources Information Center

    Monfette, Ronald J.

    1986-01-01

    Argues that college publications, including class schedules, must be accurate, timely, and easy to read and follow. Describes Schoolcraft College's unified format approach to publications marketing. Offers suggestions on the design, format, and distribution of class schedules. (DMM)

  2. Draft Genome Sequence of Ralstonia solanacearum Strain Rs-T02, Which Represents the Most Prevalent Phylotype in Guangxi, China

    PubMed Central

    Zou, Chengwu; Wang, Kaihao; Meng, Jiaorong; Yuan, Gaoqing; Lin, Wei; Peng, Haowen

    2016-01-01

    Ralstonia solanacearum strain Rs-T02 was originally isolated from a bacterial wilt of tomato plant in Nanning City of Guangxi Province, China. It represents the most prevalent phylotype in Guangxi. Here, we present the draft genome sequence of this strain, which comprises 5,225 genes and 5,976,011 nucleotides with an average G+C content of 66.79%. There are 968 different genes between this isolate and the previously reported genome sequence of Ralstonia solanacearum GMl l000 (race l, biovar 3, phylotype I), and the genome sequence information of this isolate may be useful for comparative genomic studies to determine the genetic diversity in this species. PMID:27081126

  3. Cleaning-resistant Cupriavidus and Ralstonia bacteria contaminating spacecrafts and the ultra clean rooms they are assembled in.

    NASA Astrophysics Data System (ADS)

    Leys, N.; Dams, A.; Bossus, A.; Provoost, A.; Venkateswaran, K.; Mergeay, M.

    Background Planetary Protection is preventing microbial contamination of both the target planet and the Earth when sending spacecrafts on interplanetary space mission It is important to preserve the natural conditions of other planets and to not bring with robots earthly microbes forward contamination when looking for spores of extra terrestrial life Spacecrafts and the ultra clean rooms they are assembled in are routinely monitored for microbial contamination It was shown that the floor air and surfaces of such spacecraft assembly rooms often contain Cupriavidu s and Ralstonia bacteria These bacteria not only contaminated the clean rooms but have also been found prior-to-flight on surfaces of space robots such as the Mars Odyssey Orbiter La Duc et al 2003 and even in-flight in ISS cooling water and Shuttle drinking water unpublished Aim In this study several Cupriavidus and Ralstonia strains isolated from space craft assembling rooms and spacecrafts were characterized and analysed in detail Results The analysis showed that all the Cupriavidus and Ralstonia clean-room isolates are able to use a wide variety of substrates as carbon sources including ethanol and acetone In addition they all have accumulated moderate resistances to an extraordinary collection of physical and chemical antimicrobial agents Some of the test strains were able to form biofilms on plastic and metal materials used for space robots a nutritional and

  4. Nodulation of Mimosa spp. by the beta-proteobacterium Ralstonia taiwanensis.

    PubMed

    Chen, Wen-Ming; James, Euan K; Prescott, Alan R; Kierans, Martin; Sprent, Janet I

    2003-12-01

    Several beta-proteobacteria have been isolated from legume root nodules and some of these are thought to be capable of nodulating and fixing N2. However, in no case has there been detailed studies confirming that they are the active symbionts. Here, Ralstonia taiwanensis LMG19424, which was originally isolated from Mimosa pudica nodules, was transformed to carry the green fluorescent protein (gfp) reporter gene before being used to inoculate axenically-grown seedlings of M. pudica and M. diplotricha. Plants were harvested at various intervals for 56 days after inoculation, then examined for evidence of infection and nodule formation. Nodulation of both Mimosa spp. was abundant, and acetylene reduction assays confirmed that nodules had nitrogenase activity. Confocal laser scanning microscopy (CLSM) showed that fresh M. pudica nodules with nitrogenase activity had infected cells containing bacteroids expressing gfp. In parallel, fixed and embedded nodules from both Mimosa spp. were sectioned for light and electron microscopy, followed by immunogold labeling with antibodies raised against gfp and nitrogenase Fe (nifH) protein. Significant immunolabeling with these antibodies confirmed that R. taiwanensis LMG19424 is an effective N2-fixing symbiont of Mimosa spp. Both species were infected via root hairs and, in all respects, the nodule ontogeny and development was similar to that described for other mimosoid legumes. The nodules were indeterminate with a persistent meristem, an invasion zone containing host cells being invaded via prominent infection threads, and an N2-fixing zone with infected cells containing membrane-bound symbiosomes.

  5. Extract from Maize (Zea mays L.): Antibacterial Activity of DIMBOA and Its Derivatives against Ralstonia solanacearum.

    PubMed

    Guo, Bing; Zhang, Yongqiang; Li, Shili; Lai, Ting; Yang, Liang; Chen, Juanni; Ding, Wei

    2016-10-19

    Many cereals accumulate hydroxamic acids involved in defense of plant against various fungi, bacteria, and insects. 2,4-dihydroxy-7-methoxy-1,4-benzoxazine-3-one, commonly known as DIMBOA, is one of the principal cyclic hydroxamic acids in aqueous extracts of maize. The aim of this study was to evaluate the antibacterial activity of the isolated DIMBOA and its derivatives 2-benzoxazolinone (BOA), 6-chloro-2-benzoxazolinone (CDHB), and 2-mercaptobenzothiazole (MBT) against Ralstonia solanacearum. MBT showed the strongest antibacterial activity, followed by CDHB and DIMBOA, with minimum inhibitory concentrations (MICs) of 50, 100 and 200 mg/L, respectively, better than the BOA with 300 mg/L. These compounds also significantly affect bacterial growth, reduce biofilm formation, and inhibit swarming motility within 24 h. This paper is the first to report the anti-R. solanacearum activity of DIMBOA from Z. mays. The bioassay and pot experiment results suggested that DIMBOA and its derivatives exhibit potential as a new matrix structure of designing target bactericide or elicitor for controlling tobacco bacterial wilt. Further studies must evaluate the efficacy of DIMBOA and its derivatives in controlling bacterial wilt under natural field conditions where low inoculum concentrations exist.

  6. Ralstonia solanacearum Differentially Colonizes Roots of Resistant and Susceptible Tomato Plants.

    PubMed

    Caldwell, Denise; Kim, Bong-Suk; Iyer-Pascuzzi, Anjali S

    2017-03-21

    Ralstonia solanacearum is the causal agent of bacterial wilt and infects over 200 plant species in 50 families. The soilborne bacterium is lethal to many solanaceous species, including tomato. Although resistant plants can carry high pathogen loads (between 10(5) and 10(8) CFU/g fresh weight), the disease is best controlled by the use of resistant cultivars, particularly resistant rootstocks. How these plants have latent infections yet maintain resistance is not clear. R. solanacearum first infects the plant through the root system and, thus, early root colonization events may be key to understanding resistance. We hypothesized that the distribution and timing of bacterial invasion differed in roots of resistant and susceptible tomato cultivars. Here, we use a combination of scanning electron microscopy and light microscopy to investigate R. solanacearum colonization in roots of soil-grown resistant and susceptible tomato cultivars at multiple time points after inoculation. Our results show that colonization of the root vascular cylinder is delayed in resistant 'Hawaii7996' and that, once bacteria enter the root vascular tissues, colonization in the vasculature is spatially restricted. Our data suggest that resistance is due, in part, to the ability of the resistant cultivar to restrict bacterial root colonization in space and time.

  7. Biocidal activity in plant pathogenic Acidovorax, Burkholderia, Herbaspirillum, Ralstonia and Xanthomonas spp.

    PubMed

    Hu, F P; Young, J M

    1998-02-01

    Antibacterial and antifungal activity was investigated for strains of Acidovorax spp., Burkholderia spp., Herbaspirillum rubrisubalbicans and Ralstonia solanacearum; strains representing 118 species and pathovars of Xanthomonas were also tested for phytotoxic capacity. Antibacterial activity was present in all Burkholderia spp. except B. andropogonis, in biovars II and III of R. solanacearum but not in biovars I and IV, and in two strains of Xanthomonas. Little antibacterial activity was recorded for Acidovorax spp. Antifungal activity was expressed by most strains of A. avenae ssp. avenae and A. avenae ssp. cattleyae. Weak or variable antifungal reactions were given by strains of A. avenae ssp. citrulli and no activity was expressed by A. konjaci. Most strains of B. caryophylli, B. cepacia, B. gladioli pv. agaricicola, B. gladioli pv. alliicola, B. gladioli pv. gladioli, B. glumae and B. plantari produced extensive inhibition zones against Rhodotorula mucilaginosa. Strains of H. rubrisubalbicans and R. solanacearum gave negative, weak or variable reactions. Strains of Xanthomonas spp. exhibited no antifungal activity. In all cases antifungal activity was caused by a low molecular weight toxin. Three Xanthomonas strains exhibited phytotoxic activity. The ecological implications of these data are discussed.

  8. Disruption of comA homolog in Ralstonia solanacearum does not impair its twitching motility.

    PubMed

    Barman, Anjan; Buragohain, Chandrika; Ray, Suvendra Kumar

    2017-01-13

    Ralstonia solanacearum is an important phyto-pathogenic bacterium. The bacterium exhibits type IV pili meditated twitching motility that has been implicated in the process of natural transformation in it. A comA gene homolog, alike in several other naturally competent bacteria, has been already reported in this bacterium. However, there are no report of direct link between comA and twitching motility during the natural transformation process in this pathogen. In order to figure out any connection between comA and twitching motility, we created an insertion mutation in comA gene homolog of R. solanacearum F1C1 strain. As anticipated, the insertion mutant (CBRS01 strain) was inefficient for natural transformation. CBRS01 strain was found to be proficient for twitching motility alike the wild-type F1C1. This is interesting since recent findings of Salzer et al. (2016;Environ Microbiol;18:65-74) showed deficiency of twitching motility due to comEC gene (comA homolog) mutation in another naturally competent Gram-negative bacterium Thermus thermophilus. Additionally, we also found CBRS01 strain to be proficient for extracellular cellulase activity and virulence on tomato seedlings. Our findings in this work indicate that an R. solanacearum strain inefficient in undergoing natural transformation can, however, be proficient in exhibiting twitching motility.

  9. Preventing Ralstonia solanacearum adhesion with glycans from cashew, cocoa, coffee, pumpkin, and tomato seed extract.

    PubMed

    Rachmaninov, Ofra; Zinger-Yosovich, Keren D; Gilboa-Garber, Nechama

    2012-07-01

    Ralstonia solanacearum wilts many plants, causing heavy agricultural losses. Its pathogenic strain ATCC 11696 produces 2 hemagglutinating lectins: RSL and RS-IIL. These lectins may bind to terminal l-fucose-, d-arabinose-, and d-mannose-bearing seedling xylem cell wall glycans, thus enabling pathogen adhesion to them, with devastating infection establishment. Blocking the active sites of these lectins with seed embryo-surrounding oligo- and poly-saccharides hampers binding of the lectins to the embryos. The current study shows that seeds of cashew, cocoa, coffee, pumpkin, and tomato contain low and high molecular mass glycans that block RSL and RS-IIL (like its homologous Pseudomonas aeruginosa PA-IIL lectin). The blocking of the pathogen lectins, which is attributable to the documented composition of the oligo- and poly-saccharides of these seeds, is similar to that observed with animal glycoproteins of avian egg whites (protecting their embryos from infections) and of milk and royal jelly, which likewise protect mammal and bee neonates, respectively. RSL was most strongly inhibited by cashew seed glycans, and RS-IIL by coffee seed glycans. Western blot analyses with these lectins instead of antibodies revealed the hitherto undescribed presence of lectin-binding glycoproteins in the coffee, pumpkin, tomato, and cashew (but not cocoa) seeds. The use of these lectins for unveiling potent embryo-protecting seed glycans might be helpful for seedling-bioprotection projects similar to those planned for animal protection against antibiotic-resistant infections.

  10. Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum.

    PubMed

    Elhenawy, Wael; Scott, Nichollas E; Tondo, M Laura; Orellano, Elena G; Foster, Leonard J; Feldman, Mario F

    2016-03-01

    Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can cause wilting disease in many plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the majority of the glycopeptides were modified with a pentasaccharide composed of HexNAc-(Pen)-dHex(3), similar to the O antigen subunit present in the lipopolysaccharide of multiple R. solanacearum strains. Disruption of the O-OTase led to the total loss of protein glycosylation, together with a defect in biofilm formation and reduced pathogenicity towards tomato plants. Comparative proteomic analysis revealed that the loss of glycosylation is not associated with widespread proteome changes. Only the levels of a single glycoprotein, the type IV pilin, were diminished in the absence of glycosylation. In parallel, disruption of glycosylation triggered an increase in the levels of a surface lectin homologous to Pseudomonas PA-IIL. These results reveal the important role of glycosylation in the pathogenesis of R. solanacearum.

  11. Diversity of Ralstonia solanacearum in French Guiana expands knowledge of the "emerging ecotype".

    PubMed

    Deberdt, P; Guyot, J; Coranson-Beaudu, R; Launay, J; Noreskal, M; Rivière, P; Vigné, F; Laplace, D; Lebreton, L; Wicker, E

    2014-06-01

    Although bacterial wilt remains a major plant disease throughout South America and the Caribbean, the diversity of prevalent Ralstonia solanacearum populations is largely unknown. The genetic and phenotypic diversity of R. solanacearum strains in French Guiana was assessed using diagnostic polymerase chain reactions and sequence-based (egl and mutS) genotyping on a 239-strain collection sampled on the families Solanaceae and Cucurbitaceae, revealing an unexpectedly high diversity. Strains were distributed within phylotypes I (46.9%), IIA (26.8%), and IIB (26.3%), with one new endoglucanase sequence type (egl ST) found within each group. Phylotype IIB strains consisted mostly (97%) of strains with the emerging ecotype (IIB/sequevar 4NPB). Host range of IIB/4NPB strains from French Guiana matched the original emerging reference strain from Martinique. They were virulent on cucumber; virulent and highly aggressive on tomato, including the resistant reference Hawaii 7996; and only controlled by eggplant SM6 and Surya accessions. The emerging ecotype IIB/4NPB is fully established in French Guiana in both cultivated fields and uncultivated forest, rendering the hypothesis of introduction via ornamental or banana cuttings unlikely. Thus, this ecotype may have originated from the Amazonian region and spread throughout the Caribbean region.

  12. Genetic diversity and host range variation of Ralstonia solanacearum strains entering North America.

    PubMed

    Norman, David J; Zapata, Mildred; Gabriel, Dean W; Duan, Y P; Yuen, Jeanne M F; Mangravita-Novo, Arianna; Donahoo, Ryan S

    2009-09-01

    Each year, large volumes of ornamental and food plant propagative stock are imported into the North America; occasionally, Ralstonia solanacearum is found systemically infecting this plant material. In this study, 107 new R. solanacearum strains were collected over a 10-year period from imported propagative stock and compared with 32 previously characterized R. solanacearum strains using repetitive polymerase chain reaction (rep-PCR) element (BOX, ERIC, and REP) primers. Additional strain comparisons were made by sequencing the endoglucanase and the cytochrome b561 genes. Using rep-PCR primers, populations could be distinguished by biovar and, to a limited extent, country of origin and original host. Similarity coefficients among rep-PCR clusters within biovars were relatively low in many cases, indicating that disease outbreaks over time may have been caused by different clonal populations. Similar population differentiations of R. solanacearum were obtained when comparing strain sequences using either the endoglucanase or cytochrome b561 genes. We found that most of the new biovar 1 strains of R. solanacearum entering the United States were genetically distinct from the biovar 1 strains currently found infecting vegetable production. These introduced biovar 1 strains also had a broader host range and could infect not only tomato, tobacco, and potato but also anthurium and pothos and cause symptoms on banana. All introductions into North America of race 3, biovar 2 strains in the last few years have been linked to geranium production and appeared to be clonal.

  13. Recent trends in control methods for bacterial wilt diseases caused by Ralstonia solanacearum.

    PubMed

    Yuliar; Nion, Yanetri Asi; Toyota, Koki

    2015-01-01

    Previous studies have described the development of control methods against bacterial wilt diseases caused by Ralstonia solanacearum. This review focused on recent advances in control measures, such as biological, physical, chemical, cultural, and integral measures, as well as biocontrol efficacy and suppression mechanisms. Biological control agents (BCAs) have been dominated by bacteria (90%) and fungi (10%). Avirulent strains of R. solanacearum, Pseudomonas spp., Bacillus spp., and Streptomyces spp. are well-known BCAs. New or uncommon BCAs have also been identified such as Acinetobacter sp., Burkholderia sp., and Paenibacillus sp. Inoculation methods for BCAs affect biocontrol efficacy, such as pouring or drenching soil, dipping of roots, and seed coatings. The amendment of different organic matter, such as plant residue, animal waste, and simple organic compounds, have frequently been reported to suppress bacterial wilt diseases. The combined application of BCAs and their substrates was shown to more effectively suppress bacterial wilt in the tomato. Suppression mechanisms are typically attributed to the antibacterial metabolites produced by BCAs or those present in natural products; however, the number of studies related to host resistance to the pathogen is increasing. Enhanced/modified soil microbial communities are also indirectly involved in disease suppression. New promising types of control measures include biological soil disinfection using substrates that release volatile compounds. This review described recent advances in different control measures. We focused on the importance of integrated pest management (IPM) for bacterial wilt diseases.

  14. Involvement of ralfuranone production in the virulence of Ralstonia solanacearum OE1-1.

    PubMed

    Kai, Kenji; Ohnishi, Hideyuki; Mori, Yuka; Kiba, Akinori; Ohnishi, Kouhei; Hikichi, Yasufumi

    2014-11-24

    Ralstonia solanacearum causes a destructive disease called "bacterial wilt" in numerous plant species. Its virulence is controlled by the transcriptional regulator PhcA, the activity of which is, in turn, regulated in a cell-density dependent manner, termed quorum sensing. We herein described the identification and characterization of ralfuranones J-L, new PhcA-regulated secondary metabolites, and the known derivatives, ralfuranones A and B, from R. solanacearum strain OE1-1. Their structures were determined by spectroscopic and chemical methods. These ralfuranones were also detected in vascular exudates from host plants infected with OE1-1. Deletion of ralA, which encodes an enzyme for ralfuranone biosynthesis, reduced the virulence of OE1-1 in tomato plants. Virulence was restored by complementation of the ralA gene. The results suggest that ralfuranones play important roles in the virulence of OE1-1. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Antibacterial activity of Lansiumamide B to tobacco bacterial wilt (Ralstonia solanacearum).

    PubMed

    Li, Lichun; Feng, Xiujie; Tang, Ming; Hao, Wenbo; Han, Yun; Zhang, Guobin; Wan, Shuqing

    2014-01-01

    Tobacco bacterial wilt caused by Ralstonia solanacearum is one of the most serious diseases of tobacco in the area of tobacco cultivation. As there is no effective control method for tobacco bacterial wilt diseases, developing new antibacterial agents in tobacco will make great practical sense. The antibacterial activity against R. solanacearum of Lansiumamide B which is isolated from the seeds of Clausena lansium is reported in this paper for the first time. The bioassay results indicate that Lansiumamide B could completely inhibit the growth of R. solanacearum at the concentration of 125 mg/L in vitro, the EC50 and EC90 are 48.82 mg/L and 86.26 mg/L, respectively. The result of pot experiments indicates that the control efficiency of the Lansiumamide B on tobacco bacterial wilt are 95.84%, 91.67% and 86.38% at 7 days, 14 days and 21 days after treatment at the concentration of 100mg/kg, respectively, nearly 40 times higher than Streptomycin, a special fungicide to the disease, at 21 days after treatment with root irrigation method. These results suggest that Lansiumamide B has the potential of developing as a new type of plant-type fungicide on controlling the diseases of tobacco bacterial wilt. Copyright © 2013 Elsevier GmbH. All rights reserved.

  16. Evaluation of Resistance to Ralstonia solanacearum in Tomato Genetic Resources at Seedling Stage.

    PubMed

    Kim, Sang Gyu; Hur, On-Sook; Ro, Na-Young; Ko, Ho-Cheol; Rhee, Ju-Hee; Sung, Jung Sook; Ryu, Kyoung-Yul; Lee, Sok-Young; Baek, Hyung Jin

    2016-02-01

    Bacterial wilt of tomatoes caused by Ralstonia solanacearum is a devastating disease that limits the production of tomato in Korea. The best way to control this disease is using genetically resistant tomato plant. The resistance degree to R. solanacearum was evaluated for 285 tomato accessions conserved in the National Agrobiodiversity Center of Rural Development Administration. These accessions of tomato were originated from 23 countries. Disease severity of tomato accessions was investigated from 7 days to 14 days at an interval of 7 days after inoculation of R. solanacearum under greenhouse conditions. A total of 279 accessions of tomato germplasm were susceptible to R. solanacearum, resulting in wilt and death in 70 to 90% of these plants. Two tomato accessions were moderately resistant to R. solanacearum. Only four accessions showed high resistance against R. solanacearum. No distinct symptom of bacterial wilt appeared on the resistant tomato germplasms for up to 14 days after inoculation of R. solanacearum. Microscopy of resistant tomato stems infected with R. solanacearum revealed limited bacterial spread with thickening of pit membrane and gum production. Therefore, these four resistant tomato germplasms could be used in tomato breeding program against bacterial wilt.

  17. Methyl 3-Hydroxymyristate, a Diffusible Signal Mediating phc Quorum Sensing in Ralstonia solanacearum.

    PubMed

    Kai, Kenji; Ohnishi, Hideyuki; Shimatani, Mika; Ishikawa, Shiho; Mori, Yuka; Kiba, Akinori; Ohnishi, Kouhei; Tabuchi, Mitsuaki; Hikichi, Yasufumi

    2015-11-02

    Ralstonia solanacearum, a plant pathogenic bacterium causing "bacterial wilt" on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3-hydroxypalmitate (3-OH PAME) was previously identified as the QS signal in strain AW1. However, 3-OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)-methyl 3-hydroxymyristate [(R)-3-OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)-3-OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types--(R)-3-OH MAME or (R)-3-OH PAME. These results demonstrate that (R)-3-OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Detection of Quorum Sensing Molecules and Biofilm Formation in Ralstonia solanacearum.

    PubMed

    Kumar, J Shiva; Umesha, S; Prasad, K Shiva; Niranjana, P

    2016-03-01

    Many bacteria use small diffusible signaling molecules to communicate each other termed as quorum sensing (QS). Most Gram-negative bacteria use acyl homoserine lactone (AHL) as QS signal molecules. Using these signaling molecules, bacteria are able to express specific genes in response to population density. This work aimed to detect the production of QS signal molecules and biofilm formation in Ralstonia solanacearum isolated from various diseased tomato plants with symptoms of bacterial wilt. A total of 30 R. solanacearum strains were investigated for the production of QS signal molecules using Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1 (pZLR4) biosensor systems. All 30 bacterial isolates from various bacterial wilt-affected tomato plants produced AHL molecules that induced the biosensor. The microtiter plate assay demonstrated that of the 30 bacterial isolates, 60 % formed biofilm, among which four isolates exhibited a higher degree of biofilm formation. The biofilm-inducing factor was purified from these four culture supernatants. The structure of the responsible molecule was solved using nuclear magnetic resonance and mass spectroscopy and was determined to be 2-hydroxy-4-((methylamino)(phenyl)methyl) cyclopentanone (HMCP), which was confirmed by chemical synthesis and NMR. The Confocal laser scanning microscopic analysis showed well-developed biofilm architecture of bacteria when treated with HMCP. The knowledge we obtained from this study will be useful for further researcher on the role of HMCP molecule in biofilm formation.

  19. Genomic diversity of large-plaque-forming podoviruses infecting the phytopathogen Ralstonia solanacearum.

    PubMed

    Kawasaki, Takeru; Narulita, Erlia; Matsunami, Minaho; Ishikawa, Hiroki; Shimizu, Mio; Fujie, Makoto; Bhunchoth, Anjana; Phironrit, Namthip; Chatchawankanphanich, Orawan; Yamada, Takashi

    2016-05-01

    The genome organization, gene structure, and host range of five podoviruses that infect Ralstonia solanacearum, the causative agent of bacterial wilt disease were characterized. The phages fell into two distinctive groups based on the genome position of the RNA polymerase gene (i.e., T7-type and ϕKMV-type). One-step growth experiments revealed that ϕRSB2 (a T7-like phage) lysed host cells more efficiently with a shorter infection cycle (ca. 60 min corresponding to half the doubling time of the host) than ϕKMV-like phages such as ϕRSB1 (with an infection cycle of ca. 180 min). Co-infection experiments with ϕRSB1 and ϕRSB2 showed that ϕRSB2 always predominated in the phage progeny independent of host strains. Most phages had wide host-ranges and the phage particles usually did not attach to the resistant strains; when occasionally some did, the phage genome was injected into the resistant strain's cytoplasm, as revealed by fluorescence microscopy with SYBR Gold-labeled phage particles. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. The filamentous phage ϕRSS1 enhances virulence of phytopathogenic Ralstonia solanacearum on tomato.

    PubMed

    Addy, Hardian S; Askora, Ahmed; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2012-03-01

    Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. ϕRSS1 is a filamentous phage that infects R. solanacearum strains. Upon infection, it alters the physiological state and the behavior of host cells. Here, we show that R. solanacearum infected by ϕRSS1 becomes more virulent on host plants. Some virulence and pathogenicity factors, such as extracellular polysaccharide (EPS) synthesis and twitching motility, increased in the bacterial host cells infected with ϕRSS1, resulting in early wilting. Tomato plants inoculated with ϕRSS1-infected bacteria wilted 2 to 3 days earlier than those inoculated with wild-type bacteria. Infection with ϕRSS1 induced early expression of phcA, the global virulence regulator. phcA expression was detected in ϕRSS1-infected cells at cell density as low as 10(4) CFU/ml. Filamentous phages are assembled on the host cell surface and many phage particles accumulate on the cell surface. These surface-associated phage particles (phage proteins) may change the cell surface nature (hydrophobicity) to give high local cell densities. ϕRSS1 infection also enhanced PilA and type IV pilin production, resulting in increased twitching motility.

  1. New Insights into the Antibacterial Activity of Hydroxycoumarins against Ralstonia solanacearum.

    PubMed

    Yang, Liang; Ding, Wei; Xu, Yuquan; Wu, Dousheng; Li, Shili; Chen, Juanni; Guo, Bing

    2016-04-08

    Coumarins are important plant-derived natural products with wide-ranging bioactivities and extensive applications. In this study, we evaluated for the first time the antibacterial activity and mechanisms of action of coumarins against the phytopathogen Ralstonia solanacearum, and investigated the effect of functional group substitution. We first tested the antibacterial activity of 18 plant-derived coumarins with different substitution patterns, and found that daphnetin, esculetin, xanthotol, and umbelliferone significantly inhibited the growth of R. solanacearum. Daphnetin showed the strongest antibacterial activity, followed by esculetin and umbelliferone, with MICs of 64, 192, and 256 mg/L, respectively, better than the archetypal coumarin with 384 mg/L. We further demonstrated that the hydroxylation of coumarins at the C-6, C-7 or C-8 position significantly enhanced the antibacterial activity against R. solanacearum. Transmission electron microscope (TEM) and fluorescence microscopy images showed that hydroxycoumarins may interact with the pathogen by mechanically destroying the cell membrane and inhibiting biofilm formation. The antibiofilm effect of hydroxycoumarins may relate to the repression of flagellar genes fliA and flhC. These physiological changes in R. solanacearum caused by hydroxycoumarins can provide information for integral pathogen control. The present findings demonstrated that hydroxycoumarins have superior antibacterial activity against the phytopathogen R. solanacearum, and thus have the potential to be applied for controlling plant bacterial wilt.

  2. Towards the Identification of Type III Effectors Associated with Ralstonia solanacearum Virulence on Tomato and Eggplant.

    PubMed

    Pensec, Flora; Lebeau, Aurore; Daunay, M C; Chiroleu, Frédéric; Guidot, Alice; Wicker, Emmanuel

    2015-12-01

    For the development of pathogen-informed breeding strategies, identifying the microbial genes involved in interactions with the plant is a critical step. To identify type III effector (T3E) repertoires associated with virulence of the bacterial wilt pathogen Ralstonia solanacearum on Solanaceous crops, we used an original association genetics approach combining DNA microarray data and pathogenicity data on resistant eggplant, pepper, and tomato accessions. From this first screen, 25 T3Es were further full-length polymerase chain reaction-amplified within a 35-strain field collection, to assess their distribution and allelic diversity. Six T3E repertoire groups were identified, within which 11 representative strains were chosen to challenge the bacterial wilt-resistant egg plants 'Dingras multiple Purple' and 'AG91-25', and tomato Hawaii 7996. The virulence or avirulence phenotypes could not be explained by specific T3E repertoires, but rather by individual T3E genes. We identified seven highly avirulence-associated genes, among which ripP2, primarily referenced as conferring avirulence to Arabidopsis thaliana. Interestingly, no T3E was associated with avirulence to both egg-plants. Highly virulence-associated genes were also identified: ripA5_2, ripU, and ripV2. This study should be regarded as a first step toward investigating both avirulence and virulence function of the highlighted genes, but also their evolutionary dynamics in natural R. solanacearum populations.

  3. Protein O-linked glycosylation in the plant pathogen Ralstonia solanacearum

    PubMed Central

    Elhenawy, Wael; Scott, Nichollas E; Tondo, M Laura; Orellano, Elena G; Foster, Leonard J; Feldman, Mario F

    2016-01-01

    Ralstonia solanacearum is one of the most lethal phytopathogens in the world. Due to its broad host range, it can cause wilting disease in many plant species of economic interest. In this work, we identified the O-oligosaccharyltransferase (O-OTase) responsible for protein O-glycosylation in R. solanacearum. An analysis of the glycoproteome revealed that 20 proteins, including type IV pilins are substrates of this general glycosylation system. Although multiple glycan forms were identified, the majority of the glycopeptides were modified with a pentasaccharide composed of HexNAc-(Pen)-dHex3, similar to the O antigen subunit present in the lipopolysaccharide of multiple R. solanacearum strains. Disruption of the O-OTase led to the total loss of protein glycosylation, together with a defect in biofilm formation and reduced pathogenicity towards tomato plants. Comparative proteomic analysis revealed that the loss of glycosylation is not associated with widespread proteome changes. Only the levels of a single glycoprotein, the type IV pilin, were diminished in the absence of glycosylation. In parallel, disruption of glycosylation triggered an increase in the levels of a surface lectin homologous to Pseudomonas PA-IIL. These results reveal the important role of glycosylation in the pathogenesis of R. solanacearum. PMID:26531228

  4. Oleanolic Acid Induces the Type III Secretion System of Ralstonia solanacearum

    PubMed Central

    Wu, Dousheng; Ding, Wei; Zhang, Yong; Liu, Xuejiao; Yang, Liang

    2015-01-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, can naturally infect a wide range of host plants. The type III secretion system (T3SS) is a major virulence determinant in this bacterium. Studies have shown that plant-derived compounds are able to inhibit or induce the T3SS in some plant pathogenic bacteria, though no specific T3SS inhibitor or inducer has yet been identified in R. solanacearum. In this study, a total of 50 different compounds were screened and almost half of them (22 of 50) significantly inhibited or induced the T3SS expression of R. solanacearum. Based on the strong induction activity on T3SS, the T3SS inducer oleanolic acid (OA) was chosen for further study. We found that OA induced the expression of T3SS through the HrpG-HrpB pathway. Some type III effector genes were induced in T3SS inducing medium supplemented with OA. In addition, OA targeted only the T3SS and did not affect other virulence determinants. Finally, we observed that induction of T3SS by OA accelerated disease progress on tobacco. Overall our results suggest that plant-derived compounds are an abundant source of R. solanacearum T3SS regulators, which could prove useful as tools to interrogate the regulation of this key virulence pathway. PMID:26732647

  5. Resveratrol and Coumarin: Novel Agricultural Antibacterial Agent against Ralstonia solanacearum In Vitro and In Vivo.

    PubMed

    Chen, Juanni; Yu, Yanmei; Li, Shili; Ding, Wei

    2016-11-09

    Bacterial wilt is a destructive disease caused by the phytopathogen Ralstonia solanacearum (R. solanacearum), which is widely found in various tobacco-growing areas all over the world. Botanical bactericidal substances have gradually emerged as a hot topic in modern pesticide research. In this study, the antibacterial activities of two phytochemicals (resveratrol and coumarin) against R. solanacearum and their in vivo and in vitro efficacy for controlling tobacco bacterial wilt were evaluated. We rule out significant biological effects of both phytochemicals using transmission electron microscope (TEM) and fluorescence microscope, which suppressed the growth of R. solanacearum. Furthermore, we demonstrated that the toxicity mechanisms mainly involved damaging bacterial cell membrane and preventing swarming motility and biofilm formation. A further pot experiment demonstrated that coumarin and resveratrol significantly inhibited early adhesion and colonization of R. solanacearum in tobacco plants and the corresponding control efficacies were 68% and 85% after incubation for 13 days, respectively. The findings of this study suggest that both resveratrol and coumarin have potential as non-toxic antimicrobial strategies for controlling tobacco bacterial wilt disease.

  6. Transcriptome Analysis of Quantitative Resistance-Specific Response upon Ralstonia solanacearum Infection in Tomato

    PubMed Central

    Ishihara, Takeaki; Mitsuhara, Ichiro; Takahashi, Hideki; Nakaho, Kazuhiro

    2012-01-01

    Bacterial wilt, caused by the soil-borne bacterium Ralstonia solanacearum, is a lethal disease of tomato, but the molecular mechanisms of the host resistance responses to R. solanacearum remain unclear. In this study, we report the first work describing the transcriptome of cultivar resistance and susceptible tomato cultivar after inoculation with R. solanacearum. To elucidate the characteristics of resistance early in the interaction, we analyzed microarrays for resistant cultivar LS-89 and susceptible cultivar Ponderosa 1 day after stem inoculation. No change in gene expression was detected for Ponderosa, but expression levels of over 140 genes, including pathogenesis-related, hormone signaling and lignin biosynthesis genes, increased in LS-89. Expression of β-1,3-glucanase genes increased substantially. In an immunohistochemical study, glucanase in LS-89 accumulated in the xylem and pith tissues surrounding xylem vessels filled with R. solanacearum. The expression of these genes also increased in four other resistant cultivars, but changed little in four susceptible cultivars in response to R. solanacearum, suggesting that similar reactions occur in other cultivars. These gene expression profiles will serve as fundamental information to elucidate the molecular mechanisms in the resistance response to R. solanacearum in tomato. PMID:23071630

  7. Effects of Caatinga Plant Extracts in Planktonic Growth and Biofilm Formation in Ralstonia solanacearum.

    PubMed

    Malafaia, Carolina Barbosa; Jardelino, Ana Cláudia Silva; Silva, Alexandre Gomes; de Souza, Elineide Barbosa; Macedo, Alexandre José; Correia, Maria Tereza Dos Santos; Silva, Márcia Vanusa

    2017-09-17

    This study describes the first antibiofilm and antibacterial screening for plants from Caatinga against Ralstonia solanacearum, a causal agent of bacterial wilt that presents serious difficulties in control. There were prepared 22 aqueous extracts of plants collected in the Vale do Catimbau-PE, Brazil. The potential antibacterial activity was evaluated by absorbance in OD600 and the antibiofilm activity through the crystal violet method, both of them performed in microplate against isolates of R. solanacearum biofilm formers. The results of the screening showed that Jacaranda rugosa presented antimicrobial activity higher than 90%, while Harpochilus neesianus and Myroxylon peruiferum presented antibiofilm activity higher than 50% for all tested isolates. However, Croton heliotropiifolius showed both the activities, being thus very promising for application in the control of this phytopathogen. The search for viable alternatives to the development of new bioactive compounds safe for the environment, humans, and animals from an adverse and scarce environment such as the Caatinga and encouraged us to find plants that produce effective metabolites against phytopathogenic microorganisms. This in vitro screening is important to guide the development of new products in addition to guide research studies of bioactive compounds.

  8. Comparative behavior of Ralstonia solanacearum biovar 2 in diverse plant species.

    PubMed

    Alvarez, B; Vasse, J; Le-Courtois, V; Trigalet-Démery, D; López, M M; Trigalet, A

    2008-01-01

    Ralstonia solanacearum causes bacterial wilt in numerous plant species worldwide. Although biovar 2 mostly affects solanaceous crops, identification of new hosts remains a matter of concern since there is still no clear-cut distinction between host and nonhost plants. In this work we provide data based on histological studies on the status of 20 plant species, most of them of potential interest in crop rotation. Plants were watered with a beta-glucuronidase-expressing derivative of R. solanacearum biovar 2, and after a month of incubation, sections of roots and stems were analyzed to localize the pathogen on surface, in cortex and/or xylem. Depending on whether the xylem was colonized or not, plants were classified as hosts or nonhosts, respectively. Hosts generally affected in a few xylem vessels or occasionally in all xylem bundles were classified as tolerant. These included some cabbage, kidney bean, and rutabaga cultivars, and the weed bittersweet nightshade (Solanum dulcamara). Nonhosts were the cultivars tested of alfalfa, barley, black radish, carrot, celery, colocynth, fennel, fiber flax, field bean, field pea, horseradish, maize, and zucchini. However, barley and maize, though nonhosts, may act as reservoirs for the pathogen. The present work constitutes a basis for further studies on cropping systems in fields where R. solanacearum has been detected.

  9. Biodegradation kinetics of 4-fluorocinnamic acid by a consortium of Arthrobacter and Ralstonia strains.

    PubMed

    Hasan, Syed A; Wietzes, Piet; Janssen, Dick B

    2012-02-01

    Arthrobacter sp. strain G1 is able to grow on 4-fluorocinnamic acid (4-FCA) as sole carbon source. The organism converts 4-FCA into 4-fluorobenzoic acid (4-FBA) and utilizes the two-carbon side-chain for growth with some formation of 4-fluoroacetophenone as a dead-end side product. We also have isolated Ralstonia sp. strain H1, an organism that degrades 4-FBA. A consortium of strains G1 and H1 degraded 4-FCA with Monod kinetics during growth in batch and continuous cultures. Specific growth rates of strain G1 and specific degradation rates of 4-FCA were observed to follow substrate inhibition kinetics, which could be modeled using the kinetic models of Haldane-Andrew and Luong-Levenspiel. The mixed culture showed complete mineralization of 4-FCA with quantitative release of fluoride, both in batch and continuous cultures. Steady-state chemostat cultures that were exposed to shock loadings of substrate responded with rapid degradation and returned to steady-state in 10-15 h, indicating that the mixed culture provided a robust system for continuous 4-FCA degradation.

  10. Comparative genomic analysis of Ralstonia solanacearum reveals candidate genes for host specificity.

    PubMed

    Ailloud, Florent; Lowe, Tiffany; Cellier, Gilles; Roche, David; Allen, Caitilyn; Prior, Philippe

    2015-04-08

    Ralstonia solanacearum is a vascular soil-borne plant pathogen with an unusually broad host range. This economically destructive and globally distributed bacterium has thousands of distinct lineages within a heterogeneous and taxonomically disputed species complex. Some lineages include highly host-adapted strains (ecotypes), such as the banana Moko disease-causing strains, the cold-tolerant potato brown rot strains (also known as R3bv2) and the recently emerged Not Pathogenic to Banana (NPB) strains. These distinct ecotypes offer a robust model to study host adaptation and the emergence of ecotypes because the polyphyletic Moko strains include lineages that are phylogenetically close to the monophyletic brown rot and NPB strains. Draft genomes of eight new strains belonging to these three model ecotypes were produced to complement the eleven publicly available R. solanacearum genomes. Using a suite of bioinformatics methods, we searched for genetic and evolutionary features that distinguish ecotypes and propose specific hypotheses concerning mechanisms of host adaptation in the R. solanacearum species complex. Genome-wide, few differences were identified, but gene loss events, non-synonymous polymorphisms, and horizontal gene transfer were identified among type III effectors and were associated with host range differences. This extensive comparative genomics analysis uncovered relatively few divergent features among closely related strains with contrasting biological characteristics; however, several virulence factors were associated with the emergence of Moko, NPB and brown rot and could explain host adaptation.

  11. Elicitor-Induced Defense Responses in Solanum lycopersicum against Ralstonia solanacearum

    PubMed Central

    Kar, Itishree; Mukherjee, Arup K.; Acharya, Priyambada

    2013-01-01

    We investigated on important parameters of induced resistance in hydroponic tomato (Solanum lycopersicum) against Ralstonia solanacearum using the elicitors chitosan (CHT), salicylic acid (SA), and jasmonic acid (JA). The increase in total phenolic content of roots by the elicitors was significantly higher than control. Most pronounced increase in lignin synthesis was triggered by SA followed by CHT. At 24 h post-elicitation (hpe), the activity of phenylalanine ammonia lyase was 4.5 times higher than control elicited by CHT. The peroxidase activity was about 86 nkat/mg protein at 24 hpe in case of SA and 78 nkat/mg protein in case of CHT. The activity of polyphenol oxidase increased several folds by the elicitors. Cinnamyl alcohol dehydrogenase activity increased to the maximum at 48 hpe under the influence of CHT. The results indicate that the elicitors SA and CHT induced effective defense responses in tomato plants against R. solanacearum. This was evident from reduced vascular browning and wilting symptoms of tomato plants treated with SA and CHT and challenged subsequently with R. solanacearum. This reduced disease incidence in tomato by SA and CHT may be a result of cell wall strengthening through deposition of lignin and the coincident induction of defense enzymes. PMID:24187521

  12. Characterization of the Adaptive Response to Trichloroethylene-Mediated Stresses in Ralstonia pickettii PKO1

    PubMed Central

    Park, Joonhong; Kukor, Jerome J.; Abriola, Linda M.

    2002-01-01

    In Ralstonia pickettii PKO1, a denitrifying toluene oxidizer that carries a toluene-3-monooxygenase (T3MO) pathway, the biodegradation of toluene and trichloroethylene (TCE) by the organism is induced by TCE at high concentrations. In this study, the effect of TCE preexposure was studied in the context of bacterial protective response to TCE-mediated toxicity in this organism. The results of TCE degradation experiments showed that cells induced by TCE at 110 mg/liter were more tolerant to TCE-mediated stress than were those induced by TCE at lower concentrations, indicating an ability of PKO1 to adapt to TCE-mediated stress. To characterize the bacterial protective response to TCE-mediated stress, the effect of TCE itself (solvent stress) was isolated from TCE degradation-dependent stress (toxic intermediate stress) in the subsequent chlorinated ethylene toxicity assays with both nondegradable tetrachloroethylene and degradable TCE. The results of the toxicity assays showed that TCE preexposure led to an increase in tolerance to TCE degradation-dependent stress rather than to solvent stress. The possibility that such tolerance was selected by TCE degradation-dependent stress during TCE preexposure was ruled out because a similar extent of tolerance was observed in cells that were induced by toluene, whose metabolism does not produce any toxic products. These findings suggest that the adaptation of TCE-induced cells to TCE degradation-dependent stress was caused by the combined effects of solvent stress response and T3MO pathway expression. PMID:12406709

  13. Characterization of the survival ability of Cupriavidus metallidurans and Ralstonia pickettii from space-related environments.

    PubMed

    Mijnendonckx, K; Provoost, A; Ott, C M; Venkateswaran, K; Mahillon, J; Leys, N; Van Houdt, R

    2013-02-01

    Four Cupriavidus metallidurans and eight Ralstonia pickettii isolates from the space industry and the International Space Station (ISS) were characterized in detail. Nine of the 12 isolates were able to form a biofilm on plastics and all were resistant to several antibiotics. R. pickettii isolates from the surface of the Mars Orbiter prior to flight were 2.5 times more resistant to UV-C(254nm) radiation compared to the R. pickettii type strain. All isolates showed moderate to high tolerance against at least seven different metal ions. They were tolerant to medium to high silver concentrations (0.5-4 μM), which are higher than the ionic silver disinfectant concentrations measured regularly in the drinking water aboard the ISS. Furthermore, all isolates survived a 23-month exposure to 2 μM AgNO(3) in drinking water. These resistance properties are putatively encoded by their endogenous megaplasmids. This study demonstrated that extreme resistance is not required to withstand the disinfection and sterilization procedures implemented in the ISS and space industry. All isolates acquired moderate to high tolerance against several stressors and can grow in oligotrophic conditions, enabling them to persist in these environments.

  14. Arsenite oxidase from Ralstonia sp. 22: characterization of the enzyme and its interaction with soluble cytochromes.

    PubMed

    Lieutaud, Aurélie; van Lis, Robert; Duval, Simon; Capowiez, Line; Muller, Daniel; Lebrun, Régine; Lignon, Sabrina; Fardeau, Marie-Laure; Lett, Marie-Claire; Nitschke, Wolfgang; Schoepp-Cothenet, Barbara

    2010-07-02

    We characterized the aro arsenite oxidation system in the novel strain Ralstonia sp. 22, a beta-proteobacterium isolated from soil samples of the Salsigne mine in southern France. The inducible aro system consists of a heterodimeric membrane-associated enzyme reacting with a dedicated soluble cytochrome c(554). Our biochemical results suggest that the weak association of the enzyme to the membrane probably arises from a still unknown interaction partner. Analysis of the phylogeny of the aro gene cluster revealed that it results from a lateral gene transfer from a species closely related to Achromobacter sp. SY8. This constitutes the first clear cut case of such a transfer in the Aro phylogeny. The biochemical study of the enzyme demonstrates that it can accommodate in vitro various cytochromes, two of which, c(552) and c(554,) are from the parent species. Cytochrome c(552) belongs to the sox and not the aro system. Kinetic studies furthermore established that sulfite and sulfide, substrates of the sox system, are both inhibitors of Aro activity. These results reinforce the idea that sulfur and arsenic metabolism are linked.

  15. Recent Trends in Control Methods for Bacterial Wilt Diseases Caused by Ralstonia solanacearum

    PubMed Central

    Yuliar; Nion, Yanetri Asi; Toyota, Koki

    2015-01-01

    Previous studies have described the development of control methods against bacterial wilt diseases caused by Ralstonia solanacearum. This review focused on recent advances in control measures, such as biological, physical, chemical, cultural, and integral measures, as well as biocontrol efficacy and suppression mechanisms. Biological control agents (BCAs) have been dominated by bacteria (90%) and fungi (10%). Avirulent strains of R. solanacearum, Pseudomonas spp., Bacillus spp., and Streptomyces spp. are well-known BCAs. New or uncommon BCAs have also been identified such as Acinetobacter sp., Burkholderia sp., and Paenibacillus sp. Inoculation methods for BCAs affect biocontrol efficacy, such as pouring or drenching soil, dipping of roots, and seed coatings. The amendment of different organic matter, such as plant residue, animal waste, and simple organic compounds, have frequently been reported to suppress bacterial wilt diseases. The combined application of BCAs and their substrates was shown to more effectively suppress bacterial wilt in the tomato. Suppression mechanisms are typically attributed to the antibacterial metabolites produced by BCAs or those present in natural products; however, the number of studies related to host resistance to the pathogen is increasing. Enhanced/modified soil microbial communities are also indirectly involved in disease suppression. New promising types of control measures include biological soil disinfection using substrates that release volatile compounds. This review described recent advances in different control measures. We focused on the importance of integrated pest management (IPM) for bacterial wilt diseases. PMID:25762345

  16. Ralstonia solanacearum lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues

    PubMed Central

    Spraker, Joseph E; Sanchez, Laura M; Lowe, Tiffany M; Dorrestein, Pieter C; Keller, Nancy P

    2016-01-01

    Ralstonia solanacearum is a globally distributed soil-borne plant pathogenic bacterium, which shares a broad ecological range with many plant- and soil-associated fungi. We sought to determine if R. solanacearum chemical communication directs symbiotic development of polymicrobial consortia. R. solanacearum produced a diffusible metabolite that induced conserved morphological differentiation in 34 species of fungi across three diverse taxa (Ascomycetes, Basidiomycetes and Zygomycetes). Fungi exposed to this metabolite formed chlamydospores, survival structures with thickened cell walls. Some chlamydospores internally harbored R. solanacearum, indicating a newly described endofungal lifestyle for this important plant pathogen. Using imaging mass spectrometry and peptidogenomics, we identified an undescribed lipopeptide, ralsolamycin, produced by an R. solanacearum non-ribosomal peptide synthetase-polyketide synthase hybrid. Inactivation of the hybrid non-ribosomal peptide synthetase-polyketide synthase gene, rmyA, abolished ralsolamycin synthesis. R. solanacearum mutants lacking ralsolamycin no longer induced chlamydospore development in fungal coculture and invaded fungal hyphae less well than wild-type. We propose that ralsolamycin contributes to the invasion of fungal hyphae and that the formation of chlamydospores may provide not only a specific niche for bacterial colonization but also enhanced survival for the partnering fungus. PMID:26943626

  17. Contribution of Folate Biosynthesis to Ralstonia solanacearum Proliferation in Intercellular Spaces

    PubMed Central

    Shinohara, Rena; Kanda, Ayami; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2005-01-01

    The vigorous proliferation of Ralstonia solanacearum OE1-1 in host intercellular spaces after the invasion of host plants is necessary for the virulence of this bacterium. A folate auxotroph, RM, in which a mini-Tn5 transposon was inserted into pabB encoding para-aminobenzoate synthase component I, lost its ability to vigorously proliferate in intercellular spaces along with its systemic infectivity and virulence after inoculation into roots and infiltration into leaves of tobacco plants. Complementation of RM with the pabB gene allowed the mutant to multiply in intercellular spaces and to cause disease. In tobacco plants that were pretreated with folate, RM was able to vigorously proliferate in the intercellular spaces and cause disease. Interestingly, when it was inoculated through cut stems, the mutant multiplied in the plants and was virulent. Moreover, the mutant multiplied well in stem fluids but not in intercellular fluids, suggesting that the folate concentration within intercellular spaces may be a limiting factor for bacterial proliferation. Therefore, folate biosynthesis contributes to the vigorous proliferation of bacteria in intercellular spaces and leads to systemic infectivity resulting in virulence. PMID:15640216

  18. Functional analysis of Ralstonia solanacearum PrhG regulating the hrp regulon in host plants.

    PubMed

    Zhang, Yong; Chen, Li; Yoshimochi, Takeshi; Kiba, Akinori; Hikichi, Yasufumi; Ohnishi, Kouhei

    2013-08-01

    Genes in the hrp regulon encode component proteins of the type III secretion system and are essential for the pathogenicity of Ralstonia solanacearum. The hrp regulon is controlled by HrpB. We isolated several genes regulating hrpB expression from the Japanese strain OE1-1 using minitransposon mutagenesis. Among them, we mainly focused on two genes, hrpG and prhG, which are the positive regulators of hrpB. Although the global virulence regulator PhcA negatively regulated hrpG expression via prhIR, it positively regulated prhG expression. We further investigated the contrasting regulation of hrpG and prhG by PhcA and speculated that R. solanacearum may switch from HrpG to PrhG for hrpB activation in a cell density-dependent manner. Although the prhG mutant proliferated similarly to the wild-type in leaf intercellular spaces and in xylem vessels of the host plants, it was less virulent than the wild-type. The expression of the popA operon, which belongs to the hrp regulon, was significantly reduced in the prhG mutant by more than half in the leaf intercellular spaces and more than two-thirds in the xylem vessels when compared with the wild-type.

  19. Quantitative Immunofluorescence of Regulated eps Gene Expression in Single Cells of Ralstonia solanacearum

    PubMed Central

    Kang, Yaowei; Saile, Elke; Schell, Mark A.; Denny, Timothy P.

    1999-01-01

    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 (>107 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 β-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. PMID:10347013

  20. Contrasting recombination patterns and demographic histories of the plant pathogen Ralstonia solanacearum inferred from MLSA

    PubMed Central

    Wicker, Emmanuel; Lefeuvre, Pierre; de Cambiaire, Jean-Charles; Lemaire, Christophe; Poussier, Stéphane; Prior, Philippe

    2012-01-01

    We used multilocus sequence analysis (MLSA) on a worldwide collection of the plant pathogenic Ralstonia solanacearum (Betaproteobacteria) to retrace its complex evolutionary history. Using genetic imprints left during R. solanacearum evolution, we were able to delineate distinct evolutionary complex displaying contrasting dynamics. Among the phylotypes already described (I, IIA, IIB, III, IV), eight groups of strains with distinct evolutionary patterns, named clades, were identified. From our recombination analysis, we identified 21 recombination events that occurred within and across these lineages. Although appearing the most divergent and ancestral phylotype, phylotype IV was inferred as a gene donor for the majority of the recombination events that we detected. Whereas this phylotype apparently fuelled the species diversity, ongoing diversification was mainly detected within phylotype I, IIA and III. These three groups presented a recent expanding population structure, a high level of homologous recombination and evidences of long-distance migrations. Factors such as adaptation to a specific host or intense trading of infected crops may have promoted this diversification. Whether R. solanacearum lineages will eventually evolve in distinct species remains an open question. The intensification of cropping and increase of geographical dispersion may favour situations of phylotype sympatry and promote higher exchange of key factors for host adaptation from their common genetic pool. PMID:22094345

  1. Ralstonia solanacearum lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues.

    PubMed

    Spraker, Joseph E; Sanchez, Laura M; Lowe, Tiffany M; Dorrestein, Pieter C; Keller, Nancy P

    2016-09-01

    Ralstonia solanacearum is a globally distributed soil-borne plant pathogenic bacterium, which shares a broad ecological range with many plant- and soil-associated fungi. We sought to determine if R. solanacearum chemical communication directs symbiotic development of polymicrobial consortia. R. solanacearum produced a diffusible metabolite that induced conserved morphological differentiation in 34 species of fungi across three diverse taxa (Ascomycetes, Basidiomycetes and Zygomycetes). Fungi exposed to this metabolite formed chlamydospores, survival structures with thickened cell walls. Some chlamydospores internally harbored R. solanacearum, indicating a newly described endofungal lifestyle for this important plant pathogen. Using imaging mass spectrometry and peptidogenomics, we identified an undescribed lipopeptide, ralsolamycin, produced by an R. solanacearum non-ribosomal peptide synthetase-polyketide synthase hybrid. Inactivation of the hybrid non-ribosomal peptide synthetase-polyketide synthase gene, rmyA, abolished ralsolamycin synthesis. R. solanacearum mutants lacking ralsolamycin no longer induced chlamydospore development in fungal coculture and invaded fungal hyphae less well than wild-type. We propose that ralsolamycin contributes to the invasion of fungal hyphae and that the formation of chlamydospores may provide not only a specific niche for bacterial colonization but also enhanced survival for the partnering fungus.

  2. Use of Amplified Ribosomal DNA Restriction Analysis for Identification of Ralstonia and Pandoraea Species: Interest in Determination of the Respiratory Bacterial Flora in Patients with Cystic Fibrosis

    PubMed Central

    Segonds, Christine; Paute, Sandrine; Chabanon, Gérard

    2003-01-01

    The recovery of Ralstonia and Pandoraea species from respiratory tract cultures of patients with cystic fibrosis has recently been reported. These species are difficult to identify, and especially to differentiate from Burkholderia cepacia complex organisms, with classical methods. The discriminatory power of amplified ribosomal DNA restriction analysis (ARDRA) within the two genera was assessed by comparing the restriction profiles of reference strains of each species by using a panel of six enzymes already proven suitable for the identification of Burkholderia species. ARDRA provided differentiation of all the Ralstonia species tested and of Pandoraea norimbergensis. Pandoraea species P. pnomenusa, P. sputorum, P. pulmonicola, and P. apista were not discriminated to the species level. This method allowed the identification of five clinical isolates recovered from French cystic fibrosis patients as Ralstonia mannitolilytica. PMID:12843108

  3. CaHDZ27, a Homeodomain-Leucine Zipper I (HD-Zip I) Protein, Positively Regulates the Resistance to Ralstonia solanacearum Infection in Pepper.

    PubMed

    Mou, Shaoliang; Liu, Zhiqin; Gao, Feng; Yang, Sheng; Su, Meixia; Shen, Lei; Wu, Yang; He, Shuilin

    2017-08-25

    Homeodomain-leucine zipper class I (HD-Zip I) transcription factors (TFs) have been functionally characterized in plant responses to abiotic stresses, but their roles in plant immunity are poorly understood. Here, a HD-Zip I gene, CaHZ27, was isolated from pepper (Capsicum annum) and characterized for its role in pepper immunity. Quantitative real-time PCR showed that CaHDZ27 was transcriptionally induced by Ralstonia solanacearum inoculation and exogenous application of methyl jasmonate (MeJA), salicylic acid (SA), or ethephon (ETH). The CaHDZ27-GFP (green fluorescent protein) fused protein was targeted exclusively to the nucleus. Chromatin immunoprecipitation (ChIP) demonstrated that CaHDZ27 bound to the 9-bp pseudopalindromic element (CAATAATTG) and triggered GUS expression in a CAATAATTG-dependent manner. Virus-induced gene silencing (VIGS) of CaHDZ27 significantly attenuated the resistance of pepper plants against R. solanacearum and downregulated defense-related marker genes including CaHIR1, CaACO1, CaPR1, CaPR4, CaPO2, and CaBPR1. By contrast, transient overexpression of CaHDZ27 triggered strong cell death mediated by the hypersensitive response (HR), and upregulated the tested immunity-associated marker genes. Ectopic CaHDZ27 expression in tobacco enhances its resistance against R. solanacearum. These results collectively suggest that CaHDZ27 functions as a positive regulator in pepper resistance against R. solanacearum. Bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (CoIP) assays indicate that CaHDZ27 monomers bind with each other, and this binding is enhanced significantly by R. solanacearum inoculation. We speculate that homodimerization of CaHZ27 might play a role in pepper response to R. solanacearum, further direct evidence is required to confirm it.

  4. In Vivo Detection of the Cyclic Osmoregulated Periplasmic Glucan of Ralstonia solanacearum by High-Resolution Magic Angle Spinning NMR

    NASA Astrophysics Data System (ADS)

    Wieruszeski, J.-M.; Bohin, A.; Bohin, J.-P.; Lippens, G.

    2001-07-01

    We investigate the mobility of the osmoregulated periplasmic glucans of Ralstonia solanacearum in the bacterial periplasm through the use of high-resolution (HR) NMR spectroscopy under static and magic angle spinning (MAS) conditions. Because the nature of periplasm is far from an isotropic aqueous solution, the molecules could be freely diffusing or rather associated to a periplasmic protein, a membrane protein, a lipid, or the peptidoglycan. HR MAS NMR spectroscopy leads to more reproducible results and allows the in vivo detection and characterization of the complex molecule.

  5. DNA fingerprinting of Ralstonia paucula by infrequent-restriction-site PCR and randomly amplified polymorphic DNA analysis.

    PubMed

    Moissenet, Didier; Vu-Thien, Hoang; Benzerara, Yahia; Arlet, Guillaume

    2003-12-01

    Ralstonia paucula (formerly CDC group IV c-2) is an environmental organism that can cause serious human infections, occasionally clusters of nosocomial infections. In the present work, 26 strains of R. paucula (4 from the American Centers for Disease Control and Prevention collection, 10 from the Belgian Laboratorium voor Microbiologie [LMG] collection, and 12 French clinical isolates) were analyzed with infrequent-restriction-site PCR and randomly amplified polymorphic DNA analysis. Both techniques accurately distinguished between collection strains. Two close patterns obtained for all the French isolates suggested a clonal strain. Two LMG collection strains originating from human sources in the United States also showed patterns close to those of French isolates.

  6. Isolated Pseudomonas aeruginosa strain VIH2 and antagonistic properties against Ralstonia solanacearum.

    PubMed

    Ge, Xincheng; Wei, Wei; Li, Gen; Sun, Mingming; Li, Huixin; Wu, Jun; Hu, Feng

    2017-10-01

    The aim of this study was to isolates with antagonist activity against R. solanacearum. Thirty-two bacterial isolates were obtained from samples, and they were screened for potential antagonistic activity against R. Solanacearum. Using the agar spot method, ten out of the 21 tested bacteria showed antilisterial activity. VIH2 had the highest inhibitory effect on the growth of R. Solanacearum. Based on 16S rDNA and Biolog test analysis, the strain VIH2 was identified as Pseudomonas aeruginosa. Single-factor and Response Surface Methodology experiments were used to optimize the culture medium and conditions. This study was to explore whether the hemolysin-co-regulated protein secretion island I (HSI-I)-encoded type VI secretion system (T6SS) in Pseudomonas can be used as a biological control approach against Ralstonia solanacearum under field conditions. Bacterial competition assay showed that the HSI-I type T6SS of strain VIH2 exhibited dramatic antibacterial killing activity against R. solanacearum. The HSI-I T6SS of P. aeruginosa was regulated by the ppKA gene. We disrupted the gene ppKA in VIH2 by a single crossover to yield the VIH2 (ΔppKA) mutant. The antagonism of VIH2 was significantly decreased by ppKA gene disruption. In conclusion, our data supported the idea that HSI-I T6SS plays a crucial role in the antagonistic action of strain VIH2 against R. solanacearum. This alternative approach for antagonism against R. solanacearum might help develop attenuated strains of engineered bacteria for biological control. Copyright © 2017. Published by Elsevier Ltd.

  7. A duplex PCR assay for the detection of Ralstonia solanacearum phylotype II strains in Musa spp.

    PubMed

    Cellier, Gilles; Moreau, Aurélie; Chabirand, Aude; Hostachy, Bruno; Ailloud, Florent; Prior, Philippe

    2015-01-01

    Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.

  8. Influence of native microbiota on survival of Ralstonia solanacearum phylotype II in river water microcosms.

    PubMed

    Alvarez, Belén; López, María M; Biosca, Elena G

    2007-11-01

    Ralstonia solanacearum phylotype II biovar 2 causes bacterial wilt in solanaceous hosts, producing severe economic losses worldwide. Waterways can be major dissemination routes of this pathogen, which is able to survive for long periods in sterilized water. However, little is known about its survival in natural water when other microorganisms, such as bacteriophages, other bacteria, and protozoa, are present. This study looks into the fate of a Spanish strain of R. solanacearum inoculated in water microcosms from a Spanish river, containing different microbiota fractions, at 24 degrees C and 14 degrees C, for a month. At both temperatures, R. solanacearum densities remained constant at the initial levels in control microcosms of sterile river water while, by contrast, declines in the populations of the introduced strain were observed in the nonsterile microcosms. These decreases were less marked at 14 degrees C. Lytic bacteriophages present in this river water were involved in the declines of the pathogen populations, but indigenous protozoa and bacteria also contributed to the reduced persistence in water. R. solanacearum variants displaying resistance to phage infection were observed, but only in microcosms without protozoa and native bacteria. In water microcosms, the temperature of 14 degrees C was more favorable for the survival of this pathogen than 24 degrees C, since biotic interactions were slower at the lower temperature. Similar trends were observed in microcosms inoculated with a Dutch strain. This is the first study demonstrating the influence of different fractions of water microorganisms on the survival of R. solanacearum phylotype II released into river water microcosms.

  9. Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential.

    PubMed

    Wicker, Emmanuel; Grassart, Laurence; Coranson-Beaudu, Régine; Mian, Danièle; Guilbaud, Caroline; Fegan, Mark; Prior, Philippe

    2007-11-01

    We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America.

  10. Characterization of biofumigated Ralstonia solanacearum cells using micro-Raman spectroscopy and electron microscopy.

    PubMed

    Paret, Mathews L; Sharma, Shiv K; Alvarez, Anne M

    2012-01-01

    Essential oils of palmarosa, lemongrass, and eucalyptus have shown promise as biofumigants for control of the bacterial wilt disease of edible ginger (Zingiber officinale) caused by Ralstonia solanacearum race 4 in previous potting medium studies. Biochemical changes in R. solanacearum cells were evaluated with micro-Raman spectroscopy following treatment with essential oils at different concentrations (0.04, 0.07, and 0.14% [vol/vol] of culture medium) and changes in cell structure were observed using electron microscopy. All treatments except palmarosa oil at 0.04% caused significant reductions in levels of amino acids, purine and pyrimidine bases of nucleic acids, carbohydrates, and lipids, as indicated by significant reduction in Raman peak heights at 621, 1,003, and 1,031 inverse centimeters (cm(-1)) (phenylalanine); 643, 827, 852, 1,158, and 1,172 cm(-1) (tyrosine); 758 cm(-1) (tryptophan); 725, 782, 1,337, and 1,578 cm(-1) (adenine, cytosine plus uracil, adenine, and adenine plus guanine, respectively); 1,097 cm(-1) (carbohydrates); and 1,127, 1,450, and 2,932 cm(-1) (lipids) compared with untreated controls. Lemongrass oil treatments were the most effective in degrading cellular components. Scanning electron microscopy of palmarosa and lemongrass-oil-treated cells showed rupture of cell walls and cell debris but no degradation was noted for eucalyptus-oil-treated cells. Palmarosa- and lemongrass-oil-treated cells were positively stained with uranyl acetate when viewed by transmission electron microscopy whereas controls and eucalyptus-oil-treated cells were negatively stained, indicating that the cell membranes were intact. The viability of eucalyptus-oil-treated cells was confirmed by cell culture following treatment. Micro-Raman spectroscopy is a powerful tool which can be further employed to better understand effects of fumigants and other bactericides on bacterial cells.

  11. A MotN Mutant of Ralstonia solanacearum Is Hypermotile and Has Reduced Virulence ▿ †

    PubMed Central

    Meng, Fanhong; Yao, Jian; Allen, Caitilyn

    2011-01-01

    Ralstonia solanacearum is a soil-borne plant pathogen that causes bacterial wilt disease on many plant species. We previously showed that swimming motility contributes to virulence of this bacterium in the early stages of host invasion and colonization. In this study we identified a new negative regulator of motility, named motN, that is located in a cluster of motility-related genes. A motN mutant was hypermotile both on 0.3% agar motility plates and in rich and minimal medium broth. However, like its wild-type parent, it was largely nonmotile inside plants. The motN mutant cells appeared hyperflagellated, and sheared cell protein preparations from motN contained more flagellin than preparations from wild-type cells. The motN strain was significantly reduced in virulence in a naturalistic soil soak assay on tomato plants. However, the motN mutant had wild-type virulence when it was inoculated directly into the plant vascular system. This suggests that motN makes its contribution to virulence early in disease development. The motN mutant formed weaker biofilms than the wild type, but it attached normally to tomato roots and colonized tomato stems as well as its wild-type parent. Phenotypic analysis and gene expression studies indicated that MotN directly or indirectly represses transcription of the major motility regulator FlhDC. MotN was also connected with other known motility and virulence regulators, PehSR, VsrBC, and VsrAD, via uncertain mechanisms. Together, these results demonstrate the importance of precise regulation of flagellum-mediated motility in R. solanacearum. PMID:21421761

  12. Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum

    PubMed Central

    Kong, Hyun Gi; Jo, Eun Jeong; Choi, Hye Kyung; Khan, Raees; Lee, Seon-Woo

    2016-01-01

    Ralstonia solanacearum is a soil-borne plant pathogen that infects more than 200 plant species. Its broad host range and long-term survival under different environmental stress conditions suggest that it uses a variety of mechanisms to protect itself against various types of biotic and abiotic stress. R. solanacearum produces a melanin-like brown pigment in the stationary phase when grown in minimal medium containing tyrosine. To gain deeper insight into the genetic determinants involved in melanin production, transposon-inserted mutants of R. solanacearum strain SL341 were screened for strains with defective melanin-producing capability. In addition to one mutant already known to be involved in pyomelanin production (viz., strain SL341D, with disruption of the hydroxphenylpyruvate dioxygenase gene), we identified three other mutants with disruption in the regulatory genes rpoS, hrpG, and oxyR, respectively. Wild-type SL341 produced pyomelanin in minimal medium containing tyrosine whereas the mutant strains did not. Likewise, homogentisate, a major precursor of pyomelanin, was detected in the culture filtrate of the wild-type strain but not in those of the mutant strains. A gene encoding hydroxyphenylpyruvate dioxygenase exhibited a significant high expression in wild type SL341 compared to other mutant strains, suggesting that pyomelanin production is regulated by three different regulatory proteins. However, analysis of the gene encoding homogentisate dioxygenase revealed no significant difference in its relative expression over time in the wild-type SL341 and mutant strains, except for SL341D, at 72 h incubation. The pigmented SL341 strain also exhibited a high tolerance to hydrogen peroxide stress compared with the non-pigmented SL341D strain. Our study suggests that pyomelanin production is controlled by several regulatory factors in R. solanacearum to confer protection under oxidative stress. PMID:27513990

  13. Quantitative Disease Resistance under Elevated Temperature: Genetic Basis of New Resistance Mechanisms to Ralstonia solanacearum.

    PubMed

    Aoun, Nathalie; Tauleigne, Laetitia; Lonjon, Fabien; Deslandes, Laurent; Vailleau, Fabienne; Roux, Fabrice; Berthomé, Richard

    2017-01-01

    In the context of climate warming, plants will be facing an increased risk of epidemics as well as the emergence of new highly aggressive pathogen species. Although a permanent increase of temperature strongly affects plant immunity, the underlying molecular mechanisms involved are still poorly characterized. In this study, we aimed to uncover the genetic bases of resistance mechanisms that are efficient at elevated temperature to the Ralstonia solanacearum species complex (RSSC), one of the most harmful phytobacteria causing bacterial wilt. To start the identification of quantitative trait loci (QTLs) associated with natural variation of response to R. solanacearum, we adopted a genome wide association (GWA) mapping approach using 176 worldwide natural accessions of Arabidopsis thaliana inoculated with the R. solanacearum GMI1000 strain. Following two different procedures of root-inoculation (root apparatus cut vs. uncut), plants were grown either at 27 or 30°C, with the latter temperature mimicking a permanent increase in temperature. At 27°C, the RPS4/RRS1-R locus was the main QTL of resistance detected regardless of the method of inoculation used. This highlights the power of GWA mapping to identify functionally important loci for resistance to the GMI1000 strain. At 30°C, although most of the accessions developed wilting symptoms, we identified several QTLs that were specific to the inoculation method used. We focused on a QTL region associated with response to the GMI1000 strain in the early stages of infection and, by adopting a reverse genetic approach, we functionally validated the involvement of a strictosidine synthase-like 4 (SSL4) protein that shares structural similarities with animal proteins known to play a role in animal immunity.

  14. Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment, Stem Colonization, and Virulence

    PubMed Central

    Dalsing, Beth L.

    2014-01-01

    Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium's gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum's sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production. PMID:24363343

  15. Multihost experimental evolution of the pathogen Ralstonia solanacearum unveils genes involved in adaptation to plants.

    PubMed

    Guidot, Alice; Jiang, Wei; Ferdy, Jean-Baptiste; Thébaud, Christophe; Barberis, Patrick; Gouzy, Jérôme; Genin, Stéphane

    2014-11-01

    Ralstonia solanacearum, the causal agent of a lethal bacterial wilt plant disease, infects an unusually wide range of hosts. These hosts can further be split into plants where R. solanacearum is known to cause disease (original hosts) and those where this bacterium can grow asymptomatically (distant hosts). Moreover, this pathogen is able to adapt to many plants as supported by field observations reporting emergence of strains with enlarged pathogenic properties. To investigate the genetic bases of host adaptation, we conducted evolution experiments by serial passages of a single clone of the pathogen on three original and two distant hosts over 300 bacterial generations and then analyzed the whole-genome of nine evolved clones. Phenotypic analysis of the evolved clones showed that the pathogen can increase its fitness on both original and distant hosts although the magnitude of fitness increase was greater on distant hosts. Only few genomic modifications were detected in evolved clones compared with the ancestor but parallel evolutionary changes in two genes were observed in independent evolved populations. Independent mutations in the regulatory gene efpR were selected for in three populations evolved on beans, a distant host. Reverse genetic approaches confirmed that these mutations were associated with fitness gain on bean plants. This work provides a first step toward understanding the within-host evolutionary dynamics of R. solanacearum during infection and identifying bacterial genes subjected to in planta selection. The discovery of EfpR as a determinant conditioning host adaptation of the pathogen illustrates how experimental evolution coupled with whole-genome sequencing is a potent tool to identify novel molecular players involved in central life-history traits. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Nitrate assimilation contributes to Ralstonia solanacearum root attachment, stem colonization, and virulence.

    PubMed

    Dalsing, Beth L; Allen, Caitilyn

    2014-03-01

    Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium's gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum's sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production.

  17. Genetic Determinants for Pyomelanin Production and Its Protective Effect against Oxidative Stress in Ralstonia solanacearum.

    PubMed

    Ahmad, Shabir; Lee, Seung Yeup; Kong, Hyun Gi; Jo, Eun Jeong; Choi, Hye Kyung; Khan, Raees; Lee, Seon-Woo

    2016-01-01

    Ralstonia solanacearum is a soil-borne plant pathogen that infects more than 200 plant species. Its broad host range and long-term survival under different environmental stress conditions suggest that it uses a variety of mechanisms to protect itself against various types of biotic and abiotic stress. R. solanacearum produces a melanin-like brown pigment in the stationary phase when grown in minimal medium containing tyrosine. To gain deeper insight into the genetic determinants involved in melanin production, transposon-inserted mutants of R. solanacearum strain SL341 were screened for strains with defective melanin-producing capability. In addition to one mutant already known to be involved in pyomelanin production (viz., strain SL341D, with disruption of the hydroxphenylpyruvate dioxygenase gene), we identified three other mutants with disruption in the regulatory genes rpoS, hrpG, and oxyR, respectively. Wild-type SL341 produced pyomelanin in minimal medium containing tyrosine whereas the mutant strains did not. Likewise, homogentisate, a major precursor of pyomelanin, was detected in the culture filtrate of the wild-type strain but not in those of the mutant strains. A gene encoding hydroxyphenylpyruvate dioxygenase exhibited a significant high expression in wild type SL341 compared to other mutant strains, suggesting that pyomelanin production is regulated by three different regulatory proteins. However, analysis of the gene encoding homogentisate dioxygenase revealed no significant difference in its relative expression over time in the wild-type SL341 and mutant strains, except for SL341D, at 72 h incubation. The pigmented SL341 strain also exhibited a high tolerance to hydrogen peroxide stress compared with the non-pigmented SL341D strain. Our study suggests that pyomelanin production is controlled by several regulatory factors in R. solanacearum to confer protection under oxidative stress.

  18. Ralstonia solanacearum RSp0194 Encodes a Novel 3-Keto-Acyl Carrier Protein Synthase III.

    PubMed

    Mao, Ya-Hui; Ma, Jin-Cheng; Li, Feng; Hu, Zhe; Wang, Hai-Hong

    2015-01-01

    Fatty acid synthesis (FAS), a primary metabolic pathway, is essential for survival of bacteria. Ralstonia solanacearum, a β-proteobacteria member, causes a bacterial wilt affecting more than 200 plant species, including many economically important plants. However, thus far, the fatty acid biosynthesis pathway of R. solanacearum has not been well studied. In this study, we characterized two forms of 3-keto-ACP synthase III, RsFabH and RsFabW, in R. solanacearum. RsFabH, the homologue of Escherichia coli FabH, encoded by the chromosomal RSc1050 gene, catalyzes the condensation of acetyl-CoA with malonyl-ACP in the initiation steps of fatty acid biosynthesis in vitro. The RsfabH mutant lost de novo fatty acid synthetic ability, and grows in medium containing free fatty acids. RsFabW, a homologue of Pseudomonas aeruginosa PA3286, encoded by a megaplasmid gene, RSp0194, condenses acyl-CoA (C2-CoA to C10-CoA) with malonyl-ACP to produce 3-keto-acyl-ACP in vitro. Although the RsfabW mutant was viable, RsfabW was responsible for RsfabH mutant growth on medium containing free fatty acids. Our results also showed that RsFabW could condense acyl-ACP (C4-ACP to C8-ACP) with malonyl-ACP, to produce 3-keto-acyl-ACP in vitro, which implies that RsFabW plays a special role in fatty acid synthesis of R. solanacearum. All of these data confirm that R. solanacearum not only utilizes acetyl-CoA, but also, utilizes medium-chain acyl-CoAs or acyl-ACPs as primers to initiate fatty acid synthesis.

  19. Regulation Involved in Colonization of Intercellular Spaces of Host Plants in Ralstonia solanacearum.

    PubMed

    Hikichi, Yasufumi; Mori, Yuka; Ishikawa, Shiho; Hayashi, Kazusa; Ohnishi, Kouhei; Kiba, Akinori; Kai, Kenji

    2017-01-01

    A soil-borne bacterium Ralstonia solanacearum invading plant roots first colonizes the intercellular spaces of the root, and eventually enters xylem vessels, where it replicates at high levels leading to wilting symptoms. After invasion into intercellular spaces, R. solanacearum strain OE1-1 attaches to host cells and expression of the hrp genes encoding components of the type III secretion system (T3SS). OE1-1 then constructs T3SS and secrets effectors into host cells, inducing expression of the host gene encoding phosphatidic acid phosphatase. This leads to suppressing plant innate immunity. Then, OE1-1 grows on host cells, inducing quorum sensing (QS). The QS contributes to regulation of OE1-1 colonization of intercellular spaces including mushroom-type biofilm formation on host cells, leading to its virulence. R. solanacearum strains AW1 and K60 produce methyl 3-hydroxypalmitate (3-OH PAME) as a QS signal. The methyltransferase PhcB synthesizes 3-OH PAME. When 3-OH PAME reaches a threshold level, it increases the ability of the histidine kinase PhcS to phosphorylate the response regulator PhcR. This results in elevated levels of functional PhcA, the global virulence regulator. On the other hand, strains OE1-1 and GMI1000 produce methyl 3-hydroxymyristate (3-OH MAME) as a QS signal. Among R. solanacearum strains, the deduced PhcB and PhcS amino acid sequences are related to the production of QS signals. R. solanacearum produces aryl-furanone secondary metabolites, ralfuranones, which are extracellularly secreted and required for its virulence, dependent on the QS. Interestingly, ralfuranones affect the QS feedback loop. Taken together, integrated signaling via ralfuranones influences the QS, contributing to pathogen virulence.

  20. Potential Interactions between Salmonella enterica and Ralstonia solanacearum in tomato plants.

    PubMed

    Pollard, Stephanie; Barak, Jeri; Boyer, Renee; Reiter, Mark; Gu, Ganyu; Rideout, Steven

    2014-02-01

    Over the past decade, the Eastern Shore of Virginia (ESV) has been implicated in at least four outbreaks of salmonellosis associated with tomato, all originating from the same serovar, Salmonella enterica serovar Newport. In addition to Salmonella Newport contamination, the devastating plant disease bacterial wilt, caused by the phytopathogen Ralstonia solanacearum, threatens the sustainability of ESV tomato production. Bacterial wilt is present in most ESV tomato fields and causes devastating yield losses each year. Although the connection between bacterial wilt and tomato-related salmonellosis outbreaks in ESV is of interest, the relationship between the two pathogens has never been investigated. In this study, tomato plants were root dip inoculated with one of four treatments: (i) 8 log CFU of Salmonella Newport per ml, (ii) 5 log CFU of R. solanacearum per ml, (iii) a coinoculation of 8 log CFU of Salmonella Newport per ml plus 5 log CFU of R. solanacearum per ml, and (iv) sterile water as control. Leaf, stem, and fruit samples were collected at the early-green-fruit stage, and S. enterica contamination in the internal tissues was detected. S. enterica was recovered in 1.4 and 2.9% of leaf samples from plants inoculated with Salmonella Newport only and from plants coinoculated with Salmonella Newport plus R. solanacearum, respectively. S. enterica was recovered from 1.7 and 3.5% of fruit samples from plants inoculated with Salmonella Newport only and from plants coinoculated with Salmonella Newport plus R. solanacearum, respectively. There were significantly more stem samples from plants coinoculated with Salmonella Newport plus R. solanacearum that were positive for S. enterica (18.6%) than stem samples collected from plants inoculated with Salmonella Newport only (5.7%). Results suggested that R. solanacearum could influence S. enterica survival and transportation throughout the internal tissues of tomato plants.

  1. Tropical strains of Ralstonia solanacearum Outcompete race 3 biovar 2 strains at lowland tropical temperatures.

    PubMed

    Huerta, Alejandra I; Milling, Annett; Allen, Caitilyn

    2015-05-15

    Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum

    PubMed Central

    Ailloud, Florent; Lowe, Tiffany M.; Robène, Isabelle; Cruveiller, Stéphane; Allen, Caitilyn

    2016-01-01

    Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions. Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem. Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes. PMID:26788428

  3. Comparative Transcriptome Analysis Reveals Cool Virulence Factors of Ralstonia solanacearum Race 3 Biovar 2

    PubMed Central

    Meng, Fanhong; Babujee, Lavanya; Jacobs, Jonathan M.; Allen, Caitilyn

    2015-01-01

    While most strains of the plant pathogenic bacterium Ralstonia solanacearum are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of R. solanacearum R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature in planta. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking lecM, aidA, or aidC were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The lecM and aidC mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the lecM mutant was defective in biofilm formation in vitro. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2. PMID:26445498

  4. Comparative Transcriptome Analysis Reveals Cool Virulence Factors of Ralstonia solanacearum Race 3 Biovar 2.

    PubMed

    Meng, Fanhong; Babujee, Lavanya; Jacobs, Jonathan M; Allen, Caitilyn

    2015-01-01

    While most strains of the plant pathogenic bacterium Ralstonia solanacearum are tropical, the race 3 biovar 2 (R3bv2) subgroup attacks plants in cooler climates. To identify mechanisms underlying this trait, we compared the transcriptional profiles of R. solanacearum R3bv2 strain UW551 and tropical strain GMI1000 at 20°C and 28°C, both in culture and during tomato pathogenesis. 4.2% of the ORFs in the UW551 genome and 7.9% of the GMI1000 ORFs were differentially expressed by temperature in planta. The two strains had distinct transcriptional responses to temperature change. GMI1000 up-regulated several stress response genes at 20°C, apparently struggling to cope with plant defenses. At the cooler temperature, R3bv2 strain UW551 up-regulated a cluster encoding a mannose-fucose binding lectin, LecM; a quorum sensing-dependent protein, AidA; and a related hypothetical protein, AidC. The last two genes are absent from the GMI1000 genome. In UW551, all three genes were positively regulated by the adjacent SolI/R quorum sensing system. These temperature-responsive genes were required for full virulence in R3bv2. Mutants lacking lecM, aidA, or aidC were each significantly more reduced in virulence on tomato at 20°C than at 28°C in both a naturalistic soil soak inoculation assay and when they were inoculated directly into tomato stems. The lecM and aidC mutants also survived poorly in potato tubers at the seed tuber storage temperature of 4°C, and the lecM mutant was defective in biofilm formation in vitro. Together, these results suggest novel mechanisms, including a lectin, are involved in the unique temperate epidemiology of R3bv2.

  5. Tube-Wise Diagnostic Microarray for the Multiplex Characterization of the Complex Plant Pathogen Ralstonia solanacearum

    PubMed Central

    Cellier, Gilles; Arribat, Sandrine; Chiroleu, Frédéric; Prior, Philippe; Robène, Isabelle

    2017-01-01

    Ralstonia solanacearum is a well-known agricultural and ecological threat worldwide. The complexity of the R. solanacearum species complex (Rssc) represents a challenge for the accurate characterization of epidemiological strains by official services and research laboratories. The majority of protocols only focus on a narrow range of strains; however, this species complex includes strains that represent major constraints and are under strict regulation. The main drawback associated with the current methods of detecting and characterizing Rssc strains is their reliance on combining different protocols to properly characterize the strains at the ecotype level, which require time and money. Therefore, we used microarray technology (ArrayTube) to develop a standard protocol, which characterizes 17 major groups of interest in the Rssc, in a single multiplex reaction. These 17 majors groups are linked with a phylogenetic assignation (phylotypes, sequevars), but also with an ecotype assignation associated with a range of hosts (e.g., brown rot, Moko). Probes were designed with a 50-mer length constraint and thoroughly evaluated for any flaws or secondary structures. The strains are characterized based on a DNA extraction from pure culture. Validation data showed strong intra-repeatability, inter-repeatability, and reproducibility as well as good specificity. A hierarchical analysis of the probe groups is suitable for an accurate characterization. Compared with single marker detection tests, the method described in this paper addresses efficiently the issue of combining several tests by testing a large number of phylogenetic markers in a single reaction assay. This custom microarray (RsscAT) represents a significant improvement in the epidemiological monitoring of Rssc strains worldwide, and it has the potential to provide insights for phylogenetic incongruence of Rssc strains based on the host of isolation and may be used to indicate potentially emergent strains. PMID

  6. Ralstonia solanacearum RSp0194 Encodes a Novel 3-Keto-Acyl Carrier Protein Synthase III

    PubMed Central

    Li, Feng; Hu, Zhe; Wang, Hai-Hong

    2015-01-01

    Fatty acid synthesis (FAS), a primary metabolic pathway, is essential for survival of bacteria. Ralstonia solanacearum, a β-proteobacteria member, causes a bacterial wilt affecting more than 200 plant species, including many economically important plants. However, thus far, the fatty acid biosynthesis pathway of R. solanacearum has not been well studied. In this study, we characterized two forms of 3-keto-ACP synthase III, RsFabH and RsFabW, in R. solanacearum. RsFabH, the homologue of Escherichia coli FabH, encoded by the chromosomal RSc1050 gene, catalyzes the condensation of acetyl-CoA with malonyl-ACP in the initiation steps of fatty acid biosynthesis in vitro. The RsfabH mutant lost de novo fatty acid synthetic ability, and grows in medium containing free fatty acids. RsFabW, a homologue of Pseudomonas aeruginosa PA3286, encoded by a megaplasmid gene, RSp0194, condenses acyl-CoA (C2-CoA to C10-CoA) with malonyl-ACP to produce 3-keto-acyl-ACP in vitro. Although the RsfabW mutant was viable, RsfabW was responsible for RsfabH mutant growth on medium containing free fatty acids. Our results also showed that RsFabW could condense acyl-ACP (C4-ACP to C8-ACP) with malonyl-ACP, to produce 3-keto-acyl-ACP in vitro, which implies that RsFabW plays a special role in fatty acid synthesis of R. solanacearum. All of these data confirm that R. solanacearum not only utilizes acetyl-CoA, but also, utilizes medium-chain acyl-CoAs or acyl-ACPs as primers to initiate fatty acid synthesis. PMID:26305336

  7. Diversity among Ralstonia solanacearum strains isolated from the southeastern United States.

    PubMed

    Hong, Jason C; Norman, David J; Reed, David L; Momol, M Timur; Jones, Jeffrey B

    2012-10-01

    This is the first comprehensive study of a collection of Ralstonia solanacearum strains from the southeastern United States to be characterized based on biovar, pathogenicity, hypersensitive reaction on tobacco, and phylogenetic analyses of the egl sequence. Rigorous phylogenetic analysis of the commonly used egl gene produced robust phylogenies that differed significantly from a neighbor-joining tree differed from and previously published phylogenies for R. solanacearum strains. These robust trees placed phylotype IV within the phylotype I clade, which may suggest that phylogenies based solely on egl may be misleading. As a result of phylogenetic analyses in this study, we determined that U.S. strains from Georgia, North Carolina, South Carolina, and older Florida strains isolated from solanaceous crops all belong to phylotype II sequevar 7. However, many strains recently isolated in Florida from tomato and other crops were more diverse than the southeastern United States population. These unique Florida strains grouped with strains mostly originating from the Caribbean and Central America. One of the exotic strains, which in a previous study was determined to be established in northern Florida, was characterized more extensively. Upon using Musa-specific multiplex polymerase chain reaction, this strain produced a unique banding pattern, which has not previously been reported. Inoculation of this strain into Musa spp. did not result in wilt symptoms; however, the plants were stunted and root masses were significantly reduced. Furthermore, following root inoculation, the bacterium, unlike a typical Florida race 1 biovar 1 strain, was recovered from the roots and stems, indicating systemic movement. This is the first report of an R. solanacearum strain isolated in the United States that is deleterious to the growth of Musa plants.

  8. Ralstonia solanacearum Strains from Martinique (French West Indies) Exhibiting a New Pathogenic Potential▿ †

    PubMed Central

    Wicker, Emmanuel; Grassart, Laurence; Coranson-Beaudu, Régine; Mian, Danièle; Guilbaud, Caroline; Fegan, Mark; Prior, Philippe

    2007-01-01

    We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America. PMID:17720825

  9. In planta comparative transcriptomics of host-adapted strains of Ralstonia solanacearum.

    PubMed

    Ailloud, Florent; Lowe, Tiffany M; Robène, Isabelle; Cruveiller, Stéphane; Allen, Caitilyn; Prior, Philippe

    2016-01-01

    Background. Ralstonia solanacearum is an economically important plant pathogen with an unusually large host range. The Moko (banana) and NPB (not pathogenic to banana) strain groups are closely related but are adapted to distinct hosts. Previous comparative genomics studies uncovered very few differences that could account for the host range difference between these pathotypes. To better understand the basis of this host specificity, we used RNAseq to profile the transcriptomes of an R. solanacearum Moko strain and an NPB strain under in vitro and in planta conditions. Results. RNAs were sequenced from bacteria grown in rich and minimal media, and from bacteria extracted from mid-stage infected tomato, banana and melon plants. We computed differential expression between each pair of conditions to identify constitutive and host-specific gene expression differences between Moko and NPB. We found that type III secreted effectors were globally up-regulated upon plant cell contact in the NPB strain compared with the Moko strain. Genes encoding siderophore biosynthesis and nitrogen assimilation genes were highly up-regulated in the NPB strain during melon pathogenesis, while denitrification genes were up-regulated in the Moko strain during banana pathogenesis. The relatively lower expression of oxidases and the denitrification pathway during banana pathogenesis suggests that R. solanacearum experiences higher oxygen levels in banana pseudostems than in tomato or melon xylem. Conclusions. This study provides the first report of differential gene expression associated with host range variation. Despite minimal genomic divergence, the pathogenesis of Moko and NPB strains is characterized by striking differences in expression of virulence- and metabolism-related genes.

  10. An Outbreak of Ralstonia pickettii Bloodstream Infection Associated with an Intrinsically Contaminated Normal Saline Solution.

    PubMed

    Chen, Yin-Yin; Huang, Wan-Tsuei; Chen, Chia-Ping; Sun, Shu-Mei; Kuo, Fu-Mei; Chan, Yu-Jiun; Kuo, Shu-Chen; Wang, Fu-Der

    2017-04-01

    OBJECTIVE Ralstonia pickettii has caused contamination of pharmaceutical solutions in many countries, resulting in healthcare infections or outbreak events. We determined the source of the outbreak of R. pickettii bloodstream infection (BSI). METHODS This study was conducted in a 3,000-bed tertiary referral medical center in Taiwan with >8,500 admissions during May 2015. Patients had been treated in the injection room or chemotherapy room at outpatient departments, emergency department, or hospital wards. All patients who were culture positive for R. pickettii from May 3 to June 11, 2015, were eligible for the study. The aim of the survey was to conduct clinical epidemiological and microbiological investigations to identify possible sources of infection. RESULTS We collected 57 R. pickettii-positive specimens from 30 case patients. We performed 24 blood cultures; 14 of these revealed >2 specimens and 6 used fluid withdrawn from Port-a-Cath implantable venous access devices. All patients received an injection of 20 mL 0.9% normal saline via catheter flushing. In addition, 2 unopened ampules of normal saline solution (20 mL) were confirmed positive for R. pickettii. The Taiwan Centers for Disease Control and Prevention performed sampling and testing of the same manufactured batch and identified the same strain of R. pickettii. Pulsed-field gel electrophoresis tests revealed that all clinical isolates had similarity of >90%, validating the outbreak of the same clone of R. pickettii. CONCLUSIONS R. pickettii can grow in saline solutions and cause bloodstream infections. Hospital monitoring mechanisms are extremely important measures in identifying and ending such outbreaks. Infect Control Hosp Epidemiol 2017;38:444-448.

  11. Biocontrol of Ralstonia solanacearum by Treatment with Lytic Bacteriophages ▿ †

    PubMed Central

    Fujiwara, Akiko; Fujisawa, Mariko; Hamasaki, Ryosuke; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2011-01-01

    Ralstonia solanacearum is a Gram-negative bacterium and the causative agent of bacterial wilt in many important crops. We treated R. solanacearum with three lytic phages: φRSA1, φRSB1, and φRSL1. Infection with φRSA1 and φRSB1, either alone or in combination with the other phages, resulted in a rapid decrease in the host bacterial cell density. Cells that were resistant to infection by these phages became evident approximately 30 h after phage addition to the culture. On the other hand, cells infected solely with φRSL1 in a batch culture were maintained at a lower cell density (1/3 of control) over a long period. Pretreatment of tomato seedlings with φRSL1 drastically limited penetration, growth, and movement of root-inoculated bacterial cells. All φRSL1-treated tomato plants showed no symptoms of wilting during the experimental period, whereas all untreated plants had wilted by 18 days postinfection. φRSL1 was shown to be relatively stable in soil, especially at higher temperatures (37 to 50°C). Active φRSL1 particles were recovered from the roots of treated plants and from soil 4 months postinfection. Based on these observations, we propose an alternative biocontrol method using a unique phage, such as φRSL1, instead of a phage cocktail with highly virulent phages. Using this method, φRSL1 killed some but not all bacterial cells. The coexistence of bacterial cells and the phage resulted in effective prevention of wilting. PMID:21498752

  12. Relationship between avirulence gene (avrA) diversity in Ralstonia solanacearum and bacterial wilt incidence.

    PubMed

    Robertson, Alison E; Wechter, W Patrick; Denny, Timothy P; Fortnum, Bruce A; Kluepfel, Daniel A

    2004-12-01

    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.

  13. Tropical Strains of Ralstonia solanacearum Outcompete Race 3 Biovar 2 Strains at Lowland Tropical Temperatures

    PubMed Central

    Huerta, Alejandra I.; Milling, Annett

    2015-01-01

    Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands. PMID:25769835

  14. Chemotaxis Is Required for Virulence and Competitive Fitness of the Bacterial Wilt Pathogen Ralstonia solanacearum

    PubMed Central

    Yao, Jian; Allen, Caitilyn

    2006-01-01

    Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold

  15. Negative chemotaxis of Ralstonia pseudosolanacearum to maleate and identification of the maleate chemosensory protein.

    PubMed

    Tunchai, Mattana; Hida, Akiko; Oku, Shota; Nakashimada, Yutaka; Nikata, Toshiyuki; Tajima, Takahisa; Kato, Junichi

    2017-07-22

    Ralstonia pseudosolanacearum Ps29 was repelled by maleate. Screening of a complete collection of Ps29 single-methyl-accepting chemotaxis protein (mcp) gene mutants identified the RSp0303 homolog (McpP) as a chemotaxis sensor mediating negative chemotaxis to maleate. Interestingly, the mcpP-deletion mutant was attracted to maleate, indicating that this bacterium expresses a MCP(s) for both positive and negative chemotaxis to maleate. We constructed a Ps29 derivative (designated POC14) harboring deletions in 14 individual mcp genes, including mcpP, to characterize McpP. Introduction of a plasmid harboring the mcpP gene (pPS16) restored the ability to negatively respond to maleate, confirming that McpP is a MCP for negative chemotaxis to maleate. We thought that maleate might be applied to controlling plant infection by R. pseudosolanacearum. To evaluate this possibility, we measured chemotactic responses of seven other virulent R. pseudosolanacearum strains to maleate. We confirmed that they harbored functional mcpP orthologues, but they showed no chemotactic responses to maleate. Quantitative RT-PCR analysis revealed that these seven R. pseudosolanacearum strains did not show negative chemotaxis to maleate because of negligible transcription of the mcpP genes. We compared the chemotactic responses of POC14 and POC14[pPS16] toward various chemicals and found that McpP senses inorganic phosphate as a chemoattractant. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Yao, Jian; Allen, Caitilyn

    2006-05-01

    Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold

  17. Isolation and screening of phlD (+) plant growth promoting rhizobacteria antagonistic to Ralstonia solanacearum.

    PubMed

    Ramadasappa, Srinivasamurthy; Rai, Ashwani K; Jaat, Ranjeet Singh; Singh, Aqbal; Rai, Rhitu

    2012-04-01

    Tomato (Lycopersicon esculentum) is important widely grown vegetable in India and its productivity is affected by bacterial wilt disease infection caused by Ralstonia solanacearum. To prevent this disease infection a study was conducted to isolate and screen effective plant growth promoting rhizobacteria (PGPR) antagonistic to R. solanacearum. A total 297 antagonistic bacteria were isolated through dual culture inoculation technique, out of which forty-two antagonistic bacteria were found positive for phlD gene by PCR amplification using two primer sets Phl2a:Phl2b and B2BF:BPR4. The genetic diversity of phlD (+) bacteria was studied by amplified 16S rDNA restriction analysis and demonstrated eleven groups at 65% similarity level. Out of these 42 phlD (+) antagonistic isolates, twenty exhibited significantly fair plant growth promoting activities like phosphate solubilization (0.92-5.33%), 25 produced indole acetic acid (1.63-7.78 μg ml(-1)) and few strains show production of antifungal metabolites (HCN and siderophore). The screening of PGPR (phlD (+)) for suppression of bacterial wilt disease in glass house conditions was showed ten isolated phlD (+) bacteria were able to suppress infection of bacterial wilt disease in tomato plant (var. Arka vikas) in the presence R. solanacearum. The PGPR (phlD (+)) isolates s188, s215 and s288 was observed to be effective plant growth promoter as it shows highest dry weight per plant (3.86, 3.85 and 3.69 g plant(-1) respectively). The complete absence of wilt disease symptoms in tomato crop plants was observed by these treatments compared to negative control. Therefore inoculation of tomato plant with phlD (+) isolate s188 and other similar biocontrol agents may prove to be a positive strategy for checking wilt disease and thus improving plant vigor.

  18. A Duplex PCR Assay for the Detection of Ralstonia solanacearum Phylotype II Strains in Musa spp.

    PubMed Central

    Cellier, Gilles; Moreau, Aurélie; Chabirand, Aude; Hostachy, Bruno; Ailloud, Florent; Prior, Philippe

    2015-01-01

    Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae. PMID:25811378

  19. Inventorying the molecular potential of Cupriavidus and Ralstonia strains surviving harsh space-related environments

    NASA Astrophysics Data System (ADS)

    Mijnendonckx, Kristel; van Houdt, Rob; Provoost, Ann; Bossus, Albert; Ott, C. Mark; Venkateswaran, Kasthuri; Leys, Natalie

    The craving of modern man to explore life beyond earth presents a lot of challenges. The control of microbial contamination of the confined manned spacecraft is an important aspect that has to be taken into account in this journey. Because the human body contains a huge amount of microorganisms, the crew itself is the most important contamination source. But contamination can also originate from residing environmental microorganisms or from materials that are supplied from the Earth. These microbial contaminations can cause problems for the astronauts -well documented to have a decreased immunity -and the infrastructure of the space station. In this study, 14 different Cupriavidus metallidurans and Ralstonia pickettii strains, isolated from such space-related environments, where characterised in detail. These unique strains were isolated from drinking water that returned from ISS (3), from the cooling water system of the American ISS segment (4), from a swab sample of the Mars Odyssey Orbitor surface prior to flight (4), and from an air sample taken in the space assembly facility PHSF during Mars exploration Rover assembly (3). Their resistance to heavy metals and antibiotics was screened. The C. metallidurans isolates were more resistant to Zn2+ and Hg+ but more sensitive to Ni2+ than the R. pickettii strains. The MIC values for Cu2+ ranged from 1,5mM to 12mM, for Co2+ from 1,58mM to 12,63mM and for Cd2+ from 0,25mM to 1mM. For Ni2+ , the MIC values were between 2 and 8mM, except for the strain C. metallidurans IV (0502478) that was able to grow on Ni+2 concentrations up to 48mM. A metal of special interest was Ag+ because it is used to sanitize ISS drinking water. The strains isolated from air and surface samples showed a MIC value ranging from 0,35µM to 4µM. The isolates from the water samples had MIC values from 0,3µM to 2µM, which is lower than (or comparable with) the lowest limit of the silver concentration used in the ISS (1,9µM -4,6µM). However, all

  20. Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum.

    PubMed

    Nahar, Kamrun; Matsumoto, Iyo; Taguchi, Fumiko; Inagaki, Yoshishige; Yamamoto, Mikihiro; Toyoda, Kazuhiro; Shiraishi, Tomonori; Ichinose, Yuki; Mukaihara, Takafumi

    2014-04-01

    Ralstonia solanacearum is a Gram-negative soil-borne bacterium that causes bacterial wilt disease in more than 200 plant species, including economically important Solanaceae species. In R. solanacearum, the hypersensitive response and pathogenicity (Hrp) type III secretion system is required for both the ability to induce the hypersensitive response (HR) in nonhost plants and pathogenicity in host plants. Recently, 72 effector genes, called rip (Ralstonia protein injected into plant cells), have been identified in R. solanacearum RS1000. RS1002, a spontaneous nalixidic acid-resistant derivative of RS1000, induced strong HR in the nonhost wild eggplant Solanum torvum in an Hrp-dependent manner. An Agrobacterium-mediated transient expression system revealed that Rip36, a putative Zn-dependent protease effector of R. solanacearum, induced HR in S. torvum. A mutation in the putative Zn-binding motif (E149A) completely abolished the ability to induce HR. In agreement with this result, the RS1002-derived Δrip36 and rip36E149A mutants lost the ability to induce HR in S. torvum. An E149A mutation had no effect on the translocation of Rip36 into plant cells. These results indicate that Rip36 is an avirulent factor that induces HR in S. torvum and that a putative Zn-dependent protease motif is essential for this activity.

  1. Ralstonia solanacearum iron scavenging by the siderophore staphyloferrin B is controlled by PhcA, the global virulence regulator.

    PubMed

    Bhatt, Garima; Denny, Timothy P

    2004-12-01

    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.

  2. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

    USDA-ARS?s Scientific Manuscript database

    Ralstonia solanacearum race 3 biovar 2 strains have the ability to cause brown rot of potato in temperate climates. Since these strains are not established in the U.S. and because of the potential risk they pose to the potato industry, the U.S. government has listed them as select agents. Cultivated...

  3. Cold Tolerance of some Ralstonia solanacearum strains, including Race3 Biovar2, is conferred in part by variation in cold shock gene cspD3.

    USDA-ARS?s Scientific Manuscript database

    Ralstonia solanacearum race 3 biovar 2 (R3B2) strains are one of only 10 USDA Select Agents, a category of quarantined pathogens reserved for the most serious threats to U.S. plant industry. The threat of R3B2 strains was not considered to be likely due to race (these are poorly defined) or biovar ...

  4. Development and comparison of TaqMan-based real-time PCR assays for detection and differentiation of Ralstonia solanacearum strains

    USDA-ARS?s Scientific Manuscript database

    Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperature climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is comm...

  5. A computer program for fast and easy typing of partial endoglucanase gene sequence into phylotypes and sequevars 1&2 (select agents) of Ralstonia solanacearum

    USDA-ARS?s Scientific Manuscript database

    The phytopathogen Ralstonia solanacearum is a species complex that contains a subset of strains that are quarantined or select agent pathogens. An unidentified R. solanacearum strain is considered a select agent in the US until proven otherwise, which can be done by phylogenetic analysis of a partia...

  6. Incorporation of Escherichia coli O157:H7 in biofilms with Ralstonia insidiosa, a primary localizer for the development of heterogeneous biofilms

    USDA-ARS?s Scientific Manuscript database

    It is hypothesized that the presence of strong biofilm forming microflora could potentially enhance the survival of Escherichia coli O157:H7 (EcO157) in harsh environment. In this study, a strong biofilm forming bacterium, Ralstonia insidiosa, previously isolated from a fresh-cut produce plant was c...

  7. Incorporation of Escherichia coli O157:H7 in dual-species biofilms with Ralstonia insidiosa, a primary colonizer for the development of heterogeneous biofilms

    USDA-ARS?s Scientific Manuscript database

    The presence of strong biofilm forming microflora could potentially enhance the survival of Escherichia coli O157:H7 (EcO157) in harsh environment. One strain of Ralstonia insidiosa isolated from produce processing environments, previously displayed a synergistic interaction with EcO157 in dual-spec...

  8. "Racializing" Class

    ERIC Educational Resources Information Center

    Hatt-Echeverria, Beth; Urrieta, Luis, Jr.

    2003-01-01

    In an effort to explore how racial and class oppressions intersect, the authors use their autobiographical narratives to depict cultural and experiential continuity and discontinuity in growing up white working class versus Chicano working class. They specifically focus on "racializing class" due to the ways class is often used as a copout by…

  9. Genomic and proteomic evidence supporting the division of the plant pathogen Ralstonia solanacearum into three species.

    PubMed

    Prior, Philippe; Ailloud, Florent; Dalsing, Beth L; Remenant, Benoit; Sanchez, Borja; Allen, Caitilyn

    2016-02-01

    The increased availability of genome sequences has advanced the development of genomic distance methods to describe bacterial diversity. Results of these fast-evolving methods are highly correlated with those of the historically standard DNA-DNA hybridization technique. However, these genomic-based methods can be done more rapidly and less expensively and are less prone to technical and human error. They are thus a technically accessible replacement for species delineation. Here, we use several genomic comparison methods, supported by our own proteomic analyses and metabolic characterization as well as previously published DNA-DNA hybridization analyses, to differentiate members of the Ralstonia solanacearum species complex into three species. This pathogen group consists of diverse and widespread strains that cause bacterial wilt disease on many different plants. We used three different methods to compare the complete genomes of 29 strains from the R. solanacearum species complex. In parallel we profiled the proteomes of 73 strains using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). Proteomic profiles together with genomic sequence comparisons consistently and comprehensively described the diversity of the R. solanacearum species complex. In addition, genome-driven functional phenotypic assays excitingly supported an old hypothesis (Hayward et al. (J Appl Bacteriol 69:269-80, 1990)), that closely related members of the R. solanacearum could be identified through a simple assay of anaerobic nitrate metabolism. This assay allowed us to clearly and easily differentiate phylotype II and IV strains from phylotype I and III strains. Further, genomic dissection of the pathway distinguished between proposed subspecies within the current phylotype IV. The assay revealed large scale differences in energy production within the R. solanacearum species complex, indicating coarse evolutionary distance and further supporting a

  10. Diversity, Pathogenicity, and Current Occurrence of Bacterial Wilt Bacterium Ralstonia solanacearum in Peru

    PubMed Central

    Gutarra, Liliam; Herrera, Juan; Fernandez, Elizabeth; Kreuze, Jan; Lindqvist-Kreuze, Hannele

    2017-01-01

    The current bacterial wilt infestation level in the potato fields in the Peruvian Andes was investigated by collecting stem samples from wilted plants and detecting Ralstonia solanacearum. In total 39 farmers’ fields located in the central and northern Peru between the altitudes 2111 and 3742 m above sea level were sampled. R. solanacearum was detected in 19 fields, and in 153 out of the 358 samples analyzed. Phylogenetic analysis using the partial sequence of the endoglucanase gene on strains collected in Peru between 1966 and 2016 from potato, pepper, tomato, plantain or soil, divided the strains in phylotypes I, IIA, and IIB. The Phylotype IIB isolates formed seven sequevar groups including the previously identified sequevars 1, 2, 3, 4, and 25. In addition to this, three new sequevars of phylotype IIB were identified. Phylotype IIA isolates from Peru clustered together with reference strains previously assigned to sequevars 5, 39, 41, and 50, and additionally one new sequevar was identified. The Phylotype I strain was similar to the sequevar 18. Most of the Peruvian R. solanacearum isolates were IIB-1 strains. In the old collection sampled between 1966 and 2013, 72% were IIB-1 and in the new collection at 2016 no other strains were found. The pathogenicity of 25 isolates representing the IIA and IIB sequevar groups was tested on potato, tomato, eggplant and tobacco. All were highly aggressive on potato, but differed in pathogenicity on the other hosts, especially on tobacco. All IIA strains caused latent infection on tobacco and some strains also caused wilting, while IIB strains caused only few latent infections on this species. In conclusion, high molecular diversity was found among the R. solanacearum strains in Peru. Most of the variability was found in areas that are no longer used for potato cultivation and thus these strains do not pose a real threat for potato production in the country. Compared to the previous data from the 1990s, the incidence of

  11. Genetic diversity of the bacterial wilt pathogen Ralstonia solanacearum using a RAPD marker.

    PubMed

    Nishat, Sayeda; Hamim, Islam; Khalil, M Ibrahim; Ali, Md Ayub; Hossain, Muhammed Ali; Meah, M Bahadur; Islam, Md Rashidul

    2015-11-01

    Bacterial wilt caused by Ralstonia solanacearum is a destructive disease of many economically important crop species. A significant variation in wilt incidence and severity in eggplant and potato was observed among the growing areas surveyed. R. solanacearum isolates obtained both from eggplant and potato belong to biovar III, while isolates from eggplant belong to race 1 and isolates obtained from potato belong to race 3. Random amplified polymorphic DNA (RAPD) technique was used as a tool for assessing genetic variation and relationship among seven isolate groups of R. solanacearum viz., RsB-1, RsB-2, RsB-3, RsP-1, RsP-2, RsP-3 and RsP-4, consisting in a total of 28 isolates. Out of the RAPD markers used, amplification with four decamer primers produced 70 bands with sizes ranging from 100 to 1400 bp. Out of 70 bands, 68 bands (97.06%) were polymorphic and two bands (2.94%) were monomorphic amongst the seven R. solanacearum isolates group. The Unweighted Pair Group Method of Arithmetic Means (UPGMA) dendrogram constructed from Nei's genetic distance produced two main clusters of the seven isolates of R. solanacearum. The isolates RsB-1, RsB-2, RsB-3 and R-4 grouped in cluster І, while RsP-2, RsP-3 and RsP-4 grouped in cluster ІІ. The highest intra-variety similarity index (Si) was found in RsB-1 isolate (86.35%) and the lowest one in RsP-2 (56.59%). The results indicated that relatively higher and lower levels of genetic variation were found in RsP-3 and RsB-3, respectively. The coefficient of gene differentiation (G(st)) was 0.5487, reflecting the existence of a high level of genetic variations among seven isolates of R. solanacearum. Comparatively higher genetic distance (0.4293) and lower genetic identity (0.6510) were observed between RsB-2 and RsP-4 combinations. The lowest genetic distance (0.0357) and highest genetic identity (0.9650) were found in RsB-1 vs. RsB-2 pair. Thus, RAPD offers a potentially simple, rapid and reliable method to evaluate

  12. Identification and characterization of chemosensors for d-malate, unnatural enantiomer of malate, in Ralstonia pseudosolanacearum.

    PubMed

    Tunchai, Mattana; Hida, Akiko; Oku, Shota; Nakashimada, Yutaka; Tajima, Takahisa; Kato, Junichi

    2017-02-01

    Ralstonia pseudosolanacearum Ps29 is attracted by nonmetabolizable d-malate, an unnatural enantiomer. Screening of a complete collection of single-mcp-gene deletion mutants of Ps29 revealed that the RSc1156 homologue is a chemosensor for d-malate. An RSc1156 homologue deletion mutant of Ps29 showed decreased but significant responses to d-malate, suggesting the existence of another d-malate chemosensor. McpM previously had been identified as a chemosensor for l-malate. We constructed an RSc1156 homologue mcpM double deletion mutant and noted that this mutant failed to respond to d-malate; thus, the RSc1156 homologue and McpM are the major chemosensors for d-malate in this organism. To further characterize the ligand specificities of the RSc1156 homologue and McpM, we constructed a Ps29 derivative (designated K18) harbouring deletions in 18 individual mcp genes, including mcpM and RSc1156. K18 harbouring the RSc1156 homologue responded strongly to l-tartrate and d-malate and moderately to d-tartrate, but not to l-malate or succinate. K18 harbouring mcpM responded strongly to l-malate and d-tartrate and moderately to succinate, fumarate and d-malate. Ps29 utilizes l-malate and l-tartrate, but not d-malate. We therefore concluded that l-tartrate and l-malate are natural ligands of the RSc1156 homologue and McpM, respectively, and that chemotaxis toward d-malate is a fortuitous response by the RSc1156 homologue and McpM in Ps29. We propose re-designation of the RSc1156 homologue as McpT. In tomato plant infection assays, the mcpT deletion mutant of highly virulent R. pseudosolanacearum MAFF106611 was as infectious as wild-type MAFF106611, suggesting that McpT-mediated chemotaxis does not play an important role in tomato plant infection.

  13. Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence

    PubMed Central

    2010-01-01

    Background The Ralstonia solanacearum species complex includes thousands of strains pathogenic to an unusually wide range of plant species. These globally dispersed and heterogeneous strains cause bacterial wilt diseases, which have major socio-economic impacts. Pathogenicity is an ancestral trait in R. solanacearum and strains with high genetic variation can be subdivided into four phylotypes, correlating to isolates from Asia (phylotype I), the Americas (phylotype IIA and IIB), Africa (phylotype III) and Indonesia (phylotype IV). Comparison of genome sequences strains representative of this phylogenetic diversity can help determine which traits allow this bacterium to be such a pathogen of so many different plant species and how the bacteria survive in many different habitats. Results The genomes of three tomato bacterial wilt pathogens, CFBP2957 (phy. IIA), CMR15 (phy. III) and PSI07 (phy. IV) were sequenced and manually annotated. These genomes were compared with those of three previously sequenced R. solanacearum strains: GMI1000 (tomato, phy. I), IPO1609 (potato, phy. IIB), and Molk2 (banana, phy. IIB). The major genomic features (size, G+C content, number of genes) were conserved across all of the six sequenced strains. Despite relatively high genetic distances (calculated from average nucleotide identity) and many genomic rearrangements, more than 60% of the genes of the megaplasmid and 70% of those on the chromosome are syntenic. The three new genomic sequences revealed the presence of several previously unknown traits, probably acquired by horizontal transfers, within the genomes of R. solanacearum, including a type IV secretion system, a rhi-type anti-mitotic toxin and two small plasmids. Genes involved in virulence appear to be evolving at a faster rate than the genome as a whole. Conclusions Comparative analysis of genome sequences and gene content confirmed the differentiation of R. solanacearum species complex strains into four phylotypes. Genetic

  14. Comparative Genomics of Ralstonia solanacearum Identifies Candidate Genes Associated with Cool Virulence

    PubMed Central

    Bocsanczy, Ana M.; Huguet-Tapia, Jose C.; Norman, David J.

    2017-01-01

    Strains of the Ralstonia solanacearum species complex in the phylotype IIB group are capable of causing Bacterial Wilt disease in potato and tomato at temperatures lower than 24°C. The capability of these strains to survive and to incite infection at temperatures colder than their normally tropical boundaries represents a threat to United States agriculture in temperate regions. In this work, we used a comparative genomics approach to identify orthologous genes linked to the lower temperature virulence phenotype. Six R. solanacearum cool virulent (CV) strains were compared to six strains non-pathogenic at low temperature (NPLT). CV strains can cause Bacterial Wilt symptoms at temperatures below 24°C, while NPLT cannot. Four R. solanacearum strains were sequenced for this work in order to complete the comparison. An orthologous genes comparison identified 44 genes present only in CV strains and 19 genes present only in NPLT strains. Gene annotation revealed a high percentage of genes compared with whole genomes in the transcriptional regulator and transport categories. A single nucleotide polymorphism (SNP) analysis identified 265 genes containing conserved non-synonymous SNPs in CV strains. Ten genes in the pathogenicity category were identified in this group. Comparisons of type 3 secretion system, type 6 secretion system (T6SS) clusters, and associated effectors did not indicate a correlation with the CV phenotype except for one T6SS VGR effector potentially associated with the CV phenotype. This is the first R. solanacearum genomic comparative analysis of multiple strains with different temperature related virulence. The candidate genes identified by this comparison are potential factors involved in virulence at low temperatures that need to be investigated. The high percentage of transcriptional regulators among the genes present only in CV strains supports the hypothesis that temperature dependent regulation of virulence genes explains the differential

  15. Genomes of three tomato pathogens within the Ralstonia solanacearum species complex reveal significant evolutionary divergence.

    PubMed

    Remenant, Benoît; Coupat-Goutaland, Bénédicte; Guidot, Alice; Cellier, Gilles; Wicker, Emmanuel; Allen, Caitilyn; Fegan, Mark; Pruvost, Olivier; Elbaz, Mounira; Calteau, Alexandra; Salvignol, Gregory; Mornico, Damien; Mangenot, Sophie; Barbe, Valérie; Médigue, Claudine; Prior, Philippe

    2010-06-15

    The Ralstonia solanacearum species complex includes thousands of strains pathogenic to an unusually wide range of plant species. These globally dispersed and heterogeneous strains cause bacterial wilt diseases, which have major socio-economic impacts. Pathogenicity is an ancestral trait in R. solanacearum and strains with high genetic variation can be subdivided into four phylotypes, correlating to isolates from Asia (phylotype I), the Americas (phylotype IIA and IIB), Africa (phylotype III) and Indonesia (phylotype IV). Comparison of genome sequences strains representative of this phylogenetic diversity can help determine which traits allow this bacterium to be such a pathogen of so many different plant species and how the bacteria survive in many different habitats. The genomes of three tomato bacterial wilt pathogens, CFBP2957 (phy. IIA), CMR15 (phy. III) and PSI07 (phy. IV) were sequenced and manually annotated. These genomes were compared with those of three previously sequenced R. solanacearum strains: GMI1000 (tomato, phy. I), IPO1609 (potato, phy. IIB), and Molk2 (banana, phy. IIB). The major genomic features (size, G+C content, number of genes) were conserved across all of the six sequenced strains. Despite relatively high genetic distances (calculated from average nucleotide identity) and many genomic rearrangements, more than 60% of the genes of the megaplasmid and 70% of those on the chromosome are syntenic. The three new genomic sequences revealed the presence of several previously unknown traits, probably acquired by horizontal transfers, within the genomes of R. solanacearum, including a type IV secretion system, a rhi-type anti-mitotic toxin and two small plasmids. Genes involved in virulence appear to be evolving at a faster rate than the genome as a whole. Comparative analysis of genome sequences and gene content confirmed the differentiation of R. solanacearum species complex strains into four phylotypes. Genetic distances between strains, in

  16. Interspecific Potato Breeding Lines Display Differential Colonization Patterns and Induced Defense Responses after Ralstonia solanacearum Infection

    PubMed Central

    Ferreira, Virginia; Pianzzola, María J.; Vilaró, Francisco L.; Galván, Guillermo A.; Tondo, María L.; Rodriguez, María V.; Orellano, Elena G.; Valls, Marc; Siri, María I.

    2017-01-01

    Potato (Solanum tuberosum L.) is one of the main hosts of Ralstonia solanacearum, the causative agent of bacterial wilt. This plant pathogen bacteria produce asymptomatic latent infections that promote its global spread, hindering disease control. A potato breeding program is conducted in Uruguay based on the introgression of resistance from the wild native species S. commersonii Dun. Currently, several backcrosses were generated exploiting the high genetic variability of this wild species resulting in advanced interspecific breeding lines with different levels of bacterial wilt resistance. The overall aim of this work was to characterize the interaction of the improved potato germplasm with R. solanacearum. Potato clones with different responses to R. solanacearum were selected, and colonization, dissemination and multiplication patterns after infection were evaluated. A R. solanacearum strain belonging to the phylotype IIB-sequevar 1, with high aggressiveness on potato was genetically modified to constitutively generate fluorescence and luminescence from either the green fluorescence protein gene or lux operon. These reporter strains were used to allow a direct and precise visualization of fluorescent and luminescent cells in plant tissues by confocal microscopy and luminometry. Based on wilting scoring and detection of latent infections, the selected clones were classified as susceptible or tolerant, while no immune-like resistance response was identified. Typical wilting symptoms in susceptible plants were correlated with high concentrations of bacteria in roots and along the stems. Tolerant clones showed a colonization pattern restricted to roots and a limited number of xylem vessels only in the stem base. Results indicate that resistance in potato is achieved through restriction of bacterial invasion and multiplication inside plant tissues, particularly in stems. Tolerant plants were also characterized by induction of anatomical and biochemical changes after

  17. Interspecific Potato Breeding Lines Display Differential Colonization Patterns and Induced Defense Responses after Ralstonia solanacearum Infection.

    PubMed

    Ferreira, Virginia; Pianzzola, María J; Vilaró, Francisco L; Galván, Guillermo A; Tondo, María L; Rodriguez, María V; Orellano, Elena G; Valls, Marc; Siri, María I

    2017-01-01

    Potato (Solanum tuberosum L.) is one of the main hosts of Ralstonia solanacearum, the causative agent of bacterial wilt. This plant pathogen bacteria produce asymptomatic latent infections that promote its global spread, hindering disease control. A potato breeding program is conducted in Uruguay based on the introgression of resistance from the wild native species S. commersonii Dun. Currently, several backcrosses were generated exploiting the high genetic variability of this wild species resulting in advanced interspecific breeding lines with different levels of bacterial wilt resistance. The overall aim of this work was to characterize the interaction of the improved potato germplasm with R. solanacearum. Potato clones with different responses to R. solanacearum were selected, and colonization, dissemination and multiplication patterns after infection were evaluated. A R. solanacearum strain belonging to the phylotype IIB-sequevar 1, with high aggressiveness on potato was genetically modified to constitutively generate fluorescence and luminescence from either the green fluorescence protein gene or lux operon. These reporter strains were used to allow a direct and precise visualization of fluorescent and luminescent cells in plant tissues by confocal microscopy and luminometry. Based on wilting scoring and detection of latent infections, the selected clones were classified as susceptible or tolerant, while no immune-like resistance response was identified. Typical wilting symptoms in susceptible plants were correlated with high concentrations of bacteria in roots and along the stems. Tolerant clones showed a colonization pattern restricted to roots and a limited number of xylem vessels only in the stem base. Results indicate that resistance in potato is achieved through restriction of bacterial invasion and multiplication inside plant tissues, particularly in stems. Tolerant plants were also characterized by induction of anatomical and biochemical changes after

  18. CLASS for Class.

    NASA Astrophysics Data System (ADS)

    Bluestein, Howard B.

    1993-09-01

    Faculty and students from the School of Meteorology at the University of Oklahoma and staff members from the Atmospheric Technology Division at the National Center for Atmospheric Research (NCAR) participated in a special course given during the last two weeks of May 1992. The purpose of the course was to give students the opportunity to use the NCAR mobile CLASS (Cross-Chain LORAN Atmospheric Sounding System) in the field and to interpret data they collected themselves in the context of material learned earlier in a lecture setting. Soundings were obtained in parts of Texas and Oklahoma in the environment of multicell storms, in supercells, in a gust front, and on the cold side of a cold front.

  19. [Genetic variability of the bacterium Ralstonia solanacearum (Burkholderiales: Burholderiaceae) in the banana-growing region of Uraba (Colombia)].

    PubMed

    Cardozo, Carolina; Rodríguez, Paola; Cotes, José Miguel; Marín, Mauricio

    2010-03-01

    The banana moko disease, caused by the bacterium Ralstonia solanacearum, is one of the most important phytopathological problems of the banana agribusiness in tropical countries. In Uraba and Magdalena (Colombia), the main exporting regions of banana in Colombia, this disease causes a destruction estimated in 16.5 ha/year. The bacterium presents an extremely high level of genetic variation that affects control measures. This is the first study of its variation in Colombia and was done with AFLP molecular markers on a population of 100 isolates from banana plants, soils and "weeds". The high level of genetic diversity, with Nei and Shannon indexes of h=0.32 and I=0.48, respectively, and the AMOVA, showed that this population is subestructured (Fst=0.66): the host is the main factor of differentiation. Even so, previous tests show that all varieties have pathogenicity on Musa.

  20. DNA Fingerprinting of Ralstonia paucula by Infrequent-Restriction-Site PCR and Randomly Amplified Polymorphic DNA Analysis

    PubMed Central

    Moissenet, Didier; Vu-Thien, Hoang; Benzerara, Yahia; Arlet, Guillaume

    2003-01-01

    Ralstonia paucula (formerly CDC group IV c-2) is an environmental organism that can cause serious human infections, occasionally clusters of nosocomial infections. In the present work, 26 strains of R. paucula (4 from the American Centers for Disease Control and Prevention collection, 10 from the Belgian Laboratorium voor Microbiologie [LMG] collection, and 12 French clinical isolates) were analyzed with infrequent-restriction-site PCR and randomly amplified polymorphic DNA analysis. Both techniques accurately distinguished between collection strains. Two close patterns obtained for all the French isolates suggested a clonal strain. Two LMG collection strains originating from human sources in the United States also showed patterns close to those of French isolates. PMID:14662974

  1. In silico evaluation of molecular probes for detection and identification of Ralstonia solanacearum and Clavibacter michiganensis subsp. sepedonicus.

    PubMed

    Arahal, David R; Llop, Pablo; Alonso, Manuel Pérez; López, María M

    2004-09-01

    Ralstonia solanacerum and Clavibacter michiganensis subsp. sepedonicus are the two most relevant bacterial pathogens of potato for which a large number of molecular diagnostic methods using specific DNA sequences have been developed. About one hundred oligonucleotides have been described and thoroughly tested experimentally. After having compiled and evaluated all these primers and probes in silico to check their specificity, many discrepancies were found. A detailed analysis permitted the recognition of different possible reasons for such discrepancies: sequencing errors in public sequences, wrong supposed specificity (sometimes due to more recent sequences than the oligonucleotides being evaluated) or even typing errors in the oligonucleotides. Although this study is an exercise about in silico evaluation using two potato bacterial pathogens as a model, the conclusions reflect not only information useful for phytopathologists but, in a broader scope, draw the main situations that can be found during an evaluation of probes, which can be surely found in other scenarios.

  2. Phylogeny and Functional Expression of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from the Autotrophic Ammonia-Oxidizing Bacterium Nitrosospira sp.Isolate 40KI

    PubMed Central

    Utåker, Janne B.; Andersen, Kjell; Aakra, Ågot; Moen, Birgitte; Nes, Ingolf F.

    2002-01-01

    The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO2 by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (cbb) genes and proteins from the AOB. The cbbLS genes from Nitrosospira sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the β and γ subgroups of the class Proteobacteria are also presented. All except one of the β-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the Ralstonia eutropha enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB, Nitrosococcus halophilus Nc4 and Nitrosomonas europaea Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in N. europaea was surprising, as all of the other β-subgroup AOB had red-like RubisCOs. The green-like enzyme of N. europaea Nm50 was probably acquired by horizontal gene transfer. Functional expression of Nitrosospira sp. isolate 40KI RubisCO in the chemoautotrophic host R. eutropha was demonstrated. Use of an expression vector harboring the R. eutropha cbb control region allowed regulated expression of Nitrosospira sp. isolate 40KI RubisCO in an R. eutropha cbb deletion strain. The Nitrosospira RubisCO supported autotrophic growth of R. eutropha with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB cbb genes. PMID:11751824

  3. Phylogeny and functional expression of ribulose 1,5-bisphosphate carboxylase/oxygenase from the autotrophic ammonia-oxidizing bacterium Nitrosospira sp. isolate 40KI.

    PubMed

    Utåker, Janne B; Andersen, Kjell; Aakra, Agot; Moen, Birgitte; Nes, Ingolf F

    2002-01-01

    The autotrophic ammonia-oxidizing bacteria (AOB), which play an important role in the global nitrogen cycle, assimilate CO(2) by using ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO). Here we describe the first detailed study of RubisCO (cbb) genes and proteins from the AOB. The cbbLS genes from Nitrosospira sp. isolate 40KI were cloned and sequenced. Partial sequences of the RubisCO large subunit (CbbL) from 13 other AOB belonging to the beta and gamma subgroups of the class Proteobacteria are also presented. All except one of the beta-subgroup AOB possessed a red-like type I RubisCO with high sequence similarity to the Ralstonia eutropha enzyme. All of these new red-like RubisCOs had a unique six-amino-acid insert in CbbL. Two of the AOB, Nitrosococcus halophilus Nc4 and Nitrosomonas europaea Nm50, had a green-like RubisCO. With one exception, the phylogeny of the AOB CbbL was very similar to that of the 16S rRNA gene. The presence of a green-like RubisCO in N. europaea was surprising, as all of the other beta-subgroup AOB had red-like RubisCOs. The green-like enzyme of N. europaea Nm50 was probably acquired by horizontal gene transfer. Functional expression of Nitrosospira sp. isolate 40KI RubisCO in the chemoautotrophic host R. eutropha was demonstrated. Use of an expression vector harboring the R. eutropha cbb control region allowed regulated expression of Nitrosospira sp. isolate 40KI RubisCO in an R. eutropha cbb deletion strain. The Nitrosospira RubisCO supported autotrophic growth of R. eutropha with a doubling time of 4.6 h. This expression system may allow further functional analysis of AOB cbb genes.

  4. Cutting Classes

    ERIC Educational Resources Information Center

    Hacker, Andrew

    1976-01-01

    Provides critical reviews of three books, "The Political Economy of Social Class", "Ethnicity: Theory and Experience," and "Ethnicity in the United States," focusing on the political economy of social class and ethnicity. (Author/AM)

  5. Class Size.

    ERIC Educational Resources Information Center

    Underwood, Siobhan; Lumsden, Linda S.

    1994-01-01

    The items featured in this annotated bibliography touch on several aspects of the multifaceted class-size debate. Allen Odden reviews the literature and contends that class-size reduction should be used "sparingly and strategically." C. M. Achilles and colleagues examines two different class-size situations and find student test…

  6. Class Matters

    ERIC Educational Resources Information Center

    Valdata, Patricia

    2005-01-01

    Ever since George Washington opted for the title of president rather than king, Americans have been uncomfortable with the idea of class distinctions. This article presents an interview with Dr. Janet Galligani Casey regarding the idea of class distinctions. Galligani Casey, who grew up in a working-class neighborhood in Somerville, Massachusetts,…

  7. Effect of selected environmental factors on degradation and mineralization of biaryl compounds by the bacterium Ralstonia pickettii in soil and compost.

    PubMed

    Hundt, K; Wagner, M; Becher, D; Hammer, E; Schauer, F

    1998-04-01

    By varying selected environmental factors, the degradation and mineralization of biaryl compounds by the bacterium Ralstonia pickettii in soil and compost were investigated. An optimized soil moisture and enhanced bioavailability by using the nonionic surfactant Tween 80 were of great importance for the degradation rates of biaryl compounds like biphenyl and 4-chlorobiphenyl by cells of Ralstonia picketti SBUG 290 inoculated into soil. Additionally, degradation of these compounds by the investigated strain in soil was strongly dependent upon the medium of precultivation. Also the influence of temperature and soil pH-value was tested. In contrast to the used soil, the autochthonous flora of the compost seemed to have a higher physiological activity. All investigated compounds (biphenyl, 4-chlorobiphenyl and dibenzofuran) were degraded quickly in compost. Inoculation with the investigated bacterium did not enhance the degradation rates significantly.

  8. Draft Genome Sequence of Highly Virulent Race 4/Biovar 3 of Ralstonia solanacearum CaRs_Mep Causing Bacterial Wilt in Zingiberaceae Plants in India.

    PubMed

    Kumar, Aundy; Munjal, Vibhuti; Sheoran, Neelam; Prameela, Thekkan Puthiyaveedu; Suseelabhai, Rajamma; Aggarwal, Rashmi; Jain, Rakesh Kumar; Eapen, Santhosh J

    2017-01-05

    The genome of Ralstonia solanacearum CaRs_Mep, a race 4/biovar 3/phylotype I bacterium causing wilt in small cardamom and other Zingiberaceae plants, was sequenced. Analysis of the 5.7-Mb genome sequence will aid in better understanding of the genetic determinants of host range, host jump, survival, pathogenicity, and virulence of race 4 of R. solanacearum. Copyright © 2017 Kumar et al.

  9. Complete Genome Sequences of the Plant Pathogens Ralstonia solanacearum Type Strain K60 and R. solanacearum Race 3 Biovar 2 Strain UW551.

    PubMed

    Hayes, Madeline M; MacIntyre, April M; Allen, Caitilyn

    2017-10-05

    Ralstonia solanacearum is a globally distributed plant pathogen that causes bacterial wilt diseases of many crop hosts, threatening both sustenance farming and industrial agriculture. Here, we present closed genome sequences for the R. solanacearum type strain, K60, and the cool-tolerant potato brown rot strain R. solanacearum UW551, a highly regulated U.S. select agent pathogen. Copyright © 2017 Hayes et al.

  10. Draft Genome Sequence of Highly Virulent Race 4/Biovar 3 of Ralstonia solanacearum CaRs_Mep Causing Bacterial Wilt in Zingiberaceae Plants in India

    PubMed Central

    Munjal, Vibhuti; Sheoran, Neelam; Prameela, Thekkan Puthiyaveedu; Suseelabhai, Rajamma; Aggarwal, Rashmi; Jain, Rakesh Kumar; Eapen, Santhosh J.

    2017-01-01

    ABSTRACT The genome of Ralstonia solanacearum CaRs_Mep, a race 4/biovar 3/phylotype I bacterium causing wilt in small cardamom and other Zingiberaceae plants, was sequenced. Analysis of the 5.7-Mb genome sequence will aid in better understanding of the genetic determinants of host range, host jump, survival, pathogenicity, and virulence of race 4 of R. solanacearum. PMID:28057749

  11. Comparative phylogenies of Burkholderia, Ralstonia, Comamonas, Brevundimonas and related organisms derived from rpoB, gyrB and rrs gene sequences.

    PubMed

    Tayeb, Lineda Ait; Lefevre, Martine; Passet, Virginie; Diancourt, Laure; Brisse, Sylvain; Grimont, Patrick A D

    2008-04-01

    Phylogenetic analysis of strains from Burkholderia, Ralstonia, Cupriavidus, Comamonas, Delftia, Acidovorax, Brevundimonas, Herbaspirillum huttiense and "Pseudomonas butanovora" was performed based on the protein-coding genes rpoB and gyrB and on the 16S rRNA-coding gene rrs. Overall, the phylogenies deduced from the three genes were concordant among themselves and with current taxonomy. However, a few differences among individual gene phylogenies were noted. For example, the separation of Cupriavidus from Ralstonia was not supported in the rpoB tree, as Ralstonia was nested within Cupriavidus. Similarly, the separation of Delftia from Comamonas was not supported in the gyrB tree. Based on rrs and rpoB, the genus Burkholderia contained four groups: (i) the B. cepacia complex, (ii) the B. pseudomallei-B. thailandensis group, (iii) a 6-species group including B. caledonica and B. glathei and (iv) the B. plantarii-B. glumae-B. gladioli group. However, B. caribensis and B. glathei stood as a fifth group based on gyrB. It appears that a phylogeny cannot be reliably based on a single gene. Using rpoB and gyrB, better separation of closely related species was obtained compared to rrs, indicating the potential of these two genes for identification and species definition. Nevertheless, intraspecific sequence diversity will need to be determined to fully establish the value of these genes for strain identification.

  12. Detection of Ralstonia solanacearum, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes

    PubMed Central

    Wullings, B. A.; Van Beuningen, A. R.; Janse, J. D.; Akkermans, A. D. L.

    1998-01-01

    During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas. PMID:9797321

  13. A computer program for fast and easy typing of a partial endoglucanase gene sequence into genospecies and sequevars 1&2 of the Ralstonia solanacearum species complex.

    PubMed

    Stulberg, Michael J; Huang, Qi

    2016-04-01

    The phytopathogen Ralstonia solanacearum is a species complex that contains race 3 biovar 2 strains belonging to phylotype IIB sequevars 1 and 2 that are quarantined or select agent pathogens. Recently, the R. solanacearum species complex strains have been reclassified into three genospecies: R. solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii. An unidentified R. solanacearum strain is considered a select agent in the US until proven to be a non-race 3 biovar 2 (non-phylotype IIB sequevars 1&2). Currently, sequevars of R. solanacearum species complex strains can only be determined by phylogenetic analysis of a partial endoglucanase (egl) sequence of approximately 700-bp in length. Such analysis, however, requires expert knowledge to properly trim the sequence, to include the correct reference strains, and to interpret the results. By comparing GenBank egl sequences of representative R. solanacearum species-complex strains, we identified genospecies- and sequevar 1 and 2-specific single nucleotide polymorphisms (SNPs). We also designed primers to amplify a shorter, 526-bp, egl fragment from R. solanacearum species complex strains for easy sequencing of the amplicon, and to facilitate direct and specific amplification of egl from R. solanacearum-infected plant samples without the need of bacterial isolation. We wrote a computer program (Ralstonia solanacearum typing program) that analyzes a minimum 400-bp user-input egl sequence from a R. solanacearum strain for egl homology and SNP content to determine 1) whether it belongs to the R. solanacearum species complex, 2) if so, to which genospecies, and 3) whether it is of the sequevar type (sequevars 1 and 2) associated with the select agent/quarantined R. solanacearum strain. The program correctly typed all 371 tested egl sequences with known sequevars, obtained either from GenBank or through personal communication. Additionally, the program successfully typed 25 R. solanacearum strains in our

  14. Degradation of the Plant Defense Signal Salicylic Acid Protects Ralstonia solanacearum from Toxicity and Enhances Virulence on Tobacco.

    PubMed

    Lowe-Power, Tiffany M; Jacobs, Jonathan M; Ailloud, Florent; Fochs, Brianna; Prior, Philippe; Allen, Caitilyn

    2016-06-21

    Plants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule. Plant pathogens such as the bacterial wilt agent Ralstonia solanacearum threaten food and economic security by causing significant losses for small- and large-scale growers of tomato, tobacco, banana, potato, and ornamentals. Like most plants

  15. A Ralstonia solanacearum type III effector directs the production of the plant signal metabolite trehalose-6-phosphate.

    PubMed

    Poueymiro, M; Cazalé, A C; François, J M; Parrou, J L; Peeters, N; Genin, S

    2014-12-23

    The plant pathogen Ralstonia solanacearum possesses two genes encoding a trehalose-6-phosphate synthase (TPS), an enzyme of the trehalose biosynthetic pathway. One of these genes, named ripTPS, was found to encode a protein with an additional N-terminal domain which directs its translocation into host plant cells through the type 3 secretion system. RipTPS is a conserved effector in the R. solanacearum species complex, and homologues were also detected in other bacterial plant pathogens. Functional analysis of RipTPS demonstrated that this type 3 effector synthesizes trehalose-6-phosphate and identified residues essential for this enzymatic activity. Although trehalose-6-phosphate is a key signal molecule in plants that regulates sugar status and carbon assimilation, the disruption of ripTPS did not alter the virulence of R. solanacearum on plants. However, heterologous expression assays showed that this effector specifically elicits a hypersensitive-like response on tobacco that is independent of its enzymatic activity and is triggered by the C-terminal half of the protein. Recognition of this effector by the plant immune system is suggestive of a role during the infectious process. Ralstonia solanacearum, the causal agent of bacterial wilt disease, infects more than two hundred plant species, including economically important crops. The type III secretion system plays a major role in the pathogenicity of this bacterium, and approximately 70 effector proteins have been shown to be translocated into host plant cells. This study provides the first description of a type III effector endowed with a trehalose-6-phosphate synthase enzymatic activity and illustrates a new mechanism by which the bacteria may manipulate the plant metabolism upon infection. In recent years, trehalose-6-phosphate has emerged as an essential signal molecule in plants, connecting plant metabolism and development. The finding that a bacterial pathogen could induce the production of trehalose-6

  16. The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway.

    PubMed

    Popa, Crina; Li, Liang; Gil, Sergio; Tatjer, Laura; Hashii, Keisuke; Tabuchi, Mitsuaki; Coll, Núria S; Ariño, Joaquín; Valls, Marc

    2016-06-03

    Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation.

  17. A competitive index assay identifies several Ralstonia solanacearum type III effector mutant strains with reduced fitness in host plants.

    PubMed

    Macho, Alberto P; Guidot, Alice; Barberis, Patrick; Beuzón, Carmen R; Genin, Stéphane

    2010-09-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, is a soil bacterium which can naturally infect a wide range of host plants through the root system. Pathogenicity relies on a type III secretion system which delivers a large set of approximately 75 type III effectors (T3E) into plant cells. On several plants, pathogenicity assays based on quantification of wilting symptoms failed to detect a significant contribution of R. solanacearum T3E in this process, thus revealing the collective effect of T3E in pathogenesis. We developed a mixed infection-based method with R. solanacearum to monitor bacterial fitness in plant leaf tissues as a virulence assay. This accurate and sensitive assay provides evidence that growth defects can be detected for T3E mutants: we identified 12 genes contributing to bacterial fitness in eggplant leaves and 3 of them were also implicated in bacterial fitness on two other hosts, tomato and bean. Contribution to fitness of several T3E appears to be host specific, and we show that some known avirulence determinants such as popP2 or avrA do provide competitive advantages on some susceptible host plants. In addition, this assay revealed that the efe gene, which directs the production of ethylene by bacteria in plant tissues, and hdfB, involved in the biosynthesis of the secondary metabolite 3-hydroxy-oxindole, are also required for optimal growth in plant leaf tissues.

  18. Pathogenesis and stress related, as well as metabolic proteins are regulated in tomato stems infected with Ralstonia solanacearum.

    PubMed

    Dahal, Diwakar; Heintz, Dimitri; Van Dorsselaer, Alain; Braun, Hans-Peter; Wydra, Kerstin

    2009-09-01

    A comparative proteome analysis was initiated to systematically investigate the physiological response of tomato (Solanum lycopersicum) to infection with Ralstonia solanacearum, causal agent of bacterial wilt. Plants of the susceptible tomato recombinant inbred line NHG3 and the resistant NHG13 were either infected or not infected with R. solanacearum and subsequently used for proteome analysis. Two-dimensional isoelectric focussing/sodium dodecyl-sulphate polyacrylamide gel electrophoresis (2-D IEF/SDS-PAGE) allowed the separation of about 650-690 protein spots per analysis. Twelve proteins were of differential abundance in susceptible plants in response to bacterial infection, while no differences were observed in the resistant genotype. LC-MS/MS analysis of these spots revealed 12 proteins, six of which were annotated as plant and six as bacterial proteins. Among the plant proteins, two represent pathogenesis related (PR) proteins, one stress response protein, one enzyme of carbohydrate and energy metabolism, and one hypothetical protein. A constitutive difference between resistant and susceptible lines was not found.

  19. The effector AWR5 from the plant pathogen Ralstonia solanacearum is an inhibitor of the TOR signalling pathway

    PubMed Central

    Popa, Crina; Li, Liang; Gil, Sergio; Tatjer, Laura; Hashii, Keisuke; Tabuchi, Mitsuaki; Coll, Núria S.; Ariño, Joaquín; Valls, Marc

    2016-01-01

    Bacterial pathogens possess complex type III effector (T3E) repertoires that are translocated inside the host cells to cause disease. However, only a minor proportion of these effectors have been assigned a function. Here, we show that the T3E AWR5 from the phytopathogen Ralstonia solanacearum is an inhibitor of TOR, a central regulator in eukaryotes that controls the switch between cell growth and stress responses in response to nutrient availability. Heterologous expression of AWR5 in yeast caused growth inhibition and autophagy induction coupled to massive transcriptomic changes, unmistakably reminiscent of TOR inhibition by rapamycin or nitrogen starvation. Detailed genetic analysis of these phenotypes in yeast, including suppression of AWR5-induced toxicity by mutation of CDC55 and TPD3, encoding regulatory subunits of the PP2A phosphatase, indicated that AWR5 might exert its function by directly or indirectly inhibiting the TOR pathway upstream PP2A. We present evidence in planta that this T3E caused a decrease in TOR-regulated plant nitrate reductase activity and also that normal levels of TOR and the Cdc55 homologues in plants are required for R. solanacearum virulence. Our results suggest that the TOR pathway is a bona fide T3E target and further prove that yeast is a useful platform for T3E function characterisation. PMID:27257085

  20. Ectopic expression of Ralstonia solanacearum effector protein PopA early in invasion results in loss of virulence.

    PubMed

    Kanda, Ayami; Yasukohchi, Masahiko; Ohnishi, Kouhei; Kiba, Akinori; Okuno, Tetsuro; Hikichi, Yasufumi

    2003-05-01

    Ralstonia solanacearum OE1-1 (OE1-1) is pathogenic to tobacco. The type III-secreted effector protein popA of OE1-1 showed 97.6% identity to popA of R. solanacearum GMI1000, which is not pathogenic to tobacco. Reverse transcription-polymerase chain reaction analysis showed that popA in OE1-1 was expressed at 3 h after inoculation (HAI), but not before, in infiltrated-tobacco leaves. Pathogenicity analysis using a popABC operon-deleted mutant of OE1-1 (deltaABC) showed that popABC is not directly involved in the pathogenicity of OE1-1. When Papa, which constitutively expresses popA, was infiltrated into tobacco leaves, popA was expressed by 0.5 HAI. Papa could no longer multiply or spread in tobacco leaves and was no longer virulent. Moreover, the hypersensitive response (HR) and expression of HR-related genes were not induced in Papa-infiltrated leaves. Papa was also avirulent in a tobacco root-dipping inoculation assay. These results suggest that the expression of popA in Papa immediately after invasion triggers the suppression of bacterial proliferation and movement, resulting in loss of virulence. However, Papa retained its virulence when directly inoculated into xylem vessels. This result suggests that tobacco plants can recognize PopA when it is expressed early in disease development, and respond with an effective defense in the intercellular spaces.

  1. Ralstonia solanacearum Dps contributes to oxidative stress tolerance and to colonization of and virulence on tomato plants.

    PubMed

    Colburn-Clifford, Jennifer M; Scherf, Jacob M; Allen, Caitilyn

    2010-11-01

    Ralstonia solanacearum, an economically important soilborne plant pathogen, infects host roots to cause bacterial wilt disease. However, little is known about this pathogen's behavior in the rhizosphere and early in pathogenesis. In response to root exudates from tomato, R. solanacearum strain UW551 upregulated a gene resembling Dps, a nonspecific DNA binding protein from starved cells that is critical for stress survival in other bacteria. An R. solanacearum dps mutant had increased hydrogen peroxide sensitivity and mutation rate under starvation. Furthermore, dps expression was positively regulated by the oxidative stress response regulator OxyR. These functional results are consistent with a Dps annotation. The dps mutant caused slightly delayed bacterial wilt disease in tomato after a naturalistic soil soak inoculation. However, the dps mutant had a more pronounced reduction in virulence when bacteria were inoculated directly into host stems, suggesting that Dps helps R. solanacearum adapt to conditions inside plants. Passage through a tomato plant conferred transient increased hydrogen peroxide tolerance on both wild-type and dps mutant strains, demonstrating that R. solanacearum acquires Dps-independent oxidative stress tolerance during adaptation to the host environment. The dps mutant strain was also reduced in adhesion to tomato roots and tomato stem colonization. These results indicate that Dps is important when cells are starved or in stationary phase and that Dps contributes quantitatively to host plant colonization and bacterial wilt virulence. They further suggest that R. solanacearum must overcome oxidative stress during the bacterial wilt disease cycle.

  2. Development of the sensitive lateral flow immunoassay with silver enhancement for the detection of Ralstonia solanacearum in potato tubers.

    PubMed

    Panferov, Vasily G; Safenkova, Irina V; Varitsev, Yury A; Drenova, Natalia V; Kornev, Konstantin P; Zherdev, Anatoly V; Dzantiev, Boris B

    2016-05-15

    Ralstonia solanacearum is a dangerous and economically important pathogen of potatoes and other agricultural crops. Therefore, rapid and sensitive methods for its routine diagnostics are necessary. The aim of this study was to develop a rapid control method for R. solanacearum with a low limit of detection (LOD) based on a lateral flow immunoassay (LFIA) with silver enhancement. To minimize the LOD, the membrane type, antibody amount for conjugation with gold nanoparticles, conjugate concentration and antibody concentration in the analytical zone were optimized. Silver enhancement was used to decrease the LOD of the LFIA. For silver enhancement, release fiberglass membranes with pre-absorbed silver lactate and hydroquinone were placed on the analytical zone, and a drop of silver lactate was added. The LFIA with silver enhancement was found to be 10-fold more sensitive (LOD 2×10(2) CFU/mL; 20 min) in comparison with the common analysis (LOD 2×10(3) CFU/mL; 10 min). The specificity of the developed LFIA was studied using different strains of R. solanacearum (54 samples) and other widespread bacterial pathogens (18 samples). The LFIA detected all tested strains, whereas non-specific reactions were not observed. The developed tests were used for the control of bacteria in extracts of infected and non-infected potato tubers, and the quantitative analysis results (based on the densitometry of line colouration) were confirmed by ELISA with a correlation coefficient equal to 0.965.

  3. Moko Disease-Causing Strains of Ralstonia solanacearum from Brazil Extend Known Diversity in Paraphyletic Phylotype II.

    PubMed

    Albuquerque, Greecy M R; Santos, Liliana A; Felix, Kátia C S; Rollemberg, Christtianno L; Silva, Adriano M F; Souza, Elineide B; Cellier, Gilles; Prior, Philippe; Mariano, Rosa L R

    2014-11-01

    The epidemic situation of Moko disease-causing strains in Latin America and Brazil is unclear. Thirty-seven Ralstonia solanacearum strains from Brazil that cause the Moko disease on banana and heliconia plants were sampled and phylogenetically typed using the endoglucanase (egl) and DNA repair (mutS) genes according to the phylotype and sequevar classification. All of the strains belonged to phylotype II and a portion of the strains was typed as the Moko disease-related sequevars IIA-6 and IIA-24. Nevertheless, two unsuspected sequevars also harbored the Moko disease-causing strains IIA-41 and IIB-25, and a new sequevar was described and named IIA-53. All of the strains were pathogenic to banana and some of the strains of sequevars IIA-6, IIA-24, and IIA-41 were also pathogenic to tomato. The Moko disease-causing strains from sequevar IIB-25 were pathogenic to potato but not to tomato. These results highlight the high diversity of strains of Moko in Brazil, reinforce the efficiency of the egl gene to reveal relationships among these strains, and contribute to a better understanding of the diversity of paraphyletic Moko disease-causing strains of the R. solanacearum species complex, where the following seven distinct genetic clusters have been described: IIA-6, IIA-24, IIA-41, IIA-53, IIB-3, IIB-4, and IIB-25.

  4. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China.

    PubMed

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 10(2) CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient.

  5. Loop-mediated isothermal amplification of specific endoglucanase gene sequence for detection of the bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Lenarčič, Rok; Morisset, Dany; Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

    2014-01-01

    The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes.

  6. Genomic Analysis of Phylotype I Strain EP1 Reveals Substantial Divergence from Other Strains in the Ralstonia solanacearum Species Complex

    PubMed Central

    Li, Peng; Wang, Dechen; Yan, Jinli; Zhou, Jianuan; Deng, Yinyue; Jiang, Zide; Cao, Bihao; He, Zifu; Zhang, Lianhui

    2016-01-01

    Ralstonia solanacearum species complex is a devastating group of phytopathogens with an unusually wide host range and broad geographical distribution. R. solanacearum isolates may differ considerably in various properties including host range and pathogenicity, but the underlying genetic bases remain vague. Here, we conducted the genome sequencing of strain EP1 isolated from Guangdong Province of China, which belongs to phylotype I and is highly virulent to a range of solanaceous crops. Its complete genome contains a 3.95-Mb chromosome and a 2.05-Mb mega-plasmid, which is considerably bigger than reported genomes of other R. solanacearum strains. Both the chromosome and the mega-plasmid have essential house-keeping genes and many virulence genes. Comparative analysis of strain EP1 with other 3 phylotype I and 3 phylotype II, III, IV strains unveiled substantial genome rearrangements, insertions and deletions. Genome sequences are relatively conserved among the 4 phylotype I strains, but more divergent among strains of different phylotypes. Moreover, the strains exhibited considerable variations in their key virulence genes, including those encoding secretion systems and type III effectors. Our results provide valuable information for further elucidation of the genetic basis of diversified virulences and host range of R. solanacearum species. PMID:27833603

  7. l-Histidine Induces Resistance in Plants to the Bacterial Pathogen Ralstonia solanacearum Partially Through the Activation of Ethylene Signaling.

    PubMed

    Seo, Shigemi; Nakaho, Kazuhiro; Hong, Si Won; Takahashi, Hideki; Shigemori, Hideyuki; Mitsuhara, Ichiro

    2016-09-01

    Wilt disease in plants, which is caused by the soil-borne bacterial pathogen Ralstonia solanacearum, is one of the most devastating plant diseases. We previously detected bacterial wilt disease-inhibiting activity in an extract from yeast cells. In the present study, we purified this activity and identified one of the substances responsible for the activity as the amino acid histidine. The exogenous application of l-histidine, but not d-histidine, inhibited wilt disease in tomato and Arabidopsis plants without exhibiting any antibacterial activity. l-Histidine induced the expression of genes related to ethylene (ET) biosynthesis and signaling as well as the production of ET in tomato and Arabidopsis plants. l-Histidine-induced resistance to R. solanacearum was partially abolished in ein3-1, an ET-insensitive Arabidopsis mutant line. Resistance to the fungal pathogen Botrytis cinerea, which is known to require ET biosynthesis or signaling, was also induced by exogenously applied l-histidine. These results suggest that l-histidine induces resistance to R. solanacearum and B. cinerea partially through the activation of ET signaling in plants. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis.

    PubMed

    Zhang, Bo; Tremousaygue, Dominique; Denancé, Nicolas; van Esse, H Peter; Hörger, Anja C; Dabos, Patrick; Goffner, Deborah; Thomma, Bart P H J; van der Hoorn, Renier A L; Tuominen, Hannele

    2014-09-01

    PIRIN (PRN) is a member of the functionally diverse cupin protein superfamily. There are four members of the Arabidopsis thaliana PRN family, but the roles of these proteins are largely unknown. Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum. Two prn2 mutant alleles displayed decreased disease development and bacterial growth in response to R.  solanacearum infection. We elucidated the underlying molecular mechanism by analyzing PRN2 interactions with the papain-like cysteine proteases (PLCPs) XCP2, RD21A, and RD21B, all of which bound to PRN2 in yeast two-hybrid assays and in Arabidopsis protoplast co-immunoprecipitation assays. We show that XCP2 is stabilized by PRN2 through inhibition of its autolysis on the basis of PLCP activity profiling assays and enzymatic assays with recombinant protein. The stabilization of XCP2 by PRN2 was also confirmed in planta. Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis. © 2014 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.

  9. Volatile organic compounds produced by Pseudomonas fluorescens WR-1 restrict the growth and virulence traits of Ralstonia solanacearum.

    PubMed

    Raza, Waseem; Ling, Ning; Liu, Dongyang; Wei, Zhong; Huang, Qiwei; Shen, Qirong

    2016-11-01

    The volatile organic compounds (VOCs) produced by soil microbes have a significant role in the control of plant diseases and plant growth promotion. In this study, we examined the effect of VOCs produced by Pseudomonas fluorescens strain WR-1 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum. The VOCs produced by P. fluorescens WR-1 exhibited concentration dependent bacteriostatic effect on the growth of R. solanacearum on agar medium and in infested soil. The VOCs of P. fluorescens WR-1 also significantly inhibited the virulence traits of R. solanacearum. The proteomics analysis showed that the VOCs of P. fluorescens WR-1 downregulated cellular proteins of R. solanacearum related to the antioxidant activity, virulence, inclusion body proteins, carbohydrate and amino acid synthesis and metabolism, protein folding and translation, methylation and energy transfer, while the proteins involved in the ABC transporter system, detoxification of aldehydes and ketones, protein folding and translation were upregulated. This study revealed the significance of VOCs of P. fluorescens WR-1 to control the tomato wilt pathogen R. solanacearum. Investigation of the modes of action of biocontrol agents is important to better comprehend the interactions mediated by VOCs in nature to design better control strategies for plant pathogens. Copyright © 2016 Elsevier GmbH. All rights reserved.

  10. The involvement of the PilQ secretin of type IV pili in phage infection in Ralstonia solanacearum.

    PubMed

    Narulita, Erlia; Addy, Hardian Susilo; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

    2016-01-22

    PilQ is a member of the secretin family of outer membrane proteins and specifically involved in type IV secretion. Here we report the effects of pilQ mutation in Ralstonia solanacearum on the host physiology including susceptibility to several phage types (Inoviridae, Podoviridae and Myoviridae). With three lines of cells, namely wild type, ΔpilQ and pilQ-complemented cells, the cell surface proteins, twitching motility and sensitivity to phages were compared. SDS-PAGE analysis revealed that the major TFP pilin (PilA) was specifically lost in pilQ mutants and was recovered in the complemented cells. Drastically inactivated twitching motility in pilQ mutants was recovered to the wild type level in the complemented cells. Several phages of different types including those of Inoviridae, Podoviridae, and Myoviridae that infect wild type cells could not form plaques on pilQ mutants but showed infectivity to pilQ-complemented cells. These results indicate that PilQ function is generally required for phage infection in R. solanacearum. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Loop-Mediated Isothermal Amplification of Specific Endoglucanase Gene Sequence for Detection of the Bacterial Wilt Pathogen Ralstonia solanacearum

    PubMed Central

    Pirc, Manca; Llop, Pablo; Ravnikar, Maja; Dreo, Tanja

    2014-01-01

    The increased globalization of crops production and processing industries also promotes the side-effects of more rapid and efficient spread of plant pathogens. To prevent the associated economic losses, and particularly those related to bacterial diseases where their management relies on removal of the infected material from production, simple, easy-to-perform, rapid and cost-effective tests are needed. Loop-mediated isothermal amplification (LAMP) assays that target 16S rRNA, fliC and egl genes were compared and evaluated as on-site applications. The assay with the best performance was that targeted to the egl gene, which shows high analytical specificity for diverse strains of the betaproteobacterium Ralstonia solanacearum, including its non-European and non-race 3 biovar 2 strains. The additional melting curve analysis provides confirmation of the test results. According to our extensive assessment, the egl LAMP assay requires minimum sample preparation (a few minutes of boiling) for the identification of pure cultures and ooze from symptomatic material, and it can also be used in a high-throughput format in the laboratory. This provides sensitive and reliable detection of R. solanacearum strains of different phylotypes. PMID:24763488

  12. TssB is essential for virulence and required for type VI secretion system in Ralstonia solanacearum.

    PubMed

    Zhang, Liqing; Xu, Jingsheng; Xu, Jin; Zhang, Hao; He, Liyuan; Feng, Jie

    2014-09-01

    The type VI secretion system (T6SS) is recently discovered machinery in Gram-negative bacteria for translocation of proteins and also is required for full virulence. TssB is a highly conserved protein among the T6SSs, and indispensable for composition and function of T6S. The plant pathogenic bacterium Ralstonia solanacearum also harbours T6SS gene clusters, and a homologue of TssB, hereafter designated as TssBRS, but up to date its characterization and function remain unclear. In this study, we showed that TssBRS of R. solanacearum was required for secretion of Hcp, the haemolysin coregulated protein and a hallmark of T6S pathway. Deletion of tssBRS in R. solanacearum GMI1000 strain resulted in defect of biofilm formation, and the expression of the flagella operon is decreased, leading to decreased motility. More importantly, tssBRS mutant strain had significantly attenuated its virulence on tomato plants. TssB is essential for virulence and required for type VI secretion system in R. solanacearum. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Ralstonia solanacearum Extracellular Polysaccharide Is a Specific Elicitor of Defense Responses in Wilt-Resistant Tomato Plants

    PubMed Central

    Milling, Annett; Babujee, Lavanya; Allen, Caitilyn

    2011-01-01

    Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps− mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps− mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps− mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum. PMID:21253019

  14. Rhizocompetence and antagonistic activity towards genetically diverse Ralstonia solanacearum strains--an improved strategy for selecting biocontrol agents.

    PubMed

    Xue, Qing-Yun; Ding, Guo-Chun; Li, Shi-Mo; Yang, Yang; Lan, Cheng-Zhong; Guo, Jian-Hua; Smalla, Kornelia

    2013-02-01

    Bacterial wilt caused by Ralstonia solanacearum is a serious threat for agricultural production in China. Eight soil bacterial isolates with activity against R. solanacearum TM15 (biovar 3) were tested in this study for their in vitro activity towards ten genetically diverse R. solanacearum isolates from China. The results indicated that each antagonist showed remarkable differences in its ability to in vitro antagonize the ten different R. solanacearum strains. Strain XY21 (based on 16S rRNA gene sequencing affiliated to Serratia) was selected for further studies based on its in vitro antagonistic activity and its excellent rhizocompetence on tomato plants. Under greenhouse conditions XY21 mediated biocontrol of tomato wilt caused by seven different R. solanacearum strains ranged from 19 to 70 %. The establishment of XY21 and its effects on the bacterial community in the tomato rhizosphere were monitored by denaturing gradient gel electrophoresis of 16S rRNA gene fragments PCR-amplified from total community DNA. A positive correlation of the in vitro antagonistic activities of XY21 and the actual biocontrol efficacies towards seven genetically different R. solanacearum strains was found and further confirmed by the efficacy of XY21 in controlling bacterial wilt under field conditions.

  15. The C-terminal extension of PrhG impairs its activation of hrp expression and virulence in Ralstonia solanacearum.

    PubMed

    Zhang, Yong; Luo, Feng; Hikichi, Yasufumi; Kiba, Akinori; Yasuo, Igarashi; Ohnishi, Kouhei

    2015-04-01

    Ralstonia solanacearum is the second most destructive bacterial plant pathogens worldwide and HrpG is the master regulator of its pathogenicity. PrhG is a close paralogue of HrpG and both belong to OmpR/PhoB family of two-component response regulators. Despite a high similarity (72% global identity and 96% similarity in helix-loop-helix domain), they display distinct roles in pathogenicity. HrpG is necessary for the bacterial growth in planta and pathogenicity, while PrhG is dispensable for bacterial growth in planta and contributes little to pathogenicity. The main difference between HrpG and PrhG is the 50-amino-acid-long C-terminal extension in PrhG (amino-acid residues 230-283), which is absent in HrpG. When this extension is deleted, truncated PrhGs (under the control of its native promoter) allowed complete recovery of bacterial growth in planta and wild-type virulence of hrpG mutant. This novel finding demonstrates that the extension region in PrhG is responsible for the functional difference between HrpG and PrhG, which may block the binding of PrhG to target promoters and result in impaired activation of hrp expression by PrhG and reduced virulence of R. solanacearum. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. A novel, sensitive method to evaluate potato germplasm for bacterial wilt resistance using a luminescent Ralstonia solanacearum reporter strain.

    PubMed

    Cruz, Andrea Paola Zuluaga; Ferreira, Virginia; Pianzzola, María Julia; Siri, María Inés; Coll, Núria S; Valls, Marc

    2014-03-01

    Several breeding programs are under way to introduce resistance to bacterial wilt caused by Ralstonia solanacearum in solanaceous crops. The lack of screening methods allowing easy measurement of pathogen colonization and the inability to detect latent (i.e., symptomless) infections are major limitations when evaluating resistance to this disease in plant germplasm. We describe a new method to study the interaction between R. solanacearum and potato germplasm that overcomes these restrictions. The R. solanacearum UY031 was genetically modified to constitutively generate light from a synthetic luxCDABE operon stably inserted in its chromosome. Colonization of this reporter strain on different potato accessions was followed using life imaging. Bacterial detection in planta by this nondisruptive system correlated with the development of wilting symptoms. In addition, we demonstrated that quantitative detection of the recombinant strain using a luminometer can identify latent infections on symptomless potato plants. We have developed a novel, unsophisticated, and accurate method for high-throughput evaluation of pathogen colonization in plant populations. We applied this method to compare the behavior of potato accessions with contrasting resistance to R. solanacearum. This new system will be especially useful to detect latency in symptomless parental lines before their inclusion in long-term breeding programs for disease resistance.

  17. Expressed sequence tags in cultivated peanut (Arachis hypogaea): discovery of genes in seed development and response to Ralstonia solanacearum challenge.

    PubMed

    Huang, Jiaquan; Yan, Liying; Lei, Yong; Jiang, Huifang; Ren, Xiaoping; Liao, Boshou

    2012-11-01

    Although an important oil crop, peanut has only 162,030 expressed sequence tags (ESTs) publicly available, 86,943 of which are from cultivated plants. More ESTs from cultivated peanuts are needed for isolation of stress-resistant, tissue-specific and developmentally important genes. Here, we generated 63,234 ESTs from our 5 constructed peanut cDNA libraries of Ralstonia solanacearum challenged roots, R. solanacearum challenged leaves, and unchallenged cultured peanut roots, leaves and developing seeds. Among these ESTs, there were 14,547 unique sequences with 7,961 tentative consensus sequences and 6,586 singletons. Putative functions for 47.8 % of the sequences were identified, including transcription factors, tissue-specific genes, genes involved in fatty acid biosynthesis and oil formation regulation, and resistance gene analogue genes. Additionally, differentially expressed genes, including those involved in ethylene and jasmonic acid signal transduction pathways, from both peanut leaves and roots, were identified in R. solanacearum challenged samples. This large expression dataset from different peanut tissues will be a valuable source for marker development and gene expression analysis. It will also be helpful for finding candidate genes for fatty acid synthesis and oil formation regulation as well as for studying mechanisms of interactions between the peanut host and R. solanacearum pathogen.

  18. Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Ralstonia solanacearum Phylotype I Mulberry Strains in China

    PubMed Central

    Huang, Wen; Zhang, Hao; Xu, Jingsheng; Wang, Shuai; Kong, Xiangjiu; Ding, Wei; Xu, Jin; Feng, Jie

    2017-01-01

    Ralstonia solanacearum phylotype I mulberry strains are causative agent of bacterial wilt of mulberry. Current diagnostic methods are not adopted to the mulberry wilt disease. In this study, we developed a rapid method, loop-mediated isothermal amplification (LAMP), to detect R. solanacearum phylotype I mulberry strains. A set of six primers was designed to target the clone MG67 sequence in this LAMP detection which can be completed in 20 min at 64°C. The results of the LAMP reaction could be observed with the naked eye due to magnesium pyrophosphate precipitate produced during the reaction or the color change after adding SYBR Green I. The specificity of the LAMP was confirmed using DNA from 46 representative strains of R. solanacearum and 7 other soil-borne bacteria strains. This method was also of high sensitivity and could be used to detect the presence of less than 160 fg genomic DNA or 2.2 × 102 CFU/ml of bacterial cells per 25 μl reaction volume, moreover, the presence of plant tissue fluid did not affect the sensitivity. Since it does not require expensive equipment or specialized techniques, this LAMP-based diagnostic method has the potential to be used under field conditions to make disease forecasting more accurate and efficient. PMID:28197157

  19. Identification of boric acid as a novel chemoattractant and elucidation of its chemoreceptor in Ralstonia pseudosolanacearum Ps29.

    PubMed

    Hida, Akiko; Oku, Shota; Nakashimada, Yutaka; Tajima, Takahisa; Kato, Junichi

    2017-08-17

    Chemotaxis enables bacteria to move toward more favorable environmental conditions. We observed chemotaxis toward boric acid by Ralstonia pseudosolanacearum Ps29. At higher concentrations, the chemotactic response of R. pseudosolanacearum toward boric acid was comparable to or higher than that toward L-malate, indicating that boric acid is a strong attractant for R. pseudosolanacearum. Chemotaxis assays under different pH conditions suggested that R. pseudosolanacearum recognizes B(OH)3 (or B(OH3) + B(OH)4(-)) but not B(OH)4(-) alone. Our previous study revealed that R. pseudosolanacearum Ps29 harbors homologs of all 22R. pseudosolanacearum GMI1000 mcp genes. Screening of 22 mcp single-deletion mutants identified the RS_RS17100 homolog as the boric acid chemoreceptor, which was designated McpB. The McpB ligand-binding domain (LBD) was purified in order to characterize its binding to boric acid. Using isothermal titration calorimetry, we demonstrated that boric acid binds directly to the McpB LBD with a K D (dissociation constant) of 5.4 µM. Analytical ultracentrifugation studies revealed that the McpB LBD is present as a dimer that recognizes one boric acid molecule.

  20. The Ralstonia solanacearum type III effector RipAY targets plant redox regulators to suppress immune responses.

    PubMed

    Sang, Yuying; Wang, Yaru; Ni, Hong; Cazalé, Anne-Claire; She, Yi-Min; Peeters, Nemo; Macho, Alberto P

    2016-10-21

    The subversion of plant cellular functions is essential for bacterial pathogens to proliferate in host plants and cause disease. Most bacterial plant pathogens employ a type III secretion system to inject type III effector (T3E) proteins inside plant cells, where they contribute to the pathogen-induced alteration of plant physiology. In this work, we found that the Ralstonia solanacearum T3E RipAY suppresses plant immune responses triggered by bacterial elicitors and by the phytohormone salicylic acid. Further biochemical analysis indicated that RipAY associates in planta with thioredoxins from Nicotiana benthamiana and Arabidopsis. Interestingly, RipAY displays γ-glutamyl cyclotransferase (GGCT) activity to degrade glutathione in plant cells, which is required for the reported suppression of immune responses. Given the importance of thioredoxins and glutathione as major redox regulators in eukaryotic cells, RipAY activity may constitute a novel and powerful virulence strategy employed by R. solanacearum to suppress immune responses and potentially alter general redox signalling in host cells.

  1. Phylogeny and Population Structure of Brown Rot- and Moko Disease-Causing Strains of Ralstonia solanacearum Phylotype II

    PubMed Central

    Remenant, B.; Chiroleu, F.; Lefeuvre, P.; Prior, P.

    2012-01-01

    The ancient soilborne plant vascular pathogen Ralstonia solanacearum has evolved and adapted to cause severe damage in an unusually wide range of plants. In order to better describe and understand these adaptations, strains with very similar lifestyles and host specializations are grouped into ecotypes. We used comparative genomic hybridization (CGH) to investigate three particular ecotypes in the American phylotype II group: (i) brown rot strains from phylotypes IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from phylotype IIB-4NPB that lack pathogenicity for banana but can infect many other plant species; and (iii) Moko disease-causing strains from phylotypes IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia spp. We compared the genomes of 72 R. solanacearum strains, mainly from the three major ecotypes of phylotype II, using a newly developed pangenomic microarray to decipher their population structure and gain clues about the epidemiology of these ecotypes. Strain phylogeny and population structure were reconstructed. The results revealed a phylogeographic structure within brown rot strains, allowing us to distinguish European outbreak strains of Andean and African origins. The pangenomic CGH data also demonstrated that Moko ecotype IIB-4 is phylogenetically distinct from the emerging IIB-4NPB strains. These findings improved our understanding of the epidemiology of important ecotypes in phylotype II and will be useful for evolutionary analyses and the development of new DNA-based diagnostic tools. PMID:22286995

  2. Non-Instrumented Nucleic Acid Amplification (NINA) for Rapid Detection of Ralstonia solanacearum Race 3 Biovar 2

    PubMed Central

    Kubota, Ryo; LaBarre, Paul; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard H.; Alvarez, Anne M.; Jenkins, Daniel M.

    2014-01-01

    We report on the use of a non-instrumented device for the implementation of a loop-mediated amplification (LAMP) based assay for the select-agent bacterial-wilt pathogen Ralstonia solanacearum race 3 biovar 2. Heat energy is generated within the device by the exothermic hydration of calcium oxide, and the reaction temperature is regulated by storing latent energy at the melting temperature of a renewable lipid-based engineered phase-change material. Endpoint detection of the LAMP reaction is achieved without opening the reaction tube by observing the fluorescence of an innovative FRET-based hybridization probe with a simple custom fluorometer. Non-instrumented devices could maintain reactions near the design temperature of 63°C for at least an hour. Using this approach DNA extracted from the pathogen could be detected at fewer than ten copies within a 25 μL reaction mix, illustrating the potential of these technologies for simple, powerful agricultural diagnostics in the field. Furthermore, the assay was just as reliable when implemented in a tropical environment at 31°C as it was when implemented in an air-conditioned lab maintained at 22°C, illustrating the potential value of the technology for field conditions in the tropics and subtropics. PMID:25485176

  3. Interactions with hosts at cool temperatures, not cold tolerance, explain the unique epidemiology of Ralstonia solanacearum race 3 biovar 2.

    PubMed

    Milling, Annett; Meng, Fanhong; Denny, Timothy P; Allen, Caitilyn

    2009-10-01

    Most strains of the bacterial wilt pathogen Ralstonia solanacearum are tropical, but race 3 biovar 2 (R3bv2) strains can attack plants in temperate zones and tropical highlands. The basis of this distinctive ecological trait is not understood. We compared the survival of tropical, R3bv2, and warm-temperate North American strains of R. solanacearum under different conditions. In water at 4 degrees C, North American strains remained culturable the longest (up to 90 days), whereas tropical strains remained culturable for the shortest time (approximately 40 days). However, live/dead staining indicated that cells of representative strains remained viable for >160 days. In contrast, inside potato tubers, R3bv2 strain UW551 survived >4 months at 4 degrees C, whereas North American strain K60 and tropical strain GMI1000 were undetectable after <70 days in tubers. GMI1000 and UW551 grew similarly in minimal medium at 20 and 28 degrees C and, although both strains wilted tomato plants rapidly at 28 degrees C, UW551 was much more virulent at 20 degrees C, killing all inoculated plants under conditions where GMI100 killed just over half. Thus, differences among the strains in the absence of a plant host were not predictive of their behavior in planta at cooler temperatures. These data indicate that interaction with plants is required for expression of the temperate epidemiological trait of R3bv2.

  4. Ralstonia sp. U2 naphthalene dioxygenase and Comamonas sp. JS765 nitrobenzene dioxygenase show differences in activity towards methylated naphthalenes.

    PubMed

    Tøndervik, Anne; Bruheim, Per; Berg, Laila; Ellingsen, Trond E; Kotlar, Hans K; Valla, Svein; Throne-Holst, Mimmi

    2012-02-01

    Methylsubstituted naphthalenes constitute a significant part of light gas oil fractions (LGO). These are toxic compounds with low fuel value, and can potentially be enzymatically modified to increase the fuel value and at the same time reduce toxicity. The first step in the biodegradation of naphthalene involves dioxygenation of the aromatic ring catalysed by naphthalene dioxygenase (NDO). Here we show that recombinantly produced NDO from Ralstonia sp. U2 and the related nitrobenzene dioxygenase (NBDO) from Comamonas sp. JS765 can use several mono-, di-, tri-, and tetramethylated naphthalenes as substrates. For the majority of the substrates both enzymes catalyse the formation of a mixture of mono- and dioxygenated products, and it is only dioxygenated products that are likely to be processed further, leading to ring cleavage. In some cases, like for 1-methylnaphthalene, NDO mainly generates the monooxygenated form, while with NBDO, the dioxygenated form dominates. In other cases, as for 1,4-dimethylnaphthalene, the monooxygenated product dominates with NDO, whereas NBDO generates similar amounts of both forms. Presumably, the best future strategy for bioconversion of methylated naphthalenes in LGO is to develop engineered enzyme that are optimised with respect to the specific composition of naphthalene derivatives found in a given product. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Bacterial wilt resistance in tomato, pepper, and eggplant: genetic resources respond to diverse strains in the Ralstonia solanacearum species complex.

    PubMed

    Lebeau, A; Daunay, M-C; Frary, A; Palloix, A; Wang, J-F; Dintinger, J; Chiroleu, F; Wicker, E; Prior, P

    2011-01-01

    Bacterial wilt, caused by strains belonging to the Ralstonia solanacearum species complex, inflicts severe economic losses in many crops worldwide. Host resistance remains the most effective control strategy against this disease. However, wilt resistance is often overcome due to the considerable variation among pathogen strains. To help breeders circumvent this problem, we assembled a worldwide collection of 30 accessions of tomato, eggplant and pepper (Core-TEP), most of which are commonly used as sources of resistance to R. solanacearum or for mapping quantitative trait loci. The Core-TEP lines were challenged with a core collection of 12 pathogen strains (Core-Rs2) representing the phylogenetic diversity of R. solanacearum. We observed six interaction phenotypes, from highly susceptible to highly resistant. Intermediate phenotypes resulted from the plants' ability to tolerate latent infections (i.e., bacterial colonization of vascular elements with limited or no wilting). The Core-Rs2 strains partitioned into three pathotypes on pepper accessions, five on tomato, and six on eggplant. A "pathoprofile" concept was developed to characterize the strain clusters, which displayed six virulence patterns on the whole set of Core-TEP host accessions. Neither pathotypes nor pathoprofiles were phylotype specific. Pathoprofiles with high aggressiveness were mainly found in strains from phylotypes I, IIB, and III. One pathoprofile included a strain that overcame almost all resistance sources.

  6. Ralstonia solanacearum Dps Contributes to Oxidative Stress Tolerance and to Colonization of and Virulence on Tomato Plants▿

    PubMed Central

    Colburn-Clifford, Jennifer M.; Scherf, Jacob M.; Allen, Caitilyn

    2010-01-01

    Ralstonia solanacearum, an economically important soilborne plant pathogen, infects host roots to cause bacterial wilt disease. However, little is known about this pathogen's behavior in the rhizosphere and early in pathogenesis. In response to root exudates from tomato, R. solanacearum strain UW551 upregulated a gene resembling Dps, a nonspecific DNA binding protein from starved cells that is critical for stress survival in other bacteria. An R. solanacearum dps mutant had increased hydrogen peroxide sensitivity and mutation rate under starvation. Furthermore, dps expression was positively regulated by the oxidative stress response regulator OxyR. These functional results are consistent with a Dps annotation. The dps mutant caused slightly delayed bacterial wilt disease in tomato after a naturalistic soil soak inoculation. However, the dps mutant had a more pronounced reduction in virulence when bacteria were inoculated directly into host stems, suggesting that Dps helps R. solanacearum adapt to conditions inside plants. Passage through a tomato plant conferred transient increased hydrogen peroxide tolerance on both wild-type and dps mutant strains, demonstrating that R. solanacearum acquires Dps-independent oxidative stress tolerance during adaptation to the host environment. The dps mutant strain was also reduced in adhesion to tomato roots and tomato stem colonization. These results indicate that Dps is important when cells are starved or in stationary phase and that Dps contributes quantitatively to host plant colonization and bacterial wilt virulence. They further suggest that R. solanacearum must overcome oxidative stress during the bacterial wilt disease cycle. PMID:20870795

  7. Detection of Ralstonia solanacearum Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay

    PubMed Central

    Weller, S. A.; Elphinstone, J. G.; Smith, N. C.; Boonham, N.; Stead, D. E.

    2000-01-01

    A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to ≥102 cells ml−1 was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum. PMID:10877778

  8. Overexpression of a Chinese cabbage BrERF11 transcription factor enhances disease resistance to Ralstonia solanacearum in tobacco.

    PubMed

    Lai, Yan; Dang, Fengfeng; Lin, Jing; Yu, Lu; Shi, Youliang; Xiao, Yuhua; Huang, Mukun; Lin, Jinhui; Chen, Chengcong; Qi, Aihua; Liu, Zhiqin; Guan, Deyi; Mou, Shaoliang; Qiu, Ailian; He, Shuilin

    2013-01-01

    Ethylene-responsive factors (ERFs) play diverse roles in plant growth, developmental processes and stress responses. However, the roles and underlying mechanism of ERFs remain poorly understood, especially in non-model plants. In this study, a full length cDNA of ERF gene was isolated from the cDNA library of Chinese cabbage. According to sequence alignment, we found a highly conservative AP2/ERF domain, two nuclear localization signals, and an ERF-associated Amphiphilic Repression (EAR) motif in its C-terminal region. It belonged to VIIIa group ERFs sharing the highest sequence identity with AtERF11 in all of the ERFs in Arabidopsis and designated BrERF11. BrERF11-green fluorescence protein (GFP) transient expressed in onion epidermis cells localized to the nucleus. The transcript levels of BrERF11 were induced by exogenous salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), and hydrogen peroxide (H(2)O(2)). Constitutive expression of BrERF11 enhanced tolerance to Ralstonia solanacearum infection in transgenic tobacco plants, which was coupled with hypersensitive response (HR), burst of H(2)O(2) and upregulation of defense-related genes including HR marker genes, SA-, JA-dependent pathogen-related genes and ET biosynthesis associated genes and downregulation of CAT1, suggesting BrERF11 may participate in pathogen-associated molecular pattern (PAMP)- and effector-triggered immunity (PTI and ETI) mediated by SA-, JA- and ET-dependent signaling mechanisms.

  9. Phylogeny and population structure of brown rot- and Moko disease-causing strains of Ralstonia solanacearum phylotype II.

    PubMed

    Cellier, G; Remenant, B; Chiroleu, F; Lefeuvre, P; Prior, P

    2012-04-01

    The ancient soilborne plant vascular pathogen Ralstonia solanacearum has evolved and adapted to cause severe damage in an unusually wide range of plants. In order to better describe and understand these adaptations, strains with very similar lifestyles and host specializations are grouped into ecotypes. We used comparative genomic hybridization (CGH) to investigate three particular ecotypes in the American phylotype II group: (i) brown rot strains from phylotypes IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from phylotype IIB-4NPB that lack pathogenicity for banana but can infect many other plant species; and (iii) Moko disease-causing strains from phylotypes IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia spp. We compared the genomes of 72 R. solanacearum strains, mainly from the three major ecotypes of phylotype II, using a newly developed pangenomic microarray to decipher their population structure and gain clues about the epidemiology of these ecotypes. Strain phylogeny and population structure were reconstructed. The results revealed a phylogeographic structure within brown rot strains, allowing us to distinguish European outbreak strains of Andean and African origins. The pangenomic CGH data also demonstrated that Moko ecotype IIB-4 is phylogenetically distinct from the emerging IIB-4NPB strains. These findings improved our understanding of the epidemiology of important ecotypes in phylotype II and will be useful for evolutionary analyses and the development of new DNA-based diagnostic tools.

  10. Complete factorial design to adjust pH and sugar concentrations in the inoculum phase of Ralstonia solanacearum to optimize P(3HB) production

    PubMed Central

    Alves, Mariane Igansi; Rodrigues, Amanda Ávila; Furlan, Lígia; da Silva Rodrigues, Rosane; Diaz de Oliveira, Patrícia; Vendruscolo, Claire Tondo; da Silveira Moreira, Angelita

    2017-01-01

    Poly(3-hydroxybutyrate) (P(3HB)) is a biodegradable plastic biopolymer that accumulates as lipophilic inclusions in the cytoplasm of some microorganisms. The biotechnological process by which P(3HB) is synthesized occurs in two phases. The first phase involves cell growth in a complex culture medium, while the second phase involves polymer accumulation in the presence of excess carbon sources. As such, the efficiency of the second phase depends on the first phase. The aim of this study was to evaluate culture media with different concentrations of sucrose and glucose and different pH values in the inoculum phase of Ralstonia solanacearum RS with the intention of identifying methods by which the biomass yield could be increased, subsequently enhancing the yield of P(3HB). The culture medium was formulated according to the experimental planning type of central composite rotational design 22. The independent variables were pH and sugar concentration (sucrose and glucose), and the dependent variables were OD600nm, dry cell weight (DCW), and P(3HB) yield. The highest cell growth, estimated by the OD600nm (20.6) and DCW (5.35) values, was obtained when sucrose was used in the culture medium at a concentration above 35 g.L-1 in combination with an acidic pH. High polymer (45%) accumulation was also achieved under these conditions. Using glucose, the best results for OD600nm (12.5) and DCW (2.74) were also obtained at acidic pH but with a sugar concentration at the minimum values evaluated. Due to the significant accumulation of polymer in the cells that were still in the growth phase, the accumulating microorganism P(3HB) Ralstonia solanacearum RS can be classified as having type II metabolism in relation to the polymer accumulation phase, which is different from other Ralstonia spp. studied until this time. PMID:28704411

  11. Ralstonia solanacearum uses inorganic nitrogen metabolism for virulence, ATP production, and detoxification in the oxygen-limited host xylem environment.

    PubMed

    Dalsing, Beth L; Truchon, Alicia N; Gonzalez-Orta, Enid T; Milling, Annett S; Allen, Caitilyn

    2015-03-17

    Genomic data predict that, in addition to oxygen, the bacterial plant pathogen Ralstonia solanacearum can use nitrate (NO3(-)), nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. solanacearum. Xylem fluid contained ~25 mM NO3(-), corresponding to R. solanacearum's optimal NO3(-) concentration for anaerobic growth in vitro. We tested the hypothesis that R. solanacearum uses inorganic nitrogen species to respire and grow during pathogenesis by making deletion mutants that each lacked a step in nitrate respiration (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). The ΔnarG, ΔaniA, and ΔnorB mutants grew poorly on NO3(-) compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO3(-) respiration directly enhanced growth, AniA-dependent NO2(-) reduction did not. NO2(-) and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO3(-) acts as a TEA, but the resulting NO2(-) and NO likely do not. None of the mutants grew as well as the wild type in planta, and strains lacking AniA (NO2(-) reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO3(-) to respire, grow, and cause disease. Degradation of NO2(-) and NO is also important for successful infection and depends on denitrification and NO detoxification systems. The plant-pathogenic bacterium Ralstonia solanacearum causes bacterial wilt, one of the world's most destructive crop diseases. This pathogen's explosive growth in plant vascular xylem is poorly understood. We used biochemical and genetic approaches to show

  12. The in planta transcriptome of Ralstonia solanacearum: conserved physiological and virulence strategies during bacterial wilt of tomato.

    PubMed

    Jacobs, Jonathan M; Babujee, Lavanya; Meng, Fanhong; Milling, Annett; Allen, Caitilyn

    2012-01-01

    Plant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogen Ralstonia solanacearum is well adapted to it, growing to 10(8) to 10(9) CFU/g tomato stem. To better understand how R. solanacearum succeeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinct R. solanacearum strains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 10(8) CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly altered in planta versus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in the scrABY cluster. A UW551 scrA mutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed host Solanum dulcamara. Functional scrA contributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose. scrA expression was induced by sucrose, but to a much greater degree by growth in planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulated in planta at high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle. IMPORTANCE Ralstonia solanacearum is a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical

  13. Effects of ecological factors on the survival and physiology of Ralstonia solanacearum bv. 2 in irrigation water.

    PubMed

    van Elsas, J D; Kastelein, P; de Vries, P M; van Overbeek, L S

    2001-09-01

    The fate of Ralstonia solanacearum bv. 2, the causative agent of brown rot in potato, in aquatic habitats of temperate climate regions is still poorly understood. In this study, the population dynamics and the physiological response of R. solanacearum bv. 2 were tested in sterile pure water and in agricultural drainage water obtained from waterways near potato cropping fields in The Netherlands. The behaviour of five different biovar 2 isolates in drainage water at 20 degrees C was very similar among strains. One typical isolate with consistent virulence (strain 1609) was selected for further studies. The effects of temperature, light, canal sediment, seawater salts, and the presence of competing microorganisms on the survival of strain 1609 were assessed. Moreover, the impacts of the physiological state of the inoculum and the inoculum density were analyzed. The population dynamics of strain 1609 in sterile pure water were also characterized. In sterile pure water, the fate of R. solanacearum 1609 cells depended strongly on temperature, irrespective of inoculum density or physiological state. At 4 degrees C and 44 degrees C, strain 1609 CFU numbers showed declines, whereas the strain was able to undergo several cell divisions at 12 degrees C, 20 degrees C, and 28 degrees C. At 20 degrees C and 28 degrees C, repeated growth took place when the organism was serially transferred, at low inoculum density, from grown water cultures into fresh water devoid of nutrients. Both at low and high cell densities and regardless of physiological state, R. solanacearum 1609 cells persisted as culturable cells for limited periods of time in drainage water. A major effect of temperature was found, with survival being maximal at 12 degrees C, 20 degrees C, and 28 degrees C. Temperatures of 4 degrees C, 36 degrees C, or 44 degrees C induced accelerated declines of the culturable cell numbers. The drainage water biota had a strong effect on survival at 12 degrees C, 20 degrees C, and

  14. A Single Regulator Mediates Strategic Switching between Attachment/Spread and Growth/Virulence in the Plant Pathogen Ralstonia solanacearum

    PubMed Central

    Khokhani, Devanshi; Lowe-Power, Tiffany M.; Tran, Tuan Minh

    2017-01-01

    ABSTRACT The PhcA virulence regulator in the vascular wilt pathogen Ralstonia solanacearum responds to cell density via quorum sensing. To understand the timing of traits that enable R. solanacearum to establish itself inside host plants, we created a ΔphcA mutant that is genetically locked in a low-cell-density condition. Comparing levels of gene expression of wild-type R. solanacearum and the ΔphcA mutant during tomato colonization revealed that the PhcA transcriptome includes an impressive 620 genes (>2-fold differentially expressed; false-discovery rate [FDR], ≤0.005). Many core metabolic pathways and nutrient transporters were upregulated in the ΔphcA mutant, which grew faster than the wild-type strain in tomato xylem sap and on dozens of specific metabolites, including 36 found in xylem. This suggests that PhcA helps R. solanacearum to survive in nutrient-poor environmental habitats and to grow rapidly during early pathogenesis. However, after R. solanacearum reaches high cell densities in planta, PhcA mediates a trade-off from maximizing growth to producing costly virulence factors. R. solanacearum infects through roots, and low-cell-density-mode-mimicking ΔphcA cells attached to tomato roots better than the wild-type cells, consistent with their increased expression of several adhesins. Inside xylem vessels, ΔphcA cells formed aberrantly dense mats. Possibly as a result, the mutant could not spread up or down tomato stems as well as the wild type. This suggests that aggregating improves R. solanacearum survival in soil and facilitates infection and that it reduces pathogenic fitness later in disease. Thus, PhcA mediates a second strategic switch between initial pathogen attachment and subsequent dispersal inside the host. PhcA helps R. solanacearum optimally invest resources and correctly sequence multiple steps in the bacterial wilt disease cycle. PMID:28951474

  15. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum

    PubMed Central

    Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

    2016-01-01

    Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease. PMID:27336156

  16. Differential occurrence of oxidative burst and antioxidative mechanism in compatible and incompatible interactions of Solanum lycopersicum and Ralstonia solanacearum.

    PubMed

    Mandal, Sudhamoy; Das, Rupa Kumar; Mishra, Sanjeev

    2011-02-01

    Striking increase in reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) has been demonstrated to occur in plants in response to pathogen attack. The aim of this study was to investigate the biochemical aspects of ROS generation, antioxidative mechanism and cell wall reinforcement as responses of tomato cultivars Arka Meghali (AM; susceptible) and BT-10 (BT; resistant) against Ralstonia solanacearum (Ralsol). While the oxidative burst was characterized by a single phase ROS increase in AM, there was a clear bi-phasic ROS generation in BT. The first significant increase of H(2)O(2) production was noticed at 12 h post-inoculation (hpi) followed by a sharp increase in H(2)O(2) generation after 36 hpi. Lipid peroxidation was more in roots of AM than that of BT after pathogen inoculation. Superoxide dismutase and catalase activities were continuously at very high level in Ralsol-inoculated BT plants, whereas activities of the enzymes were observed to decrease at later stage in Ralsol-inoculated AM plants. Guaiacol peroxidase activity was high in Ralsol-inoculated roots of both cultivars, but BT recorded much higher activity than AM. Higher activity of ascorbate peroxidase in inoculated BT might be an indication of better scavenging activity of the enzyme. Total phenolic content and lignin deposition were significantly higher in Ralsol-inoculated BT compared to inoculated AM. Our results indicate that increased level of ROS production coupled with more efficient antioxidative system, lower rate of lipid peroxidation and high lignin deposition in cell wall may contribute to the resistance of tomato plants to Ralsol.

  17. Ralstonia solanacearum Uses Inorganic Nitrogen Metabolism for Virulence, ATP Production, and Detoxification in the Oxygen-Limited Host Xylem Environment

    PubMed Central

    Dalsing, Beth L.; Truchon, Alicia N.; Gonzalez-Orta, Enid T.; Milling, Annett S.

    2015-01-01

    ABSTRACT Genomic data predict that, in addition to oxygen, the bacterial plant pathogen Ralstonia solanacearum can use nitrate (NO3−), nitrite (NO2−), nitric oxide (NO), and nitrous oxide (N2O) as terminal electron acceptors (TEAs). Genes encoding inorganic nitrogen reduction were highly expressed during tomato bacterial wilt disease, when the pathogen grows in xylem vessels. Direct measurements found that tomato xylem fluid was low in oxygen, especially in plants infected by R. solanacearum. Xylem fluid contained ~25 mM NO3−, corresponding to R. solanacearum’s optimal NO3− concentration for anaerobic growth in vitro. We tested the hypothesis that R. solanacearum uses inorganic nitrogen species to respire and grow during pathogenesis by making deletion mutants that each lacked a step in nitrate respiration (ΔnarG), denitrification (ΔaniA, ΔnorB, and ΔnosZ), or NO detoxification (ΔhmpX). The ΔnarG, ΔaniA, and ΔnorB mutants grew poorly on NO3− compared to the wild type, and they had reduced adenylate energy charge levels under anaerobiosis. While NarG-dependent NO3− respiration directly enhanced growth, AniA-dependent NO2− reduction did not. NO2− and NO inhibited growth in culture, and their removal depended on denitrification and NO detoxification. Thus, NO3− acts as a TEA, but the resulting NO2− and NO likely do not. None of the mutants grew as well as the wild type in planta, and strains lacking AniA (NO2− reductase) or HmpX (NO detoxification) had reduced virulence on tomato. Thus, R. solanacearum exploits host NO3− to respire, grow, and cause disease. Degradation of NO2− and NO is also important for successful infection and depends on denitrification and NO detoxification systems. PMID:25784703

  18. Necessity of OxyR for the Hydrogen Peroxide Stress Response and Full Virulence in Ralstonia solanacearum ▿ †

    PubMed Central

    Flores-Cruz, Zomary; Allen, Caitilyn

    2011-01-01

    The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence. PMID:21803891

  19. Swimming motility, a virulence trait of Ralstonia solanacearum, is regulated by FlhDC and the plant host environment.

    PubMed

    Tans-Kersten, Julie; Brown, Darby; Allen, Caitilyn

    2004-06-01

    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.

  20. The Ectopic Expression of CaRop1 Modulates the Response of Tobacco Plants to Ralstonia solanacearum and Aphids

    PubMed Central

    Qiu, Ailian; Liu, Zhiqin; Li, Jiazhi; Chen, Yanshen; Guan, Deyi; He, Shuilin

    2016-01-01

    In plants, Rho-related GTPases (Rops) are versatile molecular switches that regulate various biological processes, although their exact roles are not fully understood. Herein, we provide evidence that the ectopic expression of a Rop derived from Capsicum annuum, designated CaRop1, in tobacco plants modulates the response of these plants to Ralstonia solanacearum or aphid attack. The deduced amino acid sequence of CaRop1 harbors a conserved Rho domain and is highly homologous to Rops of other plant species. Transient expression of a CaRop1-GFP fusion protein in Nicotiana benthamiana leaf epidermal cells revealed localization of the GFP signal to the plasma membrane, cytoplasm, and nucleus. Overexpression (OE) of the wild-type CaRop1 or its dominant-negative mutant (DN-CaRop1) conferred substantial resistance to R. solanacearum infection and aphid attack, and this effect was accompanied by enhanced transcriptional expression of the hypersensitive-reaction marker gene HSR201; the jasmonic acid (JA)-responsive PR1b and LOX1; the insect resistance-associated NtPI-I, NtPI-II, and NtTPI; the ethylene (ET) production-associated NtACS1; and NPK1, a mitogen-activated protein kinase kinase kinase (MAPKKK) that interferes with N-, Bs2-, and Rx-mediated disease resistance. In contrast, OE of the constitutively active mutant of CaRop1(CA-CaRop1) enhanced susceptibility of the transgenic tobacco plants to R. solanacearum infection and aphid attack and downregulated or sustained the expression of HSR201, PR1b, NPK1, NtACS1, NtPI-I, NtPI-II, and NtTPI. These results collectively suggest that CaRop1 acts as a signaling switch in the crosstalk between Solanaceaes’s response to R. solanacearum infection and aphid attack possibly via JA/ET-mediated signaling machinery. PMID:27551287

  1. Necessity of OxyR for the hydrogen peroxide stress response and full virulence in Ralstonia solanacearum.

    PubMed

    Flores-Cruz, Zomary; Allen, Caitilyn

    2011-09-01

    The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence.

  2. The In Planta Transcriptome of Ralstonia solanacearum: Conserved Physiological and Virulence Strategies during Bacterial Wilt of Tomato

    PubMed Central

    Jacobs, Jonathan M.; Babujee, Lavanya; Meng, Fanhong; Milling, Annett; Allen, Caitilyn

    2012-01-01

    ABSTRACT Plant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogen Ralstonia solanacearum is well adapted to it, growing to 108 to 109 CFU/g tomato stem. To better understand how R. solanacearum succeeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinct R. solanacearum strains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 108 CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly altered in planta versus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in the scrABY cluster. A UW551 scrA mutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed host Solanum dulcamara. Functional scrA contributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose. scrA expression was induced by sucrose, but to a much greater degree by growth in planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulated in planta at high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle. PMID:22807564

  3. Functional assignment to positively selected sites in the core type III effector RipG7 from Ralstonia solanacearum.

    PubMed

    Wang, Keke; Remigi, Philippe; Anisimova, Maria; Lonjon, Fabien; Kars, Ilona; Kajava, Andrey; Li, Chien-Hui; Cheng, Chiu-Ping; Vailleau, Fabienne; Genin, Stéphane; Peeters, Nemo

    2016-05-01

    The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The main virulence determinants of R. solanacearum are the type III secretion system (T3SS) and its associated type III effectors (T3Es), translocated into the host cells. Of the conserved T3Es among R. solanacearum strains, the Fbox protein RipG7 is required for R. solanacearum pathogenesis on Medicago truncatula. In this work, we describe the natural ripG7 variability existing in the R. solanacearum species complex. We show that eight representative ripG7 orthologues have different contributions to pathogenicity on M. truncatula: only ripG7 from Asian or African strains can complement the absence of ripG7 in GMI1000 (Asian reference strain). Nonetheless, RipG7 proteins from American and Indonesian strains can still interact with M. truncatula SKP1-like/MSKa protein, essential for the function of RipG7 in virulence. This indicates that the absence of complementation is most likely a result of the variability in the leucine-rich repeat (LRR) domain of RipG7. We identified 11 sites under positive selection in the LRR domains of RipG7. By studying the functional impact of these 11 sites, we show the contribution of five positively selected sites for the function of RipG7CMR15 in M. truncatula colonization. This work reveals the genetic and functional variation of the essential core T3E RipG7 from R. solanacearum. This analysis is the first of its kind on an essential disease-controlling T3E, and sheds light on the co-evolutionary arms race between the bacterium and its hosts. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  4. Escaping Underground Nets: Extracellular DNases Degrade Plant Extracellular Traps and Contribute to Virulence of the Plant Pathogenic Bacterium Ralstonia solanacearum.

    PubMed

    Tran, Tuan Minh; MacIntyre, April; Hawes, Martha; Allen, Caitilyn

    2016-06-01

    Plant root border cells have been recently recognized as an important physical defense against soil-borne pathogens. Root border cells produce an extracellular matrix of protein, polysaccharide and DNA that functions like animal neutrophil extracellular traps to immobilize pathogens. Exposing pea root border cells to the root-infecting bacterial wilt pathogen Ralstonia solanacearum triggered release of DNA-containing extracellular traps in a flagellin-dependent manner. These traps rapidly immobilized the pathogen and killed some cells, but most of the entangled bacteria eventually escaped. The R. solanacearum genome encodes two putative extracellular DNases (exDNases) that are expressed during pathogenesis, suggesting that these exDNases contribute to bacterial virulence by enabling the bacterium to degrade and escape root border cell traps. We tested this hypothesis with R. solanacearum deletion mutants lacking one or both of these nucleases, named NucA and NucB. Functional studies with purified proteins revealed that NucA and NucB are non-specific endonucleases and that NucA is membrane-associated and cation-dependent. Single ΔnucA and ΔnucB mutants and the ΔnucA/B double mutant all had reduced virulence on wilt-susceptible tomato plants in a naturalistic soil-soak inoculation assay. The ΔnucA/B mutant was out-competed by the wild-type strain in planta and was less able to stunt root growth or colonize plant stems. Further, the double nuclease mutant could not escape from root border cells in vitro and was defective in attachment to pea roots. Taken together, these results demonstrate that extracellular DNases are novel virulence factors that help R. solanacearum successfully overcome plant defenses to infect plant roots and cause bacterial wilt disease.

  5. Effect of seed treatment by cold plasma on the resistance of tomato to Ralstonia solanacearum (Bacterial Wilt).

    PubMed

    Jiang, Jiafeng; Lu, Yufang; Li, Jiangang; Li, Ling; He, Xin; Shao, Hanliang; Dong, Yuanhua

    2014-01-01

    This study investigated the effect of cold plasma seed treatment on tomato bacterial wilt, caused by Ralstonia solanacearum (R. solanacearum), and the regulation of resistance mechanisms. The effect of cold plasma of 80W on seed germination, plant growth, nutrient uptake, disease severity, hydrogen peroxide (H2O2) concentration and activities of peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.2) and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) were examined in tomato plants. Plasma treatment increased tomato resistance to R. solanacearum with an efficacy of 25.0%. Plasma treatment significantly increased both germination and plant growth in comparison with the control treatment, and plasma-treated plants absorbed more calcium and boron than the controls. In addition, H2O2 levels in treated plants rose faster and reached a higher peak, at 2.579 µM gFW-1, 140% greater than that of the control. Activities of POD (421.3 U gFW-1), PPO (508.8 U gFW-1) and PAL (707.3 U gFW-1) were also greater in the treated plants than in the controls (103.0 U gFW-1, 166.0 U gFW-1 and 309.4 U gFW-1, respectively). These results suggest that plasma treatment affects the regulation of plant growth, H2O2 concentration, and POD, PPO and PAL activity in tomato, resulting in an improved resistance to R. solanacearum. Consequently, cold plasma seed treatment has the potential to control tomato bacterial wilt caused by R. solanacearum.

  6. Effect of Seed Treatment by Cold Plasma on the Resistance of Tomato to Ralstonia solanacearum (Bacterial Wilt)

    PubMed Central

    Jiang, Jiafeng; Lu, Yufang; Li, Jiangang; Li, Ling; He, Xin; Shao, Hanliang; Dong, Yuanhua

    2014-01-01

    This study investigated the effect of cold plasma seed treatment on tomato bacterial wilt, caused by Ralstonia solanacearum (R. solanacearum), and the regulation of resistance mechanisms. The effect of cold plasma of 80W on seed germination, plant growth, nutrient uptake, disease severity, hydrogen peroxide (H2O2) concentration and activities of peroxidase (POD; EC 1.11.1.7), polyphenol oxidase (PPO; EC 1.10.3.2) and phenylalanine ammonia lyase (PAL; EC 4.3.1.5) were examined in tomato plants. Plasma treatment increased tomato resistance to R. solanacearum with an efficacy of 25.0%. Plasma treatment significantly increased both germination and plant growth in comparison with the control treatment, and plasma-treated plants absorbed more calcium and boron than the controls. In addition, H2O2 levels in treated plants rose faster and reached a higher peak, at 2.579 µM gFW−1, 140% greater than that of the control. Activities of POD (421.3 U gFW−1), PPO (508.8 U gFW−1) and PAL (707.3 U gFW−1) were also greater in the treated plants than in the controls (103.0 U gFW−1, 166.0 U gFW−1 and 309.4 U gFW−1, respectively). These results suggest that plasma treatment affects the regulation of plant growth, H2O2 concentration, and POD, PPO and PAL activity in tomato, resulting in an improved resistance to R. solanacearum. Consequently, cold plasma seed treatment has the potential to control tomato bacterial wilt caused by R. solanacearum. PMID:24840508

  7. Degradation of the Plant Defense Signal Salicylic Acid Protects Ralstonia solanacearum from Toxicity and Enhances Virulence on Tobacco

    PubMed Central

    Lowe-Power, Tiffany M.; Jacobs, Jonathan M.; Ailloud, Florent; Fochs, Brianna; Prior, Philippe

    2016-01-01

    ABSTRACT Plants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum. SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule. PMID:27329752

  8. An exo-poly-alpha-D-galacturonosidase, PehB, is required for wild-type virulence of Ralstonia solanacearum.

    PubMed Central

    Huang, Q; Allen, C

    1997-01-01

    Ralstonia solanacearum, which causes bacterial wilt disease of many plant species, produces several extracellular plant cell wall-degrading enzymes that are suspected virulence factors. These include a previously described endopolygalacturonase (PG), PehA, and two exo-PGs. A gene encoding one of the exo-PGs, pehB, was cloned from R. solanacearum K60. The DNA fragment specifying PehB contained a 2,103-bp open reading frame that encodes a protein of 74.2 kDa with a typical N-terminal signal sequence. The cloned pehB gene product cleaves polygalacturonic acid into digalacturonic acid units. The amino acid sequence of pehB resembles that of pehX, an exo-PG gene from Erwinia chrysanthemi, with 47.2% identity at the amino acid level. PehB also has limited similarity to plant exo-PGs from Zea mays and Arabidopsis thaliana. The chromosomal pehB genes in R. solanacearum wild-type strain K60 and in an endo-PG PehA- strain were replaced with an insertionally inactivated copy of pehB. The resulting mutants were deficient in the production of PehB and of both PehA and PehB, respectively. The pehB mutant was significantly less virulent than the wild-type strain in eggplant virulence assays using a soil inoculation method. However, the pehA mutant was even less virulent, and the pehA pehB double mutant was the least virulent of all. These results suggest that PehB is required for a wild-type level of virulence in R. solanacearum although its individual role in wilt disease development may be minor. Together with endo-PG PehA, however, PehB contributes substantially to the virulence of R. solanacearum. PMID:9393701

  9. S-Glycoprotein-Like Protein Regulates Defense Responses in Nicotiana Plants against Ralstonia solanacearum1[C][W

    PubMed Central

    Maimbo, Milimo; Ohnishi, Kouhei; Hikichi, Yasufumi; Yoshioka, Hirofumi; Kiba, Akinori

    2010-01-01

    RsRGA4 (for Ralstonia solanacearum-responsive gene A4) encodes a polypeptide similar to S-locus glycoprotein (SGP) from Brassica rapa and SGP-like proteins from Ipomoea trifida and Medicago truncatula. Therefore, we designated RsRGA4 as NtSGLP (for Nicotiana tabacum SGP-like protein) and NbSGLP (its Nicotiana benthamiana ortholog). NbSGLP is expressed in root, leaf, petal, gynoecium, and stamen. Expression of NbSGLP was strongly induced by inoculation with an avirulent strain of R. solanacearum (Rs8107) and slightly enhanced by inoculation with virulent R. solanacearum (RsOE1-1). Expression of NbSGLP was induced by inoculation with an hrpY-deficient mutant of RsOE1-1 and Rs8107. Expression was also induced by aminocyclopropane carboxylic acid and salicylic acid. Virus-induced gene silencing of NbSGLP enhanced the growth of Rs8107. Growth of RsOE1-1 and appearance of wilt symptoms were also accelerated in silenced plants. Expression of PR-1a and EREBP was reduced, and markers for basal defense, such as callose deposition and reduced vascular flow, were compromised in NbSGLP-silenced plants. Moreover, growth of Pseudomonas cichorii, Pseudomonas syringae pv tabaci, and P. syringae pv mellea was also enhanced in the silenced plants. On the other hand, silencing of NbSGLP did not interfere with the appearance of the hypersensitive response. NbSGLP was secreted in a signal peptide-dependent manner. Agrobacterium tumefaciens-mediated expression of NbSGLP induced PR-1a and EREBP expression, callose deposition, and reduced vascular flow. NbSGLP-induced callose deposition and reduced vascular flow were not observed in salicylic acid-deficient N. benthamiana NahG plants. Taken together, SGLP might have a role in the induction of basal defense in Nicotiana plants. PMID:20118275

  10. Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles.

    PubMed

    Remenant, Benoît; de Cambiaire, Jean-Charles; Cellier, Gilles; Jacobs, Jonathan M; Mangenot, Sophie; Barbe, Valérie; Lajus, Aurélie; Vallenet, David; Medigue, Claudine; Fegan, Mark; Allen, Caitilyn; Prior, Philippe

    2011-01-01

    The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical

  11. Ralstonia syzygii, the Blood Disease Bacterium and Some Asian R. solanacearum Strains Form a Single Genomic Species Despite Divergent Lifestyles

    PubMed Central

    Cellier, Gilles; Jacobs, Jonathan M.; Mangenot, Sophie; Barbe, Valérie; Lajus, Aurélie; Vallenet, David; Medigue, Claudine; Fegan, Mark; Allen, Caitilyn; Prior, Philippe

    2011-01-01

    The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical

  12. Significant Effects Due to Peptone in Kelman Medium on Colony Characteristics and Virulence of Ralstonia solanacearum in Tomato

    PubMed Central

    Thomas, Pious; Upreti, Reshmi

    2014-01-01

    The study was taken up to assess if the media constituents played any role in governing the variable colony characteristics or pathogenicity of the bacterial wilt pathogen, Ralstonia solanacearum cultured on the widely employed Kelman medium. The effects due to the constituents 2,3,5-triphenyl tetrazolium chloride (TTC), peptone, casein hydrolysate and glucose on colony characteristics were investigated using -80°C stored culture of strain ‘NH-Av01’ (race 1, biovar 3) isolated from tomato. Comparing the pigment inducing TTC from two brands, its source or mode of storage/incorporation did not impart any significant effects. The source of peptone, on the other hand, displayed striking effects on the extent of colony growth, fluidity and red pigmentation depending on type, brand or batch / lot of manufacture as documented with 20 different formulations. Significant differences in the pathogenicity of isolate derived from different peptone sources in seedling-challenge assay on tomato were observed. The observations on peptone effects were endorsed with four other isolates belonging to distinct geographic locations, crops (eggplant, chilli, ginger) or races (race 1 or 4). The peptone source did not influence the pathogen-responses in biovar tests but notably altered the pattern of lawn formation and inhibition zone development during antagonistic assays. Casein hydrolysate displayed some variable effects while glucose source had no effect. This study brings to light the significant modifying effects by the peptone-constituent in Kelman medium on the physiology of R. solanacearum and the virulence of isolate and the need to consider the source of media components during culture maintenance, host-pathogen interaction studies or microbe-microbe interaction investigations. PMID:25408775

  13. Repertoire, unified nomenclature and evolution of the Type III effector gene set in the Ralstonia solanacearum species complex

    PubMed Central

    2013-01-01

    Background Ralstonia solanacearum is a soil-borne beta-proteobacterium that causes bacterial wilt disease in many food crops and is a major problem for agriculture in intertropical regions. R. solanacearum is a heterogeneous species, both phenotypically and genetically, and is considered as a species complex. Pathogenicity of R. solanacearum relies on the Type III secretion system that injects Type III effector (T3E) proteins into plant cells. T3E collectively perturb host cell processes and modulate plant immunity to enable bacterial infection. Results We provide the catalogue of T3E in the R. solanacearum species complex, as well as candidates in newly sequenced strains. 94 T3E orthologous groups were defined on phylogenetic bases and ordered using a uniform nomenclature. This curated T3E catalog is available on a public website and a bioinformatic pipeline has been designed to rapidly predict T3E genes in newly sequenced strains. Systematical analyses were performed to detect lateral T3E gene transfer events and identify T3E genes under positive selection. Our analyses also pinpoint the RipF translocon proteins as major discriminating determinants among the phylogenetic lineages. Conclusions Establishment of T3E repertoires in strains representatives of the R. solanacearum biodiversity allowed determining a set of 22 T3E present in all the strains but provided no clues on host specificity determinants. The definition of a standardized nomenclature and the optimization of predictive tools will pave the way to understanding how variation of these repertoires is correlated to the diversification of this species complex and how they contribute to the different strain pathotypes. PMID:24314259

  14. Repertoire, unified nomenclature and evolution of the Type III effector gene set in the Ralstonia solanacearum species complex.

    PubMed

    Peeters, Nemo; Carrère, Sébastien; Anisimova, Maria; Plener, Laure; Cazalé, Anne-Claire; Genin, Stephane

    2013-12-06

    Ralstonia solanacearum is a soil-borne beta-proteobacterium that causes bacterial wilt disease in many food crops and is a major problem for agriculture in intertropical regions. R. solanacearum is a heterogeneous species, both phenotypically and genetically, and is considered as a species complex. Pathogenicity of R. solanacearum relies on the Type III secretion system that injects Type III effector (T3E) proteins into plant cells. T3E collectively perturb host cell processes and modulate plant immunity to enable bacterial infection. We provide the catalogue of T3E in the R. solanacearum species complex, as well as candidates in newly sequenced strains. 94 T3E orthologous groups were defined on phylogenetic bases and ordered using a uniform nomenclature. This curated T3E catalog is available on a public website and a bioinformatic pipeline has been designed to rapidly predict T3E genes in newly sequenced strains. Systematical analyses were performed to detect lateral T3E gene transfer events and identify T3E genes under positive selection. Our analyses also pinpoint the RipF translocon proteins as major discriminating determinants among the phylogenetic lineages. Establishment of T3E repertoires in strains representatives of the R. solanacearum biodiversity allowed determining a set of 22 T3E present in all the strains but provided no clues on host specificity determinants. The definition of a standardized nomenclature and the optimization of predictive tools will pave the way to understanding how variation of these repertoires is correlated to the diversification of this species complex and how they contribute to the different strain pathotypes.

  15. Genomic Characterization of the Filamentous Integrative Bacteriophages φRSS1 and φRSM1, Which Infect Ralstonia solanacearum▿

    PubMed Central

    Kawasaki, Takeru; Nagata, Shoko; Fujiwara, Akiko; Satsuma, Hideki; Fujie, Makoto; Usami, Shoji; Yamada, Takashi

    2007-01-01

    The genomic DNA sequences were determined for two filamentous integrative bacteriophages, φRSS1 and φRSM1, of the phytopathogen Ralstonia solanacearum. The 6,662-base sequence of φRSS1 contained 11 open reading frames (ORFs). In the databases, this sequence showed high homology (95% identity) to the circular double-stranded DNA plasmid pJTPS1 (6,633 bp) isolated from a spontaneously occurring avirulent mutant of R. solanacearum. Two major differences between the two sequences were observed within φRSS1 ORF7, corresponding to pIII, a minor coat protein required for host adsorption, and at the φRSS1 intergenic (IG) region. The 9,004-base sequence of φRSM1 showed 12 ORFs located on the same strand (plus strand) and 2 ORFs on the opposite strand. Compared with Ff-type phages, two insertions are obvious in the φRSM1 replication module. Genomic DNA fragments containing the φRSM integration junctions were cloned and sequenced from φRSM lysogenic strain R. solanacearum MAFF211270. The att core sequence was identified as 5′-TGGCGGAGAGGGT-3′, corresponding to the 3′ end of the serine tRNA (UCG) gene. Interestingly, ORF14, located next to the attP site on the φRSM1 genome, showed high amino acid sequence homology with bacterial DNA recombinases and resolvases, different from XerCD recombinases. attP of φRSS1 is within a sequence element of the IG region. PMID:17557818

  16. Chemical forms of selenium in the metal-resistant bacterium Ralstonia metallidurans CH34 exposed to selenite and selenate.

    PubMed

    Sarret, Géraldine; Avoscan, Laure; Carrière, Marie; Collins, Richard; Geoffroy, Nicolas; Carrot, Francine; Covès, Jacques; Gouget, Barbara

    2005-05-01

    Ralstonia metallidurans CH34, a soil bacterium resistant to a variety of metals, is known to reduce selenite to intracellular granules of elemental selenium (Se(0)). We have studied the kinetics of selenite (Se(IV)) and selenate (Se(VI)) accumulation and used X-ray absorption spectroscopy to identify the accumulated form of selenate, as well as possible chemical intermediates during the transformation of these two oxyanions. When introduced during the lag phase, the presence of selenite increased the duration of this phase, as previously observed. Selenite introduction was followed by a period of slow uptake, during which the bacteria contained Se(0) and alkyl selenide in equivalent proportions. This suggests that two reactions with similar kinetics take place: an assimilatory pathway leading to alkyl selenide and a slow detoxification pathway leading to Se(0). Subsequently, selenite uptake strongly increased (up to 340 mg Se per g of proteins) and Se(0) was the predominant transformation product, suggesting an activation of selenite transport and reduction systems after several hours of contact. Exposure to selenate did not induce an increase in the lag phase duration, and the bacteria accumulated approximately 25-fold less Se than when exposed to selenite. Se(IV) was detected as a transient species in the first 12 h after selenate introduction, Se(0) also occurred as a minor species, and the major accumulated form was alkyl selenide. Thus, in the present experimental conditions, selenate mostly follows an assimilatory pathway and the reduction pathway is not activated upon selenate exposure. These results show that R. metallidurans CH34 may be suitable for the remediation of selenite-, but not selenate-, contaminated environments.

  17. Effects of compost addition and simulated solarisation on the fate of Ralstonia solanacearum biovar 2 and indigenous bacteria in soil.

    PubMed

    Schönfeld, J; Gelsomino, A; Overbeek, L S; Gorissen, A; Smalla, K; Elsas, J D

    2003-02-01

    Abstract The effects of compost addition and simulated solarisation of soil on the survival of Ralstonia solanacearum biovar 2 strain 1609, as well as on the structure of indigenous soil bacterial communities, were analysed. In addition, effects on the invasion of susceptible test plants by strain 1609 were assessed. In untreated soil in microcosms and the field, strain 1609 showed slow progressive declines, from 10(6)-10(7) to roughly 10(4)-10(5) CFU per g dry soil in around 60 days. When these soils were used in suppressiveness tests, a majority of plants developed symptoms of wilting and revealed the presence of the pathogen in their lower stem parts, as evidenced by immunofluorescence colony staining (IFC) and polymerase chain reaction (PCR). Solarisation of unamended soil did not drastically affect R. solanacearum survival or plant invasiveness. However, the addition of household compost resulted in enhanced R. solanacearum population decline rates, as well as reduced numbers of diseased plants in suppressiveness tests. Combined solarisation and compost addition yielded differential results between microcosms and the field. Some healthy-looking plants, primarily from soils treated with compost, revealed the latent presence of strain 1609 in the lower stem parts. The eubacterial and beta-subgroup proteobacterial communities in the differentially treated soil microcosms were rather stable, as evidenced by analysis of PCR-denaturing gradient gel electrophoresis (DGGE) generated molecular profiles. However, compost amendment clearly induced changes in these communities, which were detectable until the end of the experiment; two major bands, affiliated with Variovorax paradoxus and Aquaspirillum psychrophylum, were associated with the compost amendment. The decrease in abundance of R. solanacearum in the compost-amended soils was confirmed by the DGGE profiles.

  18. Class Size.

    ERIC Educational Resources Information Center

    Varner, Sherrell E.

    Two basic reasons for concern over classroom size are the desire to optimize learning conditions and the tremendous impact of class size on school finances. The first reason of concern is not as well defined as the second. Rather than looking for the optimum figure, as has been done in the past, the question should read "Best classroom size for…

  19. Project CLASS.

    ERIC Educational Resources Information Center

    McBain, Susan L.; And Others

    Project CLASS (Competency-Based Live-Ability Skills) uses a series of 60 modules to teach life survival skills to adults with low-level reading ability--especially Adult Basic Education/English as a Second Language students. Two versions of the modules have been developed: one for use with teacher-directed instruction and another for independent…

  20. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut processing plant

    USDA-ARS?s Scientific Manuscript database

    Biofilm forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been ...

  1. Sonication contribution to identifying prosthetic joint infection with Ralstonia pickettii: a case report and review of the literature.

    PubMed

    Birlutiu, Rares Mircea; Roman, Mihai Dan; Cismasiu, Razvan Silviu; Fleaca, Sorin Radu; Popa, Crina Maria; Mihalache, Manuela; Birlutiu, Victoria

    2017-07-19

    In the context of an increase number of primary and revision total hip and total knee arthroplasty performed yearly, an increased risk of complication is expected. Prosthetic joint infection (PJI) remains the most common and feared arthroplasty complication. Ralstonia pickettii is a Gram-negative bacterium, that has also been identified in biofilms. It remains an extremely rare cause of PJI. There is no report of an identification of R. pickettii on an extracted spacer loaded with antibiotic. We present the case of an 83-years-old Caucasian male patient, that underwent a right cemented total hip replacement surgery. The patient is diagnosed with an early PJI with no isolated microorganism. A debridement and change of mobile parts is performed. At the beginning of 2016, the patient in readmitted into the Orthopedic Department for sever, right abdominal and groin pain and elevated serum erythrocyte sedimentation rate and C-reactive protein. A joint aspiration is performed with a negative microbiological examination. A two-stage exchange with long interval management is adopted, and a preformed spacer loaded with gentamicin was implanted. In July 2016, based on the proinflammatory markers evolution, a shift a three-stage exchange strategy is decided. In September 2016, a debridement, and changing of the preformed spacer loaded with gentamicin with another was carried out. Bacteriological examination of the tissues sampled intraoperatively was positive for Pseudomonas aeruginosa. From the sonication fluid, no bacteria were isolated on culture or identified using the bbFISH assay. During the hospitalization period, the patient received i.v. ceftazidime 3x2g/day and p.o. ciprofloxacin 2x750mg/day, antibiotic therapy that was continued after discharge with p.o. ciprofloxacin 2x750mg/day for 6 weeks. In February 2017, a reimplantation of a revision prosthesis is performed. The retrieved spacer is sonicated, and after 4 days of incubation of the sonication fluid, R

  2. Eggplant Resistance to the Ralstonia solanacearum Species Complex Involves Both Broad-Spectrum and Strain-Specific Quantitative Trait Loci

    PubMed Central

    Salgon, Sylvia; Jourda, Cyril; Sauvage, Christopher; Daunay, Marie-Christine; Reynaud, Bernard; Wicker, Emmanuel; Dintinger, Jacques

    2017-01-01

    Bacterial wilt (BW) is a major disease of solanaceous crops caused by the Ralstonia solanacearum species complex (RSSC). Strains are grouped into five phylotypes (I, IIA, IIB, III, and IV). Varietal resistance is the most sustainable strategy for managing BW. Nevertheless, breeding to improve cultivar resistance has been limited by the pathogen’s extensive genetic diversity. Identifying the genetic bases of specific and non-specific resistance is a prerequisite to breed improvement. A major gene (ERs1) was previously mapped in eggplant (Solanum melongena L.) using an intraspecific population of recombinant inbred lines derived from the cross of susceptible MM738 (S) × resistant AG91-25 (R). ERs1 was originally found to control three strains from phylotype I, while being totally ineffective against a virulent strain from the same phylotype. We tested this population against four additional RSSC strains, representing phylotypes I, IIA, IIB, and III in order to clarify the action spectrum of ERs1. We recorded wilting symptoms and bacterial stem colonization under controlled artificial inoculation. We constructed a high-density genetic map of the population using single nucleotide polymorphisms (SNPs) developed from genotyping-by-sequencing and added 168 molecular markers [amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs), and sequence-related amplified polymorphisms (SRAPs)] developed previously. The new linkage map based on a total of 1,035 markers was anchored on eggplant, tomato, and potato genomes. Quantitative trait locus (QTL) mapping for resistance against a total of eight RSSC strains resulted in the detection of one major phylotype-specific QTL and two broad-spectrum QTLs. The major QTL, which specifically controls three phylotype I strains, was located at the bottom of chromosome 9 and corresponded to the previously identified major gene ERs1. Five candidate R-genes were underlying this QTL, with different alleles between the

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

    PubMed

    Flavier, A B; Schell, M A; Denny, T P

    1998-05-01

    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.

  4. Rapid differentiation of Ralstonia solanacearum avirulent and virulent strains by cell fractioning of an isolate using high performance liquid chromatography.

    PubMed

    Zheng, Xuefang; Zhu, Yujing; Liu, Bo; Yu, Qian; Lin, Naiquan

    2016-01-01

    Ralstonia solanacearum is one of the most destructive plant bacterial pathogens worldwide. The population dynamics and genetic stability are important issues, especially when an avirulent strain is used for biocontrol. In this study, we developed a rapid method to differentiate the virulent and avirulent strains of R. solanacearum and to predict the biocontrol efficiency of an avirulent strain using high performance liquid chromatography (HPLC). Three chromatographic peaks P1, P2 and P3 were observed on the HPLC spectra among 68 avirulent and 28 virulent R. solanacearum strains. Based on the HPLC peaks, 96 strains total were assigned to three categories. For avirulent strains, the intense peak is P1, while for virulent strains, P3 is the majority. Based on the HLPC spectra of R. solanacearum strains, a chromatography titer index (CTI) was established as CTIi = Si/(S1+S2+S3) × 100% (i represents an individual HPLC peak; S1, S2 and S3 represent peak areas of P1, P2 and P3, respectively). The avirulent strains had high values of CTI1 ranging from 63.6 to 100.0%, while the virulent strains displayed high values of CTI3 ranging from 90.2 to 100.0%. Biological inoculation studies of 68 avirulent strains revealed that the biocontrol efficacy was the best when CTI1 = 100%. The purity and genetic stability of R. solanacearum strains were confirmed in the P1 fraction of avirulent strain FJAT-1957 and P3 fraction of virulent strain FJAT-1925 after 30 generations of consecutive subculture. These results confirmed that fractioning by HPLC and their deduced CTI can be used for rapid and efficient evaluation and prediction of an isolate of R. solanacearum. To the best of our knowledge, this is the first report that HPLC fractioning can be used for rapid differentiation of virulent and avirulent strains of R. solanacearum. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Dynamics in the resistant and susceptible peanut (Arachis hypogaea L.) root transcriptome on infection with the Ralstonia solanacearum.

    PubMed

    Chen, Yuning; Ren, Xiaoping; Zhou, Xiaojing; Huang, Li; Yan, Liying; Lei, Yong; Liao, Boshou; Huang, Jinyong; Huang, Shunmou; Wei, Wenhui; Jiang, Huifang

    2014-12-07

    Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease of peanut (Arachis hypogaea L). The molecular basis of peanut response to R. solanacearum remains unknown. To understand the resistance mechanism behind peanut resistance to R. solanacearum, we used RNA-Seq to perform global transcriptome profiling on the roots of peanut resistant (R) and susceptible (S) genotypes under R. solanacearum infection. A total of 4.95 x 108 raw sequence reads were generated and subsequently assembled into 271, 790 unigenes with an average length of 890 bp and a N50 of 1, 665 bp. 179, 641 unigenes could be annotated by public protein databases. The pairwise transcriptome comparsions of time course (6, 12, 24, 48 and 72 h post inoculation) were conducted 1) between inoculated and control samples of each genotype, 2) between inoculated samples of R and S genotypes. The linear dynamics of transcriptome profile was observed between adjacent samples for each genotype, two genotypes shared similar transcriptome pattern at early time points with most significant up regulation at 12 hour, and samples from R genotype at 24 h and S genotype at 48 h showed similar transcriptome pattern, significant differences of transcriptional profile were observed in pairwise comparisons between R and S genotypes. KEGG analysis showed that the primary metabolisms were inhibited in both genotypes and stronger inhibition in R genotype post inoculation. The defense related genes (R gene, LRR-RLK, cell wall genes, etc.) generally showed a genotype-specific down regulation and different expression between both genotypes. This transcriptome profiling provided the largest data set that explores the dynamic in crosstalk between peanut and R. solanacearum. The results suggested that the down-regulation of primary metabolism is contributed to the resistance difference between R and S genotypes. The genotype-specific expression pattern of defense related DEGs also contributed to the resistance

  6. Class distinction

    NASA Astrophysics Data System (ADS)

    White, M. Catherine

    Typical 101 courses discourage many students from pursuing higher level science and math courses. Introductory classes in science and math serve largely as a filter, screening out all but the most promising students, and leaving the majority of college graduates—including most prospective teachers—with little understanding of how science works, according to a study conducted for the National Science Foundation. Because few teachers, particularly at the elementary level, experience any collegiate science teaching that stresses skills of inquiry and investigation, they simply never learn to use those methods in their teaching, the report states.

  7. Enrichment Double-Antibody Sandwich Indirect Enzyme-Linked Immunosorbent Assay That Uses a Specific Monoclonal Antibody for Sensitive Detection of Ralstonia solanacearum in Asymptomatic Potato Tubers

    PubMed Central

    Caruso, Paola; Gorris, María Teresa; Cambra, Mariano; Palomo, José Luis; Collar, Jesús; López, María M.

    2002-01-01

    Sensitive and specific routine detection of Ralstonia solanacearum in symptomless potato tubers was achieved by efficient enrichment followed by a reliable double-antibody sandwich indirect enzyme-linked immunosorbent assay based on the specific monoclonal antibody 8B-IVIA. This monoclonal antibody reacted with 168 typical R. solanacearum strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato. The optimized protocol included an initial enrichment step consisting of shaking the samples in modified Wilbrink broth for 72 h at 29°C. This step enabled specific detection by the enzyme-linked immunosorbent assay of 1 to 10 CFU of R. solanacearum per ml of initial potato extract. Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods. PMID:12089053

  8. Genome sequencing of four strains of Phylotype I, II and IV of Ralstonia solanacearum that cause potato bacterial wilt in India.

    PubMed

    Patil, Virupaksh U; Girimalla, Vanishree; Sagar, Vinay; Chauhan, Rajinder Singh; Chakrabarti, Swarup Kumar

    Ralstonia solanacearum is a heterogeneous species complex causing bacterial wilts in more than 450 plant species distributed in 54 families. The complexity of the genome and the wide diversity existing within the species has led to the concept of R. solanacearum species complex (RsSC). Here we report the genome sequence of the four strains (RS2, RS25, RS48 and RS75) belonging to three of the four phylotypes of R. solanacearum that cause potato bacterial wilt in India. The genome sequence data would be a valuable resource for the evolutionary, epidemiological studies and quarantine of this phytopathogen. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  9. Plant Genome Complexity May Be a Factor Limiting In Situ the Transfer of Transgenic Plant Genes to the Phytopathogen Ralstonia solanacearum

    PubMed Central

    Bertolla, Franck; Pepin, Regis; Passelegue-Robe, Eugenie; Paget, Eric; Simkin, Andrew; Nesme, Xavier; Simonet, Pascal

    2000-01-01

    The development of natural competence by bacteria in situ is considered one of the main factors limiting transformation-mediated gene exchanges in the environment. Ralstonia solanacearum is a plant pathogen that is also a naturally transformable bacterium that can develop the competence state during infection of its host. We have attempted to determine whether this bacterium could become the recipient of plant genes. We initially demonstrated that plant DNA was released close to the infecting bacteria. We constructed and tested various combinations of transgenic plants and recipient bacteria to show that the effectiveness of such transfers was directly related to the ratio of the complexity of the plant genome to the number of copies of the transgene. PMID:10966449

  10. Characterization of the Agrobacterium vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum.

    PubMed

    Herlache, T C; Hotchkiss, A T; Burr, T J; Collmer, A

    1997-01-01

    DNA sequencing of the Agrobacterium vitis pehA gene revealed a predicted protein with an M(r) of 58,000 and significant similarity to the polygalacturonases of two other plant pathogens, Erwinia carotovora and Ralstonia (= Pseudomonas or Burkholderia) solanacearum. Sequencing of the N terminus of the PehA protein demonstrated cleavage of a 34-amino-acid signal peptide from pre-PehA. Mature PehA accumulated primarily in the periplasm of A. vitis and pehA+ Escherichia coli cells during exponential growth. A. vitis PehA released dimers, trimers, and monomers from polygalacturonic acid and caused less electrolyte leakage from potato tuber tissue than did the E. carotovora and R. solanacearum polygalacturonases.

  11. Meta-analysis Reveals That the Genus Pseudomonas Can Be a Better Choice of Biological Control Agent against Bacterial Wilt Disease Caused by Ralstonia solanacearum

    PubMed Central

    Chandrasekaran, Murugesan; Subramanian, Dharaneedharan; Yoon, Ee; Kwon, Taehoon; Chun, Se-Chul

    2016-01-01

    Biological control agents (BCAs) from different microbial taxa are increasingly used to control bacterial wilt caused by Ralstonia solanacearum. However, a quantitative research synthesis has not been conducted on the role of BCAs in disease suppression. Therefore, the present study aimed to meta-analyze the impacts of BCAs on both Ralstonia wilt disease suppression and plant (host) growth promotion. The analysis showed that the extent of disease suppression by BCAs varied widely among studies, with effect size (log response ratio) ranging from −2.84 to 2.13. The disease incidence and severity were significantly decreased on average by 53.7% and 49.3%, respectively. BCAs inoculation also significantly increased fresh and dry weight by 34.4% and 36.1%, respectively on average. Also, BCAs inoculation significantly increased plant yield by 66%. Mean effect sizes for genus Pseudomonas sp. as BCAs were higher than for genus Bacillus spp. Among antagonists tested, P. fluorescens, P. putida, B. cereus, B. subtilis and B. amyloliquefaciens were found to be more effective in general for disease reduction. Across studies, highest disease control was found for P. fluorescens, annual plants, co-inoculation with more than one BCA, soil drench and greenhouse condition were found to be essential in understanding plant responses to R. solanacearum. Our results suggest that more efforts should be devoted to harnessing the potential beneficial effects of these antagonists, not just for plant growth promoting traits but also in mode of applications, BCAs formulations and their field studies should be considered in the future for R. solanacearum wilt disease suppression. PMID:27298597

  12. A Single Regulator Mediates Strategic Switching between Attachment/Spread and Growth/Virulence in the Plant Pathogen Ralstonia solanacearum.

    PubMed

    Khokhani, Devanshi; Lowe-Power, Tiffany M; Tran, Tuan Minh; Allen, Caitilyn

    2017-09-26

    The PhcA virulence regulator in the vascular wilt pathogen Ralstonia solanacearum responds to cell density via quorum sensing. To understand the timing of traits that enable R. solanacearum to establish itself inside host plants, we created a ΔphcA mutant that is genetically locked in a low-cell-density condition. Comparing levels of gene expression of wild-type R. solanacearum and the ΔphcA mutant during tomato colonization revealed that the PhcA transcriptome includes an impressive 620 genes (>2-fold differentially expressed; false-discovery rate [FDR], ≤0.005). Many core metabolic pathways and nutrient transporters were upregulated in the ΔphcA mutant, which grew faster than the wild-type strain in tomato xylem sap and on dozens of specific metabolites, including 36 found in xylem. This suggests that PhcA helps R. solanacearum to survive in nutrient-poor environmental habitats and to grow rapidly during early pathogenesis. However, after R. solanacearum reaches high cell densities in planta, PhcA mediates a trade-off from maximizing growth to producing costly virulence factors. R. solanacearum infects through roots, and low-cell-density-mode-mimicking ΔphcA cells attached to tomato roots better than the wild-type cells, consistent with their increased expression of several adhesins. Inside xylem vessels, ΔphcA cells formed aberrantly dense mats. Possibly as a result, the mutant could not spread up or down tomato stems as well as the wild type. This suggests that aggregating improves R. solanacearum survival in soil and facilitates infection and that it reduces pathogenic fitness later in disease. Thus, PhcA mediates a second strategic switch between initial pathogen attachment and subsequent dispersal inside the host. PhcA helps R. solanacearum optimally invest resources and correctly sequence multiple steps in the bacterial wilt disease cycle.IMPORTANCERalstonia solanacearum is a destructive soilborne crop pathogen that wilts plants by colonizing their

  13. Overture: The grid classes

    SciTech Connect

    Brislawn, K.; Brown, D.; Chesshire, G.; Henshaw, W.

    1997-01-01

    Overture is a library containing classes for grids, overlapping grid generation and the discretization and solution of PDEs on overlapping grids. This document describes the Overture grid classes, including classes for single grids and classes for collections of grids.

  14. Teachers in Class

    ERIC Educational Resources Information Center

    Van Galen, Jane

    2008-01-01

    In this article, I argue for a closer read of the daily "class work" of teachers, as posited by Reay, 1998. In developing exploratory class portraits of four teachers who occupy distinctive social positions (two from working-class homes now teaching upper-middle-class children and two from upper-middle-class homes now teaching poor children), I…

  15. Ralstonia solanacearum Type III Effector RipAY Is a Glutathione-Degrading Enzyme That Is Activated by Plant Cytosolic Thioredoxins and Suppresses Plant Immunity.

    PubMed

    Mukaihara, Takafumi; Hatanaka, Tadashi; Nakano, Masahito; Oda, Kenji

    2016-04-12

    The plant pathogen Ralstonia solanacearum uses a large repertoire of type III effector proteins to succeed in infection. To clarify the function of effector proteins in host eukaryote cells, we expressed effectors in yeast cells and identified seven effector proteins that interfere with yeast growth. One of the effector proteins, RipAY, was found to share homology with the ChaC family proteins that function as γ-glutamyl cyclotransferases, which degrade glutathione (GSH), a tripeptide that plays important roles in the plant immune system. RipAY significantly inhibited yeast growth and simultaneously induced rapid GSH depletion when expressed in yeast cells. The in vitro GSH degradation activity of RipAY is specifically activated by eukaryotic factors in the yeast and plant extracts. Biochemical purification of the yeast protein identified that RipAY is activated by thioredoxin TRX2. On the other hand, RipAY was not activated by bacterial thioredoxins. Interestingly, RipAY was activated by plant h-type thioredoxins that exist in large amounts in the plant cytosol, but not by chloroplastic m-, f-, x-, y- and z-type thioredoxins, in a thiol-independent manner. The transient expression of RipAY decreased the GSH level in plant cells and affected the flg22-triggered production of reactive oxygen species (ROS) and expression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes in Nicotiana benthamiana leaves. These results indicate that RipAY is activated by host cytosolic thioredoxins and degrades GSH specifically in plant cells to suppress plant immunity. Ralstonia solanacearum is the causal agent of bacterial wilt disease of plants. This pathogen injects virulence effector proteins into host cells to suppress disease resistance responses of plants. In this article, we report a biochemical activity of R. solanacearum effector protein RipAY. RipAY can degrade GSH, a tripeptide that plays important roles in the plant immune system, with

  16. Comparative Secretome Analysis of Ralstonia solanacearum Type 3 Secretion-Associated Mutants Reveals a Fine Control of Effector Delivery, Essential for Bacterial Pathogenicity.

    PubMed

    Lonjon, Fabien; Turner, Marie; Henry, Céline; Rengel, David; Lohou, David; van de Kerkhove, Quitterie; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2016-02-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume

  17. Comparative Secretome Analysis of Ralstonia solanacearum Type 3 Secretion-Associated Mutants Reveals a Fine Control of Effector Delivery, Essential for Bacterial Pathogenicity*

    PubMed Central

    Lonjon, Fabien; Turner, Marie; Henry, Céline; Rengel, David; Lohou, David; van de Kerkhove, Quitterie; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2016-01-01

    Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume

  18. A probable aculeacin A acylase from the Ralstonia solanacearum GMI1000 is N-acyl-homoserine lactone acylase with quorum-quenching activity.

    PubMed

    Chen, Chin-Nung; Chen, Chii-Jaan; Liao, Chen-Ting; Lee, Chia-Yin

    2009-05-09

    The infection and virulence functions of diverse plant and animal pathogens that possess quorum sensing systems are regulated by N-acylhomoserine lactones (AHLs) acting as signal molecules. AHL-acylase is a quorum quenching enzyme and degrades AHLs by removing the fatty acid side chain from the homoserine lactone ring of AHLs. This blocks AHL accumulation and pathogenic phenotypes in quorum sensing bacteria. An aac gene of undemonstrated function from Ralstonia solanacearum GMI1000 was cloned, expressed in Escherichia coli; it inactivated four AHLs that were tested. The sequence of the 795 amino acid polypeptide was considerably similar to the AHL-acylase from Ralstonia sp. XJ12B with 83% identity match and shared 39% identity with an aculeacin A acylase precursor from the gram-positive actinomycete Actinoplanes utahensis. Aculeacin A is a neutral lipopeptide antibiotic and an antifungal drug. An electrospray ionisation mass spectrometry (ESI-MS) analysis verified that Aac hydrolysed the amide bond of AHL, releasing homoserine lactone and the corresponding fatty acids. However, ESI-MS analysis demonstrated that the Aac could not catalyze the hydrolysis of the palmitoyl moiety of the aculeacin A. Moreover, the results of MIC test of aculeacin A suggest that Aac could not deacylate aculeacin A. The specificity of Aac for AHLs showed a greater preference for long acyl chains than for short acyl chains. Heterologous expression of the aac gene in Chromobacterium violaceum CV026 effectively inhibited violacein and chitinase activity, both of which were regulated by the quorum-sensing mechanism. These results indicated that Aac could control AHL-dependent pathogenicity. This is the first study to find an AHL-acylase in a phytopathogen. Our data provide direct evidence that the functioning of the aac gene (NP520668) of R. solanacearum GMI1000 is via AHL-acylase and not via aculeacin A acylase. Since Aac is a therapeutic potential quorum-quenching agent, its further

  19. New Multilocus Variable-Number Tandem-Repeat Analysis (MLVA) Scheme for Fine-Scale Monitoring and Microevolution-Related Study of Ralstonia pseudosolanacearum Phylotype I Populations

    PubMed Central

    Guinard, Jérémy; Latreille, Anne; Guérin, Fabien; Poussier, Stéphane

    2016-01-01

    ABSTRACT Bacterial wilt caused by the Ralstonia solanacearum species complex (RSSC) is considered one of the most harmful plant diseases in the world. Special attention should be paid to R. pseudosolanacearum phylotype I due to its large host range, its worldwide distribution, and its high evolutionary potential. So far, the molecular epidemiology and population genetics of this bacterium are poorly understood. Until now, the genetic structure of the RSSC has been analyzed on the worldwide and regional scales. Emerging questions regarding evolutionary forces in RSSC adaptation to hosts now require genetic markers that are able to monitor RSSC field populations. In this study, we aimed to evaluate the multilocus variable-number tandem-repeat analysis (MLVA) approach for its ability to discriminate genetically close phylotype I strains and for population genetics studies. We developed a new MLVA scheme (MLVA-7) allowing us to genotype 580 R. pseudosolanacearum phylotype I strains extracted from susceptible and resistant hosts and from different habitats (stem, soil, and rhizosphere). Based on specificity, polymorphism, and the amplification success rate, we selected seven fast-evolving variable-number tandem-repeat (VNTR) markers. The newly developed MLVA-7 scheme showed higher discriminatory power than the previously published MLVA-13 scheme when applied to collections sampled from the same location on different dates and to collections from different locations on very small scales. Our study provides a valuable tool for fine-scale monitoring and microevolution-related study of R. pseudosolanacearum phylotype I populations. IMPORTANCE Understanding the evolutionary dynamics of adaptation of plant pathogens to new hosts or ecological niches has become a key point for the development of innovative disease management strategies, including durable resistance. Whereas the molecular mechanisms underlying virulence or pathogenicity changes have been studied thoroughly, the

  20. A Conserved EAR Motif Is Required for Avirulence and Stability of the Ralstonia solanacearum Effector PopP2 In Planta.

    PubMed

    Segonzac, Cécile; Newman, Toby E; Choi, Sera; Jayaraman, Jay; Choi, Du Seok; Jung, Ga Young; Cho, Heejung; Lee, Young Kee; Sohn, Kee Hoon

    2017-01-01

    Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease in many high value Solanaceae crops. R. solanacearum secretes around 70 effectors into host cells in order to promote infection. Plants have, however, evolved specialized immune receptors that recognize corresponding effectors and confer qualitative disease resistance. In the model species Arabidopsis thaliana, the paired immune receptors RRS1 (resistance to Ralstonia solanacearum 1) and RPS4 (resistance to Pseudomonas syringae 4) cooperatively recognize the R. solanacearum effector PopP2 in the nuclei of infected cells. PopP2 is an acetyltransferase that binds to and acetylates the RRS1 WRKY DNA-binding domain resulting in reduced RRS1-DNA association thereby activating plant immunity. Here, we surveyed the naturally occurring variation in PopP2 sequence among the R. solanacearum strains isolated from diseased tomato and pepper fields across the Republic of Korea. Our analysis revealed high conservation of popP2 sequence with only three polymorphic alleles present amongst 17 strains. Only one variation (a premature stop codon) caused the loss of RPS4/RRS1-dependent recognition in Arabidopsis. We also found that PopP2 harbors a putative eukaryotic transcriptional repressor motif (ethylene-responsive element binding factor-associated amphiphilic repression or EAR), which is known to be involved in the recruitment of transcriptional co-repressors. Remarkably, mutation of the EAR motif disabled PopP2 avirulence function as measured by the development of hypersensitive response, electrolyte leakage, defense marker gene expression and bacterial growth in Arabidopsis. This lack of recognition was partially but significantly reverted by the C-terminal addition of a synthetic EAR motif. We show that the EAR motif-dependent gain of avirulence correlated with the stability of the PopP2 protein. Furthermore, we demonstrated the requirement of the PopP2 EAR motif for PTI suppression. A yeast

  1. Does Class Size Matter?

    ERIC Educational Resources Information Center

    Ehrenberg, Ronald G.; Brewer, Dominic J.; Gamoran, Adam; Willms, J. Douglas

    2001-01-01

    Reports on the significance of class size to student learning. Includes an overview of class size in various countries, the importance of teacher adaptability, and the Asian paradox of large classes allied to high test scores. (MM)

  2. The Naphthalene Catabolic (nag) Genes of Ralstonia sp. Strain U2 Are an Operon That Is Regulated by NagR, a LysR-Type Transcriptional Regulator

    PubMed Central

    Jones, Rheinallt M.; Britt-Compton, Bethan; Williams, Peter A.

    2003-01-01

    In Ralstonia sp. strain U2, the nag catabolic genes, which encode the enzymes for the pathway that catabolizes naphthalene via the alternative ring cleavage gentisate pathway, are transcribed as an operon under the same promoter. nagR, which encodes a LysR-type transcriptional regulator, is divergently transcribed compared to the nag catabolic genes. A 4-bp frameshift deletion in nagR demonstrated that NagR is required for expression of the nag operon. The transcriptional start of the nag operon was mapped, and a putative −10, −35 σ70-type promoter binding site was identified. Further upstream, a site proximal to the promoter was identified as a site that has bases which have been found to be conserved in the activator-binding motif of other naphthalene pathways. Transcriptional fusion studies demonstrated that NagR regulates the expression of the nag operon positively in the presence of salicylate and to a lesser extent in the presence of 2-nitrobenzoate. Mutation of the LysR-type activator-binding motif in the nag promoter-proximal region resulted in a loss of inducibility of a lacZ reporter gene transcriptionally fused to nagAa, the first gene of the operon. However, other mutations in the region increased the effectiveness of salicylate as an inducer. PMID:13129957

  3. Response of tomato wilt pathogen Ralstonia solanacearum to the volatile organic compounds produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9

    PubMed Central

    Raza, Waseem; Ling, Ning; Yang, Liudong; Huang, Qiwei; Shen, Qirong

    2016-01-01

    It is important to study the response of plant pathogens to the antibiosis traits of biocontrol microbes to design the efficient biocontrol strategies. In this study, we evaluated the role of volatile organic compounds (VOCs) produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum (RS). The VOCs of SQR-9 significantly inhibited the growth of RS on agar medium and in soil. In addition, the VOCs significantly inhibited the motility traits, production of antioxidant enzymes and exopolysaccharides, biofilm formation and tomato root colonization by RS. The strain SQR-9 produced 22 VOCs, but only nine VOCs showed 1–11% antibacterial activity against RS in their corresponding amounts; however, the consortium of all VOCs showed 70% growth inhibition of RS. The proteomics analysis showed that the VOCs of SQR-9 downregulated RS proteins related to the antioxidant activity, virulence, carbohydrate and amino acid metabolism, protein folding and translation, while the proteins involved in the ABC transporter system, amino acid synthesis, detoxification of aldehydes and ketones, methylation, protein translation and folding, and energy transfer were upregulated. This study describes the significance and effectiveness of VOCs produced by a biocontrol strain against tomato wilt pathogen. PMID:27103342

  4. The symbiotic transcription factor MtEFD and cytokinins are positively acting in the Medicago truncatula and Ralstonia solanacearum pathogenic interaction.

    PubMed

    Moreau, Sandra; Fromentin, Justine; Vailleau, Fabienne; Vernié, Tatiana; Huguet, Stéphanie; Balzergue, Sandrine; Frugier, Florian; Gamas, Pascal; Jardinaud, Marie-Françoise

    2014-03-01

    • A plant-microbe dual biological system was set up involving the model legume Medicago truncatula and two bacteria, the soil-borne root pathogen Ralstonia solanacearum and the beneficial symbiont Sinorhizobium meliloti. • Comparison of transcriptomes under symbiotic and pathogenic conditions highlighted the transcription factor MtEFD (Ethylene response Factor required for nodule Differentiation) as being upregulated in both interactions, together with a set of cytokinin-related transcripts involved in metabolism, signaling and response. MtRR4 (Response Regulator), a cytokinin primary response gene negatively regulating cytokinin signaling and known as a target of MtEFD in nodulation processes, was retrieved in this set of transcripts. • Refined studies of MtEFD and MtRR4 expression during M. truncatula and R. solanacearum interaction indicated differential kinetics of induction and requirement of central regulators of bacterial pathogenicity, HrpG and HrpB. Similar to MtRR4, MtEFD upregulation during the pathogenic interaction was dependent on cytokinin perception mediated by the MtCRE1 (Cytokinin REsponse 1) receptor. • The use of M. truncatula efd-1 and cre1-1 mutants evidenced MtEFD and cytokinin perception as positive factors for bacterial wilt development. These factors therefore play an important role in both root nodulation and root disease development.

  5. Transformation of Chlorinated Benzenes and Toluenes by Ralstonia sp. Strain PS12 tecA (Tetrachlorobenzene Dioxygenase) and tecB (Chlorobenzene Dihydrodiol Dehydrogenase) Gene Products

    PubMed Central

    Pollmann, Katrin; Beil, Stefan; Pieper, Dietmar H.

    2001-01-01

    The tecB gene, located downstream of tecA and encoding tetrachlorobenzene dioxygenase, in Ralstonia sp. strain PS12 was cloned into Escherichia coli DH5α together with the tecA gene. The identity of the tecB gene product as a chlorobenzene dihydrodiol dehydrogenase was verified by transformation into the respective catechols of chlorobenzene, the three isomeric dichlorobenzenes, as well as 1,2,3- and 1,2,4-trichlorobenzenes, all of which are transformed by TecA into the respective dihydrodihydroxy derivatives. Di- and trichlorotoluenes were either subject to TecA-mediated dioxygenation (the major or sole reaction observed for the 1,2,4-substituted 2,4-, 2,5-, and 3,4-dichlorotoluenes), resulting in the formation of the dihydrodihydroxy derivatives, or to monooxygenation of the methyl substituent (the major or sole reaction observed for 2,3-, 2,6-, and 3,5-dichloro- and 2,4,5-trichlorotoluenes), resulting in formation of the respective benzyl alcohols. All of the chlorotoluenes subject to dioxygenation by TecA were transformed, without intermediate accumulation of dihydrodihydroxy derivatives, into the respective catechols by TecAB, indicating that dehydrogenation is no bottleneck for chlorobenzene or chlorotoluene degradation. However, only those chlorotoluenes subject to a predominant dioxygenation were growth substrates for PS12, confirming that monooxygenation is an unproductive pathway in PS12. PMID:11526005

  6. Response of tomato wilt pathogen Ralstonia solanacearum to the volatile organic compounds produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9.

    PubMed

    Raza, Waseem; Ling, Ning; Yang, Liudong; Huang, Qiwei; Shen, Qirong

    2016-04-22

    It is important to study the response of plant pathogens to the antibiosis traits of biocontrol microbes to design the efficient biocontrol strategies. In this study, we evaluated the role of volatile organic compounds (VOCs) produced by a biocontrol strain Bacillus amyloliquefaciens SQR-9 on the growth and virulence traits of tomato wilt pathogen Ralstonia solanacearum (RS). The VOCs of SQR-9 significantly inhibited the growth of RS on agar medium and in soil. In addition, the VOCs significantly inhibited the motility traits, production of antioxidant enzymes and exopolysaccharides, biofilm formation and tomato root colonization by RS. The strain SQR-9 produced 22 VOCs, but only nine VOCs showed 1-11% antibacterial activity against RS in their corresponding amounts; however, the consortium of all VOCs showed 70% growth inhibition of RS. The proteomics analysis showed that the VOCs of SQR-9 downregulated RS proteins related to the antioxidant activity, virulence, carbohydrate and amino acid metabolism, protein folding and translation, while the proteins involved in the ABC transporter system, amino acid synthesis, detoxification of aldehydes and ketones, methylation, protein translation and folding, and energy transfer were upregulated. This study describes the significance and effectiveness of VOCs produced by a biocontrol strain against tomato wilt pathogen.

  7. Ethylene emission and PR protein synthesis in ACC deaminase producing Methylobacterium spp. inoculated tomato plants (Lycopersicon esculentum Mill.) challenged with Ralstonia solanacearum under greenhouse conditions.

    PubMed

    Yim, Woojong; Seshadri, Sundaram; Kim, Kiyoon; Lee, Gillseung; Sa, Tongmin

    2013-06-01

    Bacteria of genus Methylobacterium have been found to promote plant growth and regulate the level of ethylene in crop plants. This work is aimed to test the induction of defense responses in tomato against bacterial wilt by stress ethylene level reduction mediated by the ACC deaminase activity of Methylobacterium strains. Under greenhouse conditions, the disease index value in Methylobacterium sp. inoculated tomato plants was lower than control plants. Plants treated with Methylobacterium sp. challenge inoculated with Ralstonia solanacearum (RS) showed significantly reduced disease symptoms and lowered ethylene emission under greenhouse condition. The ACC and ACO (1-aminocyclopropane-1-carboxylate oxidase) accumulation in tomato leaves were significantly reduced with Methylobacterium strains inoculation. While ACC oxidase gene expression was found higher in plants treated with R. solanacearum than Methylobacterium sp. treatment, PR proteins related to induced systemic resistance like β-1,3-glucanase, PAL, PO and PPO were increased in Methylobacterium sp. inoculated plants. A significant increase in β-1,3-glucanase and PAL gene expression was found in all the Methylobacterium spp. treatments compared to the R. solanacearum treatment. This study confirms the activity of Methylobacterium sp. in increasing the defense enzymes by modulating the ethylene biosynthesis pathway and suggests the use of methylotrophic bacteria as potential biocontrol agents in tomato cultivation. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  8. Development of variable number of tandem repeats typing schemes for Ralstonia solanacearum, the agent of bacterial wilt, banana Moko disease and potato brown rot.

    PubMed

    N'guessan, Carine Aya; Brisse, Sylvain; Le Roux-Nio, Anne-Claire; Poussier, Stéphane; Koné, Daouda; Wicker, Emmanuel

    2013-03-01

    Ralstonia solanacearum is an important soil borne bacterial plant pathogen causing bacterial wilt on many important crops. To better monitor epidemics, efficient tools that can identify and discriminate populations are needed. In this study, we assessed variable number of tandem repeats (VNTR) genotyping as a new tool for epidemiological surveillance of R. solanacearum phylotypes, and more specifically for the monitoring of the monomorphic ecotypes "Moko" (banana-pathogenic) and "brown rot" (potato-pathogenic under cool conditions). Screening of six R. solanacearum genome sequences lead to select 36 VNTR loci that were preliminarily amplified on 24 strains. From this step, 26 single-locus primer pairs were multiplexed, and applied to a worldwide collection of 337 strains encompassing the whole phylogenetic diversity, with revelation on a capillary-electrophoresis genotype. Four loci were monomorphic within all phylotypes and were not retained; the other loci were highly polymorphic but displayed a clear phylotype-specificity. Phylotype-specific MLVA schemes were thus defined, based on 13 loci for phylotype I, 12 loci for phylotype II, 11 loci for phylotype III and 6 for phylotype IV. MLVA typing was significantly more discriminative than egl-based sequevar typing, particularly on monomorphic "brown rot" ecotype (phylotype IIB/sequevar 1) and "Moko disease" clade 4 (Phylotype IIB/sequevar 4). Our results raise promising prospects for studies of population genetic structures and epidemiological monitoring.

  9. Only One of the Five Ralstonia solanacearum Long-Chain 3-Ketoacyl-Acyl Carrier Protein Synthase Homologues Functions in Fatty Acid Synthesis

    PubMed Central

    Cheng, Juanli; Ma, Jincheng; Lin, Jinshui; Fan, Zhen-Chuan; Cronan, John E.

    2012-01-01

    Ralstonia solanacearum, a major phytopathogenic bacterium, causes a bacterial wilt disease in diverse plants. Although fatty acid analyses of total membranes of R. solanacearum showed that they contain primarily palmitic (C16:0), palmitoleic (C16:1) and cis-vaccenic (C18:1) acids, little is known regarding R. solanacearum fatty acid synthesis. The R. solanacearum GMI1000 genome is unusual in that it contains four genes (fabF1, fabF2, fabF3, and fabF4) annotated as encoding 3-ketoacyl-acyl carrier protein synthase II homologues and one gene (fabB) annotated as encoding 3-ketoacyl-acyl carrier protein synthase I. We have analyzed this puzzling apparent redundancy and found that only one of these genes, fabF1, encoded a long-chain 3-ketoacyl-acyl carrier protein synthase, whereas the other homologues did not play roles in R. solanacearum fatty acid synthesis. Mutant strains lacking fabF1 are nonviable, and thus, FabF1 is essential for R. solanacearum fatty acid biosynthesis. Moreover, R. solanacearum FabF1 has the activities of both 3-ketoacyl-acyl carrier protein synthase II and 3-ketoacyl-acyl carrier protein synthase I. PMID:22194290

  10. Modulation of Inter-kingdom Communication by PhcBSR Quorum Sensing System in Ralstonia solanacearum Phylotype I Strain GMI1000

    PubMed Central

    Li, Peng; Yin, Wenfang; Yan, Jinli; Chen, Yufan; Fu, Shuna; Song, Shihao; Zhou, Jianuan; Lyu, Mingfa; Deng, Yinyue; Zhang, Lian-Hui

    2017-01-01

    Ralstonia solanacearum is a ubiquitous soil-borne plant pathogenic bacterium, which frequently encounters and interacts with other soil cohabitants in competition for environmental niches. Ralsolamycin, which is encoded by the rmy genes, has been characterized as a novel inter-kingdom interaction signal that induces chlamydospore development in fungi. In this study, we provide the first genetic evidence that the rmy gene expression is controlled by the PhcBSR quorum sensing (QS) system in strain GMI1000. Mutation of phcB could lead to significant reduction of the expression levels of the genes involved in ralsolamycin biosynthesis. In addition, both the phcB and rmy mutants were attenuated in induction of chlamydospore formation in Fusarium oxysporum f. cubense and diminished in the ability to compete with the sugarcane pathogen Sporisorium scitamineum. Agreeable with the pattern of QS regulation, transcriptional expression analysis showed that the transcripts of the rmy genes were increased along with the increment of the bacterial population density. Taken together, the above findings provide new insights into the regulatory mechanisms that the QS system involves in governing the ralsolamycin inter-kingdom signaling system. PMID:28690607

  11. Ferulic Acid, But Not All Hydroxycinnamic Acids, Is a Novel T3SS Inducer of Ralstonia solanacearum and Promotes Its Infection Process in Host Plants under Hydroponic Condition

    PubMed Central

    Zhang, Yong; Li, Jing; Zhang, Weiqi; Wang, Rongsheng; Qiu, Qiaoqing; Luo, Feng; Hikichi, Yasufumi; Ohnishi, Kouhei; Ding, Wei

    2017-01-01

    Hydroxycinnamic acids (HCAs) are typical monocyclic phenylpropanoids, including cinnamic acid (Cin), coumaric acid (Cou), caffeic acid (Caf), ferulic acid (FA) and their isomers, and involved in the interactions between pathogens and host plants. Here, we focused on the impact of HCAs on expression of type III secretion system (T3SS) in Ralstonia solanacearum. FA significantly induced the expression of the T3SS and some type III effectors (T3Es) genes in hrp-inducing medium, while did not the other HCAs. However, exogenously supplemented FA did not affect the T3SS expression in planta and the elicitation of the hypersensitive response (HR) in tobacco leaves. Consistent with its central roles in pathogenicity, the FA-induced expression of the T3SS led to significant promotion on infection process of R. solanacearum in tomato plants under hydroponics cultivation. Moreover, the FA-induced expression of the T3SS was specifically mediated by the well-characterized signaling cascade PrhA-prhI/R-PrhJ-HrpG-HrpB, independent of the other known regulatory pathways. In summary, our results demonstrated that FA, a novel inducer of the T3SS in R. solanacearum, was able to promote its infection process in host plants under hydroponics condition. PMID:28955375

  12. Exposure to Umbelliferone Reduces Ralstonia solanacearum Biofilm Formation, Transcription of Type III Secretion System Regulators and Effectors and Virulence on Tobacco

    PubMed Central

    Yang, Liang; Li, Shili; Qin, Xiyun; Jiang, Gaofei; Chen, Juanni; Li, Bide; Yao, Xiaoyuan; Liang, Peibo; Zhang, Yong; Ding, Wei

    2017-01-01

    Ralstonia solanacearum is one of the most devastating phytopathogens and causes bacterial wilt, which leads to severe economic loss due to its worldwide distribution and broad host range. Certain plant-derived compounds (PDCs) can impair bacterial virulence by suppressing pathogenic factors of R. solanacearum. However, the inhibitory mechanisms of PDCs in bacterial virulence remain largely unknown. In this study, we screened a library of coumarins and derivatives, natural PDCs with fused benzene and α-pyrone rings, for their effects on expression of the type III secretion system (T3SS) of R. solanacearum. Here, we show that umbelliferone (UM), a 7-hydroxycoumarin, suppressed T3SS regulator gene expression through HrpG–HrpB and PrhG–HrpB pathways. UM decreased gene expression of six type III effectors (RipX, RipD, RipP1, RipR, RipTAL, and RipW) of 10 representative effector genes but did not alter T2SS expression. In addition, biofilm formation of R. solanacearum was significantly reduced by UM, though swimming activity was not affected. We then observed that UM suppressed the wilting disease process by reducing colonization and proliferation in tobacco roots and stems. In summary, the findings reveal that UM may serve as a plant-derived inhibitor to manipulate R. solanacearum T3SS and biofilm formation, providing proof of concept that these key virulence factors are potential targets for the integrated control of bacterial wilt. PMID:28713361

  13. Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages.

    PubMed

    Bhunchoth, Anjana; Blanc-Mathieu, Romain; Mihara, Tomoko; Nishimura, Yosuke; Askora, Ahmed; Phironrit, Namthip; Leksomboon, Chalida; Chatchawankanphanich, Orawan; Kawasaki, Takeru; Nakano, Miyako; Fujie, Makoto; Ogata, Hiroyuki; Yamada, Takashi

    2016-07-01

    Jumbo phages infecting Ralstonia solanacearum were isolated in Thailand (ϕRSL2) and Japan (ϕRSF1). They were similar regarding virion morphology, genomic arrangement, and host range. Phylogenetic and proteomic tree analyses demonstrate that the ϕRSL2 and ϕRSF1 belong to a group of evolutionary related phages, including Pseudomonas phages ϕKZ, 201ϕ2-1 and all previously described ϕKZ-related phages. Despite conserved genomic co-linearity between the ϕRSL2 and ϕRSF1, they differ in protein separation patterns. A major difference was seen in the detection of virion-associated-RNA polymerase subunits. All β- and β'-subunits were detected in ϕRSF1, but one β'-subunit was undetected in ϕRSL2. Furthermore, ϕRSF1 infected host cells faster (latent period: 60 and 150min for ϕRSF1 and ϕRSL2, respectively) and more efficiently than ϕRSL2. Therefore, the difference in virion-associated-RNA polymerase may affect infection efficiency. Finally, we show that ϕRSF1 is able to inhibit bacterial wilt progression in tomato plants. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Hydroxycinnamic Acid Degradation, a Broadly Conserved Trait, Protects Ralstonia solanacearum from Chemical Plant Defenses and Contributes to Root Colonization and Virulence.

    PubMed

    Lowe, Tiffany M; Ailloud, Florent; Allen, Caitilyn

    2015-03-01

    Plants produce hydroxycinnamic acid (HCA) defense compounds to combat pathogens, such as the bacterium Ralstonia solanacearum. We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a feruloyl-CoA synthetase (Δfcs) mutant that cannot degrade HCA was less virulent on tomato plants. To understand the role of HCA degradation in bacterial wilt disease, we tested the following hypotheses: HCA degradation helps the pathogen i) grow, as a carbon source; ii) spread, by reducing HCA-derived physical barriers; and iii) survive plant antimicrobial compounds. Although HCA degradation enabled R. solanacearum growth on HCA in vitro, HCA degradation was dispensable for growth in xylem sap and root exudate, suggesting that HCA are not significant carbon sources in planta. Acetyl-bromide quantification of lignin demonstrated that R. solanacearum infections did not affect the gross quantity or distribution of stem lignin. However, the Δfcs mutant was significantly more susceptible to inhibition by two HCA, namely, caffeate and p-coumarate. Finally, plant colonization assays suggested that HCA degradation facilitates early stages of infection and root colonization. Together, these results indicated that ability to degrade HCA contributes to bacterial wilt virulence by facilitating root entry and by protecting the pathogen from HCA toxicity.

  15. Isolation of Ralstonia solanacearum-infecting bacteriophages from tomato fields in Chiang Mai, Thailand, and their experimental use as biocontrol agents.

    PubMed

    Bhunchoth, A; Phironrit, N; Leksomboon, C; Chatchawankanphanich, O; Kotera, S; Narulita, E; Kawasaki, T; Fujie, M; Yamada, T

    2015-04-01

    To isolate and characterize novel bacteriophages infecting the phytopathogen, Ralstonia solanacearum, and to evaluate them as resources with potential uses in the biocontrol of bacterial wilt. Fourteen phages infecting R. solanacearum were isolated from soil samples collected in Chiang Mai, Thailand. The phages showed different host ranges when tested against 59 R. solanacearum strains isolated from Thailand and Japan. These phages were characterized as nine podoviruses and five myoviruses based on their morphology. Podovirus J2 in combination with another podovirus (φRSB2) lysed host cells very efficiently in contaminated soil. J2 treatment prevented wilting of tomato plants infected with a highly virulent R. solanacearum strain. Treatment with J2 effectively reduced the amount of the bacterial wilt pathogen in contaminated soil and prevented bacterial wilt of tomato in pot experiments. Myovirus J6 possessed jumbo phage features, giving a unique opportunity to study its utilization as a biocontrol agent. As exemplified by J2, the phages isolated in this study represent valuable resources with potential uses in biocontrol of bacterial wilt. A rare jumbo phage J6 served as a valuable subject to understand and utilize this new group of phages. © 2015 The Society for Applied Microbiology.

  16. Genome-Enabled Phylogeographic Investigation of the Quarantine Pathogen Ralstonia solanacearum Race 3 Biovar 2 and Screening for Sources of Resistance Against Its Core Effectors.

    PubMed

    Clarke, Christopher R; Studholme, David J; Hayes, Byron; Runde, Brendan; Weisberg, Alexandra; Cai, Rongman; Wroblewski, Tadeusz; Daunay, Marie-Christine; Wicker, Emmanuel; Castillo, Jose A; Vinatzer, Boris A

    2015-05-01

    Phylogeographic studies inform about routes of pathogen dissemination and are instrumental for improving import/export controls. Genomes of 17 isolates of the bacterial wilt and potato brown rot pathogen Ralstonia solanacearum race 3 biovar 2 (R3bv2), a Select Agent in the United States, were thus analyzed to get insight into the phylogeography of this pathogen. Thirteen of fourteen isolates from Europe, Africa, and Asia were found to belong to a single clonal lineage while isolates from South America were genetically diverse and tended to carry ancestral alleles at the analyzed genomic loci consistent with a South American origin of R3bv2. The R3bv2 isolates share a core repertoire of 31 type III-secreted effector genes representing excellent candidates to be targeted with resistance genes in breeding programs to develop durable disease resistance. Toward this goal, 27 R3bv2 effectors were tested in eggplant, tomato, pepper, tobacco, and lettuce for induction of a hypersensitive-like response indicative of recognition by cognate resistance receptors. Fifteen effectors, eight of them core effectors, triggered a response in one or more plant species. These genotypes may harbor resistance genes that could be identified and mapped, cloned, and expressed in tomato or potato, for which sources of genetic resistance to R3bv2 are extremely limited.

  17. Hydroxycinnamic acid degradation, a broadly conserved trait, protects Ralstonia solanacearum from chemical plant defenses and contributes to root colonization and virulence

    PubMed Central

    Lowe, Tiffany M.; Ailloud, Florent; Allen, Caitilyn

    2014-01-01

    Plants produce hydroxycinnamic acid defense compounds (HCAs) to combat pathogens, such as the bacterium Ralstonia solanacearum. We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a Δfcs (feruloyl-CoA synthetase) mutant that cannot degrade HCAs was less virulent on tomato plants. To understand the role of HCA degradation in bacterial wilt disease, we tested the following hypotheses: HCA degradation helps the pathogen (1) grow, as a carbon source; (2) spread, by reducing physical barriers HCA-derived; and (3) survive plant antimicrobial compounds. Although HCA degradation enabled R. solanacearum growth on HCAs in vitro, HCA degradation was dispensable for growth in xylem sap and root exudate, suggesting that HCAs are not significant carbon sources in planta. Acetyl-bromide quantification of lignin demonstrated that R. solanacearum infections did not affect the gross quantity or distribution of stem lignin. However, the Δfcs mutant was significantly more susceptible to inhibition by two HCAs: caffeate and p-coumarate. Finally, plant colonization assays suggested that HCA degradation facilitates early stages of infection and root colonization. Together, these results indicated that ability to degrade HCAs contributes to bacterial wilt virulence by facilitating root entry and by protecting the pathogen from HCA toxicity. PMID:25423265

  18. Bacillus thuringiensis suppresses bacterial wilt disease caused by Ralstonia solanacearum with systemic induction of defense-related gene expression in tomato.

    PubMed

    Hyakumachi, Mitsuro; Nishimura, Mitsuyoshi; Arakawa, Tatsuyuki; Asano, Shinichiro; Yoshida, Shigenobu; Tsushima, Seiya; Takahashi, Hideki

    2013-01-01

    Bacillus thuringiensis is a naturally abundant Gram-positive bacterium and a well-known, effective bio-insecticide. Recently, B. thuringiensis has attracted considerable attention as a potential biological control agent for the suppression of plant diseases. In this study, the bacterial wilt disease-suppressing activity of B. thuringiensis was examined in tomato plants. Treatment of tomato roots with B. thuringiensis culture followed by challenge inoculation with Ralstonia solanacearum suppressed the development of wilt symptoms to less than one third of the control. This disease suppression in tomato plants was reproduced by pretreating their roots with a cell-free filtrate (CF) that had been fractionated from B. thuringiensis culture by centrifugation and filtration. In tomato plants challenge-inoculated with R. solanacearum after pretreatment with CF, the growth of R. solanacearum in stem tissues clearly decreased, and expression of defense-related genes such as PR-1, acidic chitinase, and β-1,3-glucanase was induced in stem and leaf tissues. Furthermore, the stem tissues of tomato plants with their roots were pretreated with CF exhibited resistance against direct inoculation with R. solanacearum. Taken together, these results suggest that treatment of tomato roots with the CF of B. thuringiensis systemically suppresses bacterial wilt through systemic activation of the plant defense system.

  19. Complete biodegradation of 4-fluorocinnamic acid by a consortium comprising Arthrobacter sp. strain G1 and Ralstonia sp. strain H1.

    PubMed

    Hasan, Syed A; Ferreira, Maria Isabel M; Koetsier, Martijn J; Arif, Muhammad I; Janssen, Dick B

    2011-01-01

    A consortium of the newly isolated bacterial strains Arthrobacter sp. strain G1 and Ralstonia sp. strain H1 utilized 4-fluorocinnamic acid for growth under aerobic conditions. Strain G1 converted 4-fluorocinnamic acid into 4-fluorobenzoic acid and used the two-carbon side chain for growth, with some formation of 4-fluoroacetophenone as a dead-end side product. In the presence of strain H1, complete mineralization of 4-fluorocinnamic acid and release of fluoride were obtained. Degradation of 4-fluorocinnamic acid by strain G1 occurred through a β-oxidation mechanism and started with the formation of 4-fluorocinnamoyl-coenzyme A (CoA), as indicated by the presence of 4-fluorocinnamoyl-CoA ligase. Enzymes for further transformation were detected in cell extract, i.e., 4-fluorocinnamoyl-CoA hydratase, 4-fluorophenyl-β-hydroxy propionyl-CoA dehydrogenase, and 4-fluorophenyl-β-keto propionyl-CoA thiolase. Degradation of 4-fluorobenzoic acid by strain H1 proceeded via 4-fluorocatechol, which was converted by an ortho-cleavage pathway.

  20. Genetic Diversity of African and Worldwide Strains of Ralstonia solanacearum as Determined by PCR-Restriction Fragment Length Polymorphism Analysis of the hrp Gene Region

    PubMed Central

    Poussier, Stephane; Vandewalle, Peggy; Luisetti, Jacques

    1999-01-01

    The genetic diversity among a worldwide collection of 120 strains of Ralstonia solanacearum was assessed by restriction fragment length polymorphism (RFLP) analysis of amplified fragments from the hrp gene region. Five amplified fragments appeared to be specific to R. solanacearum. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the hrp gene region confirmed the results of previous studies which split the species into an “Americanum” division including biovar 1 and 2 strains and an “Asiaticum” division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the “Asiaticum” rather than to the “Americanum” division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas. PMID:10224018

  1. Bacillus volatiles adversely affect the physiology and ultra-structure of Ralstonia solanacearum and induce systemic resistance in tobacco against bacterial wilt.

    PubMed

    Tahir, Hafiz Abdul Samad; Gu, Qin; Wu, Huijun; Niu, Yuedi; Huo, Rong; Gao, Xuewen

    2017-01-16

    Volatile organic compounds (VOCs) produced by various bacteria have significant potential to enhance plant growth and to control phytopathogens. Six of the most effective antagonistic Bacillus spp. were used in this study against Ralstonia solanacearum (Rsc) TBBS1, the causal agent of bacterial wilt disease in tobacco. Bacillus amyloliquefaciens FZB42 and Bacillus artrophaeus LSSC22 had the strongest inhibitory effect against Rsc. Thirteen VOCs produced by FZB42 and 10 by LSSC22 were identified using gas chromatography-mass spectrometry analysis. Benzaldehyde, 1,2-benzisothiazol-3(2 H)-one and 1,3-butadiene significantly inhibited the colony size, cell viability, and motility of pathogens and negatively influenced chemotaxis. Transmission and scanning electron microscopy revealed severe morphological and ultra-structural changes in cells of Rsc. Furthermore, VOCs altered the transcriptional expression level of PhcA (a global virulence regulator), type III secretion system (T3SS), type IV secretion system (T4SS), extracellular polysaccharides and chemotaxis-related genes, which are major contributors to pathogenicity, resulting in decreased wilt disease. The VOCs significantly up-regulated the expression of genes related to wilt resistance and pathogen defense. Over-expression of EDS1 and NPR1 suggest the involvement of SA pathway in induction of systemic resistance. Our findings provide new insights regarding the potential of antibacterial VOCs as a biocontrol tool against bacterial wilt diseases.

  2. Bacillus volatiles adversely affect the physiology and ultra-structure of Ralstonia solanacearum and induce systemic resistance in tobacco against bacterial wilt

    PubMed Central

    Tahir, Hafiz Abdul Samad; Gu, Qin; Wu, Huijun; Niu, Yuedi; Huo, Rong; Gao, Xuewen

    2017-01-01

    Volatile organic compounds (VOCs) produced by various bacteria have significant potential to enhance plant growth and to control phytopathogens. Six of the most effective antagonistic Bacillus spp. were used in this study against Ralstonia solanacearum (Rsc) TBBS1, the causal agent of bacterial wilt disease in tobacco. Bacillus amyloliquefaciens FZB42 and Bacillus artrophaeus LSSC22 had the strongest inhibitory effect against Rsc. Thirteen VOCs produced by FZB42 and 10 by LSSC22 were identified using gas chromatography-mass spectrometry analysis. Benzaldehyde, 1,2-benzisothiazol-3(2 H)-one and 1,3-butadiene significantly inhibited the colony size, cell viability, and motility of pathogens and negatively influenced chemotaxis. Transmission and scanning electron microscopy revealed severe morphological and ultra-structural changes in cells of Rsc. Furthermore, VOCs altered the transcriptional expression level of PhcA (a global virulence regulator), type III secretion system (T3SS), type IV secretion system (T4SS), extracellular polysaccharides and chemotaxis-related genes, which are major contributors to pathogenicity, resulting in decreased wilt disease. The VOCs significantly up-regulated the expression of genes related to wilt resistance and pathogen defense. Over-expression of EDS1 and NPR1 suggest the involvement of SA pathway in induction of systemic resistance. Our findings provide new insights regarding the potential of antibacterial VOCs as a biocontrol tool against bacterial wilt diseases. PMID:28091587

  3. Ralstonia solanacearum requires PopS, an ancient AvrE-family effector, for virulence and To overcome salicylic acid-mediated defenses during tomato pathogenesis.

    PubMed

    Jacobs, Jonathan M; Milling, Annett; Mitra, Raka M; Hogan, Clifford S; Ailloud, Florent; Prior, Philippe; Allen, Caitilyn

    2013-11-26

    During bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression of popS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in the R. solanacearum species complex. The phylogeny of popS mirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. A popS mutant of R. solanacearum UW551 had reduced virulence on agriculturally important Solanum spp., including potato and tomato plants. However, the popS mutant had wild-type virulence on a weed host, Solanum dulcamara, suggesting that some species can avoid the effects of PopS. The popS mutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, the popS mutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated the popS mutant virulence defect. Finally, the popS mutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which are R. solanacearum's natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed in Nicotiana leaf tissue, unlike the AvrE homolog DspEPcc from the necrotroph Pectobacterium carotovorum subsp. carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophic R. solanacearum. The type III-secreted AvrE effector family is widely distributed in high-impact plant-pathogenic bacteria and is known to suppress plant defenses for virulence. We

  4. Phylogeny based on 16S rDNA and nifH sequences of Ralstonia taiwanensis strains isolated from nitrogen-fixing nodules of Mimosa pudica, in India.

    PubMed

    Verma, Subhash Chandra; Chowdhury, Soumitra Paul; Tripathi, Anil Kumar

    2004-05-01

    Bacterial symbionts present in the indeterminate-type nitrogen (N)-fixing nodules of Mimosa pudica grown in North and South India showed maximum similarity to Ralstonia taiwanensis on the basis of carbon-source utilization patterns and 16S rDNA sequence. Isolates from the nodules of M. pudica from North India and South India showed identical ARDRA (Amplified Ribosomal DNA Restriction Analysis) patterns with Sau3AI and RsaI, but AluI revealed dimorphy between the North Indian and South Indian isolates. Alignment of 16S rDNA sequences revealed similarity of North Indian isolates with an R. taiwanensis strain isolated from M. pudica in Taiwan, whereas South Indian isolates showed closer relatedness with the isolates from Mimosa diplotricha. Alignment of nifH sequences from both North Indian and South Indian isolates with that of the related isolates revealed their closer affinity to alpha-rhizobia, suggesting that nif genes in the beta-rhizobia might have been acquired from alpha-rhizobia via lateral transfer during co-occupancy of nodules by alpha-rhizobia and progenitors of R. taiwanensis, members of the beta-subclass of Proteobacteria. Immunological cross-reaction of the bacteroid preparation of M. pudica nodules showed strong a positive signal with anti-dinitrogenase reductase antibody, whereas a weak positive cross-reaction was observed with free-living R. taiwanensis grown microaerobically in minimal medium with and without NH4Cl. In spite of the expression of dinitrogenase reductase under free-living conditions, acetylene reduction was not observed under N-free conditions even after prolonged incubation.

  5. Salicylate 5-Hydroxylase from Ralstonia sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase

    PubMed Central

    Zhou, Ning-Yi; Al-Dulayymi, Jumáa; Baird, Mark S.; Williams, Peter A.

    2002-01-01

    The genes from the oxygenase cluster nagAaGHAbAcAd of naphthalene-degrading Ralstonia sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed nagAaGHAb or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalyzed the 5-hydroxylation of the aromatic rings of 3- and 4-substituted salicylates. However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb). NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the “classical” naphthalene degrader P. putida NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2. PMID:11872705

  6. A novel multilocus variable number tandem repeat analysis typing scheme for African phylotype III strains of the Ralstonia solanacearum species complex

    PubMed Central

    Ravelomanantsoa, Santatra; Robène, Isabelle; Chiroleu, Frédéric; Guérin, Fabien; Poussier, Stéphane; Pruvost, Olivier

    2016-01-01

    Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven microsatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more discriminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC

  7. Overexpression of CaWRKY27, a subgroup IIe WRKY transcription factor of Capsicum annuum, positively regulates tobacco resistance to Ralstonia solanacearum infection.

    PubMed

    Dang, Fengfeng; Wang, Yuna; She, Jianju; Lei, Yufen; Liu, Zhiqin; Eulgem, Thomas; Lai, Yan; Lin, Jing; Yu, Lu; Lei, Dan; Guan, Deyi; Li, Xia; Yuan, Qian; He, Shuilin

    2014-03-01

    WRKY proteins are encoded by a large gene family and are linked to many biological processes across a range of plant species. The functions and underlying mechanisms of WRKY proteins have been investigated primarily in model plants such as Arabidopsis and rice. The roles of these transcription factors in non-model plants, including pepper and other Solanaceae, are poorly understood. Here, we characterize the expression and function of a subgroup IIe WRKY protein from pepper (Capsicum annuum), denoted as CaWRKY27. The protein localized to nuclei and activated the transcription of a reporter GUS gene construct driven by the 35S promoter that contained two copies of the W-box in its proximal upstream region. Inoculation of pepper cultivars with Ralstonia solanacearum induced the expression of CaWRKY27 transcript in 76a, a bacterial wilt-resistant pepper cultivar, whereas it downregulated the expression of CaWRKY27 transcript in Gui-1-3, a bacterial wilt-susceptible pepper cultivar. CaWRKY27 transcript levels were also increased by treatments with salicylic acid (SA), methyl jasmonate (MeJA) and ethephon (ETH). Transgenic tobacco plants overexpressing CaWRKY27 exhibited resistance to R. solanacearum infection compared to that of wild-type plants. This resistance was coupled with increased transcript levels in a number of marker genes, including hypersensitive response genes, and SA-, JA- and ET-associated genes. By contrast, virus-induced gene silencing (VIGS) of CaWRKY27 increased the susceptibility of pepper plants to R. solanacearum infection. These results suggest that CaWRKY27 acts as a positive regulator in tobacco resistance responses to R. solanacearum infection through modulation of SA-, JA- and ET-mediated signaling pathways. © 2013 Scandinavian Plant Physiology Society.

  8. CaWRKY58, encoding a group I WRKY transcription factor of Capsicum annuum, negatively regulates resistance to Ralstonia solanacearum infection.

    PubMed

    Wang, Yuna; Dang, Fengfeng; Liu, Zhiqin; Wang, Xu; Eulgem, Thomas; Lai, Yan; Yu, Lu; She, Jianju; Shi, Youliang; Lin, Jinhui; Chen, Chengcong; Guan, Deyi; Qiu, Ailian; He, Shuilin

    2013-02-01

    WRKY transcription factors are encoded by large gene families across the plant kingdom. So far, their biological and molecular functions in nonmodel plants, including pepper (Capsicum annuum) and other Solanaceae, remain poorly understood. Here, we report on the functional characterization of a new group I WRKY protein from pepper, termed CaWRKY58. Our data indicate that CaWRKY58 can be localized to the nucleus and can activate the transcription of the reporter β-glucuronidase (GUS) gene driven by the 35S core promoter with two copies of the W-box in its proximal upstream region. In pepper plants infected with the bacterial pathogen Ralstonia solanacearum, CaWRKY58 transcript levels showed a biphasic response, manifested in an early/transient down-regulation and late up-regulation. CaWRKY58 transcripts were suppressed by treatment with methyl jasmonate and abscisic acid. Tobacco plants overexpressing CaWRKY58 did not show any obvious morphological phenotypes, but exhibited disease symptoms of greater severity than did wild-type plants. The enhanced susceptibility of CaWRKY58-overexpressing tobacco plants correlated with the decreased expression of hypersensitive response marker genes, as well as various defence-associated genes. Consistently, CaWRKY58 pepper plants silenced by virus-induced gene silencing (VIGS) displayed enhanced resistance to the highly virulent R. solanacearum strain FJC100301, and this was correlated with enhanced transcripts of defence-related pepper genes. Our results suggest that CaWRKY58 acts as a transcriptional activator of negative regulators in the resistance of pepper to R. solanacearum infection. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

  9. Enhanced in planta Fitness through Adaptive Mutations in EfpR, a Dual Regulator of Virulence and Metabolic Functions in the Plant Pathogen Ralstonia solanacearum

    PubMed Central

    Rengel, David; Barlet, Xavier; Gouzy, Jérôme

    2016-01-01

    Experimental evolution of the plant pathogen Ralstonia solanacearum, where bacteria were maintained on plant lineages for more than 300 generations, revealed that several independent single mutations in the efpR gene from populations propagated on beans were associated with fitness gain on bean. In the present work, novel allelic efpR variants were isolated from populations propagated on other plant species, thus suggesting that mutations in efpR were not solely associated to a fitness gain on bean, but also on additional hosts. A transcriptomic profiling and phenotypic characterization of the efpR deleted mutant showed that EfpR acts as a global catabolic repressor, directly or indirectly down-regulating the expression of multiple metabolic pathways. EfpR also controls virulence traits such as exopolysaccharide production, swimming and twitching motilities and deletion of efpR leads to reduced virulence on tomato plants after soil drenching inoculation. We studied the impact of the single mutations that occurred in efpR during experimental evolution and found that these allelic mutants displayed phenotypic characteristics similar to the deletion mutant, although not behaving as complete loss-of-function mutants. These adaptive mutations therefore strongly affected the function of efpR, leading to an expanded metabolic versatility that should benefit to the evolved clones. Altogether, these results indicated that EfpR is a novel central player of the R. solanacearum virulence regulatory network. Independent mutations therefore appeared during experimental evolution in the evolved clones, on a crucial node of this network, to favor adaptation to host vascular tissues through regulatory and metabolic rewiring. PMID:27911943

  10. Effects of mutations in the substrate-binding domain of poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 on PHB degradation.

    PubMed

    Hiraishi, Tomohiro; Hirahara, Yoko; Doi, Yoshiharu; Maeda, Mizuo; Taguchi, Seiichi

    2006-11-01

    Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZ(RpiT1)) adsorbs to denatured PHB (dPHB) via its substrate-binding domain (SBD) to enhance dPHB degradation. To evaluate the amino acid residues participating in dPHB adsorption, PhaZ(RpiT1) was subjected to a high-throughput screening system consisting of PCR-mediated random mutagenesis targeted to the SBD gene and a plate assay to estimate the effects of mutations in the SBD on dPHB degradation by PhaZ(RpiT1). Genetic analysis of the isolated mutants with lowered activity showed that Ser, Tyr, Val, Ala, and Leu residues in the SBD were replaced by other residues at high frequency. Some of the mutant enzymes, which contained the residues replaced at high frequency, were applied to assays of dPHB degradation and adsorption, revealing that those residues are essential for full activity of both dPHB degradation and adsorption. These results suggested that PhaZ(RpiT1) adsorbs on the surface of dPHB not only via hydrogen bonds between hydroxyl groups of Ser in the enzyme and carbonyl groups in the PHB polymer but also via hydrophobic interaction between hydrophobic residues in the enzyme and methyl groups in the PHB polymer. The L441H enzyme, which displayed lower dPHB degradation and adsorption abilities, was purified and applied to a dPHB degradation assay to compare it with the wild-type enzyme. The kinetic analysis of the dPHB degradation suggested that lowering the affinity of the SBD towards dPHB causes a decrease in the dPHB degradation rate without the loss of its hydrolytic activity for the polymer chain.

  11. Moderate Temperature Fluctuations Rapidly Reduce the Viability of Ralstonia solanacearum Race 3, Biovar 2, in Infected Geranium, Tomato, and Potato Plants▿

    PubMed Central

    Scherf, Jacob M.; Milling, Annett; Allen, Caitilyn

    2010-01-01

    Most Ralstonia solanacearum strains are tropical plant pathogens, but race 3, biovar 2 (R3bv2), strains can cause bacterial wilt in temperate zones or tropical highlands where other strains cannot. R3bv2 is a quarantine pathogen in North America and Europe because of its potential to damage the potato industry in cooler climates. However, R3bv2 will not become established if it cannot survive temperate winters. Previous experiments showed that in water at 4°C, R3bv2 does not survive as long as native U.S. strains, but R3bv2 remains viable longer than U.S. strains in potato tubers at 4°C. To further investigate the effects of temperature on this high-concern pathogen, we assessed the ability of R3bv2 and a native U.S. strain to survive typical temperate winter temperature cycles of 2 days at 5°C followed by 2 days at −10°C. We measured pathogen survival in infected tomato and geranium plants, in infected potato tubers, and in sterile water. The population sizes of both strains declined rapidly under these conditions in all three plant hosts and in sterile water, and no culturable R. solanacearum cells were detected after five to seven temperature cycles in plant tissue. The fluctuations played a critical role in loss of bacterial viability, since at a constant temperature of −20°C, both strains could survive in infected geranium tissue for at least 6 months. These results suggest that even when sheltered in infected plant tissue, R3bv2 is unlikely to survive the temperature fluctuations typical of a northern temperate winter. PMID:20851983

  12. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000

    PubMed Central

    Lundgren, Benjamin R.; Connolly, Morgan P.; Choudhary, Pratibha; Brookins-Little, Tiffany S.; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T.

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000. PMID:26659655

  13. Autoacetylation of the Ralstonia solanacearum Effector PopP2 Targets a Lysine Residue Essential for RRS1-R-Mediated Immunity in Arabidopsis

    PubMed Central

    Tasset, Céline; Bernoux, Maud; Jauneau, Alain; Pouzet, Cécile; Brière, Christian; Kieffer-Jacquinod, Sylvie; Rivas, Susana

    2010-01-01

    Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as “guards”. The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity. PMID:21124938

  14. Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism.

    PubMed

    Denancé, Nicolas; Ranocha, Philippe; Oria, Nicolas; Barlet, Xavier; Rivière, Marie-Pierre; Yadeta, Koste A; Hoffmann, Laurent; Perreau, François; Clément, Gilles; Maia-Grondard, Alessandra; van den Berg, Grardy C M; Savelli, Bruno; Fournier, Sylvie; Aubert, Yann; Pelletier, Sandra; Thomma, Bart P H J; Molina, Antonio; Jouanin, Lise; Marco, Yves; Goffner, Deborah

    2013-01-01

    Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  15. Application of Variable-Number Tandem-Repeat Typing To Discriminate Ralstonia solanacearum Strains Associated with English Watercourses and Disease Outbreaks

    PubMed Central

    Bryant, Ruth; Bew, Janice; Conyers, Christine; Stones, Robert; Alcock, Michael; Elphinstone, John

    2013-01-01

    Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks. PMID:23892739

  16. Development and Comparison of TaqMan-Based Real-Time PCR Assays for Detection and Differentiation of Ralstonia solanacearum strains.

    PubMed

    Stulberg, Michael J; Rascoe, John; Li, Wenbin; Yan, Zonghe; Nakhla, Mark K; Huang, Qi

    2016-10-01

    Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperate climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is commonly used in federal and state diagnostic laboratories over conventional PCR due to its speed and sensitivity. We developed the Rs16S primers and probe set and compared it with a widely used set (RS) for detecting R. solanacearum species complex strains. We also developed the RsSA3 primers and probe set and compared it with the previously published B2 and RsSA2 sets for specific detection of r3b2 strains. Both comparisons were done under standardized qPCR master mix and cycling conditions. The Rs16S and RS assays detected all 90 R. solanacearum species complex strains and none of the five outgroups, but the former was more sensitive than the latter. For r3b2 strain detection, the RsSA2 and RsSA3 sets specifically detected the 34 r3b2 strains and none of the 56 R. solanacearum non-r3b2 strains or out-group strains. The B2 set, however, detected five non-r3b2 R. solanacearum strains and was less sensitive than the other two sets under the same testing conditions. We conclude that the Rs16S, RsSA2, and RsSA3 sets are best suited under the standardized conditions for the detection of R. solanacearum species complex and r3b2 strains by TaqMan-based qPCR assays.

  17. Enhanced in planta Fitness through Adaptive Mutations in EfpR, a Dual Regulator of Virulence and Metabolic Functions in the Plant Pathogen Ralstonia solanacearum.

    PubMed

    Perrier, Anthony; Peyraud, Rémi; Rengel, David; Barlet, Xavier; Lucasson, Emmanuel; Gouzy, Jérôme; Peeters, Nemo; Genin, Stéphane; Guidot, Alice

    2016-12-01

    Experimental evolution of the plant pathogen Ralstonia solanacearum, where bacteria were maintained on plant lineages for more than 300 generations, revealed that several independent single mutations in the efpR gene from populations propagated on beans were associated with fitness gain on bean. In the present work, novel allelic efpR variants were isolated from populations propagated on other plant species, thus suggesting that mutations in efpR were not solely associated to a fitness gain on bean, but also on additional hosts. A transcriptomic profiling and phenotypic characterization of the efpR deleted mutant showed that EfpR acts as a global catabolic repressor, directly or indirectly down-regulating the expression of multiple metabolic pathways. EfpR also controls virulence traits such as exopolysaccharide production, swimming and twitching motilities and deletion of efpR leads to reduced virulence on tomato plants after soil drenching inoculation. We studied the impact of the single mutations that occurred in efpR during experimental evolution and found that these allelic mutants displayed phenotypic characteristics similar to the deletion mutant, although not behaving as complete loss-of-function mutants. These adaptive mutations therefore strongly affected the function of efpR, leading to an expanded metabolic versatility that should benefit to the evolved clones. Altogether, these results indicated that EfpR is a novel central player of the R. solanacearum virulence regulatory network. Independent mutations therefore appeared during experimental evolution in the evolved clones, on a crucial node of this network, to favor adaptation to host vascular tissues through regulatory and metabolic rewiring.

  18. Ralstonia solanacearum RSc0411 (lptC) is a determinant for full virulence and has a strain-specific novel function in the T3SS activity.

    PubMed

    Yang, Wen-Chieh; Lin, Yu-Mei; Cheng, Yi-Sheng; Cheng, Chiu-Ping

    2013-06-01

    Previously, we have identified an avirulent Ralstonia solanacearum mutant carrying a transposon insertion in RSc0411, a gene homologous to the Escherichia coli LPS-transporting protein LptC. However, how the disruption of RSc0411 affects the bacterium-plant interactions and leads to decreased pathogenicity was not known. Here we show that the disruption of RSc0411 leads to pleiotropic defects, including reducing bacterial motility, biofilm formation, root attachment, rough-form LPS production and virulence in tomato and increasing membrane permeability. Disruption of the orthologous RSc0411 present in other R. solanacearum strains proves that most of these functions are conserved in the species. In contrast, trans-complementation analyses show that only RSc0411 orthologues from closely related bacteria can rescue the defects of the disruption mutant. These results enable us to propose a function for RSc0411, and for the clustered genes, in LPS biogenesis, and for the first time, to our knowledge, also a role of a gene from the DUF1239 gene family in bacterial pathogenicity. In addition and notably, the RSc0411 mutant displays a strain-specific phenotype for hypersensitive response (HR), in which the RSc0411 disruption impairs the HR caused by strain Pss190 but not that by strain Pss1308. Consistent with this strain-specific defect, the mutation clearly affects expression of the type III secretion system (T3SS) in Pss190 but not in other strains, suggesting that the HR-deficient phenotype of the RSc0411 mutant in Pss190 is due to impairment of the T3SS and thus RSc0411 has a strain-specific role in the T3SS activity of R. solanacearum.

  19. Ralstonia solanacearum Type III Effector RipAY Is a Glutathione-Degrading Enzyme That Is Activated by Plant Cytosolic Thioredoxins and Suppresses Plant Immunity

    PubMed Central

    Hatanaka, Tadashi; Nakano, Masahito; Oda, Kenji

    2016-01-01

    ABSTRACT The plant pathogen Ralstonia solanacearum uses a large repertoire of type III effector proteins to succeed in infection. To clarify the function of effector proteins in host eukaryote cells, we expressed effectors in yeast cells and identified seven effector proteins that interfere with yeast growth. One of the effector proteins, RipAY, was found to share homology with the ChaC family proteins that function as γ-glutamyl cyclotransferases, which degrade glutathione (GSH), a tripeptide that plays important roles in the plant immune system. RipAY significantly inhibited yeast growth and simultaneously induced rapid GSH depletion when expressed in yeast cells. The in vitro GSH degradation activity of RipAY is specifically activated by eukaryotic factors in the yeast and plant extracts. Biochemical purification of the yeast protein identified that RipAY is activated by thioredoxin TRX2. On the other hand, RipAY was not activated by bacterial thioredoxins. Interestingly, RipAY was activated by plant h-type thioredoxins that exist in large amounts in the plant cytosol, but not by chloroplastic m-, f-, x-, y- and z-type thioredoxins, in a thiol-independent manner. The transient expression of RipAY decreased the GSH level in plant cells and affected the flg22-triggered production of reactive oxygen species (ROS) and expression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes in Nicotiana benthamiana leaves. These results indicate that RipAY is activated by host cytosolic thioredoxins and degrades GSH specifically in plant cells to suppress plant immunity. PMID:27073091

  20. Ralstonia solanacearum Requires PopS, an Ancient AvrE-Family Effector, for Virulence and To Overcome Salicylic Acid-Mediated Defenses during Tomato Pathogenesis

    PubMed Central

    Jacobs, Jonathan M.; Milling, Annett; Mitra, Raka M.; Hogan, Clifford S.; Ailloud, Florent; Prior, Philippe; Allen, Caitilyn

    2013-01-01

    ABSTRACT During bacterial wilt of tomato, the plant pathogen Ralstonia solanacearum upregulates expression of popS, which encodes a type III-secreted effector in the AvrE family. PopS is a core effector present in all sequenced strains in the R. solanacearum species complex. The phylogeny of popS mirrors that of the species complex as a whole, suggesting that this is an ancient, vertically inherited effector needed for association with plants. A popS mutant of R. solanacearum UW551 had reduced virulence on agriculturally important Solanum spp., including potato and tomato plants. However, the popS mutant had wild-type virulence on a weed host, Solanum dulcamara, suggesting that some species can avoid the effects of PopS. The popS mutant was also significantly delayed in colonization of tomato stems compared to the wild type. Some AvrE-type effectors from gammaproteobacteria suppress salicylic acid (SA)-mediated plant defenses, suggesting that PopS, a betaproteobacterial ortholog, has a similar function. Indeed, the popS mutant induced significantly higher expression of tomato SA-triggered pathogenesis-related (PR) genes than the wild type. Further, pretreatment of roots with SA exacerbated the popS mutant virulence defect. Finally, the popS mutant had no colonization defect on SA-deficient NahG transgenic tomato plants. Together, these results indicate that this conserved effector suppresses SA-mediated defenses in tomato roots and stems, which are R. solanacearum’s natural infection sites. Interestingly, PopS did not trigger necrosis when heterologously expressed in Nicotiana leaf tissue, unlike the AvrE homolog DspEPcc from the necrotroph Pectobacterium carotovorum subsp. carotovorum. This is consistent with the differing pathogenesis modes of necrosis-causing gammaproteobacteria and biotrophic R. solanacearum. PMID:24281716

  1. HpaP modulates type III effector secretion in Ralstonia solanacearum and harbours a substrate specificity switch domain essential for virulence.

    PubMed

    Lohou, David; Turner, Marie; Lonjon, Fabien; Cazalé, Anne-Claire; Peeters, Nemo; Genin, Stéphane; Vailleau, Fabienne

    2014-08-01

    Many pathogenic bacteria have evolved a type III secretion system (T3SS) to successfully invade their host. This extracellular apparatus allows the translocation of proteins, called type III effectors (T3Es), directly into the host cells. T3Es are virulence factors that have been shown to interfere with the host's immunity or to provide nutrients from the host to the bacteria. The Gram-negative bacterium Ralstonia solanacearum is a worldwide major crop pest whose virulence strongly relies on the T3SS. In R. solanacearum, transcriptional regulation has been extensively studied. However, very few data are available concerning the role played by type III-associated regulators, such as type III chaperones and T3SS control proteins. Here, we characterized HpaP, a putative type III secretion substrate specificity switch (T3S4) protein of R. solanacearum which is not secreted by the bacterium or translocated in the plant cells. HpaP self-interacts and interacts with the PopP1 T3E. HpaP modulates the secretion of early (HrpY pilin) and late (AvrA and PopP1 T3Es) type III substrates. HpaP is dispensable for the translocation of T3Es into the host cells. Finally, we identified two regions of five amino acids in the T3S4 domain that are essential for efficient PopP1 secretion and for HpaP's role in virulence on tomato and Arabidopsis thaliana, but not required for HpaP-HpaP and HpaP-PopP1 interactions. Taken together, our results indicate that HpaP is a putative R. solanacearum T3S4 protein important for full pathogenicity on several hosts, acting as a helper for PopP1 secretion, and repressing AvrA and HrpY secretion. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  2. Induction of the Viable but Nonculturable State of Ralstonia solanacearum by Low Temperature in the Soil Microcosm and Its Resuscitation by Catalase

    PubMed Central

    Kong, Hyun Gi; Bae, Ju Young; Lee, Hyoung Ju; Joo, Hae Jin; Jung, Eun Joo; Chung, Eunsook; Lee, Seon-Woo

    2014-01-01

    Ralstonia solanacearum is the causal agent of bacterial wilt on a wide variety of plants, and enters a viable but nonculturable (VBNC) state under stress conditions in soil and water. Here, we adopted an artificial soil microcosm (ASM) to investigate the VBNC state of R. solanacearum induced by low temperature. The culturability of R. solanacearum strains SL341 and GMI1000 rapidly decreased at 4°C in modified ASM (mASM), while it was stably maintained at 25°C in mASM. We hypothesized that bacterial cells at 4°C in mASM are viable but nonculturable. Total protein profiles of SL341 cells at 4°C in mASM did not differ from those of SL341 culturable cells at 25°C in mASM. Moreover, the VBNC cells maintained in the mASM retained respiration activity. Catalase treatment effectively restored the culturability of nonculturable cells in mASM, while temperature increase or other treatments used for resuscitation of other bacteria were not effective. The resuscitated R. solanacearum from VBNC state displayed normal level of bacterial virulence on tomato plants compared with its original culturable bacteria. Expression of omp, oxyR, rpoS, dps, and the 16S rRNA gene quantified by RT-qPCR did not differ significantly between the culturable and VBNC states of R. solanacearum. Our results suggested that the VBNC bacterial cells in mASM induced by low temperature exist in a physiologically unique state. PMID:25296177

  3. Application of variable-number tandem-repeat typing to discriminate Ralstonia solanacearum strains associated with English watercourses and disease outbreaks.

    PubMed

    Parkinson, Neil; Bryant, Ruth; Bew, Janice; Conyers, Christine; Stones, Robert; Alcock, Michael; Elphinstone, John

    2013-10-01

    Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks.

  4. CaWRKY6 transcriptionally activates CaWRKY40, regulates Ralstonia solanacearum resistance, and confers high-temperature and high-humidity tolerance in pepper.

    PubMed

    Cai, Hanyang; Yang, Sheng; Yan, Yan; Xiao, Zhuoli; Cheng, Junbin; Wu, Ji; Qiu, Ailian; Lai, Yan; Mou, Shaoliang; Guan, Deyi; Huang, Ronghua; He, Shuilin

    2015-06-01

    High temperature (HT), high humidity (HH), and pathogen infection often co-occur and negatively affect plant growth. However, these stress factors and plant responses are generally studied in isolation. The mechanisms of synergistic responses to combined stresses are poorly understood. We isolated the subgroup IIb WRKY family member CaWRKY6 from Capsicum annuum and performed quantitative real-time PCR analysis. CaWRKY6 expression was upregulated by individual or simultaneous treatment with HT, HH, combined HT and HH (HTHH), and Ralstonia solanacearum inoculation, and responded to exogenous application of jasmonic acid (JA), ethephon, and abscisic acid (ABA). Virus-induced gene silencing of CaWRKY6 enhanced pepper plant susceptibility to R. solanacearum and HTHH, and downregulated the hypersensitive response (HR), JA-, ethylene (ET)-, and ABA-induced marker gene expression, and thermotolerance-associated expression of CaHSP24, ER-small CaSHP, and Chl-small CaHSP. CaWRKY6 overexpression in pepper attenuated the HTHH-induced suppression of resistance to R. solanacearum infection. CaWRKY6 bound to and activated the CaWRKY40 promoter in planta, which is a pepper WRKY that regulates heat-stress tolerance and R. solanacearum resistance. CaWRKY40 silencing significantly blocked HR-induced cell death and reduced transcriptional expression of CaWRKY40. These data suggest that CaWRKY6 is a positive regulator of R. solanacearum resistance and heat-stress tolerance, which occurs in part by activating CaWRKY40. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  5. Induction of the viable but nonculturable state of Ralstonia solanacearum by low temperature in the soil microcosm and its resuscitation by catalase.

    PubMed

    Kong, Hyun Gi; Bae, Ju Young; Lee, Hyoung Ju; Joo, Hae Jin; Jung, Eun Joo; Chung, Eunsook; Lee, Seon-Woo

    2014-01-01

    Ralstonia solanacearum is the causal agent of bacterial wilt on a wide variety of plants, and enters a viable but nonculturable (VBNC) state under stress conditions in soil and water. Here, we adopted an artificial soil microcosm (ASM) to investigate the VBNC state of R. solanacearum induced by low temperature. The culturability of R. solanacearum strains SL341 and GMI1000 rapidly decreased at 4°C in modified ASM (mASM), while it was stably maintained at 25°C in mASM. We hypothesized that bacterial cells at 4°C in mASM are viable but nonculturable. Total protein profiles of SL341 cells at 4°C in mASM did not differ from those of SL341 culturable cells at 25°C in mASM. Moreover, the VBNC cells maintained in the mASM retained respiration activity. Catalase treatment effectively restored the culturability of nonculturable cells in mASM, while temperature increase or other treatments used for resuscitation of other bacteria were not effective. The resuscitated R. solanacearum from VBNC state displayed normal level of bacterial virulence on tomato plants compared with its original culturable bacteria. Expression of omp, oxyR, rpoS, dps, and the 16S rRNA gene quantified by RT-qPCR did not differ significantly between the culturable and VBNC states of R. solanacearum. Our results suggested that the VBNC bacterial cells in mASM induced by low temperature exist in a physiologically unique state.

  6. Deciphering the route of Ralstonia solanacearum colonization in Arabidopsis thaliana roots during a compatible interaction: focus at the plant cell wall.

    PubMed

    Digonnet, Catherine; Martinez, Yves; Denancé, Nicolas; Chasseray, Marine; Dabos, Patrick; Ranocha, Philippe; Marco, Yves; Jauneau, Alain; Goffner, Deborah

    2012-11-01

    The compatible interaction between the model plant, Arabidopsis thaliana, and the GMI1000 strain of the phytopathogenic bacterium, Ralstonia solanacearum, was investigated in an in vitro pathosystem. We describe the progression of the bacteria in the root from penetration at the root surface to the xylem vessels and the cell type-specific, cell wall-associated modifications that accompanies bacterial colonization. Within 6 days post inoculation, R. solanacearum provoked a rapid plasmolysis of the epidermal, cortical, and endodermal cells, including those not directly in contact with the bacteria. Plasmolysis was accompanied by a global degradation of pectic homogalacturonanes as shown by the loss of JIM7 and JIM5 antibody signal in the cell wall of these cell types. As indicated by immunolabeling with Rsol-I antibodies that specifically recognize R. solanacearum, the bacteria progresses through the root in a highly directed, centripetal manner to the xylem poles, without extensive multiplication in the intercellular spaces along its path. Entry into the vascular cylinder was facilitated by cell collapse of the two pericycle cells located at the xylem poles. Once the bacteria reached the xylem vessels, they multiplied abundantly and moved from vessel to vessel by digesting the pit membrane between adjacent vessels. The degradation of the secondary walls of xylem vessels was not a prerequisite for vessel colonization as LM10 antibodies strongly labeled xylem cell walls, even at very late stages in disease development. Finally, the capacity of R. solanacearum to specifically degrade certain cell wall components and not others could be correlated with the arsenal of cell wall hydrolytic enzymes identified in the bacterial genome.

  7. Evidence of aerobic utilization of di-ortho-substituted trichlorobiphenyls as growth substrates by Pseudomonas sp. SA-6 and Ralstonia sp. SA-4.

    PubMed

    Adebusoye, Sunday A; Picardal, Flynn W; Ilori, Matthew O; Amund, Olukayode O

    2008-05-01

    Robust and effective bioremediation strategies have not yet been developed for polychlorinated biphenyl (PCB)-contaminated soils. This is in part a result of the fact that ortho- or ortho- and para-substituted congeners, frequent dead-end products of reductive dechlorination of PCB mixtures, have greatly reduced aerobic biodegradability. In this study, we report substantial evidence of utilization of diortho-substituted trichlorobiphenyls (triCBs) as growth substrates by Ralstonia sp. SA-4 and Pseudomonas sp. SA-6 in which ortho-substitution resulted in no obvious patterns of recalcitrance. These stains exhibited unusual preferences for growth on congeners chlorinated on both rings. Substrate uptake studies with benzoate-grown cells revealed that the isolates attacked the 2-chlorophenyl rings of 2,2',4- and 2,2',5-triCB. Between 71% and 93% of the initial 0.23-0.34 mM dose of congeners were transformed in less than 261 h concomitant with non-stoichiometric production of respective dichlorobenzoates and chloride ion. In enzyme assays, activity of 2,3-dihydroxybiphenyl-1,2-dioxygenase was constitutive. Additionally, these strains harboured no detectable plasmids which, coupled with exponential growth on the two triCB congeners, suggested chromosomal location of PCB degradative genes. In addition to the fact that there is a paucity of information on degradation of PCBs by tropical isolates, growth on triCBs as a sole carbon and energy source has never been demonstrated for any natural or engineered microorganisms. Such isolates may help prevent accumulation of ortho-substituted congeners in natural systems and offer the hope for development of effective bioaugmentation or sequential anaerobic-aerobic bioremediation strategies.

  8. Abundance of Dioxygenase Genes Similar to Ralstonia sp. Strain U2 nagAc Is Correlated with Naphthalene Concentrations in Coal Tar-Contaminated Freshwater Sediments

    PubMed Central

    Dionisi, Hebe M.; Chewning, Christopher S.; Morgan, Katherine H.; Menn, Fu-Min; Easter, James P.; Sayler, Gary S.

    2004-01-01

    We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-μl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 ± 0.7) × 103 to (2.9 ± 0.3) × 105 copies of nagAc-like dioxygenase genes per μg of DNA extracted from sediment samples. These values corresponded to (1.2 ± 0.6) × 105 to (5.4 ± 0.4) × 107 copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene. PMID:15240274

  9. An evaluation of the wilt-causing bacterium Ralstonia solanacearum as a potential biological control agent for the alien Kahili ginger (Hedychium gardnerianum) in Hawaiian forests

    USGS Publications Warehouse

    1999-01-01

    Kahili ginger (Hedychium gardnerianum) is an invasive weed in tropical forests in Hawaii and elsewhere. Bacterial wilt caused by the ginger strain of Ralstonia(=Pseudomonas) solanacearum systemically infects edible ginger (Zingiber officinale) and ornamental gingers (Hedychium spp.), causing wilt in infected plants. The suitability of R. solanacearum as a biological control agent for kahili ginger was investigated by inoculating seedlings and rooted cuttings of native forest plants, ornamental ginger, and solanaceous species to confirm host specificity. Inoculation via stem injection or root wounding with a bacterial–water suspension was followed by observation for 8 weeks. Inoculations on H. gardnerianum were then carried out in ohia-lehua (Metrosideros polymorpha) wet forests of Hawaii Volcanoes National Park to determine the bacterium's efficacy in the field. No native forest or solanaceous species developed wilt or other symptoms during the study. The bacterium caused limited infection near the inoculation site on H. coronarium, Z. zerumbet, Heliconia latispatha, and Musa sapientum. However, infection did not become systemic in any of these species, and normal growth resumed following appearance of initial symptoms. All inoculated H. gardnerianum plants developed irreversible chlorosis and severe wilting 3–4 weeks following inoculation. Systemic infection also caused death and decay of rhizomes. Most plants were completely dead 16–20 weeks following inoculation. The destructiveness of the ginger strain of R. solanacearum to edible ginger has raised questions regarding its use for biological control. However, because locations of kahili ginger infestations are often remote, the risk of contaminating edible ginger plantings is unlikely. The ability of this bacterium to cause severe disease in H. gardnerianum in the field, together with its lack of virulence in other ginger species, contributes to its potential as a biological control agent.

  10. The vascular plant-pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces.

    PubMed

    Mori, Yuka; Inoue, Kanako; Ikeda, Kenichi; Nakayashiki, Hitoshi; Higashimoto, Chikaki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

    2016-08-01

    The mechanism of colonization of intercellular spaces by the soil-borne and vascular plant-pathogenic bacterium Ralstonia solanacearum strain OE1-1 after invasion into host plants remains unclear. To analyse the behaviour of OE1-1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1-1 were observed under a scanning electron microscope. OE1-1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1-1 cells produced mushroom-type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom-type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1-1. Mutation of lecM encoding a lectin, RS-IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom-type biofilms and virulence on tomato plants. Together, our findings indicate that OE1-1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS-IIL may contribute to biofilm formation by OE1-1, which is required for OE1-1 virulence. © 2015 BSPP AND JOHN WILEY & SONS LTD.

  11. rpoN1, but not rpoN2, is required for twitching motility, natural competence, growth on nitrate, and virulence of Ralstonia solanacearum

    PubMed Central

    Ray, Suvendra K.; Kumar, Rahul; Peeters, Nemo; Boucher, Christian; Genin, Stephane

    2015-01-01

    The plant pathogen Ralstonia solanacearum has two genes encoding for the sigma factor σ54: rpoN1, located in the chromosome and rpoN2, located in a distinct “megaplasmid” replicon. In this study, individual mutants as well as a double mutant of rpoN were created in R. solanacearum strain GMI1000 in order to determine the extent of functional overlap between these two genes. By virulence assay we observed that rpoN1 is required for virulence whereas rpoN2 is not. In addition rpoN1 controls other important functions such twitching motility, natural transformation and growth on nitrate, unlike rpoN2. The rpoN1 and rpoN2 genes have different expression pattern, the expression of rpoN1 being constitutive whereas rpoN2 expression is induced in minimal medium and in the presence of plant cells. Moreover, the expression of rpoN2 is dependent upon rpoN1. Our work therefore reveals that the two rpoN genes are not functionally redundant in R. solanacearum. A list of potential σ54 targets was identified in the R. solanacearum genome and suggests that multiple traits are under the control of these regulators. Based on these findings, we provide a model describing the functional connection between RpoN1 and the PehR pathogenicity regulator and their dual role in the control of several R. solanacearum virulence determinants. PMID:25852679

  12. Novel Gene Clusters and Metabolic Pathway Involved in 3,5,6-Trichloro-2-Pyridinol Degradation by Ralstonia sp. Strain T6

    PubMed Central

    Li, Jingquan; Huang, Yan; Hou, Ying; Li, Xiangmin; Cao, Hui

    2013-01-01

    3,5,6-Trichloro-2-pyridinol (TCP) is a widespread pollutant. Some bacteria and fungi have been reported to degrade TCP, but the gene clusters responsible for TCP biodegradation have not been characterized. In this study, a fragment of the reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase gene tcpA was amplified from the genomic DNA of Ralstonia sp. strain T6 with degenerate primers. The tcpA disruption mutant strain T6-ΔtcpA could not degrade TCP but could degrade the green intermediate metabolite 3,6-dihydroxypyridine-2,5-dione (DHPD), which was generated during TCP biodegradation by strain T6. The flanking sequences of tcpA were obtained by self-formed adaptor PCR. tcpRXA genes constitute a gene cluster. TcpR and TcpX are closely related to the LysR family transcriptional regulator and flavin reductase, respectively. T6-ΔtcpA-com, the complementation strain for the mutant strain T6-ΔtcpA, recovered the ability to degrade TCP, and the strain Escherichia coli DH10B-tcpRXA, which expressed the tcpRXA gene cluster, had the ability to transform TCP to DHPD, indicating that tcpA is a key gene in the initial step of TCP degradation and that TcpA dechlorinates TCP to DHPD. A library of DHPD degradation-deficient mutants of strain T6 was obtained by random transposon mutagenesis. The fragments flanking the Mariner transposon were amplified and sequenced, and the dhpRIJK gene cluster was cloned. DhpJ could transform DHPD to yield an intermediate product, 5-amino-2,4,5-trioxopentanoic acid (ATOPA), which was further degraded by DhpI. DhpR and DhpK are closely related to the AraC family transcriptional regulator and the MFS family transporter, respectively. PMID:24056464

  13. CaCDPK15 positively regulates pepper responses to Ralstonia solanacearum inoculation and forms a positive-feedback loop with CaWRKY40 to amplify defense signaling

    PubMed Central

    Shen, Lei; Yang, Sheng; Yang, Tong; Liang, Jiaqi; Cheng, Wei; Wen, Jiayu; Liu, Yanyan; Li, Jiazhi; Shi, Lanping; Tang, Qian; Shi, Wei; Hu, Jiong; Liu, Cailing; Zhang, Yangwen; Mou, Shaoliang; Liu, Zhiqin; Cai, Hanyang; He, Li; Guan, Deyi; Wu, Yang; He, Shuilin

    2016-01-01

    CaWRKY40 is a positive regulator of pepper (Capsicum annum) response to Ralstonia solanacearum inoculation (RSI), but the underlying mechanism remains largely unknown. Here, we functionally characterize CaCDPK15 in the defense signaling mediated by CaWRKY40. Pathogen-responsive TGA, W, and ERE boxes were identified in the CaCDPK15 promoter (pCaCDPK15), and pCaCDPK15-driven GUS expression was significantly enhanced in response to RSI and exogenously applied salicylic acid, methyl jasmonate, abscisic acid, and ethephon. Virus-induced gene silencing (VIGS) of CaCDPK15 significantly increased the susceptibility of pepper to RSI and downregulated the immunity-associated markers CaNPR1, CaPR1, and CaDEF1. By contrast, transient CaCDPK15 overexpression significantly activated hypersensitive response associated cell death, upregulated the immunity-associated marker genes, upregulated CaWRKY40 expression, and enriched CaWRKY40 at the promoters of its targets genes. Although CaCDPK15 failed to interact with CaWRKY40, the direct binding of CaWRKY40 to pCaCDPK15 was detected by chromatin immunoprecipitation, which was significantly potentiated by RSI in pepper plants. These combined results suggest that RSI in pepper induces CaCDPK15 and indirectly activates downstream CaWRKY40, which in turn potentiates CaCDPK15 expression. This positive-feedback loop would amplify defense signaling against RSI and efficiently activate strong plant immunity. PMID:26928570

  14. Defining the Metabolic Functions and Roles in Virulence of the rpoN1 and rpoN2 Genes in Ralstonia solanacearum GMI1000.

    PubMed

    Lundgren, Benjamin R; Connolly, Morgan P; Choudhary, Pratibha; Brookins-Little, Tiffany S; Chatterjee, Snigdha; Raina, Ramesh; Nomura, Christopher T

    2015-01-01

    The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

  15. A novel multilocus variable number tandem repeat analysis typing scheme for African phylotype III strains of the Ralstonia solanacearum species complex.

    PubMed

    Ravelomanantsoa, Santatra; Robène, Isabelle; Chiroleu, Frédéric; Guérin, Fabien; Poussier, Stéphane; Pruvost, Olivier; Prior, Philippe

    2016-01-01

    Background. Reliable genotyping that provides an accurate description of diversity in the context of pathogen emergence is required for the establishment of strategies to improve disease management. MultiLocus variable number tandem repeat analysis (MLVA) is a valuable genotyping method. It can be performed at small evolutionary scales where high discriminatory power is needed. Strains of the Ralstonia solanacearum species complex (RSSC) are highly genetically diverse. These destructive pathogens are the causative agent of bacterial wilt on an unusually broad range of host plants worldwide. In this study, we developed an MLVA scheme for genotyping the African RSSC phylotype III. Methods. We selected different publicly available tandem repeat (TR) loci and additional TR loci from the genome of strain CMR15 as markers. Based on these loci, a new phylotype III-MLVA scheme is presented. MLVA and multiLocus sequence typing (MLST) were compared at the global, regional, and local scales. Different populations of epidemiologically related and unrelated RSSC phylotype III strains were used. Results and Discussion. Sixteen polymorphic TR loci, which included seven microsatellites and nine minisatellites, were selected. These TR loci were distributed throughout the genome (chromosome and megaplasmid) and located in both coding and intergenic regions. The newly developed RS3-MLVA16 scheme was more discriminative than MLST. RS3-MLVA16 showed good ability in differentiating strains at global, regional, and local scales, and it especially highlighted epidemiological links between closely related strains at the local scale. RS3-MLVA16 also underlines genetic variability within the same MLST-type and clonal complex, and gives a first overview of population structure. Overall, RS3-MLVA16 is a promising genotyping method for outbreak investigation at a fine scale, and it could be used for outbreak investigation as a first-line, low-cost assay for the routine screening of RSSC

  16. Overexpression of a novel peanut NBS-LRR gene AhRRS5 enhances disease resistance to Ralstonia solanacearum in tobacco.

    PubMed

    Zhang, Chong; Chen, Hua; Cai, Tiecheng; Deng, Ye; Zhuang, Ruirong; Zhang, Ning; Zeng, Yuanhuan; Zheng, Yixiong; Tang, Ronghua; Pan, Ronglong; Zhuang, Weijian

    2017-01-01

    Bacterial wilt caused by Ralstonia solanacearum is a ruinous soilborne disease affecting more than 450 plant species. Efficient control methods for this disease remain unavailable to date. This study characterized a novel nucleotide-binding site-leucine-rich repeat resistance gene AhRRS5 from peanut, which was up-regulated in both resistant and susceptible peanut cultivars in response to R. solanacearum. The product of AhRRS5 was localized in the nucleus. Furthermore, treatment with phytohormones such as salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA) and ethephon (ET) increased the transcript level of AhRRS5 with diverse responses between resistant and susceptible peanuts. Abiotic stresses such as drought and cold conditions also changed AhRRS5 expression. Moreover, transient overexpression induced hypersensitive response in Nicotiana benthamiana. Overexpression of AhRRS5 significantly enhanced the resistance of heterogeneous tobacco to R. solanacearum, with diverse resistance levels in different transgenic lines. Several defence-responsive marker genes in hypersensitive response, including SA, JA and ET signals, were considerably up-regulated in the transgenic lines as compared with the wild type inoculated with R. solanacearum. Nonexpressor of pathogenesis-related gene 1 (NPR1) and non-race-specific disease resistance 1 were also up-regulated in response to the pathogen. These results indicate that AhRRS5 participates in the defence response to R. solanacearum through the crosstalk of multiple signalling pathways and the involvement of NPR1 and R gene signals for its resistance. This study may guide the resistance enhancement of peanut and other economic crops to bacterial wilt disease.

  17. Detection of Ralstonia solanacearum from asymptomatic tomato plants, irrigation water, and soil through non-selective enrichment medium with hrp gene-based bio-PCR.

    PubMed

    Singh, Dinesh; Sinha, Shweta; Yadav, D K; Chaudhary, Garima

    2014-08-01

    Bacterial wilt of tomato caused by Ralstonia solanacearum (Smith) Yabuuchi et al. (Microbiol Immunol 39:897-904, 1995) is a serious disease, which causes losses up to 60 % depending on environmental conditions, soil property, and cultivars. In present investigation, nucleotide sequences of virulence, hypersensitive response and pathogenicity (hrp) gene were used to design a pair of primer (Hrp_rs 2F: 5'-AGAGGTCGACGCGATACAGT-3' and Hrp_rs 2R: 5'-CATGAGCAAGGACGAAGTCA-3') for amplification of bacterial genome. The genomic DNA of 27 isolates of R. solanacearum race 1 biovar 3 & 4 was amplified at 323 bp. The specificity of primer was tested on 13 strains of R. solanacearum with other group of bacteria such as Xanthomonas oryzae pv. oryzae, X. campestris pv. campestris, and X. citri subsp. citri. Primer amplified DNA fragment of R. solanacearum at 323 bp. The sensitivity of the primer was 200 cfu/ml and improved further detection level by using non-specific enrichment medium casamino acids-pepton-glucose broth followed by PCR (BIO-PCR). Out of 130 samples of asymptomatic tomato plants, irrigation water, and soil collected from bacterial wilt infested field in different agro-climatic regions of India, R. solanacearum was detected from 86.9, 88.5, and 90.9 per cents samples using BIO-PCR, respectively. The primer was found specific for detecting viable and virulent strains of R. solanacearum and useful for the diagnosis of R. solanacearum in tomato seedlings and monitoring of pathogen in irrigation water and soil.

  18. Effects of volatile organic compounds produced by Bacillus amyloliquefaciens on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum.

    PubMed

    Raza, Waseem; Wang, Jichen; Wu, Yuncheng; Ling, Ning; Wei, Zhong; Huang, Qiwei; Shen, Qirong

    2016-09-01

    The production of volatile organic compounds (VOCs) by microbes is an important characteristic for their selection as biocontrol agents against plant pathogens. In this study, we identified the VOCs produced by the biocontrol strain Bacillus amyloliquefaciens T-5 and evaluated their impact on the growth and virulence traits of tomato bacterial wilt pathogen Ralstonia solanacearum. The results showed that the VOCs of strain T-5 significantly inhibited the growth of R. solanacearum in agar medium and in soil. In addition, VOCs significantly inhibited the motility traits, root colonization, biofilm formation, and production of antioxidant enzymes and exopolysaccharides by R. solanacearum. However, no effect of VOCs on the production of hydrolytic enzymes by R. solanacearum was observed. The strain T-5 produced VOCs, including benzenes, ketones, aldehydes, alkanes, acids, and one furan and naphthalene compound; among those, 13 VOCs showed 1-10 % antibacterial activity against R. solanacearum in their produced amounts by T-5; however, the consortium of all VOCs produced on agar medium, in sterilized soil, and in natural soil showed 75, 62, and 85 % growth inhibition of R. solanacearum, respectively. The real-time PCR analysis further confirmed the results when the expression of different virulence- and metabolism-related genes in R. solanacearum cells was decreased after exposure to the VOCs of strain T-5. The results of this study clearly revealed the significance of VOCs in the control of plant pathogens. This information would help to better comprehend the microbial interactions mediated by VOCs in nature and to develop safer strategies to control plant disease.

  19. Class Management Skills.

    ERIC Educational Resources Information Center

    Sander, Allan N.

    1989-01-01

    Effective class management in elementary physical education makes instructional time more efficient. Class management skills can be enhanced by concentrating on attention management, behavior management, and organizational techniques. Techniques for improving skills in these three areas are outlined. (IAH)

  20. Class Notes for "Class-Y-News."

    ERIC Educational Resources Information Center

    Stuart, Judy L.

    1991-01-01

    A self-contained class of students with mild to moderate disabilities published a monthly newsletter which was distributed to students' families. Students became involved in writing, typing, drawing, folding, basic editing, and disseminating. (JDD)

  1. Education and Class.

    ERIC Educational Resources Information Center

    Van Galen, Jane A.

    2000-01-01

    The working class is nearly invisible in multicultural education literature. Examines the possibilities of a more careful foregrounding of the complexities of social class in shaping life chances, focusing on the educational experiences of working class students and discussing the poor in order to promote understanding of the potential of teacher…

  2. First-Class Inquiry

    ERIC Educational Resources Information Center

    Hesser, Kathi; Buck, Gayle; Dopp, Sandra

    2005-01-01

    In the activity described in this article, students will explore how variables in a first-class lever, specifically arm length, position of the fulcrum, and placement of the load, affect the effort needed to lift the load. To begin the lesson, demonstrate to the class how a first-class lever works and review what is meant by the terms fulcrum,…

  3. First-Class Inquiry

    ERIC Educational Resources Information Center

    Hesser, Kathi; Buck, Gayle; Dopp, Sandra

    2005-01-01

    In the activity described in this article, students will explore how variables in a first-class lever, specifically arm length, position of the fulcrum, and placement of the load, affect the effort needed to lift the load. To begin the lesson, demonstrate to the class how a first-class lever works and review what is meant by the terms fulcrum,…

  4. Characterization of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid]-degrading Alcaligenes sp.CS1 and Ralstonia sp. CS2 isolated from agricultural soils.

    PubMed

    Smejkal, C W; Vallaeys, T; Seymour, F A; Burton, S K; Lappin-Scott, H M

    2001-04-01

    The herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] is widely applied to corn fields in order to control broad-leaved weeds. However, it is often detected in groundwater where it can be a persistent contaminant. Two mecoprop-degrading bacterial strains were isolated from agricultural soils through their capability to degrade (R/S)-mecoprop rapidly. 16S rDNA sequencing of the isolates demonstrated that one was closely related to the genera Alcaligenes sp. (designated CS1) and the other to Ralstonia sp. (designated CS2). Additionally, these isolates demonstrated ability to grow on other related herbicides, including 2,4-D (2,4-dichlorophenoxyacetic acid), MCPA [4-chloro-2-methyl phenoxy acetic acid] and (R/S)-2,4-DP [2-(2,4-dichlorophenoxy)propionic acid] as sole carbon sources. tfdABC gene-specific probes derived from the 2,4-D-degrading Variovorax paradoxus TV1 were used in hybridization analyses to establish whether tfd-like genes are present in mecoprop-degrading bacteria. Hybridization analysis demonstrated that both Alcaligenes sp. CS1 and Ralstonia sp. CS2 harboured tfdA, tfdB and tfdC genes on plasmids that have approximately > 60% sequence similarity to the tfdA, tfdB and tfdC genes of V. paradoxus. It is therefore likely that tfd-like genes may be involved in the degradation of mecoprop, and we are currently investigating this further.

  5. Purification and gene cloning of alpha-methylserine aldolase from Ralstonia sp. strain AJ110405 and application of the enzyme in the synthesis of alpha-methyl-L-serine.

    PubMed

    Nozaki, Hiroyuki; Kuroda, Shinji; Watanabe, Kunihiko; Yokozeki, Kenzo

    2008-12-01

    By screening microorganisms that are capable of assimilating alpha-methyl-DL-serine, we detected alpha-methylserine aldolase in Ralstonia sp. strain AJ110405, Variovorax paradoxus AJ110406, and Bosea sp. strain AJ110407. A homogeneous form of this enzyme was purified from Ralstonia sp. strain AJ110405, and the gene encoding the enzyme was cloned and expressed in Escherichia coli. The enzyme appeared to be a homodimer consisting of identical subunits, and its molecular mass was found to be 47 kDa. It contained 0.7 to 0.8 mol of pyridoxal 5'-phosphate per mol of subunit and could catalyze the interconversion of alpha-methyl-L-serine to L-alanine and formaldehyde in the absence of tetrahydrofolate. Formaldehyde was generated from alpha-methyl-L-serine but not from alpha-methyl-D-serine, L-serine, or D-serine. Alpha-methyl-L-serine synthesis activity was detected when L-alanine was used as the substrate. In contrast, no activity was detected when D-alanine was used as the substrate. In the alpha-methyl-L-serine synthesis reaction, the enzymatic activity was inhibited by an excess amount of formaldehyde, which was one of the substrates. We used cells of E. coli as a whole-cell catalyst to express the gene encoding alpha-methylserine aldolase and effectively obtained a high yield of optically pure alpha-methyl-L-serine using L-alanine and formaldehyde.

  6. Class network routing

    DOEpatents

    Bhanot, Gyan [Princeton, NJ; Blumrich, Matthias A [Ridgefield, CT; Chen, Dong [Croton On Hudson, NY; Coteus, Paul W [Yorktown Heights, NY; Gara, Alan G [Mount Kisco, NY; Giampapa, Mark E [Irvington, NY; Heidelberger, Philip [Cortlandt Manor, NY; Steinmacher-Burow, Burkhard D [Mount Kisco, NY; Takken, Todd E [Mount Kisco, NY; Vranas, Pavlos M [Bedford Hills, NY

    2009-09-08

    Class network routing is implemented in a network such as a computer network comprising a plurality of parallel compute processors at nodes thereof. Class network routing allows a compute processor to broadcast a message to a range (one or more) of other compute processors in the computer network, such as processors in a column or a row. Normally this type of operation requires a separate message to be sent to each processor. With class network routing pursuant to the invention, a single message is sufficient, which generally reduces the total number of messages in the network as well as the latency to do a broadcast. Class network routing is also applied to dense matrix inversion algorithms on distributed memory parallel supercomputers with hardware class function (multicast) capability. This is achieved by exploiting the fact that the communication patterns of dense matrix inversion can be served by hardware class functions, which results in faster execution times.

  7. Culture and social class.

    PubMed

    Miyamoto, Yuri

    2017-08-08

    A large body of research in Western cultures has demonstrated the psychological and health effects of social class. This review outlines a cultural psychological approach to social stratification by comparing psychological and health manifestations of social class across Western and East Asian cultures. These comparisons suggest that cultural meaning systems shape how people make meaning and respond to material/structural conditions associated with social class, thereby leading to culturally divergent manifestations of social class. Specifically, unlike their counterparts in Western cultures, individuals of high social class in East Asian cultures tend to show high conformity and other-orientated psychological attributes. In addition, cultures differ in how social class impacts health (i.e. on which bases, through which pathways, and to what extent). Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Universality classes of complexity

    NASA Astrophysics Data System (ADS)

    Saakian, David B.

    We give several criteria of complexity and define different universality classes. According to our classification, at the lowest class of complexity are random graph, Markov Models and Hidden Markov Models. At the next level is Sherrington-Kirkpatrick spin glass, connected with neuron-network models. On a higher level are critical theories, spin glass phase of Random Energy Model, percolation, self organized criticality (SOC). The top level class involves HOT design, error threshold in optimal coding, language, and, maybe, financial market. Alive systems are also related with the last class.

  9. Class and Gender

    ERIC Educational Resources Information Center

    Hart, Mechthild

    2005-01-01

    Everyone is dependent on caring labor. Because women's labor is financially beneficial to global capitalism, gender is inseparable from class, regardless of the specific national or cultural contexts.

  10. Class 1 Areas

    EPA Pesticide Factsheets

    A Class 1 area is a geographic area recognized by the EPA as being of the highest environmental quality and requiring maximum protection. Class I areas are areas of special national or regional scenic, recreational or historic value for which the Prevention of Significant Deterioration (PSD) regulations provide special protection. These data were compiled based on EPA's list of mandatory Class I areas and Region 8 listing of non-mandatory Class 1 areas for three tribes. Spatial data are from National Park Service, U.S. Forest Service, U.S. Fish and Wildlife, and U.S. Census Bureau.

  11. Language, Culture, Class, Gender, and Class Participation.

    ERIC Educational Resources Information Center

    Vandrick, Stephanie

    This paper explores reasons why some students with English as a Second Language (ESL) feel less entitled to speak out in class than others, discussing ways in which teachers can widen the definition of participation. The first section explains how student background can affect participation. For students who are non-native English speakers and who…

  12. Online, Bigger Classes May Be Better Classes

    ERIC Educational Resources Information Center

    Parry, Marc

    2010-01-01

    In his work as a professor, Stephen Downes used to feel that he was helping those who least needed it. His students at places like the University of Alberta already had a leg up in life and could afford the tuition. When a colleague suggested they co-teach an online class in learning theory at the University of Manitoba, in 2008, Downes welcomed…

  13. Online, Bigger Classes May Be Better Classes

    ERIC Educational Resources Information Center

    Parry, Marc

    2010-01-01

    In his work as a professor, Stephen Downes used to feel that he was helping those who least needed it. His students at places like the University of Alberta already had a leg up in life and could afford the tuition. When a colleague suggested they co-teach an online class in learning theory at the University of Manitoba, in 2008, Downes welcomed…

  14. A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum.

    PubMed

    Stulberg, Michael J; Huang, Qi

    2015-01-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.

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

    PubMed

    Flavier, A B; Ganova-Raeva, L M; Schell, M A; Denny, T P

    1997-11-01

    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

  16. Ralstonia solanacearum fatty acid composition is determined by interaction of two 3-ketoacyl-acyl carrier protein reductases encoded on separate replicons.

    PubMed

    Feng, Sai-Xiang; Ma, Jin-Cheng; Yang, Ji; Hu, Zhe; Zhu, Lei; Bi, Hong-Kai; Sun, Yi-Rong; Wang, Hai-Hong

    2015-10-22

    FabG is the only known enzyme that catalyzes reduction of the 3-ketoacyl-ACP intermediates of bacterial fatty acid synthetic pathways. However, there are two Ralstonia solanacearum genes, RSc1052 (fabG1) and RSp0359 (fabG2), annotated as encoding putative 3-ketoacyl-ACP reductases. Both FabG homologues possess the conserved catalytic triad and the N-terminal cofactor binding sequence of the short chain dehydrogenase/reductase (SDR) family. Thus, it seems reasonable to hypothesize that RsfabG1 and RsfabG2 both encode functional 3-ketoacyl-ACP reductases and play important roles in R. solanacearum fatty acid synthesis and growth. Complementation of Escherichia coli fabG temperature-sensitive mutant with R. solanacearum fabGs encoded plasmids was carried out to test the function of RsfabGs in fatty acid biosynthesis. RsFabGs proteins were purified by nickel chelate chromatography and fatty acid biosynthetic reaction was reconstituted to investigate the 3-ketoacyl-ACP reductase activity of RsFabGs in vitro. Disruption of both RsfabG genes was done via DNA homologous recombination to test the function of both RsfabG in vivo. And more we also carried out pathogenicity tests on tomato plants using RsfabG mutant strains.  We report that expression of either of the two proteins (RsFabG1 and RsFabG2) restores growth of the E. coli fabG temperature-sensitive mutant CL104 under non-permissive conditions. In vitro assays demonstrate that both proteins restore fatty acid synthetic ability to extracts of the E. coli strain. The RsfabG1 gene carried on the R. solanacearum chromosome is essential for growth of the bacterium, as is the case for fabG in E. coli. In contrast, the null mutant strain with the megaplasmid-encoded RsfabG2 gene is viable but has a fatty acid composition that differs significantly from that of the wild type strain. Our study also shows that RsFabG2 plays a role in adaptation to high salt concentration and low pH, and in pathogenesis of disease in tomato

  17. A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum

    PubMed Central

    Stulberg, Michael J.; Huang, Qi

    2015-01-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum. PMID:26426354

  18. The potato StLTPa7 gene displays a complex Ca-associated pattern of expression during the early stage of potato-Ralstonia solanacearum interaction.

    PubMed

    Gao, Gang; Jin, Li Ping; Xie, Kai Yun; Qu, Dong Yu

    2009-01-01

    Although nonspecific lipid transfer proteins (nsLTPs) are widely expressed during plant defence responses to pathogens, their functions and regulation are not fully understood. In this article, we report the isolation of a cDNA for the new nsLTP, StLTPa7, from cultivated potato (Solanum tuberosum) infected with Ralstonia solanacearum. The cDNA was predicted to encode a type 1 nsLTP containing an N-terminal signal sequence and possessing the characteristic features of nsLTPs. A phylogenetic analysis showed that the encoded amino acid sequence of the nsLTP was similar to those of other previously reported plant nsLTPs, which contain a putative calmodulin-binding site consisting of approximately 12 highly conserved amino acid residues. The expression of the StLTPa7 gene was studied during the early stages of potato-R. solanacearum interaction using real-time quantitative polymerase chain reaction (qRT-PCR) and Northern analyses, and a complex calcium (Ca2+)-associated pattern of expression was observed with the following features: (i) transcripts of the StLTPa7 gene were systemically up-regulated by infection with R. solanacearum; (ii) the StLTPa7 gene was stimulated by salicylic acid, methyl jasmonate, abscisic acid and Ca2+; (iii) qRT-PCR showed that, during the early stage of R. solanacearum infection, nsLTP transcripts accumulated over a time course that paralleled that of Ca2+ accumulation, detected using environmental scanning electron microscopy and energy-dispersive X-ray (EDAX) spectrometry; and (iv) the Ca2+ channel blocker, ruthenium red, partially blocked R. solanacearum-induced StLTPa7 expression. This report represents the first use of EDAX analysis to establish a synchrony between Ca2+ accumulation and nsLTP expression in response to potato-R. solanacearum interactions. Collectively, these results suggest that StLTPa7 may be a pathogen- and Ca(2+)-responsive plant defence gene.

  19. A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

    DOE PAGES

    Stulberg, Michael J.; Huang, Qi

    2015-10-01

    Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regionsmore » of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Moreover, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.« less

  20. Limits to Open Class Performance?

    NASA Technical Reports Server (NTRS)

    Bowers, Albion H.

    2007-01-01

    This viewgraph presentation describes the limits to open class performance. The contents include: 1) Standard Class; 2) 15m/Racing Class; 3) Open Class; and 4) Design Solutions associated with assumptions, limiting parameters, airfoil performance, current trends, and analysis.

  1. Large Classes and Student Learning.

    ERIC Educational Resources Information Center

    LoCastro, Virginia

    2001-01-01

    Surveys the main theoretical issue that a large class, more than small classes, makes salient: How much learning can take place in a class of 300, as opposed to a class of half a dozen learners. Discusses the effect of class size on learning and looks at class size as a sociocultural variable. (Author/VWL)

  2. Teaching Social Class

    ERIC Educational Resources Information Center

    Tablante, Courtney B.; Fiske, Susan T.

    2015-01-01

    Discussing socioeconomic status in college classes can be challenging. Both teachers and students feel uncomfortable, yet social class matters more than ever. This is especially true, given increased income inequality in the United States and indications that higher education does not reduce this inequality as much as many people hope. Resources…

  3. Teaching Social Class

    ERIC Educational Resources Information Center

    Tablante, Courtney B.; Fiske, Susan T.

    2015-01-01

    Discussing socioeconomic status in college classes can be challenging. Both teachers and students feel uncomfortable, yet social class matters more than ever. This is especially true, given increased income inequality in the United States and indications that higher education does not reduce this inequality as much as many people hope. Resources…

  4. Noun Classes in Tikar.

    ERIC Educational Resources Information Center

    Stanley-Thorne, Carol

    An analysis of noun classes in Tikar, a Benue-Congo language spoken in west central Cameroon, looks at patterns in the noun class system, concord system (possessives, demonstratives, demonstrative adjectives, demonstrative pronouns, third-person pronouns, relative pronouns, copula, adjectivals, and numerals) with an eye to determining whether…