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PubMed2005

A pathogen-inducible patatin-like lipid acyl hydrolase facilitates fungal and bacterial host colonization in Arabidopsis.

Genes and proteins related to patatin, the major storage protein of potato tubers, have been identified in many plant species and shown to be induced by a variety of environmental stresses. The Arabidopsis patatin-like gene family (PLPs) comprises nine members, two of which (PLP2 and PLP7) are strongly induced in leaves challenged with fungal and bacterial pathogens. Here we show that accumulation of PLP2 protein in response to Botrytis cinerea or Pseudomonas syringae pv. tomato (avrRpt2) is dependent on jasmonic acid and ethylene signaling, but is not dependent on salicylic acid. Expression of a PLP2-green fluorescent protein (GFP) fusion protein and analysis of recombinant PLP2 indicates that PLP2 encodes a cytoplasmic lipid acyl hydrolase with wide substrate specificity. Transgenic plants with altered levels of PLP2 protein were generated and assayed for pathogen resistance. Plants silenced for PLP2 expression displayed enhanced resistance to B. cinerea, whereas plants overexpressing PLP2 were much more sensitive to this necrotrophic fungus. We also established a positive correlation between the level of PLP2 expression in transgenic plants and cell death or damage in response to paraquat treatment or infection by avirulent P. syringae. Interestingly, repression of PLP2 expression increased resistance to avirulent bacteria, while PLP2-overexpressing plants multiplied avirulent bacteria close to the titers reached by virulent bacteria. Collectively, the data indicate that PLP2-encoded lipolytic activity can be exploited by pathogens with different lifestyles to facilitate host colonization. In particular PLP2 potentiates plant cell death inflicted by Botrytis and reduces the efficiency of the hypersensitive response in restricting the multiplication of avirulent bacteria. Both effects are possibly mediated by providing fatty acid precursors of bioactive oxylipins.

Amino Acid Sequence↗
PubMed2000

Identification of genes associated with copper tolerance in an adhesion-defective mutant of Aeromonas veronii biovar sobria.

TnphoA mutagenesis was used to identify adhesins of Aeromonas veronii biovar sobria 3767, a strain isolated from a diarrhoeal stool specimen. Six mutants, from a library of 154, exhibited significantly reduced levels of adhesion to HEp-2 cells. Primers to the terminal regions of TnphoA were used for inverse PCR and the product from one mutant was cloned into pBluescript and partial sequence data obtained. Scanning GenBank and EMBL data bases revealed DNA sequence similarity to the copA gene of Pseudomonas syringae pv. tomato which confers resistance to copper and other heavy metals. The transposon was located within the copA gene and the mutant exhibited a reduced tolerance to copper. Primer walking, using the inverse PCR product as a template, revealed three open reading frames (ORFs) copA, B and C in A. veronii biovar sobria 3767. The predicted amino acid sequences of ORFs A and B had significant homology (55 and 34% respectively) to the copA and B proteins of P. syringae. No amino acid or DNA sequence homology existed between ORF C of strain 3767 and any other gene in the data bases scanned. Further analysis of the nucleotide sequence failed to reveal the presence of typical copper regulatory genes within the vicinity of the Aeromonas sequence. The association between copper tolerance and adhesion in A. veronii biovar sobria requires further study.

Adhesins, Bacterial↗
PubMed1998

Role of ergosterol in growth inhibition of Saccharomyces cerevisiae by syringomycin E.

The antifungal activity of the lipodepsipeptide syringomycin E from Pseudomonas syringae pv. syringae is modulated by sterols. To study the requirement of the predominant fungal sterol, ergosterol, in syringomycin E action, the sterol composition of Saccharomyces cerevisiae sterol auxotroph strain FY-14 was modified and sensitivity to syringomycin E examined. Cells containing solely ergosterol, cholesterol, beta-sitosterol or stigmasterol were sensitive to syringomycin E with the latter two being the most sensitive. Cells containing growth-promoting cholesterol were the most sensitive and those with growth-promoting ergosterol the least sensitive. It is concluded that sensitivity to syringomycin E is modulated by growth-promoting sterols and does not necessarily require ergosterol.

Antifungal Agents↗
PubMed1994

Molecular mechanisms of copper resistance and accumulation in bacteria.

An unusual mechanism of metal resistance is found in certain plant pathogenic strains of Pseudomonas syringae that are exposed to high levels of copper compounds used in disease control on agricultural crops. These bacteria accumulate blue Cu2+ ions in the periplasm and outer membrane. At least part of this copper sequestering activity is determined by copper-binding protein products of the copper resistance operon (cop). Potential copper-binding sites of the periplasmic CopA protein show conservation with type-1, type-2, and type-3 copper sites of several eukaryotic multi-copper oxidases. In addition to compartmentalization of copper in the periplasm, two components of the cop operon, copC and copD, appear to function in copper uptake into the cytoplasm. Copper resistance operons related to cop have been described in the related plant pathogen Xanthomonas campestris and in Escherichia coli, but these resistance systems may differ functionally from the Pseudomonas syringae system.

Amino Acid Sequence↗
PubMed1983

Toxicity of smoke to epiphytic ice nucleation-active bacteria.

Wheat straw smoke aerosols and liquid smoke condensates reduced significantly both the viability and the ice-nucleating activity of Pseudomonas syringae pv. syringae and Erwinia herbicola in vitro and on leaf surfaces in vivo. Highly significant reductions in numbers of bacterial ice nuclei on the surface of both corn and almond were observed after exposure to smoke aerosols. At -5 degrees C, frost injury to corn seedlings colonized by ice nucleation-active bacteria was reduced after exposure to smoke aerosols. Effects on -9 degrees C ice nuclei, although significant, were less than on ice nuclei active at -5 degrees C. These results suggest that smoke from wildfires or smudge pots may reduce plant frost susceptibility and sources of ice nuclei important in other natural processes under some conditions.

Journal Article↗
PubMed1983

Distribution, population dynamics, and characteristics of ice nucleation-active bacteria in deciduous fruit tree orchards.

Deciduous fruit tree orchards located in the Pacific Northwest were surveyed over a 3-year period for the presence of ice nucleation-active (INA) bacteria. In the Yakima Valley, only about 30% of the fruit tree orchards contained INA bacteria (median population ca. 3 x 10 CFU/g [fresh weight]) in contrast to nearly 75% of the orchards in the Hood River Valley (median population ca. 5 x 10 CFU/g [fresh weight]). These INA populations ranged from less than 10 to over 10 CFU/g (fresh weight) of blossoms and, in Hood River Valley orchards, generally comprised over 10% of the total bacterial population. Populations of INA bacteria fluctuated during the year with highest levels developing on buds and flowers during the cool, wet spring, followed by a drop in populations during the warmer, drier, summer months and finally a gradual increase in the autumn. The INA bacteria persisted on dormant buds from which they again colonized young developing vegetative tissues. All INA bacteria were identified as Pseudomonas syringae. The frequency of ice nucleation at -5 degrees C for these strains ranged from nearly every cell being INA to less than 1 in 10 cells. The median frequency of ice nucleation at -5 degrees C was 10 cells per ice nucleus. The INA P. syringae strains from individual orchards were diverse with respect to bacteriocin typing and in ice nucleation frequency. The consistent absence of detectable INA bacteria or presence of low populations in most of the orchards surveyed during periods when critical temperatures (i.e., -2 to -5 degrees C) were common indicated a limited role for INA bacteria in frost susceptibility of most Pacific Northwest orchards.

Journal Article↗
PubMed1986

Antagonism of Lactic Acid Bacteria against Phytopathogenic Bacteria.

A variety of lactic acid bacteria, isolated from plant surfaces and plant-associated products, were found to be antagonistic to test strains of the phytopathogens Xanthomonas campestris, Erwinia carotovora, and Pseudomonas syringae. Effective "in vitro" inhibition was found both on agar plates and in broth cultures. In pot trials, treatment of bean plants with a Lactobacillus plantarum strain before inoculation with P. syringae caused a significant reduction of the disease incidence.

Journal Article↗
PubMed1987

Auxin production by plant-pathogenic pseudomonads and xanthomonads.

Pathogenic strains of Xanthomonas campestris pv. glycines which cause hypertrophy of leaf cells of susceptible soybean cultivars and nonpathogenic strains which do not cause hypertrophy were compared for their ability to produce indole compounds, including the plant hormone indole-3-acetic acid (IAA) in liquid media with or without supplementation with l-tryptophan. Several additional strains of plant-pathogenic xanthomonads and pseudomonads were also tested for IAA production to determine whether in vitro production of IAA is related to the ability to induce hypertrophic growth of host tissues. Indoles present in culture filtrates were identified by thin-layer chromatography, high-performance liquid chromatography, UV spectroscopy, mass spectroscopy, and gas chromatography-mass spectrometry and were quantitated by high-performance liquid chromatography. All strains examined produced IAA when liquid media were supplemented with l-tryptophan. The highest levels of IAA were found in culture filtrates from the common bean pathogen Pseudomonas syringae pv. syringae, and this was the only bacterium tested which produced IAA without addition of tryptophan to the medium. Additional indoles identified in culture filtrates of the various strains included indole-3-lactic acid, indole-3-aldehyde, indole-3-acetamide, and N-acetyltryptophan. Pseudomonads and xanthomonads could be distinguished by the presence of N-acetyltryptophan, which was found only in xanthomonad culture filtrates.

Journal Article↗
PubMed1989

Response of plant-colonizing pseudomonads to hydrogen peroxide.

Colonization of plant root surfaces by Pseudomonas putida may require mechanisms that protect this bacterium against superoxide anion and hydrogen peroxide produced by the root. Catalase and superoxide dismutase may be important in this bacterial defense system. Stationary-phase cells of P. putida were not killed by hydrogen peroxide (H(2)O(2)) at concentrations up to 10 mM, and extracts from these cells possessed three isozymic bands (A, B, and C) of catalase activity in native polyacrylamide gel electrophoresis. Logarithmic-phase cells exposed directly to hydrogen peroxide concentrations above 1 mM were killed. Extracts of logarithmic-phase cells displayed only band A catalase activity. Protection against 5 mM H(2)O(2) was apparent after previous exposure of the logarithmic-phase cells to nonlethal concentrations (30 to 300 muM) of H(2)O(2). Extracts of these protected cells possessed enhanced catalase activity of band A and small amounts of bands B and C. A single form of superoxide dismutase and isoforms of catalase were apparent in extracts from a foliar intercellular pathogen, Pseudomonas syringae pv. phaseolicola. The mobilities of these P. syringae enzymes were distinct from those of enzymes in P. putida extracts.

Journal Article↗
PubMed1990

Copper resistance gene homologs in pathogenic and saprophytic bacterial species from tomato.

Copper-resistant strains of Xanthomonas campestris pv. vesicatoria, Pseudomonas cichorii, Pseudomonas putida, Pseudomonas fluorescens, and a yellow Pseudomonas sp. were isolated from tomato plants or seeds. In Southern hybridizations, DNA from each strain showed homology with the copper resistance (cop) operon previously cloned from Pseudomonas syringae pv. tomato PT23. Homology was associated with plasmid and chromosomal DNA in X. compestris pv. vesicatoria, P. putida, and the yellow Pseudomonas sp. Homology was detected only in the chromosomal DNA of P. cichorii and P. fluorescens. Homology with cop was also detected in chromosomal DNA from copper-sensitive strains of P. cichorii, P. fluorescens, and P. syringae pv. tomato, suggesting that the cop homolog may be indigenous to certain Pseudomonas species and have some function other than copper resistance. No homology was detected in DNA from a copper-sensitive X. campestris pv. vesicatoria strain. Copper-inducible protein products were detected in each copper-resistant bacterium by immunoblot analysis with antibodies raised to the CopB protein from the cop operon. The role of the homologous DNA in copper resistance was confirmed for the X. campestris pv. vesicatoria strain by cloning and transferring the cop homolog to a copper-sensitive strain of X. campestris pv. vesicatoria. The possibility and implications of copper resistance gene exchange between different species and genera of pathogenic and saprophytic bacteria on tomato plants are discussed.

Journal Article↗
PubMed1992

Detection of a single genetically modified bacterial cell in soil by using charge coupled device-enhanced microscopy.

Genes encoding bioluminescence from Vibrio harveyi were cloned into Pseudomonas syringae pv. phaseoli-cola, resulting in high levels of bioluminescence. After inoculation of sterile and nonsterile soil slurries with bioluminescent P. syringae, cells could not be identified by conventional light microscopy. However, when we used charge coupled device-enhanced microscopy, bioluminescent single cells were detected easily in dark fields despite masking by soil particulate matter, and in addition, the extent of competition from indigenous soil bacteria could be monitored. The technique which we describe offers great potential for tracking and determining the spatial distribution of genetically marked microorganisms in the environment.

Journal Article↗
PubMed1992

Topical application of ice-nucleating-active bacteria decreases insect cold tolerance.

The majority of overwintering insects avoid lethal freezing by lowering the temperature at which ice spontaneously nucleates within their body fluids. We examined the effect of ice-nucleating-active bacteria on the cold-hardiness of the lady beetle, Hippodamia convergens, a freeze-intolerant species that overwinters by supercooling to ca. -16 degrees C. Topical application of the ice-nucleating-active bacteria Pseudomonas syringae increased the supercooling point to temperatures as high as -3 degrees C. This decrease in cold tolerance was maintained for at least 3 days after treatment. Various treatment doses (10, 10, and 10 bacteria per ml) and modes of action (bacterial ingestion and topical application) were also compared. At the highest concentration of topically applied P. syringae, 50% of the beetles froze between -2 and -4 degrees C. After topical application at the lowest concentration, 50% of the individuals froze by -11 degrees C. In contrast, beetles fed bacteria at this concentration did not begin to freeze until -10 degrees C, and 50% were frozen only at temperatures of -13 degrees C or less. In addition to reducing the supercooling capacity in H. convergens, ice-nucleating-active bacteria also significantly reduced the cold-hardiness of four additional insects. These data demonstrate that ice-nucleating-active bacteria can be used to elevate the supercooling point and thereby decrease insect cold tolerance. The results of this study support the proposition that ice-nucleating-active bacteria may be used as a biological insecticide for the control of insect pests during the winter.

Journal Article↗
PubMed1994

A biological sensor for iron available to bacteria in their habitats on plant surfaces.

A sensor responsive to iron was constructed by fusing a promoterless ice nucleation activity gene (inaZ) to an iron-regulated promoter of a genomic region involved in pyoverdine (fluorescent siderophore) (pvd) production in Pseudomonas syringae. Cells of Pseudomonas fluorescens and P. syringae that contained the pvd-inaZ fusion expressed iron-responsive ice nucleation activity in the bean rhizosphere and phyllosphere, respectively, and in culture. Addition of Fe(III) to leaves or soil reduced the apparent transcription of the pvd-inaZ reporter gene, as shown by a reduction in the number of ice nuclei produced, indicating that Fe(III) was primarily responsible for mediating transcription of the pvd-inaZ gene even in natural environments. A Pseudomonas sp. strain having an intact iceC gene, which conferred Fe-insensitive expression of ice nucleation activity, was included in all studies to account for small strain- or environment-dependent differences in the ability of bacterial cells to produce ice nuclei. Thus, a comparison of the ice nucleation activity conferred by pvd-inaZ with the activity conferred by iceC revealed the bioavailability of iron in culture or natural habitats. The relative ice nucleation activities expressed by strains containing iceC or pvd-inaZ indicated that, while not abundant, Fe(III) is not present at extremely low concentrations at all microsites colonized by bacteria on plant surfaces. Biological sensors that are constructed by fusing inaZ to chemically responsive promoters provide a novel way to characterize chemical constituents of microbial habitats.

Journal Article↗
PubMed1999

Two-component transcriptional regulation of N-acyl-homoserine lactone production in Pseudomonas aureofaciens.

Production of phenazine antibiotics by the biological control bacterium Pseudomonas aureofaciens 30-84 is regulated in part by the PhzI/PhzR N-acyl-homoserine lactone (AHL) response system (L. S. Pierson III, V. D. Keppenne, and D. W. Wood, J. Bacteriol. 176:3966-3974, 1994; D. W. Wood and L. S. Pierson III, Gene 168:49-53, 1996). Two mutants, 30-84W and 30-84.A2, were isolated and were found to be deficient in the production of phenazine, protease, hydrogen cyanide (HCN), and the AHL signal N-hexanoyl-homoserine lactone. These mutants were not complemented by phzI, phzR, or the phenazine biosynthetic genes (phzFABCD) (L. S. Pierson III, T. Gaffney, S. Lam, and F. Gong, FEMS Microbiol. Lett. 134:299-307, 1995). A 2.2-kb region of the 30-84 chromosome which fully restored production of all of these compounds in strain 30-84W was identified. Nucleotide sequence analysis of this region revealed a single open reading frame encoding a predicted 213-amino-acid protein which is very similar to the global response regulator GacA. Strain 30-84.A2 was not complemented by gacA or any cosmid from a genomic library of strain 30-84 but was complemented by gacS (formerly lemA) homologs from Pseudomonas fluorescens Pf-5 (N. Corbel and J. E. Loper, J. Bacteriol. 177:6230-6236, 1995) and Pseudomonas syringae pv. syringae B728a (E. M. Hrabek and D. K. Willis, J. Bacteriol. 174:3011-3020, 1992). Transcription of phzR was not altered in either mutant; however, phzI transcription was eliminated in strains 30-84W and 30-84.A2. These results indicated that the GacS/GacA two-component signal transduction system of P. aureofaciens 30-84 controls the production of AHL required for phenazine production by mediating the transcription of phzI. Addition of exogenous AHL did not complement either mutant for phenazine production, indicating that the GacS/GacA global regulatory system controls phenazine production at multiple levels. Our results reveal for the first time a mechanism by which a two-component regulatory system and an AHL-mediated regulatory system interact.

4-Butyrolactone↗
PubMed2001

Isolation and in vivo and in vitro antifungal activity of phenylacetic acid and sodium phenylacetate from Streptomyces humidus.

The antifungal substances SH-1 and SH-2 were isolated from Streptomyces humidus strain S5-55 cultures by various purification procedures and identified as phenylacetic acid and sodium phenylacetate, respectively, based on the nuclear magnetic resonance, electron ionization mass spectral, and inductively coupled plasma mass spectral data. SH-1 and SH-2 completely inhibited the growth of Pythium ultimum, Phytophthora capsici, Rhizoctonia solani, Saccharomyces cerevisiae, and Pseudomonas syringae pv. syringae at concentrations from 10 to 50 microg/ml. The two compounds were as effective as the commercial fungicide metalaxyl in inhibiting spore germination and hyphal growth of P. capsici. However, the in vivo control efficacies of the two antifungal compounds against P. capsici infection on pepper plants were similar to those of H(3)PO(3) and fosetyl-AI but less than that of metalaxyl.

Antifungal Agents↗
PubMed2002

Assessment of the importance of similarity in carbon source utilization profiles between the biological control agent and the pathogen in biological control of bacterial speck of tomato.

Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, was used to determine whether similarity in carbon source utilization between a preemptive biological control agent and the pathogen was significant in determining the ability of the bacterium to suppress disease. Similarity in carbon source utilization was quantified as the ratio of the number of tomato carbon sources utilized in vitro by the biological control agent to the number of tomato carbon sources utilized in vitro by the target pathogen (the niche overlap index [NOI]). Suppression of the disease was quantified as the percent reduction in disease severity compared to the pathogen-only control when nonpathogenic bacteria were applied to foliage 48 h prior to the pathogen. In the collection of 36 nonpathogenic bacterial strains, there was a significant (P < 0.01), but weak (r(2) = 0.25), correlation between reduction in disease severity and similarity in carbon source utilization, suggesting that similarity in carbon source use was significant in determining ability to suppress disease. The relationship was investigated further using catabolic mutants of P. syringae strain TLP2, an effective biological control agent of speck. Catabolic mutants exhibited lower levels of similarity (NOI = 0.07 to 0.90) than did wild-type TLP2 (NOI = 0.93). With these catabolic mutants there was a significant (P < 0.01), and stronger (r(2) = 0.42), correlation between reduction in disease severity and similarity in carbon source utilization. This suggests that similarity in carbon source utilization was a more important component of biological control ability for the catabolic mutants than for the nonpathogenic bacteria. Together, these studies indicate that suppression of bacterial speck of tomato was correlated with nutritional similarity between the pathogenic and nonpathogenic bacteria and suggest that preemptive utilization of carbon sources was probably involved in the biological control of the disease by both the naturally occurring nonpathogenic bacteria and the catabolic mutants.

Carbohydrate Metabolism↗
PubMed2003

Enhancement of population size of a biological control agent and efficacy in control of bacterial speck of tomato through salicylate and ammonium sulfate amendments.

Sodium salicylate and ammonium sulfate were applied to leaf surfaces along with suspensions of the biological control agents Pseudomonas syringae Cit7(pNAH7), which catabolizes salicylate, and Cit7, which does not catabolize salicylate, to determine whether enhanced biological control of bacterial speck of tomato could be achieved. Foliar amendment with salicylate alone significantly enhanced the population size and the efficacy of Cit7(pNAH7), but not of Cit7, on tomato leaves. Application of ammonium sulfate alone did not result in enhanced population size or biological control efficacy of either Cit7(pNAH7) or Cit7; however, when foliar amendments with both sodium salicylate and ammonium sulfate were applied, a trend toward further increases in population size and biological control efficacy of Cit7(pNAH7) was observed. This study demonstrates the potential of using a selective carbon source to improve the efficacy of a bacterial biological control agent in the control of a bacterial plant disease and supports previous conclusions that the growth of P. syringae in the phyllosphere is primarily carbon limited and secondarily nitrogen limited.

Ammonium Sulfate↗
PubMed2003

Freshwater bacteria can methylate selenium through the thiopurine methyltransferase pathway.

Involvement of the bacterial thiopurine methyltransferase (bTPMT) in natural selenium methylation by freshwater was investigated. A freshwater environment that had no known selenium contamination but exhibited reproducible emission of dimethyl selenide (DMSe) or dimethyl diselenide (DMDSe) when it was supplemented with an organic form of selenium [(methyl)selenocysteine] or an inorganic form of selenium (sodium selenite) was used. The distribution of the bTPMT gene (tpm) in the microflora was studied. Freshwater bacteria growing on 10 micro M sodium selenite and 10 micro M sodium selenate were isolated, and 4.5 and 10% of the strains, respectively, were shown by colony blot hybridization to hybridize with a Pseudomonas syringae tpm DNA probe. Ribotyping showed that these strains are closely related. The complete rrs sequence of one of the strains, designated Hsa.28, was obtained and analyzed. Its closest phyletic neighbor was found to be the Pseudomonas anguilliseptica rrs sequence. The Hsa.28 strain grown with sodium selenite or (methyl)selenocysteine produced significant amounts of DMSe and DMDSe. The Hsa.28 tpm gene was isolated by genomic DNA library screening and sequencing. BLASTP comparisons of the deduced Hsa.28 bTPMT sequence with P. syringae, Pseudomonas aeruginosa, Vibrio cholerae, rat, and human thiopurine methyltransferase sequences revealed that the levels of similarity were 52 to 71%. PCR-generated Escherichia coli subclones containing the Hsa.28 tpm open reading frame were constructed. E. coli cells harboring the constructs and grown with sodium selenite or (methyl)selenocysteine produced significant levels of DMSe and DMDSe, confirming that the gene plays a role in selenium methylation. The effect of strain Hsa.28 population levels on freshwater DMSe and DMDSe emission was investigated. An increase in the size of the Hsa.28 population was found to enhance significantly the emission of methyl selenides by freshwater samples supplemented with sodium selenite or (methyl)selenocysteine. These data suggest that bTPMT can play a role in natural freshwater selenium methylation processes.

Amino Acid Sequence↗