Proteomic Analysis of the GacA Response Regulator in Pseudomonas chlororaphis O6 (original) (raw)
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Regulation of GacA in Pseudomonas chlororaphis Strains Shows a Niche Specificity
PloS one, 2015
The GacS/GacA two-component system plays a central role in the regulation of a broad range of biological functions in many bacteria. In the biocontrol organism Pseudomonas chlororaphis, the Gac system has been shown to positively control quorum sensing, biofilm formation, and phenazine production, but has an overall negative impact on motility. These studies have been performed with strains originated from the rhizosphere predominantly. To investigate the level of conservation between the GacA regulation of biocontrol-related traits in P. chlororaphis isolates from different habitats, the studies presented here focused on the endophytic isolate G5 of P. chlororaphis subsp. aurantiaca. A gacA mutant deficient in the production of N-acylhomoserine lactones (AHLs) and phenazine was isolated through transposon mutagenesis. Further phenotypic characterization revealed that in strain G5, similar to other P. chlororaphis strains, a gacA mutation caused inability to produce biocontrol facto...
The plant pathology journal, 2014
The GacS/GacA system in the root colonizer Pseudomonas chlororaphis O6 is a key regulator of many traits relevant to the biocontrol function of this bacterium. Proteomic analysis revealed 12 proteins were down-regulated in a gacS mutant of P. chlororaphis O6. These GacS-regulated proteins functioned in combating oxidative stress, cell signaling, biosynthesis of secondary metabolism, and secretion. The extent of regulation was shown by real-time RT-PCR to vary between the genes. Mutants of P. chlororaphis O6 were generated in two GacS-regulated genes, trpE, encoding a protein involved in tryptophan synthesis, and prnA, required for conversion of tryptophan to the antimicrobial compound, pyrrolitrin. Failure of the trpE mutant to induce systemic resistance in tobacco against a foliar pathogen causing soft rot, Pectobacterium carotovorum SCCI, correlated with reduced colonization of root surfaces implying an inadequate supply of tryptophan to support growth. Although colonization was n...
BMC Microbiology, 2014
Background: Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of suppressing the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces the antibiotics phenazine and pyrrolnitrin together with other metabolites believed to contribute to biocontrol. A mutant no longer capable of inhibiting fungal growth was identified harboring a transposon insertion in a gene encoding a LysR-type transcriptional regulator (LTTR), designated ptrA (Pseudomonas transcriptional regulator). Isobaric tag for relative and absolute quantitation (iTRAQ) based protein analysis was used to reveal changes in protein expression patterns in the ptrA mutant compared to the PA23 wild type. Results: Relative abundance profiles showed 59 differentially-expressed proteins in the ptrA mutant, which could be classified into 16 clusters of orthologous groups (COGs) based on their predicted functions. The largest COG category was the unknown function group, suggesting that many yet-to-be identified proteins are involved in the loss of fungal activity. In the secondary metabolite biosynthesis, transport and catabolism COG, seven proteins associated with phenazine biosynthesis and chitinase production were downregulated in the mutant. Phenotypic assays confirmed the loss of phenazines and chitinase activity. Upregulated proteins included a lipoprotein involved in iron transport, a flagellin and hook-associated protein and four proteins categorized into the translation, ribosome structure and biogenesis COG. Phenotypic analysis revealed that the mutant exhibited increased siderophore production and flagellar motility and an altered growth profile, supporting the proteomic findings. Conclusion: PtrA is a novel LTTR that is essential for PA23 fungal antagonism. Differential protein expression was observed across 16 COG categories suggesting PtrA is functioning as a global transcriptional regulator. Changes in protein expression were confirmed by phenotypic assays that showed reduced phenazine and chitinase expression, elevated flagellar motility and siderophore production, as well as early entrance into log phase growth.
The Gac/Rsm Signaling Pathway of a Biocontrol Bacterium, Pseudomonas chlororaphis O6
Research in Plant Disease, 2017
Pseudomonas chlororaphis O6, isolated from the roots of dryland, field-grown commercial wheat in the USA, enhances plant health and therefore it is used in agriculture as a biofertilizer and biocontrol agent. The metabolites produced by this pseudomonad stimulate plant growth through direct antagonism of pathogens and by inducing systemic resistance in the plant. Studies upon P. chlororaphis O6 identify the pathways through which defined bacterial metabolites generate protection against pathogenic microbes, insects, and nematodes. P. chlororaphis O6 also triggers plant resistance to drought and salinity stresses. The beneficial determinants are produced from bacterial cells as they form biofilms during root colonization. Molecular control these processes in P. chlororaphis O6 involves the global regulatory Gac/Rsm signaling cascade with cross-talk between other global regulatory pathways. The Gac/Rsm regulon allows for coordinate phasing of expression of the genes that encode these beneficial traits among a community of cells. This review provides insights on the Gac/Rsm regulon in expression of beneficial traits of the P. chlororaphis O6 which can contribute to help yield enhancement and quality in agricultural production.
Journal of Bacteriology, 2000
The GacS-GacA two-component signal transduction system, which is highly conserved in gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in Pseudomonas spp. Screening of a Pseudomonas fluorescens F113 gene bank led to the isolation of a previously undefined locus which could restore secondary metabolite production to both gacS and gacA mutants of F113. Sequence analysis of this locus demonstrated that it did not contain any obvious Pseudomonas protein-coding open reading frames or homologues within available databases. Northern analysis indicated that the locus encodes an RNA (PrrB RNA) which is able to phenotypically complement gacS and gacA mutants and is itself regulated by the GacS-GacA two-component signal transduction system. Primer extension analysis of the 132-base transcript identified the transcription start site located downstream of a 70 promoter sequence from positions ؊10 to ؊35. Inactivation of the prrB gene in F113 resulted in a significant reduction of 2,4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) production, while increased metabolite production was observed when prrB was overexpressed. The prrB gene sequence contains a number of imperfect repeats of the consensus sequence 5-AGGA-3, and sequence analysis predicted a complex secondary structure featuring multiple putative stem-loops with the consensus sequences predominantly positioned at the single-stranded regions at the ends of the stem-loops. This structure is similar to the CsrB and RsmB regulatory RNAs in Escherichia coli and Erwinia carotovora, respectively. Results suggest that a regulatory RNA molecule is involved in GacA-GacSmediated regulation of Phl and HCN production in P. fluorescens F113. Pseudomonas fluorescens F113 was isolated as a biocontrol agent for the control of Pythium ultimum-mediated dampingoff of sugar beet (35). Inhibition of Pythium ultimum has been attributed to the production of the antimicrobial agent 2,4diacetylphloroglucinol (Phl) (11). However, the strain also synthesizes hydrogen cyanide (HCN) and an exoprotease. These secondary metabolites and exoprotease have previously been shown to be positively regulated by the GacS (previously LemA) and GacA two-component signal transduction system (8) common to numerous Pseudomonas spp., including P. syringae (31), P. viridiflava (18), P. aeruginosa (30), and P. fluorescens (6, 13, 17, 32). Sensor proteins such as GacS are typically transmembrane proteins that respond to environmental stimuli by autophosphorylation, followed by transfer of the phosphate to the cognate response regulator, in this case GacA. The GacA response regulator contains a DNA binding motif and is thought to activate or repress genes directly by binding to the target gene promoter. However, direct binding of GacA to putative target promoters has yet to be demonstrated. Recent research in P. aeruginosa PAO (30) has revealed that the GacS-GacA signal transduction system contributes to a larger regulatory cascade involving acyl-homoserine lactonemediated quorum sensing and alternate sigma factors. Indeed, Reimmann et al. (30) demonstrated that GacA positively controls the production of N-butyryl-homoserine lactone.
Molecular Plant Pathology
GacS/GacA is a conserved two-component system that functions as a master regulator of virulence-associated traits in many bacterial pathogens, including Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/ GacA-regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogen Pseudomonas aeruginosa, GacS/GacA negatively regulates the expression of type III secretion system (T3SS)-encoding genes. However, in the plant pathogenic bacterium Pseudomonas syringae, strain-to-strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we reevaluated the function of GacA in P. syringae pv. tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence inside Arabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface-inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulating P. syringae virulence.
Canadian Journal of Microbiology, 2013
Pseudomonas chlororaphis O6 possesses many beneficial traits involved in biocontrol of plant diseases. In this paper, we examined the effect of a mutation in rpoS encoding a stress-related alternative sigma factor to better understand the regulation of these traits. Biochemical studies indicated that production of acyl homoserine lactones was altered and phenazine was increased in the P. chlororaphis O6 rpoS mutant. The rpoS mutation reduced hydrogen cyanide levels, but the rpoS mutant still displayed a level of in vitro antifungal activity against Fusarium graminearum and Alternaria alternata. Tomato root colonization by the rpoS mutant was lower than that by the wild type at 5, 7, and 13 days after inoculation. The rpoS mutant was less effective than the wild type in induction of systemic resistance to two foliar pathogens after root inoculation of the tomato plants. Our findings demonstrate that the stationary-phase sigma factor RpoS regulates production of several key factors in...
Journal compilation, 1865
The alternative sigma factor RpoN is a key regulator in the acclimation of Pseudomonas to complex natural environments. In this study we show that RpoN is required for efficient colonization of sugar beet seedlings by the plant growth-promoting bacterium Pseudomonas fluorescens SBW25, and use phenotypic and bioinformatic approaches to profile the RpoN-dependent traits and genes of P. fluorescens SBW25. RpoN is required for flagellar biosynthesis and for assimilation of a wide variety of nutrient sources including inorganic nitrogen, amino acids, sugar alcohols and dicarboxylic acids. Chemosensitivity assays indicate that RpoN-regulated genes contribute to acid tolerance and resistance to some antibiotics, including tetracyclines and aminoglycosides. Gain of function changes associated with loss of RpoN included increased tolerance to hydroxyurea and Guanazole. Bioinformatic predictions of RpoN-regulated genes show a close correspondence with phenotypic analyses of RpoNregulated traits and suggest novel functions for RpoN in P. fluorescens, including regulation of poly(A) polymerase. The reduced plant colonization ability observed for an rpoN mutant of P. fluorescens is therefore likely to be due to defects in multiple traits including nutrient assimilation, protein secretion and stress tolerance.
Using Molecular Techniques to Understand and Enhance Biological Control by Pseudomonas spp
The health and environmental impact of agrochemicals make alternative methods of disease control more attractive for sustainable crop production. Biocontrol strains of bacteria that are able to inhibit plant pathogenic fungi and suppress plant diseases represent one of the most promising alternatives. Various mechanisms of biological control have been recognized including effective root colonization, production of AF metabolites, interference with fungal pathogenic factors, and elicitation of induced systemic resistance in the plant. These mechanisms were initially demonstrated through microbiological and/or biochemical techniques. The advent of molecular techniques to study the genetic basis of biological control has shed light on genes responsible for production of antibiotics and degradative enzymes. Through inactivation, overexpression, and analysis of gene expression patterns, their role in biocontrol has been more clearly defined. In this review we describe the work done in Canada, using molecular techniques, to understand biological control in Pseudomonas chlororaphis PA23. The end goal of this research is to develop more reliable products that demonstrate enhanced performance in the field.
Applied and Environmental Microbiology, 2009
The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the ⌬gcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.