Dataset Information

Vfr, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in Pseudomonas chlororaphis G05.

ABSTRACT: In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant G05?phz?prn::lacZ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant G05?vfr and G05?phz?prn::lacZ?vfr. By quantifying ?-galactosidase activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant G05?vfr, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

SUBMITTER: Wu X

PROVIDER: S-EPMC6706016 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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vfr, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in Pseudomonas chlororaphis G05.

Wu Xia X Chi Xiaoyan X Wang Yanhua Y Zhang Kailu K Kai Le L He Qiuning Q Tang Jinxiu J Wang Kewen K Sun Longshuo L Hao Xiuying X Xie Weihai W Ge Yihe Y

The plant pathology journal 20190801 4

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant ...[more]

PMID: 31481858

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Project description:BackgroundPhenazine-1-carboxamide (PCN), a phenazine derivative, is strongly antagonistic to fungal phytopathogens. The high PCN biocontrol activity fascinated researcher's attention in isolating and identifying novel bacterial strains combined with engineering strategies to target PCN as a lead molecule. The chemical route for phenazines biosynthesis employs toxic chemicals and display low productivities, require harsh reaction conditions, and generate toxic by-products. Phenazine biosynthesis using some natural phenazine-producers represent remarkable advantages of non-toxicity and possibly high yield in environmentally-friendlier settings.ResultsA biocontrol bacterium with antagonistic activity towards fungal plant pathogens, designated as strain HT66, was isolated from the rice rhizosphere. The strain HT66 was identified as Pseudomonas chlororaphis based on the colony morphology, gas chromatography of cellular fatty acids and 16S rDNA sequence analysis. The secondary metabolite produced by HT66 strain was purified and identified as PCN through mass spectrometry, and 1H, 13C nuclear magnetic resonance spectrum. The yield of PCN by wild-type strain HT66 was 424.87 mg/L at 24 h. The inactivation of psrA and rpeA increased PCN production by 1.66- and 3.06-fold, respectively, which suggests that psrA and rpeA are PCN biosynthesis repressors. qRT-PCR analysis showed that the expression of phzI, phzR, and phzE was markedly increased in the psrA and rpeA double mutant than in psrA or rpeA mutant. However, the transcription level of rpeA and rpeB in strain HT66ΔpsrA increased by 3.52- and 11.58-folds, respectively. The reduced psrA expression in HT66ΔrpeA strain evidenced a complex regulation mechanism for PCN production in HT66.ConclusionIn conclusion, the results evidence that P. chlororaphis HT66 could be modified as a potential cell factory for industrial-scale biosynthesis of PCN and other phenazine derivatives by metabolic engineering strategies.

Project description:Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ; PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay's Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and phaI, were compared with wild type PA23 by quantitative real time polymerase chain reaction (qPCR). The qPCR studies indicated that there was no change in levels of transcription of the PHA synthase genes phaC1 and phaC2 in the phz? (PA23-63) and phz? prn? (PA23-63-1) mutants in glucose medium. There was a significant increase in expression of phaC2 in octanoate medium. Transcription of phaD, phaF and phaI increased significantly in the phz? prn? (PA23-63-1) mutant. Mutations in regulatory genes like gacS, rpoS, and relA/spoT, which affect PHZ and PRN production, also resulted in altered gene expression. The expression of phaC1, phaC2, phaF, and phaI genes was down-regulated significantly in gacS and rpoS mutants. Thus, it appears that PHZ, PRN, and PHA production is regulated by common mechanisms. Higher PHA production in the phz? (PA23-63), prn- (PA23-8), and phz? prn? (PA23-63-1) mutants in octanoic medium could be correlated with higher expression of phaC2. Further, the greater PHA production observed in the phz? and prn? mutants was not due to increased transcription of PHA synthase genes in glucose medium, but due to more accessibility of carbon substrates and reducing power, which were otherwise used for the synthesis of PHZ and PRN.

Project description:The GacS/GacA two-component regulatory system activates the production of secondary metabolites including phenazines crucial for biological control activity in Pseudomonas chlororaphis 30-84. To better understand the role of the Gac system on phenazine regulation, transcriptomic analyses were conducted by comparing the wild-type strain to a gacA mutant. RNA-seq analysis identified 771 genes under GacA control, including many novel genes. Consistent with previous findings, phenazine biosynthetic genes were significantly downregulated in a gacA mutant. The transcript abundances of phenazine regulatory genes such as phzI, phzR, iopA, iopB, rpoS, and pip also were reduced. Moreover, the transcript abundance of three noncoding RNAs (ncRNAs) including rsmX, rsmY, and rsmZ was significantly decreased by gacA mutation consistent with the presence of consensus GacA-binding sites associated with their promoters. Our results also demonstrated that constitutive expression of rsmZ from a non-gac regulated promoter resulted in complete restoration of N-acyl-homoserine lactone (AHL) and phenazine production as well as the expression of other gac-dependent secondary metabolites in gac mutants. The role of RsmA and RsmE in phenazine production also was investigated. Overexpression of rsmE, but not rsmA, resulted in decreased AHL and phenazine production in P. chlororaphis, and only a mutation in rsmE bypassed the requirement for GacA in phenazine gene expression. In contrast, constitutive expression of the phzI/phzR quorum sensing system did not rescue phenazine production in the gacA mutant, indicating the direct posttranscriptional control by Gac on the phenazine biosynthetic genes. On the basis of these results, we propose a model to illustrate the hierarchic role of phenazine regulators modulated by Gac in the control of phenazine production. The transcriptomic analysis also was used to identify additional genes regulated by GacA that may contribute to the biological control capability of strain 30-84.

Project description:Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.

Dataset Information

Vfr, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in Pseudomonas chlororaphis G05.

Publications

<i>vfr</i>, A Global Regulatory Gene, is Required for Pyrrolnitrin but not for Phenazine-1-carboxylic Acid Biosynthesis in <i>Pseudomonas chlororaphis</i> G05.

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