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1.
Appl Microbiol Biotechnol ; 105(20): 7825-7839, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34562115

RESUMO

Phenazine-1-carboxylic acid and pyrrolnitrin, the two secondary metabolites produced by Pseudomonas chlororaphis G05, serve as biocontrol agents that mainly contribute to the growth repression of several fungal phytopathogens. Although some regulators of phenazine-1-carboxylic acid biosynthesis have been identified, the regulatory pathway involving phenazine-1-carboxylic acid synthesis is not fully understood. We isolated a white conjugant G05W03 on X-Gal-containing LB agar during our screening of novel regulator candidates using transposon mutagenesis with a fusion mutant G05Δphz::lacZ as a recipient. By cloning of DNA adjacent to the site of the transposon insertion, we revealed that a LysR-type transcriptional regulator (LTTR) gene, finR, was disrupted in the conjugant G05W03. To confirm the regulatory function of FinR, we constructed the finR-knockout mutant G05ΔfinR, G05Δphz::lacZΔfinR, and G05Δprn::lacZΔfinR, using the wild-type strain G05 and its fusion mutant derivatives as recipient strains, respectively. We found that the expressions of phz and prn operons were dramatically reduced in the finR-deleted mutant. With quantification of the production of antifungal metabolites biosynthesized by the finR-negative strain G05ΔfinR, it was shown that FinR deficiency also led to decreased yield of phenazine-1-carboxylic acid and pyrrolnitrin. In addition, the pathogen inhibition assay confirmed that the production of phenazine-1-carboxylic acid was severely reduced in the absence of FinR. Transcriptional fusions and qRT-PCR verified that FinR could positively govern the transcription of the phz and prn operons. Taken together, FinR is required for antifungal metabolite biosynthesis and crop protection against some fungal pathogens.Key points• A novel regulator FinR was identified by transposon mutagenesis.• FinR regulates antifungal metabolite production.• FinR regulates the phz and prn expression by binding to their promoter regions.


Assuntos
Pseudomonas chlororaphis , Pirrolnitrina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Óperon , Fenazinas , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo
2.
Sci Rep ; 11(1): 16451, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385485

RESUMO

Trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) is a cyclic ß-amino acid used for the synthesis of non-natural peptides and chiral materials. And it is an intermediate product of phenazine production in Pseudomonas spp. Lzh-T5 is a P. chlororaphis strain isolated from tomato rhizosphere found in China. It can synthesize three antifungal phenazine compounds. Disruption the phzF gene of P. chlororaphis Lzh-T5 results in DHHA accumulation. Several strategies were used to improve production of DHHA: enhancing the shikimate pathway by overexpression, knocking out negative regulatory genes, and adding metal ions to the medium. In this study, three regulatory genes (psrA, pykF, and rpeA) were disrupted in the genome of P. chlororaphis Lzh-T5, yielding 5.52 g/L of DHHA. When six key genes selected from the shikimate, pentose phosphate, and gluconeogenesis pathways were overexpressed, the yield of DHHA increased to 7.89 g/L. Lastly, a different concentration of Fe3+ was added to the medium for DHHA fermentation. This genetically engineered strain increased the DHHA production to 10.45 g/L. According to our result, P. chlororaphis Lzh-T5 could be modified as a microbial factory to produce DHHA. This study laid a good foundation for the future industrial production and application of DHHA.


Assuntos
Ácido 3-Hidroxiantranílico/metabolismo , Pseudomonas chlororaphis/genética , Ácido 3-Hidroxiantranílico/química , Meios de Cultura , Fermentação , Compostos Férricos/metabolismo , Técnicas de Silenciamento de Genes , Genes Bacterianos , Genes Reguladores , Fenazinas/metabolismo
3.
World J Microbiol Biotechnol ; 37(6): 99, 2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-33978868

RESUMO

The Pseudomonas fluorescens complex contains at least eight phylogenetic groups and each of these includes several bacterial species sharing ecological and physiological traits. Pseudomonas chlororaphis classified in a separate group is represented by three different subspecies that show distinctive traits exploitable for phytostimulation and biocontrol of phytopathogens. The high level of microbial competitiveness in soil as well as the effectiveness in controlling several plant pathogens and pests can be related to the P. chlororaphis ability to implement different stimulating and toxic mechanisms in its interaction with plants and the other micro- and macroorganisms. Pseudomonas chlororaphis strains produce antibiotics, such as phenazines, pyrrolnitrine, 2-hexyl, 5-propyl resorcinol and hydrogen cyanide, siderophores such as pyoverdine and achromobactine and a complex blend of volatile organic compounds (VOCs) that effectively contribute to the control of several plant pathogens, nematodes and insects. Phenazines and some VOCs are also involved in the induction of systemic resistance in plants. This complex set of beneficial strategies explains the high increasing interest in P. chlororaphis for commercial and biotechnological applications. The aim of this review is to highlight the role of the different mechanisms involved in the biocontrol activity of P. chlororaphis strains.


Assuntos
Agentes de Controle Biológico/farmacologia , Desenvolvimento Vegetal/efeitos dos fármacos , Pseudomonas chlororaphis/química , Resistência à Doença , Filogenia , Metabolismo Secundário
4.
Can J Microbiol ; 67(6): 476-490, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34057367

RESUMO

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola against the fungal pathogen Sclerotinia sclerotiorum. In addition to producing antifungal compounds, this bacterium synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds. Because the role of PHA in PA23 physiology is currently unknown, we investigated the impact of this polymer on stress resistance, adherence to surfaces, and interaction with the protozoan predator Acanthamoeba castellanii. Three PHA biosynthesis mutants were created, PA23phaC1, PA23phaC1ZC2, and PA23phaC1ZC2D, which accumulated reduced PHA. Our phenotypic assays revealed that PA23phaC1ZC2D produced less phenazine (PHZ) compared with the wild type (WT) and the phaC1 and phaC1ZC2 mutants. All three mutants exhibited enhanced sensitivity to UV irradiation, starvation, heat stress, cold stress, and hydrogen peroxide. Moreover, motility, exopolysaccharide production, biofilm formation, and root attachment were increased in strains with reduced PHA levels. Interaction studies with the amoeba A. castellanii revealed that the WT and the phaC1 and phaC1ZC2 mutants were consumed less than the phaC1ZC2D mutant, likely due to decreased PHZ production by the latter. Collectively these findings indicate that PHA accumulation enhances PA23 resistance to a number of stresses in vitro, which could improve the environmental fitness of this bacterium in hostile environments.


Assuntos
Acanthamoeba castellanii/fisiologia , Biofilmes/crescimento & desenvolvimento , Poli-Hidroxialcanoatos/metabolismo , Pseudomonas chlororaphis/fisiologia , Estresse Fisiológico/fisiologia , Aderência Bacteriana , Brassica napus/microbiologia , Mutação , Fenazinas/metabolismo , Poli-Hidroxialcanoatos/genética , Polissacarídeos Bacterianos/metabolismo , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo
5.
J Agric Food Chem ; 69(16): 4778-4784, 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33848158

RESUMO

The take-all disease of wheat is one of the most serious diseases in the field of food security in the world. There is no effective biological pesticide to prevent the take-all disease of wheat. 2-Hydroxyphenazine (2-OH-PHZ) was reported to possess a better inhibitory effect on the take-all disease of wheat than phenazine-1-carboxylic acid, which was registered as "Shenqinmycin" in China in 2011. The aim of this study was to construct a 2-OH-PHZ high-producing strain by strain screening, genome sequencing, genetic engineering, and fermentation optimization. First, the metabolites of the previously screened new phenazine-producing Pseudomonas sp. strain were identified, and the taxonomic status of the new Pseudomonas sp. strain was confirmed through 16S rRNA and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Then, the new Pseudomonas sp. strain was named Pseudomonas chlororaphis subsp. aurantiaca LX24, which is a new subspecies of P. chlororaphis that can synthesize 2-OH-PHZ. Next, the draft genome of strain LX24 was determined, and clusters of orthologous group (COG) analysis, KEGG analysis, and gene ontology (GO) analysis of strain LX24 were performed. Furthermore, the production of 2-OH-PHZ increased to 351.7 from 158.6 mg/L by deletion of the phenazine synthesis negative regulatory genes rpeA and rsmE in strain LX24. Finally, the 2-OH-PHZ production of strain LX24 reached 677.1 mg/L after fermentation optimization, which is the highest production through microbial fermentation reported to date. This work provides a reference for the efficient production of other pesticides and antibiotics.


Assuntos
Pseudomonas chlororaphis , Proteínas de Bactérias/genética , China , Fenazinas , Pseudomonas , Pseudomonas chlororaphis/genética , RNA Ribossômico 16S
6.
World J Microbiol Biotechnol ; 37(5): 82, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33855623

RESUMO

Many volatile compounds secreted by bacteria play an important role in the interactions of microorganisms, can inhibit the growth of phytopathogenic bacteria and fungi, can suppress or stimulate plant growth and serve as infochemicals presenting a new type of interspecies communication. In this work, we investigated the effect of total pools of volatile substances and individual volatile organic compounds (VOCs) synthesized by the rhizosphere bacteria Pseudomonas chlororaphis 449 and Serratia plymuthica IC1270, the soil-borne strain P. fluorescens B-4117 and the spoiled meat isolate S. proteamaculans 94 on Arabidopsis thaliana plants. We showed that total gas mixtures secreted by these strains during their growth on Luria-Bertani agar inhibited A. thaliana growth. Hydrogen cyanide synthesis was unnecessary for the growth suppression. A decrease in the inhibition level was observed for the strain P. chlororaphis 449 with a mutation in the gacS gene, while inactivation of the rpoS gene had no effect. Individual VOCs synthesized by these bacteria (1-indecene, ketones 2-nonanone, 2-heptanone, 2-undecanone, and dimethyl disulfide) inhibited the growth of plants or killed them. Older A. thaliana seedlings were more resistant to VOCs than younger seedlings. The results indicated that the ability of some volatiles emitted by the rhizosphere and soil bacteria to inhibit plant growth should be considered when assessing the potential of such bacteria for the biocontrol of plant diseases.


Assuntos
Arabidopsis/efeitos dos fármacos , Pseudomonas chlororaphis/química , Pseudomonas chlororaphis/genética , Pseudomonas fluorescens/química , Serratia/química , Compostos Orgânicos Voláteis/toxicidade , Arabidopsis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cianeto de Hidrogênio/metabolismo , Carne/microbiologia , Mutação , Pseudomonas chlororaphis/metabolismo , Pseudomonas fluorescens/metabolismo , Rizosfera , Plântula/efeitos dos fármacos , Serratia/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Microbiologia do Solo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Compostos Orgânicos Voláteis/química
7.
J Agric Food Chem ; 69(13): 3895-3903, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33759523

RESUMO

Polyhydroxyalkanoates (PHAs) have been reported with agricultural and medical applications in virtue of their biodegradable and biocompatible properties. Here, we systematically engineered three modules for the enhanced biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA) in Pseudomonas chlororaphis HT66. The phzE, fadA, and fadB genes were deleted to block the native phenazine pathway and weaken the fatty acid ß-oxidation pathway. Additionally, a PHA depolymerase gene phaZ was knocked out to prevent the degradation of mcl-PHA. Three genes involved in the mcl-PHA biosynthesis pathway were co-overexpressed to increase carbon flux. The engineered strain HT4Δ::C1C2J exhibited an 18.2 g/L cell dry weight with 84.9 wt % of mcl-PHA in a shake-flask culture, and the 3-hydroxydodecanoate (3HDD) monomer was increased to 71.6 mol %. Thermophysical and mechanical properties of mcl-PHA were improved with an enriched ratio of 3HDD. This study demonstrated a rational metabolic engineering approach to enhance the production of mcl-PHA with the enriched dominant monomer and improved material properties.


Assuntos
Poli-Hidroxialcanoatos , Pseudomonas chlororaphis , Engenharia Metabólica , Pseudomonas chlororaphis/genética
8.
Appl Environ Microbiol ; 87(7)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33452032

RESUMO

Pseudomonas chlororaphis YL-1 has extensive antimicrobial activities against phytopathogens, and its genome harbors a pyoverdine (PVD) biosynthesis gene cluster. The alternative sigma factor PvdS in Pseudomonas aeruginosa PAO1 acts as a critical regulator in response to iron starvation. The assembly of the PVD backbone starts with peptide synthetase enzyme PvdL. PvdF catalyzes formylation of l-OH-Orn to produce l-N 5-hydroxyornithine. Here, we describe the characterization of PVD production in YL-1 and its antimicrobial activity in comparison with that of its PVD-deficient ΔpvdS, ΔpvdF, and ΔpvdL mutants, which were obtained using a sacB-based site-specific mutagenesis strategy. Using in vitro methods, we examined the effect of exogenous iron under low-iron conditions and an iron-chelating agent under iron-sufficient conditions on PVD production, antibacterial activity, and the relative expression of the PVD transcription factor gene pvdS in YL-1. We found that strain YL-1, the ΔpvdF mutant, and the ΔpvdS(pUCP26-pvdS) complemented strain produced visible PVDs and demonstrated a wide range of inhibitory effects against Gram-negative and Gram-positive bacteria in vitro under low-iron conditions and that with the increase of iron, its PVD production and antibacterial activity were reduced. The antibacterial compounds produced by strain YL-1 under low-iron conditions were PVDs based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Moreover, the antibacterial activity observed in vitro was correlated with in vivo control efficacies of strain YL-1 against rice bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae. Collectively, PVDs are responsible for the antibacterial activities of strain YL-1 under both natural and induced low-iron conditions.IMPORTANCE The results demonstrated that PVDs are essential for the broad-spectrum antibacterial activities of strain YL-1 against both Gram-positive and Gram-negative bacteria under low-iron conditions. Our findings also highlight the effect of exogenous iron on the production of PVD and the importance of this bacterial product in bacterial interactions. As a biocontrol agent, PVDs can directly inhibit the proliferation of the tested bacteria in addition to participating in iron competition.


Assuntos
Antibacterianos/farmacologia , Ferro/metabolismo , Oligopeptídeos/farmacologia , Pseudomonas chlororaphis/metabolismo , Antibacterianos/química , Cromatografia Líquida , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Oligopeptídeos/química , Pseudomonas chlororaphis/química , Pseudomonas chlororaphis/genética , Espectrometria de Massas em Tandem
9.
J Gen Appl Microbiol ; 67(1): 24-32, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33162426

RESUMO

Pseudomonas chlororaphis B23 yields nitrile hydratase (NHase) used for the production of 5-cyanovaleramide at the industrial level. Although the nhpC gene (known as P47K) located just downstream of the NHase structural genes (nhpAB) has been important for efficient NHase expression, the key role of nhpC remains poorly studied. Here, we purified two NHases expressed in the presence and absence of nhpC, respectively, and characterized them. The purified NHase expressed with nhpC proved to be an iron-containing holo-NHase, while the purified one expressed without nhpC was identified as an apo-NHase, which was iron-deficient. These findings indicated that nhpC would play a crucial role in the post-translational incorporation of iron into the NHase active site as a metal chaperone. In the overall amino acid sequence of NhpC, only the N-terminus exhibited similarities to the CobW protein involved in cobalamin biosynthesis, the UreG and HypB proteins essential for the metallocenter biosynthesis of urease and hydrogenase, respectively. NhpC contains a P-loop motif known as a nucleotide-binding site, and Lys23 and Thr24 are conserved in the P-loop motif in NhpC. Expression analysis of NHase formed in the presence of each mutant NhpC (i.e., K23A and T24A) resulted in immunodetectable production of a mutant NhpC and remarkable expression of NHase lacking the enzyme activity. These findings suggested that an intact P-loop containing Lys23 and Thr24 would be essential for the NhpC function in vivo for the post-translational metallocenter assembly of NHase.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidroliases/biossíntese , Hidroliases/genética , Pseudomonas chlororaphis/enzimologia , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Ferro , Mutagênese Sítio-Dirigida , Proteínas Recombinantes , Urease/metabolismo
10.
Environ Microbiol ; 23(4): 2086-2101, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33314481

RESUMO

Pseudomonas chlororaphis PCL1606 (PcPCL1606) displays plant-colonizing features and exhibits antagonistic traits against soil-borne phytopathogenic fungi. Biofilm formation could be relevant for the PcPCL1606 lifestyle, and in this study the role of some putative extracellular matrix components (EMC; Fap-like fibre, alginate and Psl-like polysaccharides) in the biofilm architecture and biocontrol activity of this bacterium were determined. EMC such as the Fap-like fibre and alginate polysaccharide play secondary roles in biofilm formation in PcPCL1606, because they are not fundamental to its biofilm architecture in flow cell chamber, but synergistically they have shown to favour bacterial competition during biofilm formation. Conversely, studies on Psl-like polysaccharide have revealed that it may contain mannose, and that it is strongly involved in the PcPCL1606 biofilm architecture and niche competition. Furthermore, the Fap-like fibre and Psl-like exopolysaccharide play roles in early surface attachment and contribute to biocontrol activity against the white root rot disease caused by Rosellinia necatrix in avocado plants. These results constitute the first report regarding the study of the extracellular matrix of the PcPCL1606 strain and highlight the importance of a putative Fap-like fibre and Psl-like exopolysaccharide produced by PcPCL1606 in the biofilm formation process and interactions with the host plant root.


Assuntos
Pseudomonas chlororaphis , Xylariales , Ascomicetos , Biofilmes , Matriz Extracelular , Polissacarídeos Bacterianos , Pseudomonas aeruginosa
11.
J Agric Food Chem ; 68(50): 14832-14840, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33287542

RESUMO

Phenazine-1-carboxylic acid (PCA), the primary active ingredient of Shenqinmycin, was awarded the China Pesticide Certificate in 2011 due to its excellent antibacterial action. Phenazine-1-carboxamide (PCN) is a derivative of PCA, which is modified by the phzH gene, and its anti-bacterial effect is better than that of PCA. At present, PCN can be produced via Pseudomonas fermentation using an opportunistic pathogen, Pseudomonas aeruginosa. Qlu-1 is an environmentally friendly strain of Pseudomonas chlororaphis that can produce phenazine derivatives. We replaced the phzO gene with the phzH gene from P. aeruginosa to achieve PCN accumulation. Different strategies were used to enhance PCN production: knocking out of negative regulatory factors, enhancing the shikimate pathway by gene overexpression and gene knocking, and using fed-batch fermentation. Finally, an engineered strain of P. chlororaphis was produced, which produced 11.45 g/L PCN. This achievement indicates that Qlu-1 could be modified as a potential microbial cell factory for PCN production by metabolic engineering.


Assuntos
Proteínas de Bactérias/genética , Fenazinas/metabolismo , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Proteínas de Bactérias/metabolismo , Engenharia Metabólica , Família Multigênica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo
12.
Sci Rep ; 10(1): 21289, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277560

RESUMO

Outer membrane vesicles (OMVs) produced by Gram-negative bacteria have roles in cell-to-cell signaling, biofilm formation, and stress responses. Here, the effects of abiotic stressors on OMV contents and composition from biofilm cells of the plant health-promoting bacterium Pseudomonas chlororaphis O6 (PcO6) are examined. Two stressors relevant to this root-colonizing bacterium were examined: CuO nanoparticles (NPs)-a potential fertilizer and fungicide- and H2O2-released from roots during plant stress responses. Atomic force microscopy revealed 40-300 nm diameter OMVs from control and stressed biofilm cells. Raman spectroscopy with linear discriminant analysis (LDA) was used to identify changes in chemical profiles of PcO6 cells and resultant OMVs according to the cellular stressor with 84.7% and 83.3% accuracies, respectively. All OMVs had higher relative concentrations of proteins, lipids, and nucleic acids than PcO6 cells. The nucleic acid concentration in OMVs exhibited a cellular stressor-dependent increase: CuO NP-induced OMVs > H2O2-induced OMVs > control OMVs. Biochemical assays confirmed the presence of lipopolysaccharides, nucleic acids, and protein in OMVs; however, these assays did not discriminate OMV composition according to the cellular stressor. These results demonstrate the sensitivity of Raman spectroscopy using LDA to characterize and distinguish cellular stress effects on OMVs composition and contents.


Assuntos
Membrana Externa Bacteriana/metabolismo , Vesículas Extracelulares/metabolismo , Pseudomonas chlororaphis/metabolismo , Estresse Fisiológico , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/ultraestrutura , Vesículas Extracelulares/química , Vesículas Extracelulares/ultraestrutura , Pseudomonas chlororaphis/química , Pseudomonas chlororaphis/ultraestrutura , Análise Espectral Raman
13.
J Basic Microbiol ; 60(11-12): 950-961, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33025611

RESUMO

Plant-growth-promoting rhizobacteria (PGPR) should effectively colonize along the plant root to enhance the plant and soil health. The present investigation aims to improve the PGPR-mediated plant health benefits through above-ground foliar management. A green fluorescent protein-tagged PGPR strain, Pseudomonas chlororaphis (ZSB15-M2) was inoculated in a nonautoclaved agricultural soil before rice culturing. Salicylic acid and cell extracts of Corynebacterium glutamicum and Saccharomyces cerevisiae as a supply of hormonal and inducer compounds were applied on the foliage of the 10-days-old rice plants and subsequently observed the colonizing ability of ZSB15-M2. The cell extracts of Corynebacteria and yeast showed a 100-fold increase in the ZSB15-M2 population in the rhizosphere of rice, whereas salicylic acid had a 10-fold increase in relation to mock control. The rice root exudates collected after the spraying of salicylic acid and microbial extracts showed significantly enhanced release of total carbon, total protein, total sugar, total amino nitrogen, total nitrogen, and phenol content. In vitro assays revealed that these root exudates collected after exogenous spray of these chemicals enhanced the chemotactic motility and biofilm formation of ZSB15-M2 compared to the control plant's root exudate. Metabolomic analysis of root exudates collected from these rice plants by gas chromatography-mass spectrometry revealed that the Corynebacteria and yeast cell extracts enhanced the divergence of metabolites of rice root exudate. Further, due to these cumulative effects in the rice rhizosphere, the total chlorophyll, total protein, total nitrogen, and total phosphorus of rice were significantly improved. These observations provide insights into the rhizosphere functioning of rice plants as modulated by above-ground treatments with improved colonization of inoculant strains as well as the plant growth.


Assuntos
Inoculantes Agrícolas/efeitos dos fármacos , Oryza/crescimento & desenvolvimento , Reguladores de Crescimento de Plantas/farmacologia , Ácido Salicílico/farmacologia , Inoculantes Agrícolas/fisiologia , Biofilmes/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Corynebacterium glutamicum/química , Metaboloma/efeitos dos fármacos , Oryza/efeitos dos fármacos , Oryza/metabolismo , Oryza/microbiologia , Exsudatos de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Pseudomonas chlororaphis/efeitos dos fármacos , Pseudomonas chlororaphis/fisiologia , Rizosfera , Saccharomyces cerevisiae/química , Microbiologia do Solo
14.
Appl Microbiol Biotechnol ; 104(23): 10119-10132, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32984920

RESUMO

Pseudomonas chlororaphis is a plant-associated bacterium with reported antagonistic activity against different organisms and plant growth-promoting properties. P. chlororaphis possesses exciting biotechnological features shared with another Pseudomonas with a nonpathogenic phenotype. Part of the antagonistic role of P. chlororaphis is due to its production of a wide variety of phenazines. To expand the knowledge of the metabolic traits of this organism, we constructed the first experimentally validated genome-scale model of P. chlororaphis ATCC 9446, containing 1267 genes and 2289 reactions, and analyzed strategies to maximize its potential for the production of phenazine-1-carboxamide (PCN). The resulting model also describes the capability of P. chlororaphis to carry out the denitrification process and its ability to consume sucrose (Scr), trehalose, mannose, and galactose as carbon sources. Additionally, metabolic network analysis suggested fatty acids as the best carbon source for PCN production. Moreover, the optimization of PCN production was performed with glucose and glycerol. The optimal PCN production phenotype requires an increased carbon flux in TCA and glutamine synthesis. Our simulations highlight the intrinsic H2O2 flux associated with PCN production, which may generate cellular stress in an overproducing strain. These results suggest that an improved antioxidative strategy could lead to optimal performance of phenazine-producing strains of P. chlororaphis. KEY POINTS : • This is the first publication of a metabolic model for a strain of P. chlororaphis. • Genome-scale model is worthy tool to increase the knowledge of a non model organism. • Fluxes simulations indicate a possible effect of H2O2 on phenazines production. • P. chlororaphis can be a suitable model for a wide variety of compounds.


Assuntos
Pseudomonas chlororaphis , Peróxido de Hidrogênio , Fenazinas , Pseudomonas/genética , Pseudomonas chlororaphis/genética
15.
Ecotoxicol Environ Saf ; 202: 110854, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32585484

RESUMO

Atrazine as a kind of herbicide could cause damage to the sensitive plants. Though plant growth promoting rhizobacteria (PGPR) have been proven with the potential to enhance the resistance of plants against various abiotic stresses, whether it could alleviate phytotoxicity caused by atrazine is sill unclear. In present study, the effects of strain Pseudomonas chlororaphis PAS18, a kind of PGPR enable to produce indole-3-acetic acid (IAA), on the growth and physiological responses of Pennisetum americanum (L.) K.Schum seedlings were investigated under three different levels (0, 20 and 100 mg kg-1) of atrazine in pot experiment. The results suggest that strain PAS18 could alleviate the growth and physiological interference caused by 20 mg kg-1 of atrazine. Physiological analysis showed strain PAS18 could further decrease the damaged extent of photosystem II, superoxide radical level and malondialdehyde content of test plant via up-regulating psbA expression, enhancing superoxide dismutase activity and reducing atrazine accumulation in the test plant. Moreover, ion flux measurements suggest that IAA could alleviate the Ca2+ exflux state of the test plant which caused by atrazine stress. Hence, it is plausible that strain PAS18 could alleviate atrazine-induced stress to P. americanum by enhancing the photosystem II repair and antioxidant defense ability as well as balancing the Ca2+ flux.


Assuntos
Atrazina/toxicidade , Ácidos Indolacéticos/metabolismo , Pennisetum/fisiologia , Pseudomonas chlororaphis/fisiologia , Antioxidantes/metabolismo , Atrazina/metabolismo , Tolerância a Medicamentos , Herbicidas/metabolismo , Malondialdeído/metabolismo , Pennisetum/efeitos dos fármacos , Fotossíntese , Pseudomonas chlororaphis/metabolismo , Plântula/efeitos dos fármacos , Estresse Fisiológico
16.
Microbiol Res ; 239: 126517, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32535393

RESUMO

Three morphological mutants (M71a, M71b, M71c) of the antagonist Pseudomonas chlororaphis M71, naturally arose during a biocontrol trial against the phytopathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersisci. In this study, the three mutants were investigated to elucidate their role in the biocontrol of plant pathogens. M71a and M71b phenotypes were generated by a mutation in the two-component system GacS/GacA. The mutation determined an increase in siderophore production and an impaired ability to release proteases, to swarm, to produce phenazine and AHLs and to colonize tomato roots. In vitro antagonistic activity against different plant pathogens was partially reduced in M71a, while M71b resulted effective only against Pythium ultimum. Biocontrol efficacy against Fusarium oxysporum f.sp. radicis-lycopersisci, was partially reduced in M71a and completely lost in M71b. M71c phenotype was impaired in swarming motility, did not produce biofilms and its antagonistic activity was similar to the parental M71 strain. M71c showed an enhanced ability to colonize tomato roots, on which its progeny in part reverted to the M71 parental phenotype. Volatile organic compounds (VOCs) emitted by all four strains, inhibited the growth of Clavibacter michiganensis subsp. michiganensis and Seiridium cardinale in vitro. Real-time screening of VOCs by PTR-MS combined with GC-MS analysis, showed that methanethiol was the main component of the blend produced by all four M71 strains. However, the emissions of hydrogen cyanide, dimethyl disulfide, 1,3-butadiene and acetone were significantly affected by the three different mutations. These findings highlight that the simultaneous presence of different M71 phenotypes may improve, through the integration of different mechanisms, the ecological fitness and biocontrol efficacy of P. chlororaphis M71.


Assuntos
Agentes de Controle Biológico/metabolismo , Mutação , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/fisiologia , Proteínas de Bactérias/genética , Agentes de Controle Biológico/farmacologia , Fusarium/efeitos dos fármacos , Lycopersicon esculentum/microbiologia , Controle Biológico de Vetores , Fenazinas/metabolismo , Fenazinas/farmacologia , Fenótipo , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , Pseudomonas chlororaphis/química , Sideróforos/fisiologia , Compostos Orgânicos Voláteis/metabolismo , Compostos Orgânicos Voláteis/farmacologia
17.
Microb Cell Fact ; 19(1): 105, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32430011

RESUMO

BACKGROUND: 2-Acetamidophenol (AAP) is an aromatic compound with the potential for antifungal, anti-inflammatory, antitumor, anti-platelet, and anti-arthritic activities. Due to the biosynthesis of AAP is not yet fully understood, AAP is mainly produced by chemical synthesis. Currently, metabolic engineering of natural microbial pathway to produce valuable aromatic compound has remarkable advantages and exhibits attractive potential. Thus, it is of paramount importance to develop a dominant strain to produce AAP by elucidating the AAP biosynthesis pathway. RESULT: In this study, the active aromatic compound AAP was first purified and identified in gene phzB disruption strain HT66ΔphzB, which was derived from Pseudomonas chlororaphis HT66. The titer of AAP in the strain HT66ΔphzB was 236.89 mg/L. Then, the genes involved in AAP biosynthesis were determined. Through the deletion of genes phzF, Nat and trpE, AAP was confirmed to have the same biosynthesis route as phenazine-1-carboxylic (PCA). Moreover, a new arylamine N-acetyltransferases (NATs) was identified and proved to be the key enzyme required for generating AAP by in vitro assay. P. chlororaphis P3, a chemical mutagenesis mutant strain of HT66, has been demonstrated to have a robust ability to produce antimicrobial phenazines. Therefore, genetic engineering, precursor addition, and culture optimization strategies were used to enhance AAP production in P. chlororaphis P3. The inactivation of phzB in P3 increased AAP production by 92.4%. Disrupting the phenazine negative regulatory genes lon and rsmE and blocking the competitive pathway gene pykA in P3 increased AAP production 2.08-fold, which also confirmed that AAP has the same biosynthesis route as PCA. Furthermore, adding 2-amidophenol to the KB medium increased AAP production by 64.6%, which suggested that 2-amidophenol is the precursor of AAP. Finally, by adding 5 mM 2-amidophenol and 2 mM Fe3+ to the KB medium, the production of AAP reached 1209.58 mg/L in the engineered strain P3ΔphzBΔlonΔpykAΔrsmE using a shaking-flask culture. This is the highest microbial-based AAP production achieved to date. CONCLUSION: In conclusion, this study clarified the biosynthesis process of AAP in Pseudomonas and provided a promising host for industrial-scale biosynthesis of AAP from renewable resources.


Assuntos
Acetaminofen/metabolismo , Arilamina N-Acetiltransferase/metabolismo , Vias Biossintéticas , Engenharia Metabólica , Pseudomonas chlororaphis/enzimologia , Arilamina N-Acetiltransferase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Microbiologia Industrial , Pseudomonas chlororaphis/genética
18.
ACS Synth Biol ; 9(4): 883-892, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32197042

RESUMO

Aromatic N-oxides are valuable due to their versatile chemical, pharmaceutical, and agricultural applications. Natural phenazine N-oxides possess potent biological activities and can be applied in many ways; however, few N-oxides have been identified. Herein, we developed a microbial system to synthesize phenazine N-oxides via an artificial pathway. First, the N-monooxygenase NaphzNO1 was predicted and screened in Nocardiopsis sp. 13-12-13 through a product comparison and gene sequencing. Subsequently, according to similarities in the chemical structures of substrates, an artificial pathway for the synthesis of a phenazine N-oxide in Pseudomonas chlororaphis HT66 was designed and established using three heterologous enzymes, a monooxygenase (PhzS) from P. aeruginosa PAO1, a monooxygenase (PhzO) from P. chlororaphis GP72, and the N-monooxygenase NaphzNO1. A novel phenazine derivative, 1-hydroxyphenazine N'10-oxide, was obtained in an engineered strain, P. chlororaphis HT66-SN. The phenazine N-monooxygenase NaphzNO1 was identified by metabolically engineering the phenazine-producing platform P. chlororaphis HT66. Moreover, the function of NaphzNO1, which can catalyze the conversion of 1-hydroxyphenazine but not that of 2-hydroxyphenazine, was confirmed in vitro. Additionally, 1-hydroxyphenazine N'10-oxide demonstrated substantial cytotoxic activity against two human cancer cell lines, MCF-7 and HT-29. Furthermore, the highest microbial production of 1-hydroxyphenazine N'10-oxide to date was achieved at 143.4 mg/L in the metabolically engineered strain P3-SN. These findings demonstrate that P. chlororaphis HT66 has the potential to be engineered as a platform for phenazine-modifying gene identification and derivative production. The present study also provides a promising alternative for the sustainable synthesis of aromatic N-oxides with unique chemical structures by N-monooxygenase.


Assuntos
Engenharia Metabólica/métodos , Óxidos/metabolismo , Fenazinas/metabolismo , Pseudomonas chlororaphis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Pseudomonas chlororaphis/metabolismo
19.
World J Microbiol Biotechnol ; 36(3): 49, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32157439

RESUMO

Glycerol is a by-product of biodiesel, and it has a great application prospect to be transformed to synthesize high value-added compounds. Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere is a plant growth promoting rhizobacteria that can utilize amount of glycerol to synthesize phenazine-1-carboxylic acid (PCA). PCA has been commercially registered as "Shenqinmycin" in China due to its characteristics of preventing pepper blight and rice sheath blight. The aim of this study was to engineer glycerol utilization pathway in P. chlororaphis GP72. First, the two genes glpF and glpK from the glycerol metabolism pathway were overexpressed in GP72ANO separately. Then, the two genes were co-expressed in GP72ANO, improving PCA production from 729.4 mg/L to 993.4 mg/L at 36 h. Moreover, the shunt pathway was blocked to enhance glycerol utilization, resulting in 1493.3 mg/L PCA production. Additionally, we confirmed the inhibition of glpR on glycerol metabolism pathway in P. chlororaphis GP72. This study provides a good example for improving the utilization of glycerol to synthesize high value-added compounds in Pseudomonas.


Assuntos
Glicerol/metabolismo , Engenharia Metabólica/métodos , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Aquaporinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capsicum/microbiologia , China , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Glicerolfosfato Desidrogenase/genética , Redes e Vias Metabólicas/genética , Fenazinas/metabolismo , Proteínas Repressoras/genética , Rizosfera
20.
J Agric Food Chem ; 68(8): 2373-2380, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32013409

RESUMO

Pseudomonas chlororaphis have been demonstrated to be environmentally friendly biocontrol strains, and most of them can produce phenazine compounds. Phenazine-1,6-dicarboxylic acid (PDC), with a potential antibacterial activity, is generally found in Streptomyces but not in Pseudomonas. The present study aimed to explore the feasibility of PDC synthesis and the function of PhzG in Pseudomonas. A PDC producer was constructed by replacing phzG in P. chlororaphis with lphzG from Streptomyces lomondensis. Through gene deletion, common start codon changing, gene silence, and in vitro assay, our result revealed that the yield of PDC in P. chlororaphis is associated with the relative expression of phzG to phzA and phzB. In addition, it is found that PDC can be spontaneously synthesized without PhzG. This study provides an efficient way for PDC production and promotes a better understanding of PhzG function in PDC biosynthesis. Moreover, this study gives an alternative opportunity for developing new antibacterial biopesticides.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Fenazinas/metabolismo , Pseudomonas chlororaphis/genética , Pseudomonas chlororaphis/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Deleção de Genes , Dados de Sequência Molecular , Pseudomonas chlororaphis/enzimologia , Alinhamento de Sequência
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