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Microbial Cell Factories Dec 20211-Hydroxyphenazine (1-OH-PHZ) is a phenazine microbial metabolite with broad-spectrum antibacterial activities against a lot of plant pathogens. However, its use is...
BACKGROUND
1-Hydroxyphenazine (1-OH-PHZ) is a phenazine microbial metabolite with broad-spectrum antibacterial activities against a lot of plant pathogens. However, its use is hampered by the low yield all along. Metabolic engineering of microorganisms is an increasingly powerful method for the production of valuable organisms at high levels. Pseudomonas chlororaphis is recognized as a safe and effective plant rhizosphere growth-promoting bacterium, and faster growth rate using glycerol or glucose as a renewable carbon source. Therefore, Pseudomonas chlororaphis is particularly suitable as the chassis cell for the modification and engineering of phenazines.
RESULTS
In this study, enzyme PhzS (monooxygenase) was heterologously expressed in a phenazine-1-carboxylic acid (PCA) generating strain Pseudomonas chlororaphis H18, and 1-hydroxyphenazine was isolated, characterized in the genetically modified strain. Next, the yield of 1-hydroxyphenazine was systematically engineered by the strategies including (1) semi-rational design remodeling of crucial protein PhzS, (2) blocking intermediate PCA consumption branch pathway, (3) enhancing the precursor pool, (4) engineering regulatory genes, etc. Finally, the titer of 1-hydroxyphenazine reached 3.6 g/L in 5 L fermenter in 54 h.
CONCLUSIONS
The 1-OH-PHZ production of Pseudomonas chlororaphis H18 was greatly improved through systematically engineering strategies, which is the highest, reported to date. This work provides a promising platform for 1-hydroxyphenazine engineering and production.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Biosynthetic Pathways; Fermentation; Genetic Engineering; Metabolic Engineering; Mixed Function Oxygenases; Phenazines; Pseudomonas chlororaphis
PubMed: 34965873
DOI: 10.1186/s12934-021-01731-y -
BMC Microbiology Jan 2022Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this...
BACKGROUND
Peanut stem rot is a serious plant disease that causes great economic losses. At present, there are no effective measures to prevent or control the occurrence of this plant disease. Biological control is one of the most promising plant disease control measures. In this study, Pseudomonas chlororaphis subsp. aurantiaca strain zm-1, a bacterial strain with potential biocontrol properties isolated by our team from the rhizosphere soil of Anemarrhena asphodeloides, was studied to control this plant disease.
METHODS
We prepared extracts of Pseudomonas chloroaphis zm-1 extracellular antibacterial compounds (PECEs), determined their antifungal activities by confrontation assay, and identified their components by UPLC-MS/MS. The gene knockout strains were constructed by homologous recombination, and the biocontrol efficacy of P. chlororaphis zm-1 and its mutant strains were evaluated by pot experiments under greenhouse conditions and plot experiments, respectively.
RESULTS
P. chlororaphis zm-1 could produce extracellular antifungal substances and inhibit the growth of Sclerotium rolfsii, the main pathogenic fungus causing peanut stem rot. The components of PECEs identified by UPLC-MS/MS showed that three kinds of phenazine compounds, i.e., 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), and the core phenazine, were the principal components. In particular, 1-hydroxyphenazine produced by P. chlororaphis zm-1 showed antifungal activities against S. rolfsii, but 2-hydroxyphenazine did not. This is quite different with the previously reported. The extracellular compounds of two mutant strains, ΔphzH and ΔphzE, was analysed and showed that ΔphzE did not produce any phenazine compounds, and ΔphzH no longer produced 1-hydroxyphenazine but could still produce PCA and phenazine. Furthermore, the antagonistic ability of ΔphzH declined, and that of ΔphzE was almost completely abolished. According to the results of pot experiments under greenhouse conditions, the biocontrol efficacy of ΔphzH dramatically declined to 47.21% compared with that of wild-type P. chlororaphis zm-1 (75.63%). Moreover, ΔphzE almost completely lost its ability to inhibit S. rolfsii (its biocontrol efficacy was reduced to 6.19%). The results of the larger plot experiments were also consistent with these results.
CONCLUSIONS
P. chlororaphis zm-1 has the potential to prevent and control peanut stem rot disease. Phenazines produced and secreted by P. chlororaphis zm-1 play a key role in the control of peanut stem rot caused by S. rolfsii. These findings provide a new idea for the effective prevention and treatment of peanut stem rot.
Topics: Antibiosis; Antifungal Agents; Arachis; Bacterial Proteins; Basidiomycota; Biological Control Agents; Mutation; Phenazines; Plant Diseases; Pseudomonas
PubMed: 34986788
DOI: 10.1186/s12866-021-02420-x -
Microbial Biotechnology Nov 2023Virulence factor modulating (VFM) is a quorum sensing (QS) signal shared by and specific to Dickeya bacteria, regulating the production of plant cell wall degrading...
Virulence factor modulating (VFM) is a quorum sensing (QS) signal shared by and specific to Dickeya bacteria, regulating the production of plant cell wall degrading enzymes (PCWDEs) and virulence of Dickeya. High polarity and trace of VFM signal increase the difficulty of signal separation and structure identification, and thus limit the development of quorum quenching strategy to biocontrol bacterial soft rot diseases caused by Dickeya. In order to high-throughput screen VFM quenching bacteria, a vfmE-gfp biosensor VR2 (VFM Reporter) sensitive to VFM signal was first constructed. Subsequently, two bacterial strains with high quenching efficiency were screened out by fluorescence intensity measurement and identified as Pseudomonas chlororaphis L5 and Enterobacter asburiae L95 using multilocus sequence analysis (MLSA). L5 and L95 supernatants reduced the expression of vfm genes, and both strains also decreased the production of PCWDEs of D. zeae MS2 and significantly reduced the virulence of D. oryzae EC1 on rice seedlings, D. zeae MS2 on banana seedlings, D. dadantii 3937 on potato and D. fangzhongdai CL3 on taro. Findings in this study provide a method to high-throughput screen VFM quenching bacteria and characterize novel functions of P. chlororaphis and E. asburiae in biocontrolling plant diseases through quenching VFM QS signal.
Topics: Virulence Factors; Dickeya; Quorum Sensing; Pseudomonas chlororaphis; Enterobacteriaceae; Plant Diseases
PubMed: 37815509
DOI: 10.1111/1751-7915.14351 -
Frontiers in Bioengineering and... 2022The outstanding metabolic and bioprotective properties of the bacterial genus make these species a potentially interesting source for the search of hydrolytic...
The outstanding metabolic and bioprotective properties of the bacterial genus make these species a potentially interesting source for the search of hydrolytic activities that could be useful for the degradation of plastics. We identified two genes encoding the intracellular lipases LIP1 and LIP2 of the biocontrol bacterium PA23 and subsequently performed cloning and expression in . The gene has an open reading frame of 828 bp and encodes a protein of 29.7 kDa whereas the consists of 834 bp and has a protein of 30.2 kDa. Although secondary structure analyses of LIP1 and LIP2 indicate a dominant α/β-hydrolase-fold, the two proteins differ widely in their amino acid sequences (15.39% identity), substrate specificities, and hydrolysis rates. Homology modeling indicates the catalytic serine in both enzymes located in a GXSXG sequence motif (lipase box). However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with a GGX-type oxyanion pocket, whereas LIP2 has Ser138-His249-Asp221 in its active site and a GX-type of oxyanion hole residues. However, LIP1 has a catalytic triad of Ser152-His253-Glu221 with an oxyanion pocket of GGX-type, whereas LIP2 has Ser138-His249-Asp221 in its active site and a GX-type of oxyanion hole residues. Our three-dimensional models of LIP1 and LIP2 complexed with a 3-hydroxyoctanoate dimer revealed the core α/β hydrolase-type domain with an exposed substrate binding pocket in LIP1 and an active-site capped with a closing lid domain in LIP2. The recombinant LIP1 was optimally active at 45°C and pH 9.0, and the activity improved in the presence of Ca. LIP2 exhibited maximum activity at 40°C and pH 8.0, and was unaffected by Ca. Despite different properties, the enzymes exhibited broadsubstrate specificity and were able to hydrolyze short chain length and medium chain length polyhydroxyalkanoates (PHAs), polylactic acid (PLA), and para-nitrophenyl (pNP) alkanoates. Gel Permeation Chromatography (GPC) analysis showed a decrease in the molecular weight of the polymers after incubation with LIP1 and LIP2. The enzymes also manifested some polymer-degrading activity on petroleum-based polymers such as poly(ε-caprolactone) (PCL) and polyethylene succinate (PES), suggesting that these enzymes could be useful for biodegradation of synthetic polyester plastics. The study will be the first report of the complete characterization of intracellular lipases from bacterial and/or species. The lipases, LIP1 and LIP2 are different from other bacterial lipases/esterases in having broad substrate specificity for polyesters.
PubMed: 35519608
DOI: 10.3389/fbioe.2022.854298 -
Plants (Basel, Switzerland) Jul 2021The development of biotechnologies based on beneficial microorganisms for improving soil fertility and crop yields could help to address many current agriculture...
The development of biotechnologies based on beneficial microorganisms for improving soil fertility and crop yields could help to address many current agriculture challenges, such as food security, climate change, pest control, soil depletion while decreasing the use of chemical fertilizers and pesticides. Plant growth-promoting (PGP) microbes can be used as probiotics in order to increase plant tolerance/resistance to abiotic/biotic stresses and in this context strains belonging to the group have shown to have potential as PGP candidates. In this study a new isolate is reported and tested for (i) in vitro PGP features, (ii) whole-genome sequence analysis, and (iii) its effects on the rhizosphere microbiota composition, plant growth, and different plant genes expression levels in greenhouse experiments. Results showed that ST9 is an efficient rice root colonizer which integrates into the plant resident-microbiota and affects the expression of several plant genes. The potential use of this strain as a plant probiotic is discussed.
PubMed: 34371669
DOI: 10.3390/plants10071466 -
Frontiers in Microbiology 2023Phenazine-1-carboxylic acid (PCA) is a biologically active substance with the ability to prevent and control crop diseases. It was certified as a pesticide by the...
Phenazine-1-carboxylic acid (PCA) is a biologically active substance with the ability to prevent and control crop diseases. It was certified as a pesticide by the Ministry of Agriculture of China in 2011 and was named "Shenzimycin." Lzh-T5 is a strain found in the rhizosphere of tomatoes. This strain can produce only 230 mg/L of PCA. We used LDA-4, which produces the phenazine synthetic intermediate trans-2,3-dihydro-3-hydroxyanthranilic acid in high amounts, as the starting strain. By restoring and knocking out , we achieved PCA accumulation. Moreover, PCA production was enhanced after knocking out negative regulators, enhancing the shikimate pathway, and performing fed-batch fermentation, thus resulting in the production of 10,653 mg/L of PCA. It suggested that Lzh-T5 has the potential to become an efficiency cell factory of biologically active substances.
PubMed: 37168109
DOI: 10.3389/fmicb.2023.1186052 -
Pathogens (Basel, Switzerland) Jul 2021Wheat is a worldwide staple food crop, and take-all caused by var. can lead to a tremendous decrease in wheat yield and quality. In this study, strain YB-10 was...
Wheat is a worldwide staple food crop, and take-all caused by var. can lead to a tremendous decrease in wheat yield and quality. In this study, strain YB-10 was isolated from wheat rhizospheric soil and identified as by morphology and 16S rRNA gene sequencing. YB-10 had extracellular protease and cellulase activities and strongly inhibited the mycelium growth of var. in dual cultures. Up to 87% efficacy of YB-10 in controlling the take-all of seedlings was observed in pot experiments when wheat seed was coated with the bacterium. YB-10 was also positive for indole acetic acid (IAA) and siderophore production, and coating wheat seed with the bacterium significantly promoted the growth of seedlings at 10 and 10 CFU/mL. Furthermore, treatment with YB-10 increased activities of the wheat defense-related enzymes POD, SOD, CAT, PAL and PPO in seedlings, indicating induced resistance against pathogens. Overall, YB-10 is a promising new seed-coating agent to both promote wheat growth and suppress take-all.
PubMed: 34358053
DOI: 10.3390/pathogens10070903 -
Transgenic Research Feb 2018Genetically modified crops undergo extensive evaluation to characterize their food, feed and environmental safety prior to commercial introduction, using a... (Review)
Review
Genetically modified crops undergo extensive evaluation to characterize their food, feed and environmental safety prior to commercial introduction, using a well-established, science-based assessment framework. One component of the safety assessment includes an evaluation of each introduced trait, including its source organism, for potential adverse pathogenic, toxic and allergenic effects. Several Pseudomonas species have a history of safe use in agriculture and certain species represent a source of genes with insecticidal properties. The ipd072Aa gene from P. chlororaphis encodes the IPD072Aa protein, which confers protection against certain coleopteran pests when expressed in maize plants. P. chlororaphis is ubiquitous in the environment, lacks known toxic or allergenic properties, and has a history of safe use in agriculture and in food and feed crops. This information supports, in part, the safety assessment of potential traits, such as IPD072Aa, that are derived from this source organism.
Topics: Bacterial Proteins; Biological Control Agents; Crops, Agricultural; Insecticides; Phylogeny; Plants, Genetically Modified; Pseudomonas chlororaphis; Zea mays
PubMed: 29427161
DOI: 10.1007/s11248-018-0061-6 -
Applied and Environmental Microbiology Aug 2017R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many species. In this study, analysis of...
R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many species. In this study, analysis of the 30-84 R-tailocin gene cluster revealed that it contains the structural components to produce two R-tailocins of different ancestral origins. Two distinct R-tailocin populations differing in length were observed in UV-induced lysates of 30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to species. The spectra of pseudomonads killed by the two R-tailocins differed, although a few species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of 30-84 to compete with an R-tailocin-sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that species can produce more than one functional R-tailocin particle sharing the same lysis cassette but differing in their killing spectra. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated in biofilms and the rhizosphere. Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated species in bacterial population dynamics within surface-attached biofilms and on roots.
Topics: Antibiosis; Bacterial Proteins; Bacteriocins; Biofilms; Plant Roots; Pseudomonas; Pseudomonas chlororaphis; Rhizosphere; Soil Microbiology
PubMed: 28526791
DOI: 10.1128/AEM.00706-17 -
Frontiers in Microbiology 2021Members of the genus are metabolically versatile and capable of adapting to a wide variety of environments. Stress physiology of strains has been extensively studied... (Review)
Review
Members of the genus are metabolically versatile and capable of adapting to a wide variety of environments. Stress physiology of strains has been extensively studied because of their biotechnological potential in agriculture as well as their medical importance with regards to pathogenicity and antibiotic resistance. This versatility and scientific relevance led to a substantial amount of information regarding the stress response of a diverse set of species such as , , , , and . In this review, environmental and industrial stressors including desiccation, heat, and cold stress, are cataloged along with their corresponding mechanisms of survival in . Mechanisms of survival are grouped by the type of inducing stress with a focus on adaptations such as synthesis of protective substances, biofilm formation, entering a non-culturable state, enlisting chaperones, transcription and translation regulation, and altering membrane composition. The strategies strains utilize for survival can be leveraged during the development of beneficial strains to increase viability and product efficacy.
PubMed: 34040596
DOI: 10.3389/fmicb.2021.660134