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World Journal of Microbiology &... May 2021The Pseudomonas fluorescens complex contains at least eight phylogenetic groups and each of these includes several bacterial species sharing ecological and physiological... (Review)
Review
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.
Topics: Biological Control Agents; Disease Resistance; Phylogeny; Plant Development; Pseudomonas chlororaphis; Secondary Metabolism
PubMed: 33978868
DOI: 10.1007/s11274-021-03063-w -
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 -
Journal of Agricultural and Food... Apr 2021The 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...
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 sp. strain were identified, and the taxonomic status of the new sp. strain was confirmed through 16S rRNA and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Then, the new sp. strain was named subsp. LX24, which is a new subspecies of 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 and 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.
Topics: Bacterial Proteins; China; Phenazines; Pseudomonas; Pseudomonas chlororaphis; RNA, Ribosomal, 16S
PubMed: 33848158
DOI: 10.1021/acs.jafc.1c00434 -
Journal of Bioscience and Bioengineering Jun 2022Quorum sensing is a population density-dependent gene regulation mechanism. N-Acyl-l-homoserine lactone (AHL) has been identified as a signal compound in quorum sensing...
Regulation of phenazine-1-carboxamide production by quorum sensing in type strains of Pseudomonas chlororaphis subsp. chlororaphis and Pseudomonas chlororaphis subsp. piscium.
Quorum sensing is a population density-dependent gene regulation mechanism. N-Acyl-l-homoserine lactone (AHL) has been identified as a signal compound in quorum sensing in gram-negative bacteria. Phenazine derivatives are bacterial secondary metabolites known for their broad-spectrum antifungal activity. Pseudomonas chlororaphis has been demonstrated to be a biocontrol strain, and most of its species can produce phenazine derivatives under AHL-mediated quorum sensing. Although P. chlororaphis is divided into four subspecies, the relationship between phenazine production and quorum sensing has not been investigated in two of the subspecies, P. chlororaphis subsp. chlororaphis and piscium. Two luxI/luxR homolog gene sets, phzI and phzR and csaI and csaR, were found in the complete genome sequences of the type strains of P. chlororaphis subsp. chlororaphis JCM 2778 and P. chlororaphis subsp. piscium DSM 21509. Two major AHLs, N-(3-hydroxyhexanoyl)-l-homoserine lactone and N-(3-hydroxyoctanoyl)-l-homoserine lactone, were detected in JCM 2778 and DSM 21509 samples. PhzI synthesized all AHLs; however, CsaI could not perform AHL biosynthesis in JCM 2778 and DSM 21509. In both strains, disruption of the phzI caused complete disappearance of phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN) production; however, disruption of csaI did not induce significant changes in PCA and PCN production. Phenazine derivatives produced by JCM 2778 and DSM 21509 under quorum sensing are crucial for the control of the plant pathogenic fungi, Rhizoctonia solani, Fusarium graminearum, and Fusarium nirenbergiae. These results demonstrated that PhzI/PhzR quorum-sensing system play an important role in production of phenazine derivatives and biocontrol activity.
Topics: Acyl-Butyrolactones; Bacterial Proteins; Phenazines; Pseudomonas; Quorum Sensing
PubMed: 35365429
DOI: 10.1016/j.jbiosc.2022.03.004 -
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 -
Chemosphere Dec 2023Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, Rox), a widely used organoarsenical feed additive, can enter soils and be further biotransformed into various arsenic...
Roxarsone (3-nitro-4-hydroxyphenylarsonic acid, Rox), a widely used organoarsenical feed additive, can enter soils and be further biotransformed into various arsenic species that pose human health and ecological risks. However, the pathway and molecular mechanism of Rox biotransformation by soil microbes are not well studied. Therefore, in this study, we isolated a Rox-transforming bacterium from manure-fertilized soil and identified it as Pseudomonas chlororaphis through morphological analysis and 16S rRNA gene sequencing. Pseudomonas chlororaphis was able to biotransform Rox to 3-amino-4-hydroxyphenylarsonic acid (3-AHPAA), N-acetyl-4-hydroxy-m-arsanilic acid (N-AHPAA), arsenate [As(V)], arsenite [As(III)], and dimethylarsenate [DMAs(V)]. The complete genome of Pseudomonas chlororaphis was sequenced. PcmdaB, encoding a nitroreductase, and PcnhoA, encoding an acetyltransferase, were identified in the genome of Pseudomonas chlororaphis. Expression of PcmdaB and PcnhoA in E. coli Rosetta was shown to confer Rox(III) and 3-AHPAA(III) resistance through Rox nitroreduction and 3-AHPAA acetylation, respectively. The PcMdaB and PcNhoA enzymes were further purified and functionally characterized in vitro. The kinetic data of both PcMdaB and PcNhoA were well fit to the Michaelis-Menten equation, and nitroreduction catalyzed by PcMdaB is the rate-limiting step for Rox transformation. Our results provide new insights into the environmental risk assessment and bioremediation of Rox(V)-contaminated soils.
Topics: Humans; Roxarsone; Pseudomonas chlororaphis; Soil; Acetyltransferases; RNA, Ribosomal, 16S; Escherichia coli; Arsenic; Biotransformation; Nitroreductases
PubMed: 37898462
DOI: 10.1016/j.chemosphere.2023.140558 -
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 -
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