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Microbial Cell Factories May 2024Aminopyrrolnitrin (APRN), a natural halogenated phenylpyrrole derivative (HPD), has strong antifungal and antiparasitic activities. Additionally, it showed 2.8-fold...
Aminopyrrolnitrin (APRN), a natural halogenated phenylpyrrole derivative (HPD), has strong antifungal and antiparasitic activities. Additionally, it showed 2.8-fold increased photostability compared to pyrrolnitrin, a commercially available HPD with antimicrobial activity. For microbial production of APRN, we first engineered anthranilate phosphoribosyltransferase encoded by trpD from Corynebacterium glutamicum, resulting in a TrpD mutation that exhibits feedback-resistant against L-tryptophan and higher substrate affinity compared to wild-type TrpD. Plasmid-borne expression of trpD in C. glutamicum TP851 strain with two copies of trpD in the genome led to the production of 3.1 g/L L-tryptophan in flask culture. Subsequent step for L-tryptophan chlorination into 7-chloro-L-tryptophan was achieved by introducing diverse sources of genes encoding tryptophan 7-halogenase (PrnA or RebH) and flavin reductase (Fre, PrnF, or RebF). The combined expression of prnA from Serratia grimesii or Serratia plymuthica with flavin reductase gene from Escherichia coli, Pseudomonas fluorescens, or Lechevalieria aerocolonigenes yielded higher production of 7-chloro-L-tryptophan in comparison to other sets of two-component systems. In the next step, production of putative monodechloroaminopyrrolnitrin (MDAP) from 7-chloro-L-tryptophan was achieved through the expression of prnB encoding MDAP synthase from S. plymuthica or P. fluorescens. Finally, an artificial APRN biosynthetic pathway was constructed by simultaneously expressing genes coding for tryptophan 7-halogenase, flavin reductase, MDAP synthase, and MDAP halogenase (PrnC) from different microbial sources within the L-tryptophan-producing TP851 strain. As prnC from S. grimesii or S. plymuthica was introduced into the host strain, which carried plasmids expressing prnA from S. plymuthica, fre from E. coli, and prnB from S. plymuthica, APN3639 and APN3638 accumulated 29.5 mg/L and 28.1 mg/L of APRN in the culture broth. This study represents the first report on the fermentative APRN production by metabolically engineered C. glutamicum.
Topics: Corynebacterium glutamicum; Metabolic Engineering; Pyrrolnitrin; Fermentation; Bacterial Proteins; Tryptophan; Escherichia coli; Oxidoreductases
PubMed: 38783320
DOI: 10.1186/s12934-024-02424-y -
Access Microbiology 2023Several species of soil-dwelling nematodes are used in the biocontrol of crop pests, due to their natural capacity to kill diverse lepidopteran species. Although this...
Several species of soil-dwelling nematodes are used in the biocontrol of crop pests, due to their natural capacity to kill diverse lepidopteran species. Although this insect-killing trait is known to be augmented by the nematodes' endosymbionts, the role of other steinernematid-associated bacterial genera in the nematode lifecycle remains unclear. This genomic study aimed to determine the potential of to contribute to the entomopathogenicity of its host. Insect larvae were infected with three separate cultures. From each of the three treatments, the prevalent bacteria in the haemocoel of cadavers, four days post-infection, were isolated. These three bacterial isolates were morphologically characterised. DNA was extracted from each of the three bacterial isolates and used for long-read genome sequencing and assembly. Assemblies were used to delineate species and identify genes that encode insect toxins, antimicrobials, and confer antibiotic resistance. We assembled three complete genomes. Through digital DNA-DNA hybridisation analyses, we ascertained that the haemocoels of insect cadavers previously infected with sp. Kalro, sp. 75, and sp. 97 were dominated by Kalro, 75, and 97, respectively. Kalro and 97 formed a subspecies with other symbionts of steinernematids from Kenya. 75 phylogenetically clustered with pseudomonads that are characterised by high insecticidal activity. The 75 genome encoded the production pathway of insect toxins such as orfamides and rhizoxins, antifungals such as pyrrolnitrin and pyoluteorin, and the broad-spectrum antimicrobial 2,4-diacetylphloroglucinol. The 75 genome encoded resistance to over ten classes of antibiotics, including cationic lipopeptides. Steinernematid-associated bacteria hence have the biosynthetic potential to contribute to nematode entomopathogenicity.
PubMed: 37970093
DOI: 10.1099/acmi.0.000659.v3 -
Data in Brief Jun 2023sp. IMCC1007 is a gram-negative, aerobic bacterium affiliated with class Betaproteobacteria, which was successfully isolated from maize rhizospheric soil sample in UTM...
sp. IMCC1007 is a gram-negative, aerobic bacterium affiliated with class Betaproteobacteria, which was successfully isolated from maize rhizospheric soil sample in UTM research plot, Pagoh, Malaysia by using enrichment method. Strain IMCC1007 utilized 50 mgL fusaric acid as its carbon source and degraded it completely within 14 h. Genome sequencing was performed using Illumina NovaSeq platform. The assembled genome was annotated using RAST (Rapid Annotation Subsystem Technology) server. The genome size was approximately 8,568,405 base pairs (bp) in 147 contigs with a G+C content of 66.04%. The genome includes 8,733 coding sequences and 68 RNAs. The genome sequence has been deposited at GenBank with the accession number of JAPVQY000000000. In the pairwise genome-to-genome comparisons, the strain IMCC1007 had an average nucleotide identity (ANI) of 91.9% and digital DNA-DNA hybridization (dDDH) value of 55.2% with DSM 16086 respectively. Interestingly, fusaric acid resistance gene (C) and ABCDFXT gene clusters (hydroxylation of pyridine compound) were found in the genome. Additionally, preliminary genome annotation analysis of strain IMCC1007 identified tryptophan halogenase (A) gene responsible for antifungal pyrrolnitrin biosynthesis. This dataset herein provides further insights into the fusaric acid degradation mechanism of the genus .
PubMed: 37383771
DOI: 10.1016/j.dib.2023.109204 -
3 Biotech Jun 2023The recent advances in omics and computational analysis have enabled the capacity to identify the exclusive strain-specific metabolites and novel biosynthetic gene...
UNLABELLED
The recent advances in omics and computational analysis have enabled the capacity to identify the exclusive strain-specific metabolites and novel biosynthetic gene clusters. This study analyzed eight strains of including GS1, GS3, GS4, GS6, GS7, FS2, ARS38, PBSt2, one strain of RP4, one strain of (At1RP4), and one strain of (RS1) for the production of rhamnolipids, quorum-sensing signals, and osmolytes. Seven rhamnolipid derivatives were variably detected in fluorescent pseudomonads. These rhamnolipids included Rha-C-C, Rha-Rha-C-C, Rha-C-Cdb, Rha-C-C, Rha-Rha-C-C Rha-C-C, and Rha-Rha-C-Cdb. spp. also showed the variable production of osmoprotectants including N-acetyl glutaminyl glutamine amide (NAGGN), betaine, ectoine, and trehalose. Betaine and ectoine were produced by all pseudomonads, however, NAGGN and trehalose were observed by five and three strains, respectively. Four strains including (RP4), (At1RP4), (RS1), and (PBSt2) were exposed to 1- 4% NaCl concentrations and evaluated for the changes in phenazine production profile which were negligible. AntiSMASH 5.0 platform showed 50 biosynthetic gene clusters in PB-St2, of which 23 (45%) were classified as putative gene clusters with ClusterFinder algorithm, five (10%) were classified as non-ribosomal peptides synthetases (NRPS), five (10%) as saccharides, and four (8%) were classified as putative fatty acids. The genomic attributes and comprehensive insights into the metabolomic profile of these spp. strains showcase their phytostimulatory, phyto-protective, and osmoprotective effects of diverse crops grown in normal and saline soils.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s13205-023-03607-x.
PubMed: 37193329
DOI: 10.1007/s13205-023-03607-x -
Frontiers in Microbiology 2022Plant growth-promoting bacteria (PGPB) have been considered promising biological agents to increase crop yields for years. However, the successful application of PGPB...
Plant growth-promoting bacteria (PGPB) have been considered promising biological agents to increase crop yields for years. However, the successful application of PGPB for biocontrol of sharp eyespot in wheat has been limited, partly by the lack of knowledge of the ecological/environmental factors affecting the colonization, prevalence, and activity of beneficial bacteria on the crop. In this study, an endophytic bacterium XN08 with antagonistic activity against (wheat sharp eyespot pathogenic fungus), isolated from healthy wheat plants, was identified as according to the sequence analysis of 16S rRNA. The antibiotic synthesis gene amplification and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) analyses were used to characterize the secondary metabolites. The results showed that the known powerful antifungal compound named pyrrolnitrin was produced by the strain XN08. In addition, XN08 also showed the capacity for phosphate solubilization, indole-3-acetic acid (IAA), protease, and siderophore production . In the pot experiments, a derivate strain carrying the green fluorescent protein () gene was used to observe its colonization in wheat plants. The results showed that GFP-tagged could colonize wheat tissues effectively. This significant colonization was accompanied by an enhancement of wheat plants' growth and an induction of immune resistance for wheat seedlings, which was revealed by the higher activities of polyphenol oxidase (PPO), peroxidase (POD), and phenylalanine ammonia-lyase (PAL). As far as we know, this is the first report describing the colonization traits of in wheat plants. In addition, our results indicated that XN08 might serve as a new effective biocontrol agent against wheat sharp eyespot disease caused by .
PubMed: 35966702
DOI: 10.3389/fmicb.2022.906724 -
International Microbiology : the... Nov 2022The biocontrol rhizobacterium Pseudomonas chlororaphis is one of the bacterial species of the P. fluorescens group where insecticide fit genes have been found. Fit...
The biocontrol rhizobacterium Pseudomonas chlororaphis is one of the bacterial species of the P. fluorescens group where insecticide fit genes have been found. Fit toxin, supported with other antimicrobial compounds, gives the bacterial the ability to repel and to fight against eukaryotic organisms, such as nematodes and insect larvae, thus protecting the plant host and itself. Pseudomonas chlororaphis PCL1606 is an antagonistic rhizobacterium isolated from avocado roots and show efficient biocontrol against fungal soil-borne disease. The main antimicrobial compound produced by P. chlororaphis PCL606 is 2-hexyl-5-propyl resorcinol (HPR), which plays a crucial role in effective biocontrol against fungal pathogens. Further analysis of the P. chlororaphis PCL1606 genome showed the presence of hydrogen cyanide (HCN), pyrrolnitrin (PRN), and homologous fit genes. To test the insecticidal activity and to determine the bases for such activity, single and double mutants on the biosynthetic genes of these four compounds were tested in a Galleria mellonella larval model using inoculation by injection. The results revealed that Fit toxin and HPR in combination are involved in the insecticide phenotype of P. chlororaphis PCL1606, and additional compounds such as HCN and PRN could be considered supporting compounds.
Topics: Anti-Infective Agents; Hydrogen Cyanide; Insecticides; Pseudomonas chlororaphis; Pyrrolnitrin; Resorcinols; Soil
PubMed: 35670867
DOI: 10.1007/s10123-022-00253-w -
Microbiological Research Jul 2022Pseudomonas chlororaphis G05 has the capability to repress the mycelial growth of many phytopathogenic fungi by producing and secreting certain antifungal compounds,...
Pseudomonas chlororaphis G05 has the capability to repress the mycelial growth of many phytopathogenic fungi by producing and secreting certain antifungal compounds, including phenazines and pyrrolnitrin. Although some regulatory genes have been identified to be involved in antifungal metabolite production, the regulatory mechanism and pathway of phenazine-1-carboxylic acid biosynthesis remain poorly defined. To identify more new regulatory genes, we applied transposon mutagenesis with the chromosomal lacZ fusion strain G05Δphz::lacZ as an acceptor. In the white conjugant colony G05W05, a novel transcriptional regulator gene, eppR, was verified to be interrupted by the transposon mini-Tn5Kan. To evaluate the specific function of eppR, we created a set of eppR-deletion mutants, including G05ΔeppR, G05Δphz::lacZΔeppR and G05Δprn::lacZΔeppR. By quantifying the production of antifungal compounds and β-galactosidase expression, we found that the expression of the phenazine biosynthetic gene cluster (phz) and the production of phenazine-1-carboxylic acid were markedly reduced in the absence of EppR. Moreover, the pathogen suppression test verified that the yield of phenazine-1-carboxylic acid was significantly decreased when eppR was deleted in frame. At the same time, no changes in the expression of the phzI/phzR quorum-sensing (QS) system and the production of N-acyl homoserine lactones (AHLs) and pyrrolnitrin were found in the EppR-deficient mutant. In addition, chromosomal fusion analyses and quantitative real-time polymerase chain reaction (qRT-PCR) results also showed that EppR could positively mediate the expression of the phz cluster at the posttranscriptional level. In summary, EppR is specifically essential for phenazine biosynthesis but not for pyrrolnitrin biosynthesis in P. chlororaphis.
Topics: Antifungal Agents; Bacterial Proteins; Gene Expression Regulation, Bacterial; Phenazines; Pseudomonas chlororaphis; Pyrrolnitrin
PubMed: 35504237
DOI: 10.1016/j.micres.2022.127050 -
Frontiers in Plant Science 2022Plants and rhizobacteria are coexisting since the beginning, but the exact mechanism of communication between them remains enigmatic. The PsoR protein of...
Identification of Putative Elicitors From Plant Root Exudates Responsible for PsoR Activation in Plant-Beneficial spp. by Docking and Molecular Dynamics Simulation Approaches to Decipher Plant-Microbe Interaction.
Plants and rhizobacteria are coexisting since the beginning, but the exact mechanism of communication between them remains enigmatic. The PsoR protein of plant-beneficial spp., a group of root-associated bacteria, is known to produce a range of antifungal and insecticidal secondary metabolites like 2,4-diacetyl phloroglucinol (DAPG), pyrrolnitrin, and chitinase making them great biocontrol agents and thus helping in plant growth promotion. To better understand the inter-kingdom signaling between plants and plant growth-promoting rhizobacteria (PGPR), the interaction of PsoR with various root exudates was investigated computationally. For this, we first modeled the PsoR protein and confirmed it using the Ramachandran plot. A total of 59 different low molecular weight phytochemicals, secreted as root exudates by plants, were identified by extensive text mining. They were virtually screened with the PsoR protein by molecular docking. Based on the lowest binding energy, ranging from -7.1 to -6.3 kcal mol, the top five exudates were chosen. To analyze the stability of the docked protein-ligand complex, a molecular dynamics (MD) simulation of 100 nanoseconds was done. Two root exudates, saponarin and 2-benzoxazolinone (BOA), showed suitable binding with PsoR by forming hydrogen, hydrophobic, and Van der Waals interactions. To confirm the MD simulation results, RMSF, RG, SASA, and interaction energy were calculated. This computational study first time reports that saponarin and 2-BOA, predominantly present in the root exudates of barley and wheat, respectively, demonstrate effective binding with the modeled PsoR protein and are likely of showing cross-kingdom interactions.
PubMed: 35463415
DOI: 10.3389/fpls.2022.875494 -
Pathogens (Basel, Switzerland) Mar 2022(strain DSMZ 13134) is a biocontrol agent with promising antagonistic activity hinging on antibiosis against the fungal forest pathogens spp. Here, by using...
(strain DSMZ 13134) is a biocontrol agent with promising antagonistic activity hinging on antibiosis against the fungal forest pathogens spp. Here, by using High-Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS), we assessed whether monocultures of (strain DSMZ 13134) produce the three major determinants of biocontrol activity known for the genus : 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT), and pyrrolnitrin (PRN). At the tested culture conditions, we observed the production of PLT at concentrations ranging from 0.01 to 10.21 mg/L and 2,4-DAPG at a concentration not exceeding 0.5 mg/L. Variations of culture conditions involving culture medium, incubation temperature, and incubation period had no consistent influence on PLT production by the bacterium. Assays using culture medium amended with PLT at the same concentration of that present in cell-free filtrate of the bacterium, i.e., 3.77 mg/L, previously documented as effective against spp., showed a remarkable activity of PLT against genotypes of all the four species present in Europe, including the non-native invasive . However, such antifungal activity decreased over time, and this may be a constraint for using this molecule as a pesticide against spp. When the bacterium was co-cultured in liquid medium with genotypes of the different species, an increased production of PLT was observed at 4 °C, suggesting the bacterium may perform better as a PLT producer in field applications under similar environmental conditions, i.e., at low temperatures. Our results demonstrated the role of PLT in the inhibition of spp., although all lines of evidence suggest that antibiosis does not rely on a single constitutively produced metabolite, but rather on a plethora of secondary metabolites. Findings presented in this study will help to optimize treatments based on (strain DSMZ 13134) against spp.
PubMed: 35456066
DOI: 10.3390/pathogens11040391 -
Frontiers in Microbiology 2022Nematicidal potential of the common plant pathogen has been recently identified against . The current study was designed to investigate the detailed genetic mechanism...
Nematicidal potential of the common plant pathogen has been recently identified against . The current study was designed to investigate the detailed genetic mechanism of the bacterial pathogenicity by applying comparative genomics, transcriptomics, mutant library screening, and protein expression. Results showed that strain MB03 could kill in the liquid assay by gut colonization. The genome of MB03 was sequenced and comparative analysis including multi locus sequence typing, and genome-to-genome distance placed MB03 in phylogroup II of . Furthermore, comparative genomics of MB03 with nematicidal strains of (PAO1 and PA14) predicted 115 potential virulence factors in MB03. However, genes for previously reported nematicidal metabolites, such as phenazine, pyochelin, and pyrrolnitrin, were found absent in the MB03 genome. Transcriptomics analysis showed that the growth phase of the pathogen considerably affected the expression of virulence factors, as genes for the flagellum, glutamate ABC transporter, /, /, type VI secretion system, and serralysin were highly up-regulated when stationary phase MB03 cells interacted with Additionally, screening of a transposon insertion mutant library led to the identification of other nematicidal genes such as , and . Finally, the nematicidal activity of selected proteins was confirmed by heterologous expression in .
PubMed: 35356513
DOI: 10.3389/fmicb.2022.826962