Biosynthetic Cluster Research Articles

DEEPENED GENOMICS-METABOLOMICS CHARACTERISTICS OF THE BACTERIA OF THE BACILLUS GENUS ISOLATED FROM DEEP-SEA

Marine bacteria from the genus Bacillus are attracting increasing attention as a source of large amounts of bioactive metabolites. The biosynthetic potential of marine bacteria from the genus Bacillus is not sufficiently studied. The previous study of authors could not give a complete result, so it was decided to reanalyze the metabolites not identified at that time, taking into account the development of existing databases. Therefore, the work aimed to reanalyze the metabolome already performed on Bacillus velezensis ONU 553, Bacillus pumilus ONU 554, and Bacillus subtilis ONU 559 strains isolated from the Black Sea sediments. Methods. The general genome identification was performed using the online version of the Dictionary of Natural Products database, and search for biosynthetic clusters in genome of Bacillus velezensis ONU 553 - with antiSMASH 7.0. Results and Conclusion. Genomic-metabolic profiling of strains led to the identification of several new biological compounds – 7 and 1 metabolites in Bacillus velezensis ONU 553 and Bacillus pumilus ONU 554 strains, respectively, and to identify biosynthetic clusters of iturin- and helipeptin-like peptides in Bacillus velezensis ONU 553 strain for the first time. A new biosynthetic cluster was found in the Bacillus velezensis ONU 553 strain.

Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration

The causes of nigrostriatal cell death in idiopathic Parkinson’s disease are unknown, but exposure to toxic chemicals may play some role. We followed up here on suggestions that bacterial secondary metabolites might be selectively cytotoxic to dopaminergic neurons. Extracts from Streptomyces venezuelae were found to kill human dopaminergic neurons (LUHMES cells). Utilizing this model system as a bioassay, we identified a bacterial metabolite known as aerugine (C10H11NO2S; 2-[4-(hydroxymethyl)-4,5-dihydro-1,3-thiazol-2-yl]phenol) and confirmed this finding by chemical re-synthesis. This 2-hydroxyphenyl-thiazoline compound was previously shown to be a product of a wide-spread biosynthetic cluster also found in the human microbiome and in several pathogens. Aerugine triggered half-maximal dopaminergic neurotoxicity at 3–4 µM. It was less toxic for other neurons (10–20 µM), and non-toxic (at <100 µM) for common human cell lines. Neurotoxicity was completely prevented by several iron chelators, by distinct anti-oxidants and by a caspase inhibitor. In the Caenorhabditis elegans model organism, general survival was not affected by aerugine concentrations up to 100 µM. When transgenic worms, expressing green fluorescent protein only in their dopamine neurons, were exposed to aerugine, specific neurodegeneration was observed. The toxicant also exerted functional dopaminergic toxicity in nematodes as determined by the “basal slowing response” assay. Thus, our research has unveiled a bacterial metabolite with a remarkably selective toxicity toward human dopaminergic neurons in vitro and for the dopaminergic nervous system of Caenorhabditis elegans in vivo. These findings suggest that microbe-derived environmental chemicals should be further investigated for their role in the pathogenesis of Parkinson's disease.

Whole-genome sequencing and bacteriocin purification of Lactiplantibacillus plantarum HY41 confirms bactericidal and probiotic potential

In this study, the genomic tools were used to characterize the probiotic and bactericidal potential of Lactiplantibacillus plantarum HY41. Firstly, the strain was found to carry two insertion sequences and prophage regions, and there was no CRISPR/Cas system by whole-genome sequencing. At the same time, functional genes related to probiotics such as encoding exopolysaccharides synthesize proteins, stress tolerance, flavor protease, and terpenoids were found in HY41. The putative bacteriocin biosynthetic cluster was identified using BAGEL4, and the bacteriocin HY41 was purified, it was discovered that Lactiplantibacillus plantarum HY41 harbored a silent gene cluster responsible for producing pediocin PA-1. Along with the pedABCD operon, this gene cluster also contained the gene encoding a toxin-antitoxin (TA) system, which acted as a defense mechanism against phage infection, and it was not found in other studies. Activation of the silent genes can produce novel and with multiple functions bacteriocin. The mode of action of bacteriocin was studied by scanning electron microscopy, and the results showed that bacteriocin HY41 caused cell death by disrupting the cell membrane of Listeria monocytogenes. The analysis of the genome of Lactiplantibacillus plantarum HY41 has confirmed its probiotic and bactericidal properties, demonstrating its potential for industrial probiotic applications.

Identification and whole-genome sequencing of a bacterial strain isolated from healthy rice plants antagonistic to Magnaporthe oryzae

Rice blast is a globally devastating fungal disease that affects the production of rice (Oryza sativa), and the screening of excellent biocontrol strains is an important direction for the biological control of the fungus that causes rice blast disease (Magnaporthe oryzae). The objectives were to obtain strains that were highly antagonistic to rice blast, analyze the genetic information of the antagonistic bacterium YN-917, and explore the resources of its antagonistic gene cluster. The antagonistic bacteria were isolated and identified by the plate confrontation method, morphological observation, physiological and biochemical identification, and molecular biology methods. In addition, the strains were subjected to whole-genome sequencing, and their sequences were analyzed. Strain YN-917 was screened from healthy rice plants of the variety Xiangzaoxian 24, which is susceptible to rice blast, and it inhibited M. oryzae by 72.63% ± 1.30%. Additionally, the strain had different degrees of inhibitory effects on various plant pathogenic fungi and was highly resistant to stress. The morphological observation, analysis of physiological and biochemical features, 16S rRNA homology analysis, and whole-genome sequencing analysis revealed that the strain YN-917 was Bacillus cereus (GenBank No.: PRJNA687285). The total length of its whole genome was 5326162 bp, and its average G + C content was 35.37%. It was composed of one circular chromosome and one endoplasmic plasmid. There were 5483 genes encoded on average. They included 105 tRNA genes, 42 self-replicating RNA (sRNA), 178 tandem repeat sequences, three prophages, and nine genomic islands. The prediction of antagonistic gene cluster demonstrated that the genome sequence of YN-917 had six secondary metabolic gene biosynthetic clusters, including those for bacteriocins, siderophores, non-ribosomal peptide synthetases (NRPs), and terpene. This study provides a theoretical basis to further explore microbial resources and their metabolic gene clusters for agricultural biological control.

Identification of 2,4-diacetylphloroglucinol production in the genus Chromobacterium

The compound 2,4-diacetylphloroglucinol (DAPG) is a broad-spectrum antibiotic that is primarily produced by Pseudomonas spp. DAPG plays an important role in the biocontrol disease suppressing activity of Pseudomonas spp. In the current study, we report the discovery of the DAPG biosynthetic cluster in strains of Chromobacterium vaccinii isolated from Brazilian aquatic environments and the distribution of the biosynthetic cluster in the Chromobacterium genus. Phylogenetic analysis of the phlD protein suggests the biosynthetic cluster probably entered the genus of Chromobacterium after a horizontal gene transfer event with a member of the Pseudomonas fluorescens group. We were able to detect trace amounts of DAPG in wild type cultures and confirm the function of the cluster with heterologous expression in Escherichia coli. In addition, we identified and verified the presence of other secondary metabolites in these strains. We also confirmed the ability of C. vaccinii strains to produce bioactive pigment violacein and bioactive cyclic depsipeptide FR900359. Both compounds have been reported to have antimicrobial and insecticidal activities. These compounds suggest strains of C. vaccinii should be further explored for their potential as biocontrol agents.

(Invited) Enhancing the Performance of Microbial Fuel Cells Via Metabolic Engineering of Escherichia coli for Phenazine Production

Establishing an efficient and successful extracellular electron transfer (EET) between microorganisms and electrode surfaces plays a critical role in the design, development, and application of mediated microbial electrochemical technologies, such as microbial fuel cells (MFCs). Most microbial-based systems require the use of artificial, redox-active mediator systems in order to facilitate and/or increase electron transfer. Our previous work established an exogenous phenazine-based library as a mediator system to enable electron transfer from the model microorganism Escherichia coli. However, the addition of exogenous mediators to a microbial electrochemical system has certain limiting downsides, specifically with regard to mediator toxicity to cells and increased operational expenses. Herein, we demonstrate the metabolic and genetic engineering of E. coli to self-generate phenazine metabolites endogenously by introducing the phenazine biosynthetic pathway from Pseudomonas aeruginosa into E. coli. This pathway consists of a seven-gene phenazine biosynthetic cluster phzA-G responsible for the synthesis of phenazine-1-carboxylic acid (PCA) and two phenazine accessory genes phzM and phzS to produce pyocyanin (PYO). We present the characterization of the engineered E. coli cells via electrochemical measurements, RNA sequencing, and microscopy imaging. Finally, the engineered E. coli cells were used for the design of a microbial fuel cell with enhanced performances, demonstrating a maximum power density increase from 305 mW/m2 with non-engineered E. coli cells to 1555 mW/m2 with the genetically engineered, phenazine-producing E. coli. Our results indicate that introducing a heterologous electron shuttle into E. coli can be an efficient approach to establishing efficacious mediation in living bioelectrochemical systems and improving the performance of MFCs.

New targets of TetR-type regulator SLCG_2919 for controlling lincomycin biosynthesis in Streptomyces lincolnensis.

The transcription factor (TF)-mediated regulatory network controlling lincomycin production in Streptomyces lincolnensis is yet to be fully elucidated despite several types of associated TFs having been reported. SLCG_2919, a tetracycline repressor (TetR)-type regulator, was the first TF to be characterized outside the lincomycin biosynthetic cluster to directly suppress the lincomycin biosynthesis in S. lincolnensis. In this study, improved genomic systematic evolution of ligands by exponential enrichment (gSELEX), an in vitro technique, was adopted to capture additional SLCG_2919-targeted sequences harboring the promoter regions of SLCG_6675, SLCG_4123-4124, SLCG_6579, and SLCG_0139-0140. The four DNA fragments were confirmed by electrophoretic mobility shift assays (EMSAs). Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) showed that the corresponding target genes SLCG_6675 (anthranilate synthase), SLCG_0139 (LysR family transcriptional regulator), SLCG_0140 (beta-lactamase), SLCG_6579 (cytochrome P450), SLCG_4123 (bifunctional DNA primase/polymerase), and SLCG_4124 (magnesium or magnesium-dependent protein phosphatase) in ΔSLCGL_2919 were differentially increased by 3.3-, 4.2-, 3.2-, 2.5-, 4.6-, and 2.2-fold relative to those in the parental strain S. lincolnensis LCGL. Furthermore, the individual inactivation of these target genes in LCGL reduced the lincomycin yield to varying degrees. This investigation expands on the known DNA targets of SLCG_2919 to control lincomycin production and lays the foundation for improving industrial lincomycin yields via genetic engineering of this regulatory network.

The Potential of Arctic Pseudogymnoascus Fungi in the Biosynthesis of Natural Products

Scarce research into the secondary metabolites of the fungi Pseudogymnoascus spp. has shown a hidden biosynthetic potential for biologically active compounds. This work investigated the biosynthesis of secondary metabolites by two Pseudogymnoascus fungal strains, VKM F-4518 and VKM F-4519, isolated from the surface soil layer of the Kolyma Lowland, Russia, in the Arctic. In these strains, 16-membered trilactone macrolides, (+)-macrosphelides A and B, were identified using 1D and 2D NMR, UHRMS, and optical rotation data. In the fungi of this genus, these metabolites were found for the first time. The studied strains are highly active producers of macrosphelide A, which is being considered as a promising agent for the cure of cancer. Using the antiSMASH secondary metabolite analysis tool, we found that the genome of strain VKM F-4518 contained 32 of the biosynthetic clusters of the secondary metabolite genes (BGC) and that of VKM F-4519 had 17 BGCs. Based on the comparison of the cluster of macrotriolide genes from the fungus Paraphaeosphaeria sporulosa, we found the complete supposed cluster BGCs of macrosphelides in the genomes of two Pseudogymnoascus strains using the BLAST+ program.

Sphingomonas abietis sp. nov., an Endophytic Bacterium Isolated from Korean Fir.

PAMB 00755T , a bacterial strain, was isolated from Korean fir leaves. The strain exhibits yellow colonies and consists of Gram-negative, non-motile, short rods or ovoid-shaped cells. It displays optimal growth conditions at 20°C, 0% NaCl, and pH 6.0. Results of 16S rRNA gene-based phylogenetic analyses showed that strain PAMB 00755T was most closely related to Sphingomonas chungangi MAH-6T (97.7%) and Sphingomonas polyaromaticivorans B2-7T (97.4%), and ≤96.5% sequence similarity to other members of the genus Sphingomonas. The values of average nucleotide identity (79.9-81.3%), average amino acid identity (73.3-75.9%), and digital DNA-DNA hybridization (73.3- 75.9%) were significantly lower than the threshold values for species boundaries; these overall genome-related indexes (OGRI) analyses indicated that the strain represents a novel species. Genomic analysis revealed that the strain has a 4.4-Mbp genome encoding 4,083 functional genes, while the DNA G+C content of the whole genome is 66.1%. The genome of strain PAMB 00755T showed a putative carotenoid biosynthetic cluster responsible for its antioxidant activity. The respiratory quinone was identified as ubiquinone 10 (Q-10), while the major fatty acids in the profile were identified as C18:1ω7c and/or C18:1ω6c (summed feature 8). The major polar lipids of strain PAMB 00755T were diphosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, and phosphatidylcholine. Based on a comprehensive analysis of genomic, phenotypic, and chemotaxonomic characteristics, we proposed the name Sphingomonas abietis sp. nov. for this novel species, with PAMB 00755T as the type strain (= KCTC 92781T = GDMCC 1.3779T ).

Expanding the Utility of Bioinformatic Data for the Full Stereostructural Assignments of Marinolides A and B, 24- and 26-Membered Macrolactones Produced by a Chemically Exceptional Marine-Derived Bacterium.

Marinolides A and B, two new 24- and 26-membered bacterial macrolactones, were isolated from the marine-derived actinobacterium AJS-327 and their stereostructures initially assigned by bioinformatic data analysis. Macrolactones typically possess complex stereochemistry, the assignments of which have been one of the most difficult undertakings in natural products chemistry, and in most cases, the use of X-ray diffraction methods and total synthesis have been the major methods of assigning their absolute configurations. More recently, however, it has become apparent that the integration of bioinformatic data is growing in utility to assign absolute configurations. Genome mining and bioinformatic analysis identified the 97 kb mld biosynthetic cluster harboring seven type I polyketide synthases. A detailed bioinformatic investigation of the ketoreductase and enoylreductase domains within the multimodular polyketide synthases, coupled with NMR and X-ray diffraction data, allowed for the absolute configurations of marinolides A and B to be determined. While using bioinformatics to assign the relative and absolute configurations of natural products has high potential, this method must be coupled with full NMR-based analysis to both confirm bioinformatic assignments as well as any additional modifications that occur during biosynthesis.

Genome‐based analysis of Pseudomonas paracarnisRQ057, a strain responsible for blue discoloration spoilage in processed cheese

A strain of Pseudomonas causing blue discoloration was isolated from processed cheese ‘Requeijão em Barra’ and studied by whole‐genome sequencing. The strain RQ057 was identified as Pseudomonas paracarnis using the TYGS web server and also by in silico DNA–DNA hybridisation and ANIb calculation. Pyoverdine biosynthetic clusters, integrative and conjugative elements (ICE), prophage regions and genomic islands (GIs) were predicted. Furthermore, we observed that copies of genes related to the tryptophan biosynthetic pathway (trpCBAFD), which are exclusive to strains causing blue discoloration, are contained in the region identified as ICE and which was also identified in GIs. This genomic region also contains genes for transposase, type IV transport system (T4SS) and pili construction. Thereby, we can assume that this region contains potential conjugative‐transposon‐like mobile genetic elements and it has plasmid‐like conjugative properties.

LaeA-Regulated Fungal Traits Mediate Bacterial Community Assembly.

Potent antimicrobial metabolites are produced by filamentous fungi in pure culture, but their ecological functions in nature are often unknown. Using an antibacterial Penicillium isolate and a cheese rind microbial community, we demonstrate that a fungal specialized metabolite can regulate the diversity of bacterial communities. Inactivation of the global regulator, LaeA, resulted in the loss of antibacterial activity in the Penicillium isolate. Cheese rind bacterial communities assembled with the laeA deletion strain had significantly higher bacterial abundances than the wild-type strain. RNA-sequencing and metabolite profiling demonstrated a striking reduction in the expression and production of the natural product pseurotin in the laeA deletion strain. Inactivation of a core gene in the pseurotin biosynthetic cluster restored bacterial community composition, confirming the role of pseurotins in mediating bacterial community assembly. Our discovery demonstrates how global regulators of fungal transcription can control the assembly of bacterial communities and highlights an ecological role for a widespread class of fungal specialized metabolites. IMPORTANCE Cheese rinds are economically important microbial communities where fungi can impact food quality and aesthetics. The specific mechanisms by which fungi can regulate bacterial community assembly in cheeses, other fermented foods, and microbiomes in general are largely unknown. Our study highlights how specialized metabolites secreted by a Penicillium species can mediate cheese rind development via differential inhibition of bacterial community members. Because LaeA regulates specialized metabolites and other ecologically relevant traits in a wide range of filamentous fungi, this global regulator may have similar impacts in other fungus-dominated microbiomes.

Search for Novel Halophilic and Halotolerant Producers of Antimicrobial Compounds in Various Extreme Ecosystems

Abstract—The recent increase of antibiotic resistance in pathogenic microorganisms stimulated interest in the search for new antimicrobial compounds and their producers. Among the latter, halophilic microorganisms are considered to be the most promising group, since actinomycetes, producers of the majority of the known clinically important classes of antibiotics, are widely represented in this group. The present work reports the results of the search with three different approaches for new antimicrobial compounds in halophilic and halotolerant microorganisms inhabiting three different types of extreme ecosystems. Metagenomic analysis of microbial communities of subsurface thermal mineral waters revealed biosynthetic clusters of putative antimicrobial compounds, which belong to bacteria and archaea of uncultured lineages. Enrichment cultures with antimicrobial activity were obtained from the cold, deep oceanic sediments; analysis of their phylogenetic diversity resulted in identification of the potential producers of antimicrobial compounds as the members of class Desulfuromonadia. Finally, antimicrobial activity of a new species of soil natronophilic streptomycetes, Streptomyces sp. ACA25, was characterized; it was active only against gram-positive bacteria. The genome of this organism was sequenced, and the pathways for biosynthesis of polypeptide, polyketide, and beta-lactam antibiotics were identified. Active fractions of the ACA25 culture, containing antimicrobial compounds of polyketide and beta-lactam nature, were obtained. The active polyketide was identified as rosamycin, an antibiotic of the macrolide structural group. However, the fact that it did not inhibit bacterial translation highlighted structural differences between the new polyketide and rosamycin.

Mining Chromodoris quadricolor symbionts for biosynthesis of novel secondary metabolites.

Many secondary metabolites with medicinal potential are produced by various animals, plants, and microorganisms. Because marine creatures have a greater proportion of unexplored biodiversity than their terrestrial counterparts, they have emerged as a key research focus for the discovery of natural product drugs. Several studies have revealed that bacteria isolated from Chromodoris quadricolor (C. quadricolor) have antibiotic and anticancer properties. In this study, meta-transcriptomics and meta-proteimic analysis were combined to identify biosynthetic gene clusters (BGCs) in the symbiotic bacteria of the C. quadricolor mantle. Symbiotic bacteria were separated from the host by differential pelleting, and then total RNA was extracted, purified, and sequenced. Meta-transcriptomic analysis was done using different natural product mining tools to identify biosynthetic transcript clusters (BTCs). Furthermore, proteins were extracted from the same cells and then analyzed by LC-MS. A meta-proteomic analysis was performed to find proteins that are translated from BCGs. Finally, only 227 proteins have been translated from 40,742 BTCs. The majority of these clusters were polyketide synthases (PKSs) with antibacterial activity. Ten novel potential metabolic clusters with the ability to produce antibiotics have been identified in Novosphingobium and Microbacteriaceae, including members of the ribosomal synthesized and post-translationally modified peptides (RiPPs), polyketide synthases, and others. We realized that using a meta-proteomic approach to identify BGCs that have already been translated makes it easier to concentrate on BGCs that are utilized by bacteria. The symbiotic bacteria associated with C. quadricolor could be a source of novel antibiotics.

Genome Sequence Resources for Verticillium dahliae-Inhibiting Streptomyces spp. Isolated from Agricultural Soil

Gram-positive Streptomyces bacteria are common in soil, where they can inhibit soilborne plant pathogens by producing antimicrobial secondary metabolites. This study generated genome assemblies of Streptomyces isolates from soil that inhibit the soilborne plant pathogen Verticillium dahliae. Five bacterial isolates were cultured from soil collected from the Salinas Valley region of California. In vitro tests showed that three bacterial isolates inhibited V. dahliae growth when applied at low and high spore concentrations, whereas two isolates only inhibited V. dahliae when applied at high spore concentrations. Generation of hybrid Illumina-Nanopore assemblies showed that isolate genomes ranged from 7.45 to 8.95 Mbs. The contig number per assembly ranged from 1 to 54. All assemblies had 100% completeness values based on a standard set of conserved orthologs found in all bacteria. The number of predicted genes ranged from 6,594 to 8,098. Over 25 putative secondary metabolite biosynthetic clusters were identified in each assembly. Calculation of digital DNA:DNA hybridization and average nucleotide identity values using additional genome assemblies from type specimens of Streptomyces showed that the five isolates are likely novel Streptomyces spp. The results and data generated from this study will provide a basis for future research on the use of Streptomyces for managing common soilborne pathogens such as V. dahliae. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Metabolomic Analysis Demonstrates the Impacts of Polyketide Synthases PKS14 and PKS15 on the Production of Beauvericins, Bassianolide, Enniatin A, and Ferricrocin in Entomopathogen Beauveria bassiana.

Beauveria bassiana is a globally distributed entomopathogenic fungus that produces various secondary metabolites to support its pathogenesis in insects. Two polyketide synthase genes, pks14 and pks15, are highly conserved in entomopathogenic fungi and are important for insect virulence. However, understanding of their mechanisms in insect pathogenicity is still limited. Here, we overexpressed these two genes in B. bassiana and compared the metabolite profiles of pks14 and pks15 overexpression strains to those of their respective knockout strains in culture and in vivo using tandem liquid chromatography-mass spectrometry (LC-MS/MS) with Global Natural Products Social Molecular Networking (GNPS). The pks14 and pks15 clusters exhibited crosstalk with biosynthetic clusters encoding insect-virulent metabolites, including beauvericins, bassianolide, enniatin A, and the intracellular siderophore ferricrocin under certain conditions. These secondary metabolites were upregulated in the pks14-overexpressing strain in culture and the pks15-overexpressing strain in vivo. These data suggest that pks14 and pks15, their proteins or their cluster components might be directly or indirectly associated with key pathways in insect pathogenesis of B. bassiana, particularly those related to secondary metabolism. Information about interactions between the polyketide clusters and other biosynthetic clusters improves scientific understanding about crosstalk among biosynthetic pathways and mechanisms of pathogenesis.

A Novel Biosynthetic Gene Cluster Across the Pantoea Species Complex Is Important for Pathogenicity in Onion.

Onion center rot is caused by at least four species of genus Pantoea (P. ananatis, P. agglomerans, P. allii, and P. stewartii subsp. indologenes). Critical onion pathogenicity determinants for P. ananatis were recently described, but whether those determinants are common among other onion-pathogenic Pantoea species remains unknown. In this work, we report onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. We identified two distinct secondary metabolite biosynthetic gene clusters present separately in different strains of onion-pathogenic P. stewartii subsp. indologenes. One cluster is similar to the previously described HiVir phosphonate biosynthetic cluster identified in P. ananatis and another is a novel putative phosphonate biosynthetic gene cluster, which we named Halophos. The Halophos gene cluster was also identified in P. allii strains. Both clusters are predicted to be phosphonate biosynthetic clusters based on the presence of a characteristic phosphoenolpyruvate phosphomutase (pepM) gene. The deletion of the pepM gene from either HiVir or Halophos clusters in P. stewartii subsp. indologenes caused loss of necrosis on onion leaves and red onion scales and resulted in significantly lower bacterial populations compared with the corresponding wild-type and complemented strains. Seven (halB to halH) of 11 genes (halA to halK) in the Halophos gene cluster are required for onion necrosis phenotypes. The onion nonpathogenic strain PNA15-2 (P. stewartii subsp. indologenes) gained the capacity to cause foliar necrosis on onion via exogenous expression of a minimal seven-gene Halophos cluster (genes halB to halH). Furthermore, cell-free culture filtrates of PNA14-12 expressing the intact Halophos gene cluster caused necrosis on onion leaves consistent with the presence of a secreted toxin. Based on the similarity of proteins to those with experimentally determined functions, we are able to predict most of the steps in Halophos biosynthesis. Together, these observations indicate that production of the toxin phosphonate seems sufficient to account for virulence of a variety of different Pantoea strains, although strains differ in possessing a single but distinct phosphonate biosynthetic cluster. Overall, this is the first report of onion pathogenicity determinants in P. stewartii subsp. indologenes and P. allii. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The natural pyrazolotriazine pseudoiodinine from Pseudomonas mosselii 923 inhibits plant bacterial and fungal pathogens

Natural products largely produced by Pseudomonads-like soil-dwelling microorganisms are a consistent source of antimicrobial metabolites and pesticides. Herein we report the isolation of Pseudomonas mosselii strain 923 from rice rhizosphere soils of paddy fields, which specifically inhibit the growth of plant bacterial pathogens Xanthomonas species and the fungal pathogen Magnaporthe oryzae. The antimicrobial compound is purified and identified as pseudoiodinine using high-resolution mass spectra, nuclear magnetic resonance and single-crystal X-ray diffraction. Genome-wide random mutagenesis, transcriptome analysis and biochemical assays define the pseudoiodinine biosynthetic cluster as psdABCDEFG. Pseudoiodinine biosynthesis is proposed to initiate from guanosine triphosphate and 1,6-didesmethyltoxoflavin is a biosynthetic intermediate. Transposon mutagenesis indicate that GacA is the global regulator. Furthermore, two noncoding small RNAs, rsmY and rsmZ, positively regulate pseudoiodinine transcription, and the carbon storage regulators CsrA2 and CsrA3, which negatively regulate the expression of psdA. A 22.4-fold increase in pseudoiodinine production is achieved by optimizing the media used for fermentation, overexpressing the biosynthetic operon, and removing the CsrA binding sites. Both of the strain 923 and purified pseudoiodinine in planta inhibit the pathogens without affecting the rice host, suggesting that pseudoiodinine can be used to control plant diseases.

Exploring the Relationship between Biosynthetic Gene Clusters and Constitutive Production of Mycosporine-like Amino Acids in Brazilian Cyanobacteria.

Cyanobacteria are oxygenic phototrophic prokaryotes that have evolved to produce ultraviolet-screening mycosporine-like amino acids (MAAs) to lessen harmful effects from obligatory exposure to solar UV radiation. The cyanobacterial MAA biosynthetic cluster is formed by a gene encoding 2-epi-5-epi-valiolone synthase (EVS) located immediately upstream from an O-methyltransferase (OMT) encoding gene, which together biosynthesize the expected MAA precursor 4-deoxygadusol. Accordingly, these genes are typically absent in non-producers. In this study, the relationship between gene cluster architecture and constitutive production of MAAs was evaluated in cyanobacteria isolated from various Brazilian biomes. Constitutive production of MAAs was only detected in strains where genes formed a co-linear cluster. Expectedly, this production was enhanced upon exposure of the strains to UV irradiance and by using distinct culture media. Constitutive production of MAAs was not detected in all other strains and, unexpectedly, production could not be induced by exposure to UV irradiation or changing growth media. Other photoprotection strategies which might be employed by these MAA non-producing strains are discussed. The evolutionary and ecological significance of gene order conservation warrants closer experimentation, which may provide a first insight into regulatory interactions of genes encoding enzymes for MAA biosynthesis.

A Zn(II)(2)Cys(6) transcription factor MPsGeI suppresses pigment biosynthesis in Monascus

High-quality natural edible pigments known as monascus pigments (MPs) are widely used in food, medicine, and chemical industries as active functional ingredients. At the transcriptional level, the expression of MPs genes are tightly controlled, limiting their productivity and color value. Hitherto our understanding of the regulation of expression of MPs genes has been rather limited. Here, we describe a pathway-specific Zn(II)(2)Cys(6) transcription factor involved in the MPs biosynthetic cluster named MPsGeI, which encodes a 813-amino-acid protein with six introns. Expression of all MPs biosynthetic genes and accumulation of MPs were remarkably increased in ΔMPsGeI strain, and MPs production was significantly reduced in MPsGeI over-expressing strain. Results clearly demonstrated that MPsGeI negatively regulates MPs accumulation via transcriptional regulation of MPs biosynthetic genes, and plays a central repressive role in MPs' biosynthesis. Transcriptomic analyses revealed that MPsGeI disruptant regulated higher concentrations of precursors flowing to pigment and resulted in accumulation of a large amount of red MPs in hyphae. This work offers an efficient method for increasing MPs's productivity and color value and provides novel insights into the regulatory mechanisms of fungal cellular processes, which will assist to enhance MPs production and application.