Secondary Metabolite Biosynthetic Clusters Research Articles

"Revealing the genetic arsenal of Bacillus firmus TNAU1: Unleashing nematicidal and plant growth promotion traits"

Bacillus firmus is a promising nematicidal bacterium that also confers plant growth promotion, nutrient acquisition and antimicrobial activities. In the present study, attempts were made for whole genome sequencing of B. firmus TNAU1 isolated from the rhizosphere region of cucumber. The results confirmed that the isolate was B. firmus which contained a single circular genome of 5,370,919 bp (5.3 Mb) in length with an average GC content of 41.16 %. Annotation was performed using PATRIC tool to explore the subsystem analysis, specialty genes, Enzyme Commission (EC) numbers, Gene Ontology (GO) mappings, KEGG pathways of B. firmus TNAU1. A total of 5804 coding sequence (CDS) genes that encode proteins were predicted based on the KEGG and clusters of orthologous groups (COGs) database. Comparative genomic analysis was performed to discover the unique features and their relationship with other available B. firmus strains. Blastp analysis revealed that TNAU 1 genome assemblies had 26 homologs corresponding to nematode-virulent proteases and various genes responsible for plant growth promotion which includes indole acetic acid (IAA) production, nitrate transport, phosphorus and potassium solubilization. Four secondary metabolite biosynthetic clusters encoding antibiotics and siderophore production were confirmed through comparative genomics. B. firmus TNAU1 was evaluated for its antagonistic activity against root-knot nematode Meloidogyne incognita of tomato. The results exhibited that B. firmus inhibited the egg hatching and mortality of juveniles by 96% over control under in vitro conditions and greenhouse experiments confirmed that the TNAU1 strain not only had the potential to suppress the soil and root population of M. incognita (70% reduction over control), but also triggered the plant growth parameters of tomato.

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.

Characterization of Bacillus velezensis AK-0 as a biocontrol agent against apple bitter rot caused by Colletotrichum gloeosporioides

Bacillus genus produces several secondary metabolites with biocontrol ability against various phytopathogens. Bacillus velezensis AK-0 (AK-0), an antagonistic strain isolated from Korean ginseng rhizospheric soil, was found to exhibit antagonistic activity against several phytopathogens. To further display the genetic mechanism of the biocontrol traits of AK-0, we report the complete genome sequence of AK-0 and compared it with complete genome sequences of closely related strains. We report the biocontrol activity of AK-0 against apple bitter rot caused by Colletotrichum gloeosporioides, which could lead to commercialization of this strain as a microbial biopesticide in Korea. To retain its biocontrol efficacy for a longer period, AK-0 has been formulated with ingredients for commercialization, named AK-0 product formulation (AK-0PF). AK-0PF played a role in the suppression of the mycelial growth of the fungicide-resistant pathogen C. gloeosporioides YCHH4 at a greater level than the non-treated control. Moreover, AK-0PF exhibited greater disease suppression of bitter rot in matured under field conditions. Here, we report the complete genome sequence of the AK-0 strain, which has a 3,969,429 bp circular chromosome with 3808 genes and a G+C content of 46.5%. The genome sequence of AK-0 provides a greater understanding of the Bacillus species, which displays biocontrol activity via secondary metabolites. The genome has eight potential secondary metabolite biosynthetic clusters, among which, ituD and bacD genes were expressed at a greater level than other genes. This work provides a better understanding of the strain AK-0, as an effective biocontrol agent (BCA) against phytopathogens, including bitter rot in apple.

Genomic and transcriptomic insights into Raffaelea lauricola pathogenesis

BackgroundLaurel wilt caused by Raffaelea lauricola is a lethal vascular disease of North American members of the Lauraceae plant family. This fungus and its primary ambrosia beetle vector Xyleborus glabratus originated from Asia; however, there is no report of laurel wilt causing widespread mortality on native Lauraceae trees in Asia. To gain insight into why R. lauricola is a tree-killing plant pathogen in North America, we generated and compared high quality draft genome assemblies of R. lauricola and its closely related non-pathogenic species R. aguacate.ResultsRelative to R. aguacate, the R. lauricola genome uniquely encodes several small-secreted proteins that are associated with virulence in other pathogens and is enriched in secondary metabolite biosynthetic clusters, particularly polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS) and PKS-NRPS anchored gene clusters. The two species also exhibit significant differences in secreted proteins including CAZymes that are associated with polysaccharide binding including the chitin binding CBM50 (LysM) domain. Transcriptomic comparisons of inoculated redbay trees and in vitro-grown fungal cultures further revealed a number of secreted protein genes, secondary metabolite clusters and alternative sulfur uptake and assimilation pathways that are coordinately up-regulated during infection.ConclusionsThrough these comparative analyses we have identified potential adaptations of R. lauricola that may enable it to colonize and cause disease on susceptible hosts. How these adaptations have interacted with co-evolved hosts in Asia, where little to no disease occurs, and non-co-evolved hosts in North America, where lethal wilt occurs, requires additional functional analysis of genes and pathways.

Whole Genome Sequencing and Comparative Genomics of Two Nematicidal Bacillus Strains Reveals a Wide Range of Possible Virulence Factors

Bacillus firmus nematicidal bacterial strains are used to control plant parasitic nematode infestation of crops in agricultural production. Proteases are presumed to be the primary nematode virulence factors in nematicidal B. firmus degrading the nematode cuticle and other organs. We determined and compared the whole genome sequences of two nematicidal strains. Comparative genomics with a particular focus on possible virulence determinants revealed a wider range of possible virulence factors in a B. firmus isolate from a commercial bionematicide and a wild type Bacillus sp. isolate with nematicidal activity. The resulting 4.6 Mb B. firmus I-1582 and 5.3 Mb Bacillus sp. ZZV12-4809 genome assemblies contain respectively 18 and 19 homologs to nematode-virulent proteases, two nematode-virulent chitinase homologs in ZZV12-4809 and 28 and 36 secondary metabolite biosynthetic clusters, projected to encode antibiotics, small peptides, toxins and siderophores. The results of this study point to the genetic capability of B. firmus and related species for nematode virulence through a range of direct and indirect mechanisms.

Streptomyces albulus yields ε-poly-L-lysine and other products from salt-contaminated glycerol waste.

Actinomycetes are the most important microorganisms for the industrial production of secondary metabolites with antimicrobial and anticancer properties. However, they have not been implicated in biorefineries. Here, we study the ability of the ε-poly-L-lysine producing Streptomyces albulus BCRC 11814 to utilize biodiesel-derived crude glycerol. S. albulus was cultured in a mineral medium supplemented with up to 10% w/v sodium chloride or potassium chloride, and with crude glycerol as the sole carbohydrate source. Under these conditions, the strain produced 0.1g ε-poly-L-lysine per 1g of biomass. RNA sequencing revealed upregulation of the ectoine biosynthetic pathway of S. albulus, which provides proof of halotolerance. S. albulus has several silent secondary metabolite biosynthetic clusters predicted within the genome. Based on the results, we conclude that S. albulus BCRC 11814 is a halotolerant microorganism capable of utilizing biodiesel-derived crude glycerol better than other actinomycetes included in the present study. S. albulus has the potential to be established as microbial platform production host for a range of high-value biological products.

Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein complexes

The Fusarium oxysporum species complex (FOSC) is an economically important group of pathogenic filamentous fungi that are able to infect both animals and plants. Reverse genetic techniques, including gene disruption/deletion methods, to study these fungi are available although limitations exist resulting in decreased efficiency. Herein we describe a gene editing system developed using a F. oxysporum-optimized Cas9 ribonucleoprotein (RNP) and protoplast transformation method. The Cas9 protein and sgRNA were assembled to form a stable RNP in vitro and this complex was transferred into fungal protoplasts for gene editing with PEG-mediated transformation. In order to determine if the Cas9 RNP system is functional in the FOSC protoplasts and assess the efficacy of the system, two genes, URA5 and URA3, were selected for targeted disruption generating uracil auxotroph mutants that are resistant to 5-fluoroorotic acid, 5-FOA. In addition, a gene in a secondary metabolite biosynthetic cluster, the ortholog of BIK1, was mutated using this system and the maximum efficiency of this gene disruption was about 50%. Further analysis of the bik1 mutant confirmed that this polyketide synthase was involved in the synthesis of the red pigment, bikaverin. The mutants generated in this study displayed the strong expected phenotypes, demonstrating this F. oxysporum-optimized CRISPR/Cas9 system is stable and can efficiently disrupt the genes of interest.

A systems approach using OSMAC, Log P and NMR fingerprinting: An approach to novelty

The growing number of sequenced microbial genomes has revealed a remarkably large number of secondary metabolite biosynthetic clusters for which the compounds are still unknown. The aim of the present work was to apply a strategy to detect newly induced natural products by cultivating microorganisms in different fermentation conditions. The metabolomic analysis of 4160 fractions generated from 13 actinomycetes under 32 different culture conditions was carried out by 1H NMR spectroscopy and multivariate analysis. The principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples within PC1 and PC2. The fractions with induced metabolites that are only produced under specific growth conditions was identified by PCA analysis. This method allows an efficient differentiation within a large dataset with only one fractionation step. This work demonstrates the potential of NMR spectroscopy in combination with metabolomic data analysis for the screening of large sets of fractions.

Draft Genome Sequence of Phytopathogenic Fungus Fusarium proliferatum [corrected] CF-295141, Isolated from Pinus sylvestris.

Here, we report the draft genome sequence of a new strain of Fusarium fujikuroi, isolated from Pinus sylvestris, which was also found to produce the mycotoxin beauvericin. The Illumina-based sequence analysis revealed an approximate genome size of 44.2 Mbp, containing 164 secondary metabolite biosynthetic clusters.

Discovery of pentangular polyphenols hexaricins A–C from marine Streptosporangium sp. CGMCC 4.7309 by genome mining

Many novel microbial nature products were discovered from Actinobacteria by genome mining methods. However, only a few number of genome mining works were carried out in rare actinomycetes. An important reason precluding the genome mining efforts in rare actinomycetes is that most of them are recalcitrant to genetic manipulation. Herein, we chose the rare marine actinomycete Streptosporangium sp. CGMCC 4.7309 to explore its secondary metabolite diversity by genome mining. The genetic manipulation method has never been established for Streptosporangium strains. At first, we set up the genetic system of Streptosporangium sp. CGMCC 4.7309 unprecedentedly. The draft genome sequencing of Streptosporangium sp. CGMCC 4.7309 revealed that it contains more than 20 cryptic secondary metabolite biosynthetic clusters. A type II polyketide synthases-containing cluster (the hex cluster) was predicted to encode compounds with a pentangular polyphenol scaffold by in silico analysis. The products of the hex cluster were uncovered by comparing the metabolic profile of Streptosporangium sp. CGMCC 4.7309 with that of the hex30 inactivated mutant, in which a key ketoreductase gene was disrupted. Finally, three pentangular polyphenols were isolated and named as hexaricins A (1), B (2), and C (3). The inconsistency of the stereochemistry of C-15 in hexaricins A, B, and C indicates a branch point in their biosynthesis. Finally, the biosynthetic pathway of the hexaricins was proposed based on bioinformatics analysis.

A genome-wide transcriptomic analysis reveals diverse roles of the two-component system DraR-K in the physiological and morphological differentiation of Streptomyces coelicolor.

A novel two-component system (TCS) of DraR-K was previously identified as playing differential roles in the biosynthesis of antibiotics (blue-pigmented type II polyketide actinorhodin (ACT), red-pigmented tripyrrole undecylprodigiosin (RED), and yellow-pigmented type I polyketide (yCPK)) in Streptomyces coelicolor M145 under the conditions of minimal medium (MM) supplemented with a high concentration of different nitrogen sources (e.g., 75 mM glutamine). To assess whether DraR-K has more globalized roles, a genome-wide transcriptomic analysis of the parental strain M145 and a ΔdraR-K mutant under the condition of MM supplemented with 75 mM glutamine was performed using DNA microarray analysis combined with real-time reverse transcriptase PCR (RT-qPCR). The analyses showed that deletion of the draR-K genes led to the differential expression not only of the biosynthetic gene clusters of ACT, RED, and yCPK but also of other five secondary metabolite biosynthetic clusters. In addition, a number of primary metabolism-related genes in the ΔdraR-K mutant, such as ureA/B/C/D/G/F, the pstSCAB operon, and the chb gene, exhibited altered expression, which might enable the organism to balance the C/N/P ratio under the condition of a high concentration of glutamine. We also found that the expression of many developmental genes, including ramR, chpA/D/E, and the whiE gene cluster, was affected by the draR-K deletion. Furthermore, the direct role of DraR-K on the transcription of several genes, including chb and pepA/pepA2, was validated using electrophoretic mobility shift assays (EMSAs). In summary, our transcriptomic analyses revealed that DraR-K plays global regulatory roles in the physiological and morphological differentiation of S. coelicolor.

Improved Lanthipeptide Detection and Prediction for antiSMASH

Lanthipeptides are a class of ribosomally synthesised and post-translationally modified peptide (RiPP) natural products from the bacterial secondary metabolism. Their name is derived from the characteristic lanthionine or methyl-lanthionine residues contained in the processed peptide. Lanthipeptides that possess an antibacterial activity are called lantibiotics. Whereas multiple tools exist to identify lanthipeptide gene clusters from genomic data, no programs are available to predict the post-translational modifications of lanthipeptides, such as the proteolytic cleavage of the leader peptide part or tailoring modifications based on the analysis of the gene cluster sequence. antiSMASH is a software pipeline for the identification of secondary metabolite biosynthetic clusters from genomic input and the prediction of products produced by the identified clusters. Here we present a novel antiSMASH module using a rule-based approach to combine signature motifs for biosynthetic enzymes and lanthipeptide-specific cleavage site motifs to identify lanthipeptide clusters in genomic data, assign the specific lanthipeptide class, predict prepeptide cleavage, tailoring reactions, and the processed molecular weight of the mature peptide products.

Evolutionary concepts in natural products discovery: what actinomycetes have taught us

Actinomycetes are a very important source of natural products for the pharmaceutical industry and other applications. Most of the strains belong to Streptomyces or related genera, partly because they are particularly amenable to growth in the laboratory and industrial fermenters. It is unlikely that chemical synthesis can fulfil the needs of the pharmaceutical industry for novel compounds so there is a continuing need to find novel natural products. An evolutionary perspective can help this process in several ways. Genome mining attempts to identify secondary metabolite biosynthetic clusters in DNA sequences, which are likely to produce interesting chemical entities. There are often technical problems in assembling the DNA sequences of large modular clusters in genome and metagenome projects, which can be overcome partially using information about the evolution of the domain sequences. Understanding the evolutionary mechanisms of modular clusters should allow simulation of evolutionary pathways in the laboratory to generate novel compounds.

Transcriptional Profiling Identifies a Role for BrlA in the Response to Nitrogen Depletion and for StuA in the Regulation of Secondary Metabolite Clusters in Aspergillus fumigatus

Conidiation (asexual sporulation) is a key developmental process in filamentous fungi. We examined the gene regulatory roles of the Aspergillus fumigatus developmental transcription factors StuAp and BrlAp during conidiation. Conidiation was completely abrogated in an A. fumigatus DeltabrlA mutant and was severely impaired in a DeltastuA mutant. We determined the full genome conidiation transcriptomes of wild-type and DeltabrlA and DeltastuA mutant A. fumigatus and found that BrlAp and StuAp governed overlapping but distinct transcriptional programs. Six secondary metabolite biosynthetic clusters were found to be regulated by StuAp, while only one cluster exhibited BrlAp-dependent expression. The DeltabrlA mutant, but not the DeltastuA mutant, had impaired downregulation of genes encoding ribosomal proteins under nitrogen-limiting, but not carbon-limiting, conditions. Interestingly, inhibition of the target of rapamycin (TOR) pathway also caused downregulation of ribosomal protein genes in both the wild-type strain and the DeltabrlA mutant. Downregulation of these genes by TOR inhibition was associated with conidiation in the wild-type strain but not in the DeltabrlA mutant. Therefore, BrlAp-mediated repression of ribosomal protein gene expression is not downstream of the TOR pathway. Furthermore, inhibition of ribosomal protein gene expression is not sufficient to induce conidiation in the absence of BrlAp.