scholarly journals Genomic and Chemical Diversity of Bacillus subtilis Secondary Metabolites against Plant Pathogenic Fungi

mSystems ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Heiko T. Kiesewalter ◽  
Carlos N. Lozano-Andrade ◽  
Mario Wibowo ◽  
Mikael L. Strube ◽  
Gergely Maróti ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Secondary Metabolites ◽  
Pathogenic Fungus ◽  
Gene Clusters ◽  
Chemical Diversity ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Wide Range

ABSTRACT Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant growth-promoting and biocontrol abilities. These secondary metabolites include nonribosomal peptides with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates, and therefore, a comparative overview of secondary metabolites from various environmental B. subtilis strains is missing. In this study, we isolated 23 B. subtilis strains from 11 sampling sites, compared the fungal inhibition profiles of wild types and their nonribosomal peptide mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that nonribosomal peptide production varied among B. subtilis strains coisolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium spp., a combination of plipastatin and surfactin is required to hinder growth of Botrytis cinerea. Detailed genomic analysis revealed that altered nonribosomal peptide production profiles in specific isolates are due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical nonribosomal peptide detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis. IMPORTANCE Secondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which nonribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate the prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack the production of certain secondary metabolites.

scholarly journals Genomic and chemical diversity of Bacillus subtilis secondary metabolites against plant pathogenic fungi

2020 ◽  
Author(s):  
Heiko T. Kiesewalter ◽  
Carlos N. Lozano-Andrade ◽  
Mario Wibowo ◽  
Mikael L. Strube ◽  
Gergely Maróti ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Secondary Metabolites ◽  
Mutational Analysis ◽  
Pathogenic Fungus ◽  
Pathogenic Fungi ◽  
Gene Clusters ◽  
Chemical Diversity ◽  
Biosynthetic Gene ◽  
Wide Range

ABSTRACTBacillus subtilis produces a wide range of secondary metabolites providing diverse plant-growth-promoting and biocontrol abilities. These secondary metabolites include non-ribosomal peptides (NRPs) with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates and therefore, a comparative overview of B. subtilis secondary metabolites is missing.In this study, we have isolated 23 B. subtilis strains from eleven sampling sites, compared the fungal inhibition profiles of wild types and their NRPs mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that non-ribosomal peptide production varied among B. subtilis strains co-isolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium sp., a combination of plipastatin and surfactin is required to hinder the growth of Botrytis cinerea. Detailed genomic analysis revealed that altered NRP production profiles in certain isolates is due to missing core genes, nonsense mutation, or potentially altered gene regulation.Our study combines microbiological antagonism assays with chemical NRPs detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis.IMPORTANCESecondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which non-ribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack production of certain secondary metabolites.

scholarly journals Phylogenetic Distribution of Secondary Metabolites in the Bacillus subtilis Species Complex

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Kat Steinke ◽  
Omkar S. Mohite ◽  
Tilmann Weber ◽  
Ákos T. Kovács
Keyword(s):  
Bacillus Subtilis ◽  
Secondary Metabolites ◽  
Secondary Metabolite ◽  
Species Complex ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Content Type ◽  
Frameshift Mutations ◽  
Metabolite Production

ABSTRACT Microbes produce a plethora of secondary (or specialized) metabolites that, although not essential for primary metabolism, benefit them to survive in the environment, communicate, and influence cell differentiation. Biosynthetic gene clusters (BGCs), responsible for the production of these secondary metabolites, are readily identifiable on bacterial genome sequences. Understanding the phylogeny and distribution of BGCs helps us to predict the natural product synthesis ability of new isolates. Here, we examined 310 genomes from the Bacillus subtilis group, determined the inter- and intraspecies patterns of absence/presence for all BGCs, and assigned them to defined gene cluster families (GCFs). This allowed us to establish patterns in the distribution of both known and unknown products. Further, we analyzed variations in the BGC structures of particular families encoding natural products, such as plipastatin, fengycin, iturin, mycosubtilin, and bacillomycin. Our detailed analysis revealed multiple GCFs that are species or clade specific and a few others that are scattered within or between species, which will guide exploration of the chemodiversity within the B. subtilis group. Surprisingly, we discovered that partial deletion of BGCs and frameshift mutations in selected biosynthetic genes are conserved within phylogenetically related isolates, although isolated from around the globe. Our results highlight the importance of detailed genomic analysis of BGCs and the remarkable phylogenetically conserved erosion of secondary metabolite biosynthetic potential in the B. subtilis group. IMPORTANCE Members of the B. subtilis species complex are commonly recognized producers of secondary metabolites, among those, the production of antifungals, which makes them promising biocontrol strains. While there are studies examining the distribution of well-known secondary metabolites in Bacilli, intraspecies clade-specific distribution has not been systematically reported for the B. subtilis group. Here, we report the complete biosynthetic potential within the B. subtilis group to explore the distribution of the biosynthetic gene clusters and to reveal an exhaustive phylogenetic conservation of secondary metabolite production within Bacillus that supports the chemodiversity within this species complex. We identify that certain gene clusters acquired deletions of genes and particular frameshift mutations, rendering them inactive for secondary metabolite biosynthesis, a conserved genetic trait within phylogenetically conserved clades of certain species. The overview guides the assignment of the secondary metabolite production potential of newly isolated Bacillus strains based on genome sequence and phylogenetic relatedness.

scholarly journals An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases inMyxococcus xanthusDK1622

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Karla J. Esquilín-Lebrón ◽  
Tye O. Boynton ◽  
Lawrence J. Shimkets ◽  
Michael G. Thomas
Keyword(s):  
Myxococcus Xanthus ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

ABSTRACTOne mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 fromMyxococcus xanthusDK1622 for characterization. TheM. xanthusDK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination ofin vivoandin vitroassays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping ofM. xanthusDK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a “universal” MLP for generating functional hybrid NRPSs.IMPORTANCEMbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP fromMyxococcus xanthusDK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a “universal” MLP during the construction of functional hybrid NRPSs.

scholarly journals A Two-Step Mechanism for the Activation of Actinorhodin Export and Resistance in Streptomyces coelicolor

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ye Xu ◽  
Andrew Willems ◽  
Catherine Au-yeung ◽  
Kapil Tahlan ◽  
Justin R. Nodwell
Keyword(s):  
Secondary Metabolites ◽  
Pathogenic Bacteria ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistance Mechanisms ◽  
Biosynthetic Gene ◽  
Content Type

ABSTRACT Many microorganisms produce secondary metabolites that have antibiotic activity. To avoid self-inhibition, the producing cells often encode cognate export and/or resistance mechanisms in the biosynthetic gene clusters for these molecules. Actinorhodin is a blue-pigmented antibiotic produced by Streptomyces coelicolor. The actAB operon, carried in the actinorhodin biosynthetic gene cluster, encodes two putative export pumps and is regulated by the transcriptional repressor protein ActR. In this work, we show that normal actinorhodin yields require actAB expression. Consistent with previous in vitro work, we show that both actinorhodin and its 3-ring biosynthetic intermediates [e.g., (S)-DNPA] can relieve repression of actAB by ActR in vivo. Importantly, an ActR mutant that interacts productively with (S)-DNPA but not with actinorhodin responds to the actinorhodin biosynthetic pathway with the induction of actAB and normal yields of actinorhodin. This suggests that the intermediates are sufficient to trigger the export genes in actinorhodin-producing cells. We further show that actinorhodin-producing cells can induce actAB expression in nonproducing cells; however, in this case actinorhodin is the most important signal. Finally, while the “intermediate-only” ActR mutant permits sufficient actAB expression for normal actinorhodin yields, this expression is short-lived. Sustained culture-wide expression requires a subsequent actinorhodin-mediated signaling step, and the defect in this response causes widespread cell death. These results are consistent with a two-step model for actinorhodin export and resistance where intermediates trigger initial expression for export from producing cells and actinorhodin then triggers sustained export gene expression that confers culture-wide resistance. IMPORTANCE Understanding the links between antibiotic resistance and biosynthesis is important for our efforts to manipulate secondary metabolism. For example, many secondary metabolites are produced at low levels; our work suggests that manipulating export might be one way to enhance yields of these molecules. It also suggests that understanding resistance will be relevant to the generation of novel secondary metabolites through the creation of synthetic secondary metabolic gene clusters. Finally, these cognate resistance mechanisms are related to mechanisms that arise in pathogenic bacteria, and understanding them is relevant to our ability to control microbial infections clinically.

scholarly journals Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy

2020 ◽  
Vol 117 (16) ◽  
pp. 8850-8858
Author(s):  
Kyle L. Dunbar ◽  
Maria Dell ◽  
Finn Gude ◽  
Christian Hertweck
Keyword(s):  
Secondary Metabolites ◽  
Anaerobic Bacteria ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Peptide Backbone ◽  
Nonribosomal Peptide ◽  
Bacterial Phyla ◽  
Biochemical Assays

Closthioamide (CTA) is a rare example of a thioamide-containing nonribosomal peptide and is one of only a handful of secondary metabolites described from obligately anaerobic bacteria. Although the biosynthetic gene cluster responsible for CTA production and the thioamide synthetase that catalyzes sulfur incorporation were recently discovered, the logic for peptide backbone assembly has remained a mystery. Here, through the use of in vitro biochemical assays, we demonstrate that the amide backbone of CTA is assembled in an unusual thiotemplated pathway involving the cooperation of a transacylating member of the papain-like cysteine protease family and an iteratively acting ATP-grasp protein. Using the ATP-grasp protein as a bioinformatic handle, we identified hundreds of such thiotemplated yet nonribosomal peptide synthetase (NRPS)-independent biosynthetic gene clusters across diverse bacterial phyla. The data presented herein not only clarify the pathway for the biosynthesis of CTA, but also provide a foundation for the discovery of additional secondary metabolites produced by noncanonical biosynthetic pathways.

scholarly journals Complete Genome Sequences of Three Bacillus amyloliquefaciens Strains That Inhibit the Growth of Listeria monocytogenes In Vitro

2018 ◽  
Vol 6 (25) ◽  
Author(s):  
Thao D. Tran ◽  
Steven Huynh ◽  
Craig T. Parker ◽  
Robert Hnasko ◽  
Lisa Gorski ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Secondary Metabolites ◽  
Growth Inhibition ◽  
Complete Genome ◽  
Bacillus Amyloliquefaciens ◽  
Gene Clusters ◽  
Genome Sequences ◽  
Multiple Gene ◽  
Content Type

ABSTRACT Here, we report the complete genome sequences of three Bacillus amyloliquefaciens strains isolated from alfalfa, almond drupes, and grapes that inhibited the growth of Listeria monocytogenes strain 2011L-2857 in vitro. We also report multiple gene clusters encoding secondary metabolites that may be responsible for the growth inhibition of L. monocytogenes.

scholarly journals Complete Genome Sequence of the Lignocellulose-Degrading Actinomycete Streptomyces albus CAS922

2020 ◽  
Vol 9 (21) ◽  
Author(s):  
Anna Tippelt ◽  
Markus Nett ◽  
M. Soledad Vela Gurovic
Keyword(s):  
Secondary Metabolites ◽  
Complete Genome Sequence ◽  
Sunflower Seed ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Carbohydrate Active Enzymes ◽  
Content Type ◽  
Bioactive Secondary Metabolites ◽  
Streptomyces Albus

ABSTRACT Streptomyces albus CAS922 was isolated from sunflower seed hulls. Its fully sequenced genome harbors a multitude of genes for carbohydrate-active enzymes, which likely facilitate growth on lignocellulosic biomass. Furthermore, the presence of 27 predicted biosynthetic gene clusters indicates a significant potential for the production of bioactive secondary metabolites.

scholarly journals Draft Genome Sequence of Bacillus subtilis SB-14, an Antimicrobially Active Isolate from Namibian Social Spiders (Stegodyphus dumicola)

2019 ◽  
Vol 8 (25) ◽  
Author(s):  
Stine Sofie Frank Nielsen ◽  
Simone Weiss ◽  
Seven Nazipi ◽  
Ian P. G. Marshall ◽  
Trine Bilde ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Genome Sequence ◽  
Draft Genome ◽  
Gene Clusters ◽  
Draft Genome Sequence ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
High Quality ◽  
Social Spider ◽  
Content Type

We present the high-quality draft genome sequence of Bacillus subtilis SB-14, isolated from the Namibian social spider Stegodyphus dumicola. In accordance with its antimicrobial activity, both known and potentially novel antimicrobial biosynthetic gene clusters were identified in the genome of SB-14.

scholarly journals Nonribosomal Peptide Synthase Gene Clusters for Lipopeptide Biosynthesis in Bacillus subtilis 916 and Their Phenotypic Functions

2014 ◽  
Vol 81 (1) ◽  
pp. 422-431 ◽  
Author(s):  
Chuping Luo ◽  
Xuehui Liu ◽  
Huafei Zhou ◽  
Xiaoyu Wang ◽  
Zhiyi Chen
Keyword(s):  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Hemolytic Activity ◽  
Biofilm Formation ◽  
Gene Clusters ◽  
Colony Morphology ◽  
Swarming Motility ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Phenotypic Features

ABSTRACTBacilluscyclic lipopeptides (LPs) have been well studied for their phytopathogen-antagonistic activities. Recently, research has shown that these LPs also contribute to the phenotypic features ofBacillusstrains, such as hemolytic activity, swarming motility, biofilm formation, and colony morphology.Bacillus subtilis916 not only coproduces the three families of well-known LPs, i.e., surfactins, bacillomycin Ls (iturin family), and fengycins, but also produces a new family of LP called locillomycins. The genome ofB. subtilis916 contains four nonribosomal peptide synthase (NRPS) gene clusters,srf,bmy,fen, andloc, which are responsible for the biosynthesis of surfactins, bacillomycin Ls, fengycins, and locillomycins, respectively. By studyingB. subtilis916 mutants lacking production of one, two, or three LPs, we attempted to unveil the connections between LPs and phenotypic features. We demonstrated that bacillomycin Ls and fengycins contribute mainly to antifungal activity. Although surfactins have weak antifungal activityin vitro, the strain mutated insrfAAhad significantly decreased antifungal activity. This may be due to the impaired productions of fengycins and bacillomycin Ls. We also found that the disruption of any LP gene cluster other thanfenresulted in a change in colony morphology. While surfactins and bacillomycin Ls play very important roles in hemolytic activity, swarming motility, and biofilm formation, the fengycins and locillomycins had little influence on these phenotypic features. In conclusion,B. subtilis916 coproduces four families of LPs which contribute to the phenotypic features ofB. subtilis916 in an intricate way.

scholarly journals In vitro Antibacterial Potential of Halophilic Bacterial Secondary Metabolites from Salted Fish

2016 ◽  
Vol 8 (04) ◽  
Author(s):  
G. Prakash Williams ◽  
Anju Anand ◽  
Parvathy A. ◽  
Rakky C. Raj ◽  
Robert Raju ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Secondary Metabolites ◽  
Xanthomonas Campestris ◽  
Halophilic Bacteria ◽  
Resistance Pattern ◽  
Salted Fish ◽  
Wide Range ◽  
Fish Samples

Living organisms can be found over a wide range of extreme conditions. Most of the organisms living in extreme environments (i.e, extremophiles) belong to the prokaryotes. Halophiles are interesting class of extremophilic organisms that have adapted to harsh, hypersaline conditions. They are able to compete successfully for water and resist the denaturing effects of salts. The present study was an investigation on the in vitro antibacterial effect of secondary metabolites from halophilic bacteria isolated from salted fish samples. The cured salted fish samples were collected and enumerated using halophilic Nutrient Agar supplemented with 4% NaCl. The isolated and purified bacterial cultures are numbered as SF1, SF2, SF3, SF4 and SF5 are further identified using VITEK 2 system as Bacillus vallismortis, Ralstonia mannitolytica, Bacillus subtilis, Rhizoboum radiobacter and Kocuria kristina. Growth kinetics of halobacterial isolates were determined by spectrophotometric assay. The antibiotic resistance pattern of tested pathogenic microorganisms using the commercial antibiotics was screened and almost all the tested microorganisms are resistant to Penicillin. The antimicrobial activity of secondary metabolites of halophilic bacteria against drug resistant microbes was assessed using the Agar well diffusion assay. Among the different extracts of the halophilic bacteria, the chloroform extracts of R. mannitolytica showed maximum antibacterial activity against Bacillus subtilis MTCC 441 and Xanthomonas campestris MTCC 2286. The results of antimicrobial activity are considerable because it enables the identification of potential secondary metabolites present in marine halophilic bacteria, which act as source of innumerable therapeutic agents. Further research is highly warranted to find out the active principle responsible for the antibacterial property and to elucidate the structure of particular compound.

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