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

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.

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

Journal of Bacteriology ◽

10.1128/jb.00346-18 ◽

2018 ◽

Vol 200 (21) ◽

Cited By ~ 8

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.

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Genome-wide analysis of the role of the antibiotic biosynthesis regulator AbsA2 inStreptomyces coelicolorA3(2)

10.1101/361360 ◽

2018 ◽

Author(s):

Richard A. Lewis ◽

Abdul Wahab ◽

Giselda Bucca ◽

Emma E. Laing ◽

Carla Möller-Levet ◽

...

Keyword(s):

Large Scale ◽

Time Course ◽

Streptomyces Coelicolor ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Negative Influence ◽

Antibiotic Biosynthesis ◽

Biosynthetic Gene

AbstractThe AbsA1-AbsA2 two component signalling system ofStreptomyces coelicolorhas long been known to exert a powerful negative influence on the production of the antibiotics actinorhodin, undecylprodiginine and the Calcium-Dependent Antibiotic (CDA). Here we report the analysis of aΔabsA2deletion strain, which exhibits the classic precocious antibiotic hyper-production phenotype, and its complementation by an N-terminal triple-FLAG-tagged version of AbsA2. The complemented and non-complementedΔabsA2mutant strains were used in large-scale microarray-based time-course experiments to investigate the effect of deletingabsA2on gene expression and to identify thein vivoAbsA2 DNA-binding target sites using ChIP-on chip. We show that in addition to binding to the promoter regions ofredZandactII-orfIVAbsA2 binds to several previously unidentified sites within thecdabiosynthetic gene cluster within and/or upstream ofSCO3215-SCO3216,SCO3217,SCO3229-SCO3230, andSCO3226, and we relate the pattern of AbsA2 binding to the results of the transcriptomic study and antibiotic phenotypic assays. Interestingly, dual ‘biphasic’ ChIP peaks were observed with AbsA2 binding across the regulatory genesactII-orfIVandredZand theabsA2gene itself, while more conventional single promoter-proximal peaks were seen at the CDA biosynthetic genes suggesting a different mechanism of regulation of the former loci. Taken together the results shed light on the complex mechanism of regulation of antibiotic biosynthesis inStreptomyces coelicolorand the important role of AbsA2 in controlling the expression of three antibiotic biosynthetic gene clusters.

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Heterologous Expression of Pseudouridimycin and Description of the Corresponding Minimal Biosynthetic Gene Cluster

Molecules ◽

10.3390/molecules26020510 ◽

2021 ◽

Vol 26 (2) ◽

pp. 510

Author(s):

Nils Böhringer ◽

Maria A. Patras ◽

Till F. Schäberle

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Streptomyces Coelicolor ◽

Biosynthetic Gene Cluster ◽

Infection Model ◽

Biosynthetic Gene ◽

Mouse Infection Model ◽

Production Platform

Pseudouridimycin (PUM) was recently discovered from Streptomyces sp. DSM26212 as a novel bacterial nucleoside analog that competes with UTP for access to the RNA polymerase (RNAP) active site, thereby inhibiting bacterial RNAP by blocking transcription. This represents a novel antibacterial mode of action and it is known that PUM inhibits bacterial RNAP in vitro, inhibits bacterial growth in vitro, and was active in vivo in a mouse infection model of Streptococcus pyogenes peritonitis. The biosynthetic gene cluster (BGC) was previously identified and characterized by knockout experiments. However, the minimal set of genes necessary for PUM production was not proposed. To identify the minimal BGC and to create a plug-and-play production platform for PUM and its biosynthetic precursors, several versions of a redesigned PUM BGC were generated and expressed in the heterologous host Streptomyces coelicolor M1146 under control of strong promotors. Heterologous expression allowed identification of the putative serine/threonine kinase PumF as an enzyme essential for heterologous PUM production and thus corroboration of the PUM minimal BGC.

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Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1918759117 ◽

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.

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

mSystems ◽

10.1128/msystems.00770-20 ◽

2021 ◽

Vol 6 (1) ◽

Cited By ~ 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.

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Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705016114 ◽

2017 ◽

Vol 114 (27) ◽

pp. 7025-7030 ◽

Cited By ~ 31

Author(s):

Nicholas C. Harris ◽

Michio Sato ◽

Nicolaus A. Herman ◽

Frederick Twigg ◽

Wenlong Cai ◽

...

Keyword(s):

Gene Cluster ◽

Acyl Chain ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Nonribosomal Peptide Synthetase ◽

Biosynthetic Gene ◽

Nonribosomal Peptide ◽

Peptide Synthetase

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

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Self-Resistance during Muraymycin Biosynthesis: a Complementary Nucleotidyltransferase and Phosphotransferase with Identical Modification Sites and Distinct Temporal Order

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00193-18 ◽

2018 ◽

Vol 62 (7) ◽

Cited By ~ 7

Author(s):

Zheng Cui ◽

Xia-Chang Wang ◽

Xiaodong Liu ◽

Anke Lemke ◽

Stefan Koppermann ◽

...

Keyword(s):

Temporal Order ◽

Biochemical Characterization ◽

Antibacterial Activities ◽

Biosynthetic Gene Cluster ◽

Resistance Mechanisms ◽

Biosynthetic Gene ◽

Content Type ◽

Inhibitory Activities ◽

Nucleoside Inhibitors

ABSTRACTMuraymycins are antibacterial natural products fromStreptomycesspp. that inhibit translocase I (MraY), which is involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5′-C-glycyluridine (GlyU) appended to a 5″-amino-5″-deoxyribose (ADR), forming a disaccharide core that is found in several peptidyl nucleoside inhibitors of MraY. For muraymycins, the GlyU-ADR disaccharide is further modified with an aminopropyl-linked peptide to generate the simplest structures, annotated as the muraymycin D series. Two enzymes encoded in the muraymycin biosynthetic gene cluster, Mur29 and Mur28, were functionally assignedin vitroas a Mg·ATP-dependent nucleotidyltransferase and a Mg·ATP-dependent phosphotransferase, respectively, both modifying the 3″-OH of the disaccharide. Biochemical characterization revealed that both enzymes can utilize several nucleotide donors as cosubstrates and the acceptor substrate muraymycin also behaves as an inhibitor. Single-substrate kinetic analyses revealed that Mur28 preferentially phosphorylates a synthetic GlyU-ADR disaccharide, a hypothetical biosynthetic precursor of muraymycins, while Mur29 preferentially adenylates the D series of muraymycins. The adenylated or phosphorylated products have significantly reduced (170-fold and 51-fold, respectively) MraY inhibitory activities and reduced antibacterial activities, compared with the respective unmodified muraymycins. The results are consistent with Mur29-catalyzed adenylation and Mur28-catalyzed phosphorylation serving as complementary self-resistance mechanisms, with a distinct temporal order during muraymycin biosynthesis.

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Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

Applied and Environmental Microbiology ◽

10.1128/aem.03254-12 ◽

2013 ◽

Vol 79 (7) ◽

pp. 2349-2357 ◽

Cited By ~ 9

Author(s):

Li Li ◽

Jun Wu ◽

Zixin Deng ◽

T. Mark Zabriskie ◽

Xinyi He

Keyword(s):

Gene Cluster ◽

Genetic Manipulation ◽

Biosynthetic Gene Cluster ◽

Streptomyces Lividans ◽

Dna Uptake ◽

Biosynthetic Gene ◽

Content Type ◽

Blasticidin S

ABSTRACTBlasticidin S is a peptidyl nucleoside antibiotic produced byStreptomyces griseochromogenesthat exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesisin vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome ofStreptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, forS. lividansblasticidin S deaminase) was identified inS. lividansand shown to govern thisin vivoconversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin Sin vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene inS. lividansLL2 led to successful production of active blasticidin S in the resultant mutant,S. lividansWJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster,blsE,blsF, andblsL, encoding a predicted radicalS-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis.

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

Genome Announcements ◽

10.1128/genomea.00579-18 ◽

2018 ◽

Vol 6 (25) ◽

Cited By ~ 2

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.

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Phylogenetic Distribution of Secondary Metabolites in the Bacillus subtilis Species Complex

mSystems ◽

10.1128/msystems.00057-21 ◽

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.

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