The N-terminal substrate specificity of the SurE peptide cyclase.

SurE is a standalone peptide cyclase essential for the production of surugamide antibiotics. Although SurE catalyses the cyclisation of varied nonribosomal peptides in vivo, its substrate specificity is poorly understood. To address this issue, an on-resin SurE cyclisation assay was developed and in combination with SNAC thioesters and kinetic measurements was used to define the chemical space of the N-terminal substrate residue.

A chromatogram-simplified Streptomyces albus host for heterologous production of natural products.

Cloning natural product biosynthetic gene clusters from cultured or uncultured sources and their subsequent expression by genetically tractable heterologous hosts is an essential strategy for the elucidation and characterisation of novel microbial natural products. The availability of suitable expression hosts is a critical aspect of this workflow. In this work, we mutagenised five endogenous biosynthetic gene clusters from Streptomyces albus S4, which reduced the complexity of chemical extracts generated from the strain and eliminated antifungal and antibacterial bioactivity. We showed that the resulting quintuple mutant can express foreign biosynthetic gene clusters by heterologously producing actinorhodin, cinnamycin and prunustatin. We envisage that our strain will be a useful addition to the growing suite of heterologous expression hosts available for exploring microbial secondary metabolism.

Regulation of antibiotic production in Actinobacteria: new perspectives from the post-genomic era.

Covering: 2000 to 2018 The antimicrobial activity of many of their natural products has brought prominence to the Streptomycetaceae, a family of Gram-positive bacteria that inhabit both soil and aquatic sediments. In the natural environment, antimicrobial compounds are likely to limit the growth of competitors, thereby offering a selective advantage to the producer, in particular when nutrients become limited and the developmental programme leading to spores commences. The study of the control of this secondary metabolism continues to offer insights into its integration with a complex lifecycle that takes multiple cues from the environment and primary metabolism. Such information can then be harnessed to devise laboratory screening conditions to discover compounds with new or improved clinical value. Here we provide an update of the review we published in NPR in 2011. Besides providing the essential background, we focus on recent developments in our understanding of the underlying regulatory networks, ecological triggers of natural product biosynthesis, contributions from comparative genomics and approaches to awaken the biosynthesis of otherwise silent or cryptic natural products. In addition, we highlight recent discoveries on the control of antibiotic production in other Actinobacteria, which have gained considerable attention since the start of the genomics revolution. New technologies that have the potential to produce a step change in our understanding of the regulation of secondary metabolism are also described.

A phylogenetic and evolutionary analysis of antimycin biosynthesis.

Streptomyces species and other Actinobacteria are ubiquitous in diverse environments worldwide and are the source of, or inspiration for, the majority of antibiotics. The genomic era has enhanced biosynthetic understanding of these valuable chemical entities and has also provided a window into the diversity and distribution of natural product biosynthetic gene clusters. Antimycin is an inhibitor of mitochondrial cytochrome c reductase and more recently was shown to inhibit Bcl-2/Bcl-XL-related anti-apoptotic proteins commonly overproduced by cancerous cells. Here we identify 73 putative antimycin biosynthetic gene clusters (BGCs) in publicly available genome sequences of Actinobacteria and classify them based on the presence or absence of cluster-situated genes antP and antQ, which encode a kynureninase and a phosphopantetheinyl transferase (PPTase), respectively. The majority of BGCs possess either both antP and antQ (L-form) or neither (S-form), while a minority of them lack either antP or antQ (IQ- or IP-form, respectively). We also evaluate the biogeographical distribution and phylogenetic relationships of antimycin producers and BGCs. We show that antimycin BGCs occur on five of the seven continents and are frequently isolated from plants and other higher organisms. We also provide evidence for two distinct phylogenetic clades of antimycin producers and gene clusters, which delineate S-form from L- and I-form BGCs. Finally, our findings suggest that the ancestral antimycin producer harboured an L-form gene cluster which was primarily propagated by vertical transmission and subsequently diversified into S-, IQ- and IP-form biosynthetic pathways.

The MtrAB two-component system controls antibiotic production in Streptomyces coelicolor A3(2).

MtrAB is a highly conserved two-component system implicated in the regulation of cell division in the Actinobacteria. It coordinates DNA replication with cell division in the unicellular Mycobacterium tuberculosis and links antibiotic production to sporulation in the filamentous Streptomyces venezuelae. Chloramphenicol biosynthesis is directly regulated by MtrA in S. venezuelae and deletion of mtrB constitutively activates MtrA and results in constitutive over-production of chloramphenicol. Here we report that in Streptomyces coelicolor, MtrA binds to sites upstream of developmental genes and the genes encoding ActII-1, ActII-4 and RedZ, which are cluster-situated regulators of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red). Consistent with this, deletion of mtrB switches on the production of Act, Red and streptorubin B, a product of the Red pathway. Thus, we propose that MtrA is a key regulator that links antibiotic production to development and can be used to upregulate antibiotic production in distantly related streptomycetes.

The regulation and biosynthesis of antimycins.

Antimycins (>40 members) were discovered nearly 65 years ago but the discovery of the gene cluster encoding antimycin biosynthesis in 2011 has facilitated rapid progress in understanding the unusual biosynthetic pathway. Antimycin A is widely used as a piscicide in the catfish farming industry and also has potent killing activity against insects, nematodes and fungi. The mode of action of antimycins is to inhibit cytochrome c reductase in the electron transport chain and halt respiration. However, more recently, antimycin A has attracted attention as a potent and selective inhibitor of the mitochondrial anti-apoptotic proteins Bcl-2 and Bcl-xL. Remarkably, this inhibition is independent of the main mode of action of antimycins such that an artificial derivative named 2-methoxyantimycin A inhibits Bcl-xL but does not inhibit respiration. The Bcl-2/Bcl-xL family of proteins are over-produced in cancer cells that are resistant to apoptosis-inducing chemotherapy agents, so antimycins have great potential as anticancer drugs used in combination with existing chemotherapeutics. Here we review what is known about antimycins, the regulation of the ant gene cluster and the unusual biosynthetic pathway.

Antibiotics from rare actinomycetes, beyond the genus Streptomyces.

Throughout the golden age of antibiotic discovery, Streptomyces have been unsurpassed for their ability to produce bioactive metabolites. Yet, this success has been hampered by rediscovery. As we enter a new stage of biodiscovery, omics data and existing scientific repositories can enable informed choices on the biodiversity that may yield novel antibiotics. Here, we focus on the chemical potential of rare actinomycetes, defined as bacteria within the order Actinomycetales, but not belonging to the genus Streptomyces. They are named as such due to their less-frequent isolation under standard laboratory practices, yet there is increasing evidence to suggest these biologically diverse genera harbour considerable biosynthetic and chemical diversity. In this review, we focus on examples of successful isolation and genera that have been the focus of more concentrated biodiscovery efforts, we survey the representation of rare actinomycete taxa, compared with Streptomyces, across natural product data repositories in addition to its biosynthetic potential. This is followed by an overview of clinically useful drugs produced by rare actinomycetes and considerations for future biodiscovery efforts. There is much to learn about these underexplored taxa, and mounting evidence suggests that they are a fruitful avenue for the discovery of novel antimicrobials.

Fungus-growing Allomerus ants are associated with antibiotic-producing actinobacteria.

Fungus-growing attine ants use natural-product antibiotics produced by mutualist actinobacteria as 'weedkillers' in their fungal gardens. Here we report for the first time that fungus-growing Allomerus ants, which lie outside the tribe Attini, are associated with antifungal-producing actinobacteria, which offer them protection against non-cultivar fungi isolated from their ant-plants.

Antibiotics made to order.

New lipopeptide antibiotics provide hope in the fight against multidrug-resistant bacteria.

ActinoBase: tools and protocols for researchers working on Streptomyces and other filamentous actinobacteria.

Actinobacteria is an ancient phylum of Gram-positive bacteria with a characteristic high GC content to their DNA. The ActinoBase Wiki is focused on the filamentous actinobacteria, such as Streptomyces species, and the techniques and growth conditions used to study them. These organisms are studied because of their complex developmental life cycles and diverse specialised metabolism which produces many of the antibiotics currently used in the clinic. ActinoBase is a community effort that provides valuable and freely accessible resources, including protocols and practical information about filamentous actinobacteria. It is aimed at enabling knowledge exchange between members of the international research community working with these fascinating bacteria. ActinoBase is an anchor platform that underpins worldwide efforts to understand the ecology, biology and metabolic potential of these organisms. There are two key differences that set ActinoBase apart from other Wiki-based platforms: [1] ActinoBase is specifically aimed at researchers working on filamentous actinobacteria and is tailored to help users overcome challenges working with these bacteria and [2] it provides a freely accessible resource with global networking opportunities for researchers with a broad range of experience in this field.

Draft genome sequence of Streptomyces strain S4, a symbiont of the leaf-cutting ant Acromyrmex octospinosus.

Streptomyces spp. are common symbionts of the leaf-cutting ant species Acromyrmex octospinosus, which feeds on basidiomycete fungus leaf matter and harvests the lipid- and carbohydrate-rich gongylidia as a food source. A. octospinosus and other ant genera use antifungal compounds produced by Streptomyces spp. and other actinomycetes in order to help defend their fungal gardens from parasitic fungi. Herein, we report the draft genome sequence of Streptomyces strain S4, an antifungal-producing symbiont of A. octospinosus.

The plant pathogen Streptomyces scabies 87-22 has a functional pyochelin biosynthetic pathway that is regulated by TetR- and AfsR-family proteins.

Siderophores are high-affinity iron-chelating compounds produced by bacteria for iron uptake that can act as important virulence determinants for both plant and animal pathogens. Genome sequencing of the plant pathogen Streptomyces scabies 87-22 revealed the presence of a putative pyochelin biosynthetic gene cluster (PBGC). Liquid chromatography (LC)-MS analyses of culture supernatants of S. scabies mutants, in which expression of the cluster is upregulated and which lack a key biosynthetic gene from the cluster, indicated that pyochelin is a product of the PBGC. LC-MS comparisons with authentic standards on a homochiral stationary phase confirmed that pyochelin and not enantio-pyochelin (ent-pyochelin) is produced by S. scabies. Transcription of the S. scabies PBGC occurs via ~19 kb and ~3 kb operons and transcription of the ~19 kb operon is regulated by TetR- and AfsR-family proteins encoded by the cluster. This is the first report, to our knowledge, of pyochelin production by a Gram-positive bacterium; interestingly regulation of pyochelin production is distinct from characterized PBGCs in Gram-negative bacteria. Though pyochelin-mediated iron acquisition by Pseudomonas aeruginosa is important for virulence, in planta bioassays failed to demonstrate that pyochelin production by S. scabies is required for development of disease symptoms on excised potato tuber tissue or radish seedlings.

A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus.

BACKGROUND: Attine ants live in an intensely studied tripartite mutualism with the fungus Leucoagaricus gongylophorus, which provides food to the ants, and with antibiotic-producing actinomycete bacteria. One hypothesis suggests that bacteria from the genus Pseudonocardia are the sole, co-evolved mutualists of attine ants and are transmitted vertically by the queens. A recent study identified a Pseudonocardia-produced antifungal, named dentigerumycin, associated with the lower attine Apterostigma dentigerum consistent with the idea that co-evolved Pseudonocardia make novel antibiotics. An alternative possibility is that attine ants sample actinomycete bacteria from the soil, selecting and maintaining those species that make useful antibiotics. Consistent with this idea, a Streptomyces species associated with the higher attine Acromyrmex octospinosus was recently shown to produce the well-known antifungal candicidin. Candicidin production is widespread in environmental isolates of Streptomyces, so this could either be an environmental contaminant or evidence of recruitment of useful actinomycetes from the environment. It should be noted that the two possibilities for actinomycete acquisition are not necessarily mutually exclusive. RESULTS: In order to test these possibilities we isolated bacteria from a geographically distinct population of A. octospinosus and identified a candicidin-producing Streptomyces species, which suggests that they are common mutualists of attine ants, most probably recruited from the environment. We also identified a Pseudonocardia species in the same ant colony that produces an unusual polyene antifungal, providing evidence for co-evolution of Pseudonocardia with A. octospinosus. CONCLUSIONS: Our results show that a combination of co-evolution and environmental sampling results in the diversity of actinomycete symbionts and antibiotics associated with attine ants.

A Standalone ß-Ketoreductase Acts Concomitantly with Biosynthesis of the Antimycin Scaffold.

Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. The biosynthesis of these compounds is well characterized, with the exception of the standalone ß-ketoreductase enzyme AntM that is proposed to catalyze the reduction of the C8 carbonyl of the antimycin scaffold. Inactivation of antM and structural characterization suggested that rather than functioning as a post-PKS tailoring enzyme, AntM acts upon the terminal biosynthetic intermediate while it is tethered to the PKS acyl carrier protein. Mutational analysis identified two amino acid residues (Tyr185 and Phe223) that are proposed to serve as checkpoints controlling substrate access to the AntM active site. Aromatic checkpoint residues are conserved in uncharacterized standalone ß-ketoreductases, indicating that they may also act concomitantly with synthesis of the scaffold. These data provide novel mechanistic insights into the functionality of standalone ß-ketoreductases and will enable their reprogramming for combinatorial biosynthesis.

Streptomyces scabies 87-22 contains a coronafacic acid-like biosynthetic cluster that contributes to plant-microbe interactions.

Plant-pathogenic Streptomyces spp. cause scab disease on economically important root and tuber crops, the most important of which is potato. Key virulence determinants produced by these species include the cellulose synthesis inhibitor, thaxtomin A, and the secreted Nec1 protein that is required for colonization of the plant host. Recently, the genome sequence of Streptomyces scabies 87-22 was completed, and a biosynthetic cluster was identified that is predicted to synthesize a novel compound similar to coronafacic acid (CFA), a component of the virulence-associated coronatine phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Southern analysis indicated that the cfa-like cluster in S. scabies 87-22 is likely conserved in other strains of S. scabies but is absent from two other pathogenic streptomycetes, S. turgidiscabies and S. acidiscabies. Transcriptional analyses demonstrated that the cluster is expressed during plant-microbe interactions and that expression requires a transcriptional regulator embedded in the cluster as well as the bldA tRNA. A knockout strain of the biosynthetic cluster displayed a reduced virulence phenotype on tobacco seedlings compared with the wild-type strain. Thus, the cfa-like biosynthetic cluster is a newly discovered locus in S. scabies that contributes to host-pathogen interactions.

Cryptic or Silent? The Known Unknowns, Unknown Knowns, and Unknown Unknowns of Secondary Metabolism.

Microbial natural products, particularly those produced by filamentous Actinobacteria, underpin the majority of clinically used antibiotics. Unfortunately, only a few new antibiotic classes have been discovered since the 1970s, which has exacerbated fears of a postapocalyptic world in which antibiotics have lost their utility. Excitingly, the genome sequencing revolution painted an entirely new picture, one in which an average strain of filamentous Actinobacteria harbors 20 to 50 natural product biosynthetic pathways but expresses very few of these under laboratory conditions. Development of methodology to access this "hidden" biochemical diversity has the potential to usher in a second Golden Era of antibiotic discovery. The proliferation of genomic data has led to inconsistent use of "cryptic" and "silent" when referring to biosynthetic gene clusters identified by bioinformatic analysis. In this Perspective, we discuss this issue and propose to formalize the use of this terminology.

The Desotamide Family of Antibiotics.

Microbial natural products underpin the majority of antimicrobial compounds in clinical use and the discovery of new effective antibacterial treatments is urgently required to combat growing antimicrobial resistance. Non-ribosomal peptides are a major class of natural products to which many notable antibiotics belong. Recently, a new family of non-ribosomal peptide antibiotics were discovered-the desotamide family. The desotamide family consists of desotamide, wollamide, surugamide, ulleungmycin and noursamycin/curacomycin, which are cyclic peptides ranging in size between six and ten amino acids in length. Their biosynthesis has attracted significant attention because their highly functionalised scaffolds are cyclised by a recently identified standalone cyclase. Here, we provide a concise review of the desotamide family of antibiotics with an emphasis on their biosynthesis.

Regulation of Antimycin Biosynthesis Is Controlled by the ClpXP Protease.

The survival of any microbe relies on its ability to respond to environmental change. Use of extracytoplasmic function (ECF) RNA polymerase sigma (σ) factors is a major strategy enabling dynamic responses to extracellular signals. Streptomyces species harbor a large number of ECF σ factors, nearly all of which are uncharacterized, but those that have been characterized generally regulate genes required for morphological differentiation and/or response to environmental stress, except for σAntA, which regulates starter-unit biosynthesis in the production of antimycin, an anticancer compound. Unlike a canonical ECF σ factor, whose activity is regulated by a cognate anti-σ factor, σAntA is an orphan, raising intriguing questions about how its activity may be controlled. Here, we reconstituted in vitro ClpXP proteolysis of σAntA but not of a variant lacking a C-terminal di-alanine motif. Furthermore, we show that the abundance of σAntAin vivo was enhanced by removal of the ClpXP recognition sequence and that levels of the protein rose when cellular ClpXP protease activity was abolished. These data establish direct proteolysis as an alternative and, thus far, unique control strategy for an ECF RNA polymerase σ factor and expands the paradigmatic understanding of microbial signal transduction regulation.IMPORTANCE Natural products produced by Streptomyces species underpin many industrially and medically important compounds. However, the majority of the ∼30 biosynthetic pathways harbored by an average species are not expressed in the laboratory. This unrevealed biochemical diversity is believed to comprise an untapped resource for natural product drug discovery. Major roadblocks preventing the exploitation of unexpressed biosynthetic pathways are a lack of insight into their regulation and limited technology for activating their expression. Our findings reveal that the abundance of σAntA, which is the cluster-situated regulator of antimycin biosynthesis, is controlled by the ClpXP protease. These data link proteolysis to the regulation of natural product biosynthesis for the first time to our knowledge, and we anticipate that this will emerge as a major strategy by which actinobacteria regulate production of their natural products. Further study of this process will advance understanding of how expression of secondary metabolism is controlled and will aid pursuit of activating unexpressed biosynthetic pathways.

Hopanoids are not essential for growth of Streptomyces scabies 87-22.

Hopanoids are triterpenoic, pentacyclic compounds that are structurally similar to sterols, which are required for normal cell function in eukaryotes. Hopanoids are thought to be an important component of bacterial cell membranes because they control membrane fluidity and diminish passive diffusion of ions, and a few taxons modulate their hopanoid content in response to environmental stimuli. However, to our knowledge, mutational studies to assess the importance of hopanoids in bacterial physiology have never been performed. Genome sequencing of the potato scab pathogen, Streptomyces scabies 87-22, revealed a hopanoid biosynthetic gene cluster (HBGC) that is predicted to synthesize hopene and aminotrihydroxybacteriohopane products. Hopene was produced by fully sporulated cultures of S. scabies on solid ISP4 (International Streptomyces Project 4) medium as well as by submerged mycelia grown in liquid minimal medium. The elongated hopanoid aminotrihydroxybacteriohopane was not detected under either growth condition. Transcription of the S. scabies HBGC was upregulated during aerial growth, which suggests a link between hopanoid production and morphological development. Functional analysis of the S. scabies Delta hop615-1 and Delta hop615-7 mutant strains, the first hopanoid mutants created in any bacterial taxon, revealed that hopanoids are not required for normal growth or for tolerance of ethanol, osmotic and oxidative stress, high temperature, or low pH. This suggests that hopanoids are not essential for normal streptomycete physiology.

A trans-Acting Cyclase Offloading Strategy for Nonribosomal Peptide Synthetases.

The terminal step in the biosynthesis of nonribosomal peptides is the hydrolytic release and, frequently, macrocyclization of an aminoacyl-S-thioester by an embedded thioesterase. The surugamide biosynthetic pathway is composed of two nonribosomal peptide synthetase (NRPS) assembly lines in which one produces surugamide A, which is a cyclic octapeptide, and the other produces surugamide F, a linear decapeptide. The terminal module of each system lacks an embedded thioesterase, which led us to question how the peptides are released from the assembly line (and cyclized in the case of surugamide A). We characterized a cyclase belonging to the ß-lactamase superfamily in vivo, established that it is a trans-acting release factor for both compounds, and verified this functionality in vitro with a thioester mimic of linear surugamide A. Using bioinformatics, we estimate that ∼11% of filamentous Actinobacteria harbor an NRPS system lacking an embedded thioesterase and instead employ a trans-acting cyclase. This study improves the paradigmatic understanding of how nonribosomal peptides are released from the terminal peptidyl carrier protein and adds a new dimension to the synthetic biology toolkit.