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

A single Streptomyces symbiont makes multiple antifungals to support the fungus farming ant Acromyrmex octospinosus

Attine ants are dependent on a cultivated fungus for food and use antibiotics produced by symbiotic Actinobacteria as weedkillers in their fungus gardens. Actinobacterial species belonging to the genera Pseudonocardia, Streptomyces and Amycolatopsis have been isolated from attine ant nests and shown to confer protection against a range of microfungal weeds. In previous work on the higher attine Acromyrmex octospinosus we isolated a Streptomyces strain that produces candicidin, consistent with another report that attine ants use Streptomyces-produced candicidin in their fungiculture. Here we report the genome analysis of this Streptomyces strain and identify multiple antibiotic biosynthetic pathways. We demonstrate, using gene disruptions and mass spectrometry, that this single strain has the capacity to make candicidin and multiple antimycin compounds. Although antimycins have been known for > 60 years we report the sequence of the biosynthetic gene cluster for the first time. Crucially, disrupting the candicidin and antimycin gene clusters in the same strain had no effect on bioactivity against a co-evolved nest pathogen called Escovopsis that has been identified in similar to 30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibit Escovopsis. However, candicidin and antimycins likely offer protection against other microfungal weeds that infect the attine fungal gardens. Thus, we propose that the selection of this biosynthetically prolific strain from the natural environment provides A. octospinosus with broad spectrum activity against Escovopsis and other microfungal weeds.Publisher PDFPeer reviewe

Complete genome sequence of Streptomyces formicae KY5, the formicamycin producer

Here we report the complete genome of the new species Streptomyces formicae KY5 isolated from Tetraponera fungus growing ants. S. formicae was sequenced using the PacBio and 454 platforms to generate a single linear chromosome with terminal inverted repeats. Illumina MiSeq sequencing was used to correct base changes resulting from the high error rate associated with PacBio. The genome is 9.6 Mbps, has a GC content of 71.38% and contains 8162 protein coding sequences. Predictive analysis shows this strain encodes at least 45 gene clusters for the biosynthesis of secondary metabolites, including a type 2 polyketide synthase encoding cluster for the antibacterial formicamycins. Streptomyces formicae KY5 is a new, taxonomically distinct Streptomyces species and this complete genome sequence provides an important marker in the genus of Streptomyces

The conserved actinobacterial two-component system MtrAB coordinates chloramphenicol production with sporulation in Streptomyces venezuelae NRRL B-65442

Streptomyces bacteria make numerous secondary metabolites, including half of all known antibiotics. Production of antibiotics is usually coordinated with the onset of sporulation but the cross regulation of these processes is not fully understood. This is important because most Streptomyces antibiotics are produced at low levels or not at all under laboratory conditions and this makes large scale production of these compounds very challenging. Here we characterise the highly conserved actinobacterial two-component system MtrAB in the model organism Streptomyces venezuelae and provide evidence that it coordinates production of the antibiotic chloramphenicol with sporulation. MtrAB are known to coordinate DNA replication and cell division in Mycobacterium tuberculosis where TB-MtrA is essential for viability. We were unable to delete mtrA in S. venezuelae unless another copy was present in trans but deletion of mtrB resulted in a global shift in the metabolome, including constitutive, high-level production of chloramphenicol. We found that chloramphenicol is detectable in the wild type strain, but only at very low levels and only after it has sporulated. ChIP-seq showed that MtrA binds upstream of DNA replication and cell division genes and genes required for chloramphenicol production. dnaA, dnaN, oriC and wblE (whiB1) appear to be targets for MtrA in both M. tuberculosis and S. venezuelae. Intriguingly, over-expression of TB-MtrA and gain of function TB- and Sv-MtrA proteins in S. venezuelae also switched on high level production of chloramphenicol. Given the conservation of MtrAB, these constructs might be useful tools for manipulating antibiotic production in other filamentous actinomycetes

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

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

Biosynthesis of the 15-membered ring depsipeptide neoantimycin

Antimycins are a family of natural products possessing outstanding biological activities and unique structures, which have intrigued chemists for over a half century. Of particular interest are the ring-expanded antimycins that show promising anti-cancer potential and whose biosynthesis remains uncharacterized. Specifically, neoantimycin and its analogs have been shown to be effective regulators of the oncogenic proteins GRP78/BiP and K-Ras. The neoantimycin structural skeleton is built on a 15-membered tetralactone ring containing one methyl, one hydroxy, one benzyl and three alkylmoieties, as well as an amide linkage to a conserved 3-formamidosalicylic acid moiety. Although the biosynthetic gene cluster for neoantimycins was recently identified, the enzymatic logic that governs the synthesis of neoantimycins has not yet been revealed. In this work, the neoantimycin gene cluster is identified and an updated sequence and annotation is provided delineating a non-ribosomal peptide synthetase/polyketide synthase (NRPS/PKS) hybrid scaffold. Using cosmid expression and CRISPR/Cas-based genome editing, several heterologous expression strains for neoantimycin production are constructed in two separate Streptomyces species. A combination of in vivo and in vitro analysis is further used to completely characterize the biosynthesis of neoantimycins including the megasynthases and trans-acting domains. This work establishes a set of highly tractable hosts for producing and engineering neoantimycins and their C11 oxidized analogs, paving the way for neoantimycin-based drug discovery and development

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

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

Minimum Information about a Biosynthetic Gene cluster

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog

The core secondary metabolome of <i>Streptomyces albus</i>.

<p>*denotes genomic coordinates were edited manually</p><p>** denotes a gene cluster which was not annotated by antiSMASH 2.0</p><p>***denotes a gene cluster which is spread over multiple contigs (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116457#pone.0116457.s002" target="_blank">S2 Table</a>).</p><p>The core secondary metabolome of <i>Streptomyces albus</i>.</p