Antimicrobial resistance is a global health crisis and few novel antimicrobials have been discovered in recent decades. Natural products, particularly from Streptomyces, are the source of most antimicrobials, yet discovery campaigns focusing on Streptomyces from the soil largely rediscover known compounds. Investigation of understudied and symbiotic sources has seen some success, yet no studies have systematically explored microbiomes for antimicrobials. Here we assess the distinct evolutionary lineages of Streptomyces from insect microbiomes as a source of new antimicrobials through large-scale isolations, bioactivity assays, genomics, metabolomics, and in vivo infection models. Insect-associated Streptomyces inhibit antimicrobial-resistant pathogens more than soil Streptomyces. Genomics and metabolomics reveal their diverse biosynthetic capabilities. Further, we describe cyphomycin, a new molecule active against multidrug resistant fungal pathogens. The evolutionary trajectories of Streptomyces from the insect microbiome influence their biosynthetic potential and ability to inhibit resistant pathogens, supporting the promise of this source in augmenting future antimicrobial discovery.

Streptomyces strains are well known as promising source of bioactive secondary metabolites, important in ecology, biotechnology and medicine. In this study, we present the draft genome of the new type strain Streptomyces tunisialbus DSM 105760T (= JCM 32165T), a rhizospheric bacterium with antimicrobial activity. The genome is 6,880,753 bp in size (average GC content, 71.85%) and encodes 5802 protein-coding genes. Preliminary analysis with antiSMASH 5.1.2. reveals 34 predicted gene clusters for the synthesis of potential secondary metabolites, which was compared with those of Streptomyces varsoviensis NRRL ISP-5346.

Whilst being closely related to the model actinomycete Streptomyces coelicolor A3(2), S. lividans 66 differs from it in several significant and phenotypically observable ways, including antibiotic production. Previous comparative gene hybridization studies investigating such differences have used low-density (one probe per gene) PCR-based spotted arrays. Here we use new experimentally optimised 104,000 × 60-mer probe arrays to characterize in detail the genomic differences between wild-type S. lividans 66, a derivative industrial strain, TK24, and S. coelicolor M145.

A polyphasic study was undertaken to determine the taxonomic status of a Streptomyces strain which had been isolated from a high altitude Atacama Desert soil and shown to have bioactive properties. The strain, isolate H9T, was found to have chemotaxonomic, cultural and morphological properties that place it in the genus Streptomyces. 16S rRNA gene sequence analyses showed that the isolate forms a distinct branch at the periphery of a well-delineated subclade in the Streptomyces 16S rRNA gene tree together with the type strains of Streptomyces crystallinus, Streptomyces melanogenes and Streptomyces noboritoensis. Multi-locus sequence analysis (MLSA) based on five house-keeping gene alleles showed that isolate H9T is closely related to the latter two type strains and to Streptomyces polyantibioticus NRRL B-24448T. The isolate was distinguished readily from the type strains of S. melanogenes, S. noboritoensis and S. polyantibioticus using a combination of phenotypic properties. Consequently, the isolate is considered to represent a new species of Streptomyces for which the name Streptomyces aridus sp. nov. is proposed; the type strain is H9T (=NCIMB 14965T=NRRL B65268T). In addition, the MLSA and phenotypic data show that the S. melanogenes and S. noboritoensis type strains belong to a single species, it is proposed that S. melanogenes be recognised as a heterotypic synonym of S. noboritoensis for which an emended description is given.

The central player in bacterial cell division, FtsZ, is essential in almost all organisms in which it has been tested, with the most notable exception being Streptomyces. Streptomycetes differ from many bacteria in growing from the cell tip and undergoing branching, similar to filamentous fungi. Here we show that limited cell damage, either mechanical or enzymatic, leads to near complete destruction of mycelial microcolonies of a Streptomyces venezuelae ftsZ mutant. This result is consistent with a lack of ftsZ-dependent cross-walls and may be inconsistent with a recently proposed role for membrane structures in the proliferation of ftsZ mutants in other Streptomyces species. Rare surviving fragments of mycelium, usually around branches, appear to be the preferred sites of resealing. Restoration of growth in hyphal fragments of both wild-type and ftsZ mutant hyphae can occur at multiple sites, via branch-like outgrowths containing DivIVA protein at their tips. Thus, our results highlight branching as a means of FtsZ-independent cell proliferation.

Streptomycetes are highly metabolically gifted bacteria with the abilities to produce bioproducts that have profound economic and societal importance. These bioproducts are produced by metabolic pathways including those for the biosynthesis of secondary metabolites and catabolism of plant biomass constituents. Advancements in genome sequencing technologies have revealed a wealth of untapped metabolic potential from Streptomyces genomes. Here, we report the largest Streptomyces pangenome generated by using 205 complete genomes. Metabolic potentials of the pangenome and individual genomes were analyzed, revealing degrees of conservation of individual metabolic pathways and strains potentially suitable for metabolic engineering. Of them, Streptomyces bingchenggensis was identified as a potent degrader of plant biomass. Polyketide, non-ribosomal peptide, and gamma-butyrolactone biosynthetic enzymes are primarily strain specific while ectoine and some terpene biosynthetic pathways are highly conserved. A large number of transcription factors associated with secondary metabolism are strain-specific while those controlling basic biological processes are highly conserved. Although the majority of genes involved in morphological development are highly conserved, there are strain-specific varieties which may contribute to fine tuning the timing of cellular differentiation. Overall, these results provide insights into the metabolic potential, regulation and physiology of streptomycetes, which will facilitate further exploitation of these important bacteria.

Streptomyces remains one of the prolific sources of structural diversity, and a reservoir to mine for novel natural products. Continued screening for new Streptomyces strains in our laboratory led to the isolation of Streptomyces sp. RK44 from the underexplored areas of Kintampo waterfalls, Ghana, Africa. Preliminary screening of the metabolites from this strain resulted in the characterization of a new 2-alkyl-4-hydroxymethylfuran carboxamide (AHFA) 1 together with five known compounds, cyclo-(L-Pro-Gly) 2, cyclo-(L-Pro-L-Phe) 3, cyclo-(L-Pro-L-Val) 4, cyclo-(L-Leu-Hyp) 5, and deferoxamine E 6. AHFA 1, a methylenomycin (MMF) homolog, exhibited anti-proliferative activity (EC50 = 89.6 µM) against melanoma A2058 cell lines. This activity, albeit weak is the first report amongst MMFs. Furthermore, the putative biosynthetic gene cluster (ahfa) was identified for the biosynthesis of AHFA 1. DFO-E 6 displayed potent anti-plasmodial activity (IC50 = 1.08µM) against P. falciparum 3D7. High-resolution electrospray ionization mass spectrometry (HR ESIMS) and molecular network assisted the targeted-isolation process, and tentatively identified six AHFA analogues, 7-12 and six siderophores 13-18.

Chemical investigation of the cultures of marine Streptomyces sp. 182SMLY led to the discovery of two new polycyclic anthraquinones, which were elucidated as N-acetyl-N-demethylmayamycin (1) and streptoanthraquinone A (2) based on the extensive spectroscopic analysis including 2D NMR, HRESIMS, and an electronic circular dichroism (ECD) calculation. Both anthraquinones remarkably suppressed the proliferation of four different glioma cell lines with IC50 values in a range from 0.5 to 7.3 μM and induced apoptosis in the glioma cells. The ratios of IC50 for normal human astrocytes to IC50 for glioma cells were 6.4-53 for 1 and >14-31 for 2. N-acetyl-N-demethylmayamycin (1) also inhibited the growth of methicillin-resistant Staphylococcus aureus with MIC 20.0 μM.

Streptomyces clavuligerus is an industrially important producer of clavulanic acid (CA), a β-lactamase inhibitor which is used together with amoxicillin in one of the most widely prescribed antibacterial medicines, the co-amoxiclav. In a mid-eighties ATCC vial of S. clavuligerus ATCC 27064 culture, we have found a new genotype, which was apparently lost from the subsequent ATCC collection stocks, and has remained obscure to the scientific community. Most importantly, this genotype harbors teleocidin (lyngbyatoxin) biosynthetic genes, which are located on an enigmatic 138 kb chromosomal region and support accumulation of significant amounts of these highly toxic, tumor-promoting secondary metabolites in cultures of S. clavuligerus. While this genomic region is completely absent from all published sequences for S. clavuligerus ATCC strain, at least one of the industrial strains for commercial production of CA, originating from ATCC 27064, retained the genetic potential for production of teleocidins. The origin of teleocidin biosynthetic cluster can now be traced back to early S. clavuligerus stocks at the ATCC. Our work provides a genome sequence and a deposited monoisolate of this genotype. Given the scale of industrial use of S. clavuligerus world-wide and toxicity of teleocidins, we also discuss the environmental and safety implications and provide a method of abolishing teleocidin production without affecting productivity of CA. KEY POINTS: • Early stocks of S. clavuligerus ATCC 27064 produce toxic teleocidins • Teleocidin biosynthetic genes were found within a distinct S. clavuligerus genotype • The genotype has been passed on to some industrial clavulanic acid producer strains.

3'-O-β-Glucosyl-4',5'-didehydro-5'-deoxyadenosine 13 is identified as a natural product of Streptomyces calvus and Streptomyces virens. It is also generated in vitro by direct β-glucosylation of 4',5'-didehydro-5'-deoxyadenosine 12 with the enzyme NucGT. The intact incorporation of oxygen-18 and deuterium isotopes from (±)[1-18O,1-2H2]-glycerol 14 into C-5' of nucleocidin 1 and its related metabolites precludes 3'-O-β-glucosyl-4',5'-didehydro-5'-deoxyadenosine 13 as a biosynthetic precursor to nucleocidin 1.

Acyl-homoserine lactones (AHLs), mediating pivotal physiological activities through quorum sensing (QS), have conventionally been considered limited to Gram-negative bacteria. However, few reports on the existence of AHLs in Gram-positive bacteria have questioned this conception. Streptomyces, as Gram-positive bacteria already utilizing a lactone-based QS molecule (i.e., gamma-butyrolactones), are yet to be explored for producing AHLs, considering their metabolic capacity and physiological distinction. In this regard, our study examined the potential production of AHLs within Streptomyces by deploying HPLC-MS/MS methods, which resulted in the discovery of multiple AHL productions by S. griseus, S. lavendulae FRI-5, S. clavuligerus, S. nodosus, S. lividans, and S. coelicolor A3(2). Each of these Streptomyces species possesses a combination of AHLs of different size ranges, possibly due to their distinct properties and regulatory roles. In light of additional lactone molecules, we further confirm that AHL- and GBL-synthases (i.e., LuxI and AfsA enzyme families, respectively) and their receptors (i.e., LuxR and ArpA) are evolutionarily distinct. To this end, we searched for the components of the AHL signaling circuit, i.e., AHL synthases and receptors, in the Streptomyces genus, and we have identified multiple potential LuxI and LuxR homologs in all 2,336 Streptomyces species included in this study. The 6 Streptomyces of interest in this study also had at least 4 LuxI homologs and 97 LuxR homologs. In conclusion, AHLs and associated gene regulatory systems could be more widespread within the prokaryotic realm than previously believed, potentially contributing to the control of secondary metabolites (e.g., antibiotics) and their complex life cycle, which leads to substantial industrial and clinical applications.

The Streptomyces albus J1074 strain is one of the most widely used chassis for the heterologous production of bioactive natural products. The fast growth and an efficient genetic system make this strain an attractive model for expressing cryptic biosynthetic pathways to aid drug discovery.

The undesired attachment of micro and macroorganisms on water-immersed surfaces, known as marine biofouling, results in severe prevention and maintenance costs (billions €/year) for aquaculture, shipping and other industries that rely on coastal and off-shore infrastructures. To date, there are no sustainable, cost-effective and environmentally safe solutions to address this challenging phenomenon. Therefore, we investigated the antifouling activity of napyradiomycin derivatives that were isolated from actinomycetes from ocean sediments collected off the Madeira Archipelago. Our results revealed that napyradiomycins inhibited ≥80% of the marine biofilm-forming bacteria assayed, as well as the settlement of Mytilus galloprovincialis larvae (EC50 < 5 µg/ml and LC50/EC50 >15), without viability impairment. In silico prediction of toxicity end points are of the same order of magnitude of standard approved drugs and biocides. Altogether, napyradiomycins disclosed bioactivity against marine micro and macrofouling organisms, and non-toxic effects towards the studied species, displaying potential to be used in the development of antifouling products.

Nine sesquiterpenes, including eight pentalenenes (1-8) and one bolinane derivative (9), were isolated from the culture broth of a marine-derived actinobacterium Streptomyces qinglanensis 213DD-006. Among them, 1, 4, 7, and 9 were new compounds. Their planar structures were determined by spectroscopic methods (HRMS, 1D, and 2D NMR), and the absolute configuration was established by biosynthesis consideration and electronic-circular-dichroism (ECD) calculations. All the isolated compounds were screened for their cytotoxicity against six solid and seven blood cancer cell lines. Compounds 4-6 and 8 showed a moderate activity against all of the tested solid cell lines, with GI50 values ranging from 1.97 to 3.46 µM.

Pristinamycin, produced by Streptomyces pristinaespiralis Pr11, is a streptogramin antibiotic consisting of two chemically unrelated compounds, pristinamycin I and pristinamycin II. The semi-synthetic derivatives of these compounds are used in human medicine as therapeutic agents against methicillin-resistant Staphylococcus aureus strains. Only the partial sequence of the pristinamycin biosynthetic gene cluster has been previously reported. To complete the sequence, overlapping cosmids were isolated from a S. pristinaespiralis Pr11 gene library and sequenced. The boundaries of the cluster were deduced, limiting the cluster size to approximately 210 kb. In the central region of the cluster, previously unknown pristinamycin biosynthetic genes were identified. Combining the current and previously identified sequence information, we propose that all essential pristinamycin biosynthetic genes are included in the 210 kb region. A pristinamycin biosynthetic pathway was established. Furthermore, the pristinamycin gene cluster was found to be interspersed by a cryptic secondary metabolite cluster, which probably codes for a glycosylated aromatic polyketide. Gene inactivation experiments revealed that this cluster has no influence on pristinamycin production. Overall, this work provides new insights into pristinamycin biosynthesis and the unique genetic organization of the pristinamycin gene region, which is the largest antibiotic 'supercluster' known so far.

Zincophorin is a polyketide antibiotic that possesses potent activity against Gram-positive bacteria, including human pathogens. While a number of total syntheses of this highly functionalized natural product were reported since its initial discovery, the genetic basis for the biosynthesis of zincophorin has remained unclear. In this study, the co-linearity inherent to polyketide pathways was used to identify the zincophorin biosynthesis gene cluster in the genome of the natural producer Streptomyces griseus HKI 0741. Interestingly, the same locus is fully conserved in the streptomycin-producing actinomycete S. griseus IFO 13350, suggesting that the latter bacterium is also capable of zincophorin biosynthesis. Biological profiling of zincophorin revealed a dose-dependent inhibition of the Gram-positive bacterium Streptococcus pneumoniae. The antibacterial effect, however, is accompanied by cytotoxicity. Antibiotic and cytotoxic activities were completely abolished upon esterification of the carboxylic acid group in zincophorin.

Streptomyces coelicolor is a model organism for the study of Streptomyces, a genus of Gram-positive bacteria that undergoes a complex life cycle and produces a broad repertoire of bioactive metabolites and extracellular enzymes. This study investigated the production and characterization of membrane vesicles (MVs) in liquid cultures of S. coelicolor M145 from a structural and biochemical point of view; this was achieved by combining microscopic, physical and -omics analyses. Two main populations of MVs, with different sizes and cargos, were isolated and purified. S. coelicolor MV cargo was determined to be complex, containing different kinds of proteins and metabolites. In particular, a total of 166 proteins involved in cell metabolism/differentiation, molecular processing/transport, and stress response were identified in MVs, the latter functional class also being important for bacterial morpho-physiological differentiation. A subset of these proteins was protected from degradation following treatment of MVs with proteinase K, indicating their localization inside the vesicles. Moreover, S. coelicolor MVs contained an array of metabolites, such as antibiotics, vitamins, amino acids, and components of carbon metabolism. In conclusion, this analysis provides detailed information on S. coelicolor MVs under basal conditions and on their corresponding content, which may be useful in the near future to elucidate vesicle biogenesis and functions. IMPORTANCE Streptomycetes are widely distributed in nature and characterized by a complex life cycle that involves morphological differentiation. They are very relevant in industry because they produce about half of all clinically used antibiotics, as well as other important pharmaceutical products of natural origin. Streptomyces coelicolor is a model organism for the study of bacterial differentiation and bioactive molecule production. S. coelicolor produces extracellular vesicles that carry many molecules, such as proteins and metabolites, including antibiotics. The elucidation of S. coelicolor extracellular vesicle cargo will help us to understand different aspects of streptomycete physiology, such as cell communication during differentiation and response to environmental stimuli. Moreover, the capability of these vesicles for carrying different kinds of biomolecules opens up new biotechnological possibilities related to drug delivery. Indeed, decoding the molecular mechanisms involved in cargo selection may lead to the customization of extracellular vesicle content.

The genus Streptomyces demonstrates enormous promise in promoting plant growth and protecting plants against various pathogens. Single and consortium treatments of two selected Streptomyces strains (Streptomyces shenzhenensis TKSC3 and Streptomyces sp. SS8) were evaluated for their growth-promoting potential on rice, and biocontrol efficiency through induced systemic resistance (ISR) mediation against Xanthomonas oryzae pv. oryzicola (Xoc), the causal agent of rice bacterial leaf streak (BLS) disease. Seed bacterization by Streptomyces strains improved seed germination and vigor, relative to the untreated seed. Under greenhouse conditions, seed bacterization with consortium treatment TKSC3 + SS8 increased seed germination, root length, and dry weight by 20%, 23%, and 33%, respectively. Single and consortium Streptomyces treatments also successfully suppressed Xoc infection. The result was consistent with defense-related enzyme quantification wherein single and consortium Streptomyces treatments increased peroxidase (POX), polyphenol oxidase, phenylalanine ammonia-lyase, and β,1-3 glucanase (GLU) accumulation compared to untreated plant. Within all Streptomyces treatments, consortium treatment TKSC3 + SS8 showed the highest disease suppression efficiency (81.02%) and the lowest area under the disease progress curve value (95.79), making it the best to control BLS disease. Consortium treatment TKSC3 + SS8 induced the highest POX and GLU enzyme activities at 114.32 μmol/min/mg protein and 260.32 abs/min/mg protein, respectively, with both enzymes responsible for plant cell wall reinforcement and resistant interaction. Our results revealed that in addition to promoting plant growth, these Streptomyces strains also mediated ISR in rice plants, thereby, ensuring protection from BLS disease.

ArgR is the regulator of arginine biosynthesis genes in Streptomyces species. Transcriptomic comparison by microarrays has been made between Streptomyces coelicolor M145 and its mutant S. coelicolor ΔargR under control, unsupplemented conditions, and in the presence of arginine. Expression of 459 genes was different in transcriptomic assays, but only 27 genes were affected by arginine supplementation. Arginine and pyrimidine biosynthesis genes were derepressed by the lack of ArgR, while no strong effect on expression resulted on arginine supplementation. Several nitrogen metabolism genes expression as glnK, glnA and glnII, were downregulated in S. coelicolor ΔargR. In addition, downregulation of genes for the yellow type I polyketide CPK antibiotic and for the antibiotic regulatory genes afsS and scbR was observed. The transcriptomic data were validated by either reverse transcription-PCR, expression of the gene-promoter coupled to the luciferase gene, proteomic or by electrophoresis mobility shift assay (EMSA) using pure Strep-tagged ArgR. Two ARG-boxes in the arginine operon genes suggest that these genes are more tightly controlled. Other genes, including genes encoding regulatory proteins, possess a DNA sequence formed by a single ARG-box which responds to ArgR, as validated by EMSA.

Fluorescent proteins are a major cell biology tool to analyze protein sub-cellular topology. Here we have applied this technology to study protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, a widely used host for heterologous protein secretion biotechnology. Green and monomeric red fluorescent proteins were fused behind Sec (SPSec) or Tat (SPTat) signal peptides to direct them through the respective export pathway. Significant secretion of fluorescent eGFP and mRFP was observed exclusively through the Tat and Sec pathways, respectively. Plasmid over-expression was compared to a chromosomally integrated spSec-mRFP gene to allow monitoring secretion under high and low level synthesis in various media. Fluorimetric detection of SPSec-mRFP recorded folded states, while immuno-staining detected even non-folded topological intermediates. Secretion of SPSec-mRFP is unexpectedly complex, is regulated independently of cell growth phase and is influenced by the growth regime. At low level synthesis, highly efficient secretion occurs until it is turned off and secretory preforms accumulate. At high level synthesis, the secretory pathway overflows and proteins are driven to folding and subsequent degradation. High-level synthesis of heterologous secretory proteins, whether secretion competent or not, has a drastic effect on the endogenous secretome, depending on their secretion efficiency. These findings lay the foundations of dissecting how protein targeting and secretion are regulated by the interplay between the metabolome, secretion factors and stress responses in the S. lividans model.