Evidence of a role for CutRS and actinorhodin in the secretion stress response in Streptomyces coelicolor M145.

CutRS was the first two-component system to be identified in Streptomyces species and is highly conserved in this genus. It was reported >25 years ago that deletion of cutRS increases the production of the antibiotic actinorhodin in Streptomyces coelicolor. However, despite this early work, the function of CutRS has remained enigmatic until now. Here we show that deletion of cutRS upregulates the production of the actinorhodin biosynthetic enzymes up to 300-fold, explaining the increase in actinorhodin production. However, while ChIP-seq identified 85 CutR binding sites in S. coelicolor none of these are in the actinorhodin biosynthetic gene cluster, meaning the effect is indirect. The directly regulated CutR targets identified in this study are implicated in extracellular protein folding, including two of the four highly conserved HtrA-family foldases: HtrA3 and HtrB, and a putative VKOR enzyme, which is predicted to recycle DsbA following its catalysis of disulphide bond formation in secreted proteins. Thus, we tentatively propose a role for CutRS in sensing and responding to protein misfolding outside the cell. Since actinorhodin can oxidise cysteine residues and induce disulphide bond formation in proteins, its over production in the ∆cutRS mutant may be a response to protein misfolding on the extracellular face of the membrane.

Heterologous Expression of the Formicamycin Biosynthetic Gene Cluster Unveils Glycosylated Fasamycin Congeners.

Formicamycins and their biosynthetic intermediates the fasamycins are polyketide antibiotics produced by Streptomyces formicae KY5 from a pathway encoded by the for biosynthetic gene cluster. In this work the ability of Streptomyces coelicolor M1146 and the ability of Saccharopolyspora erythraea Δery to heterologously express the for biosynthetic gene cluster were assessed. This led to the identification of eight new glycosylated fasamycins modified at different phenolic groups with either a monosaccharide (glucose, galactose, or glucuronic acid) or a disaccharide comprised of a proximal hexose (either glucose or galactose), with a terminal pentose (arabinose) moiety. In contrast to the respective aglycones, minimal inhibitory screening assays showed these glycosylated congeners lacked antibacterial activity.

Competition-based screening helps to secure the evolutionary stability of a defensive microbiome.

BACKGROUND: The cuticular microbiomes of Acromyrmex leaf-cutting ants pose a conundrum in microbiome biology because they are freely colonisable, and yet the prevalence of the vertically transmitted bacteria Pseudonocardia, which contributes to the control of Escovopsis fungus garden disease, is never compromised by the secondary acquisition of other bacterial strains. Game theory suggests that competition-based screening can allow the selective recruitment of antibiotic-producing bacteria from the environment, by providing abundant resources to foment interference competition between bacterial species and by using Pseudonocardia to bias the outcome of competition in favour of antibiotic producers. RESULTS: Here, we use RNA-stable isotope probing (RNA-SIP) to confirm that Acromyrmex ants can maintain a range of microbial symbionts on their cuticle by supplying public resources. We then used RNA sequencing, bioassays, and competition experiments to show that vertically transmitted Pseudonocardia strains produce antibacterials that differentially reduce the growth rates of other microbes, ultimately biassing the bacterial competition to allow the selective establishment of secondary antibiotic-producing strains while excluding non-antibiotic-producing strains that would parasitise the symbiosis. CONCLUSIONS: Our findings are consistent with the hypothesis that competition-based screening is a plausible mechanism for maintaining the integrity of the co-adapted mutualism between the leaf-cutting ant farming symbiosis and its defensive microbiome. Our results have broader implications for explaining the stability of other complex symbioses involving horizontal acquisition.

Defining the regulon of genes controlled by σE , a key regulator of the cell envelope stress response in Streptomyces coelicolor.

The extracytoplasmic function (ECF) σ factor, σE , is a key regulator of the cell envelope stress response in Streptomyces coelicolor. Although its role in maintaining cell wall integrity has been known for over a decade, a comprehensive analysis of the genes under its control has not been undertaken. Here, using a combination of chromatin immunoprecipitation-sequencing (ChIP-seq), microarray transcriptional profiling and bioinformatic analysis, we attempt to define the σE regulon. Approximately half of the genes identified encode proteins implicated in cell envelope function. Seventeen novel targets were validated by S1 nuclease mapping or in vitro transcription, establishing a σE -binding consensus. Subsequently, we used bioinformatic analysis to look for conservation of the σE target promoters identified in S. coelicolor across 19 Streptomyces species. Key proteins under σE control across the genus include the actin homolog MreB, three penicillin-binding proteins, two L,D-transpeptidases, a LytR-CpsA-Psr-family protein predicted to be involved in cell wall teichoic acid deposition and a predicted MprF protein, which adds lysyl groups to phosphatidylglycerol to neutralize membrane surface charge. Taken together, these analyses provide biological insight into the σE -mediated cell envelope stress response in the genus Streptomyces.

Streptomyces Endophytes Promote Host Health and Enhance Growth across Plant Species.

Streptomyces bacteria are ubiquitous in soils and are well known for producing secondary metabolites, including antimicrobials. Increasingly, they are being isolated from plant roots, and several studies have shown they are specifically recruited to the rhizosphere and the endosphere of the model plant Arabidopsis thaliana Here, we test the hypothesis that Streptomyces bacteria have a beneficial effect on A. thaliana growth and could potentially be used as plant probiotics. To do this, we selectively isolated streptomycetes from surface-washed A. thaliana roots and generated high-quality genome sequences for five strains, which we named L2, M2, M3, N1, and N2. Reinfection of A. thaliana plants with L2, M2, and M3 significantly increased plant biomass individually and in combination, whereas N1 and N2 had a negative effect on plant growth, likely due to their production of polyene natural products which can bind to phytosterols and reduce plant growth. N2 exhibits broad-spectrum antimicrobial activity and makes filipin-like polyenes, including 14-hydroxyisochainin which inhibits the take-all fungus, Gaeumannomyces graminis var. tritici N2 antifungal activity as a whole was upregulated ∼2-fold in response to indole-3-acetic acid (IAA), suggesting a possible role during competition in the rhizosphere. Furthermore, coating wheat seeds with N2 spores protected wheat seedlings against take-all disease. We conclude that at least some soil-dwelling streptomycetes confer growth-promoting benefits on A. thaliana, while others might be exploited to protect crops against disease.IMPORTANCE We must reduce reliance on agrochemicals, and there is increasing interest in using bacterial strains to promote plant growth and protect against disease. Our study follows up reports that Arabidopsis thaliana specifically recruits Streptomyces bacteria to its roots. We test the hypotheses that they offer benefits to their A. thaliana hosts and that strains isolated from these plants might be used as probiotics. We isolated Streptomyces strains from A. thaliana roots and genome sequenced five phylogenetically distinct strains. Genome mining and bioassays indicated that all five have plant growth-promoting properties, including production of indole-3-acetic acid (IAA), siderophores, and aminocyclopropane-1-carboxylate (ACC) deaminase. Three strains significantly increased A. thaliana growth in vitro and in combination in soil. Another produces potent filipin-like antifungals and protected germinating wheat seeds against the fungal pathogen Gaeumannomyces graminis var. tritici (wheat take-all fungus). We conclude that introducing Streptomyces strains into the root microbiome provides significant benefits to plants.

In Situ Activation and Heterologous Production of a Cryptic Lantibiotic from an African Plant Ant-Derived Saccharopolyspora Species.

Most clinical antibiotics are derived from actinomycete natural products discovered at least 60 years ago. However, the repeated rediscovery of known compounds led the pharmaceutical industry to largely discard microbial natural products (NPs) as a source of new chemical diversity. Recent advances in genome sequencing have revealed that these organisms have the potential to make many more NPs than previously thought. Approaches to unlock NP biosynthesis by genetic manipulation of strains, by the application of chemical genetics, or by microbial cocultivation have resulted in the identification of new antibacterial compounds. Concomitantly, intensive exploration of coevolved ecological niches, such as insect-microbe defensive symbioses, has revealed these to be a rich source of chemical novelty. Here, we report the new lanthipeptide antibiotic kyamicin, which was generated through the activation of a cryptic biosynthetic gene cluster identified by genome mining Saccharopolyspora species found in the obligate domatium-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium Transcriptional activation of this silent gene cluster was achieved by ectopic expression of a pathway-specific activator under the control of a constitutive promoter. Subsequently, a heterologous production platform was developed which enabled the purification of kyamicin for structural characterization and bioactivity determination. This strategy was also successful for the production of lantibiotics from other genera, paving the way for a synthetic heterologous expression platform for the discovery of lanthipeptides that are not detected under laboratory conditions or that are new to nature.IMPORTANCE The discovery of novel antibiotics to tackle the growing threat of antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic potential of microorganisms. The development of new tools to unlock the biosynthesis of cryptic bacterial natural products will greatly increase the repertoire of natural product scaffolds. Here, we report a strategy for the ectopic expression of pathway-specific positive regulators that can be rapidly applied to activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This allowed the discovery of a new lanthipeptide antibiotic directly from the native host and via heterologous expression.

Diet, Gut Microbes and Host Mate Choice: Understanding the significance of microbiome effects on host mate choice requires a case by case evaluation.

All organisms live in close association with microbes. However, not all such associations are meaningful in an evolutionary context. Current debate concerns whether hosts and microbes are best described as communities of individuals or as holobionts (selective units of hosts plus their microbes). Recent reports that assortative mating of hosts by diet can be mediated by commensal gut microbes have attracted interest as a potential route to host reproductive isolation (RI). Here, the authors discuss logical problems with this line of argument. The authors briefly review how microbes can affect host mating preferences and evaluate recent findings from fruitflies. Endosymbionts can potentially influence host RI given stable and recurrent co-association of hosts and microbes over evolutionary time. However, observations of co-occurrence of microbes and hosts are ripe for misinterpretation and such associations will rarely represent a meaningful holobiont. A framework in which hosts and their microbes are independent evolutionary units provides the only satisfactory explanation for the observed range of effects and associations.

Gut microbiomes and reproductive isolation in Drosophila.

Experimental studies of the evolution of reproductive isolation (RI) in real time are a powerful way in which to reveal fundamental, early processes that initiate divergence. In a classic speciation experiment, populations of Drosophila pseudoobscura were subjected to divergent dietary selection and evolved significant positive assortative mating by diet. More recently, a direct role for the gut microbiome in determining this type of RI in Drosophila melanogaster has been proposed. Manipulation of the diet, and hence the gut microbiome, was reported to result in immediate assortative mating by diet, which could be eliminated by reducing gut microbes using antibiotics and recreated by adding back Lactobacillus plantarum We suggest that the evolutionary significance of this result is unclear. For example, in D. melanogaster, the microbiome is reported as flexible and largely environmentally determined. Therefore, microbiome-mediated RI would be transient and would break down under dietary variation. In the absence of evolutionary coassociation or recurrent exposure between host and microbiome, there are no advantages for the gut bacteria or host in effecting RI. To explore these puzzling effects and their mechanisms further, we repeated the tests for RI associated with diet-specific gut microbiomes in D. melanogaster Despite observing replicable differences in the gut microbiomes of flies maintained on different diets, we found no evidence for diet-associated RI, for any role of gut bacteria, or for L. plantarum specifically. The results suggest that there is no general role for gut bacteria in driving the evolution of RI in this species and resolve an evolutionary riddle.

Crystal Structure of the Transcription Regulator RsrR Reveals a [2Fe-2S] Cluster Coordinated by Cys, Glu, and His Residues.

The recently discovered Rrf2 family transcriptional regulator RsrR coordinates a [2Fe-2S] cluster. Remarkably, binding of the protein to RsrR-regulated promoter DNA sequences is switched on and off through the facile cycling of the [2Fe-2S] cluster between +2 and +1 states. Here, we report high resolution crystal structures of the RsrR dimer, revealing that the [2Fe-2S] cluster is asymmetrically coordinated across the RsrR monomer-monomer interface by two Cys residues from one subunit and His and Glu residues from the other. To our knowledge, this is the first example of a protein bound [Fe-S] cluster with three different amino acid side chains as ligands, and of Glu acting as ligand to a [2Fe-2S] cluster. Analyses of RsrR structures revealed a conformational change, centered on Trp9, which results in a significant shift in the DNA-binding helix-turn-helix region.

Sensing and responding to diverse extracellular signals: an updated analysis of the sensor kinases and response regulators of Streptomyces species.

Streptomyces venezuelae is a Gram-positive, filamentous actinomycete with a complex developmental life cycle. Genomic analysis revealed that S. venezuelae encodes a large number of two-component systems (TCSs): these consist of a membrane-bound sensor kinase (SK) and a cognate response regulator (RR). These proteins act together to detect and respond to diverse extracellular signals. Some of these systems have been shown to regulate antimicrobial biosynthesis in Streptomyces species, making them very attractive to researchers. The ability of S. venezuelae to sporulate in both liquid and solid cultures has made it an increasingly popular model organism in which to study these industrially and medically important bacteria. Bioinformatic analysis identified 58 TCS operons in S. venezuelae with an additional 27 orphan SK and 18 orphan RR genes. A broader approach identified 15 of the 58 encoded TCSs to be highly conserved in 93 Streptomyces species for which high-quality and complete genome sequences are available. This review attempts to unify the current work on TCS in the streptomycetes, with an emphasis on S. venezuelae.

Differentiated, Promoter-specific Response of [4Fe-4S] NsrR DNA Binding to Reaction with Nitric Oxide.

NsrR is an iron-sulfur cluster protein that regulates the nitric oxide (NO) stress response of many bacteria. NsrR from Streptomyces coelicolor regulates its own expression and that of only two other genes, hmpA1 and hmpA2, which encode HmpA enzymes predicted to detoxify NO. NsrR binds promoter DNA with high affinity only when coordinating a [4Fe-4S] cluster. Here we show that reaction of [4Fe-4S] NsrR with NO affects DNA binding differently depending on the gene promoter. Binding to the hmpA2 promoter was abolished at ∼2 NO per cluster, although for the hmpA1 and nsrR promoters, ∼4 and ∼8 NO molecules, respectively, were required to abolish DNA binding. Spectroscopic and kinetic studies of the NO reaction revealed a rapid, multi-phase, non-concerted process involving up to 8-10 NO molecules per cluster, leading to the formation of several iron-nitrosyl species. A distinct intermediate was observed at ∼2 NO per cluster, along with two further intermediates at ∼4 and ∼6 NO. The NsrR nitrosylation reaction was not significantly affected by DNA binding. These results show that NsrR regulates different promoters in response to different concentrations of NO. Spectroscopic evidence indicates that this is achieved by different NO-FeS complexes.

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.

NsrR from Streptomyces coelicolor is a nitric oxide-sensing [4Fe-4S] cluster protein with a specialized regulatory function.

The Rrf2 family transcription factor NsrR controls expression of genes in a wide range of bacteria in response to nitric oxide (NO). The precise form of the NO-sensing module of NsrR is the subject of controversy because NsrR proteins containing either [2Fe-2S] or [4Fe-4S] clusters have been observed previously. Optical, Mössbauer, resonance Raman spectroscopies and native mass spectrometry demonstrate that Streptomyces coelicolor NsrR (ScNsrR), previously reported to contain a [2Fe-2S] cluster, can be isolated containing a [4Fe-4S] cluster. ChIP-seq experiments indicated that the ScNsrR regulon is small, consisting of only hmpA1, hmpA2, and nsrR itself. The hmpA genes encode NO-detoxifying flavohemoglobins, indicating that ScNsrR has a specialized regulatory function focused on NO detoxification and is not a global regulator like some NsrR orthologues. EMSAs and DNase I footprinting showed that the [4Fe-4S] form of ScNsrR binds specifically and tightly to an 11-bp inverted repeat sequence in the promoter regions of the identified target genes and that DNA binding is abolished following reaction with NO. Resonance Raman data were consistent with cluster coordination by three Cys residues and one oxygen-containing residue, and analysis of ScNsrR variants suggested that highly conserved Glu-85 may be the fourth ligand. Finally, we demonstrate that some low molecular weight thiols, but importantly not physiologically relevant thiols, such as cysteine and an analogue of mycothiol, bind weakly to the [4Fe-4S] cluster, and exposure of this bound form to O2 results in cluster conversion to the [2Fe-2S] form, which does not bind to DNA. These data help to account for the observation of [2Fe-2S] forms of NsrR.

Biochemical properties of Paracoccus denitrificans FnrP: reactions with molecular oxygen and nitric oxide.

In Paracoccus denitrificans, three CRP/FNR family regulatory proteins, NarR, NnrR and FnrP, control the switch between aerobic and anaerobic (denitrification) respiration. FnrP is a [4Fe-4S] cluster-containing homologue of the archetypal O2 sensor FNR from E. coli and accordingly regulates genes encoding aerobic and anaerobic respiratory enzymes in response to O2, and also NO, availability. Here we show that FnrP undergoes O2-driven [4Fe-4S] to [2Fe-2S] cluster conversion that involves up to 2 O2 per cluster, with significant oxidation of released cluster sulfide to sulfane observed at higher O2 concentrations. The rate of the cluster reaction was found to be ~sixfold lower than that of E. coli FNR, suggesting that FnrP can remain transcriptionally active under microaerobic conditions. This is consistent with a role for FnrP in activating expression of the high O2 affinity cytochrome c oxidase under microaerobic conditions. Cluster conversion resulted in dissociation of the transcriptionally active FnrP dimer into monomers. Therefore, along with E. coli FNR, FnrP belongs to the subset of FNR proteins in which cluster type is correlated with association state. Interestingly, two key charged residues, Arg140 and Asp154, that have been shown to play key roles in the monomer-dimer equilibrium in E. coli FNR are not conserved in FnrP, indicating that different protomer interactions are important for this equilibrium. Finally, the FnrP [4Fe-4S] cluster is shown to undergo reaction with multiple NO molecules, resulting in iron nitrosyl species and dissociation into monomers.

Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes.

The reaction of protein-bound iron-sulfur (Fe-S) clusters with nitric oxide (NO) plays key roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe-S clusters has been hampered by a lack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe-4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe2 (NO)4 (Cys)2 ]) and Roussin's Black Salt (RBS, [Fe4 (NO)7 S3 ]. In the latter case, the absence of 32 S/34 S shifts in the Fe-S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

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.

Stabilisation of the RirA [4Fe-4S] cluster results in loss of iron-sensing function.

RirA is a global iron regulator in diverse Alphaproteobacteria that belongs to the Rrf2 superfamily of transcriptional regulators, which can contain an iron-sulfur (Fe-S) cluster. Under iron-replete conditions, RirA contains a [4Fe-4S] cluster, enabling high-affinity binding to RirA-regulated operator sequences, thereby causing the repression of cellular iron uptake. Under iron deficiency, one of the cluster irons dissociates, generating an unstable [3Fe-4S] form that subsequently degrades to a [2Fe-2S] form and then to apo RirA, resulting in loss of high-affinity DNA-binding. The cluster is coordinated by three conserved cysteine residues and an unknown fourth ligand. Considering the lability of one of the irons and the resulting cluster fragility, we hypothesized that the fourth ligand may not be an amino acid residue. To investigate this, we considered that the introduction of an amino acid residue that could coordinate the cluster might stabilize it. A structural model of RirA, based on the Rrf2 family nitrosative stress response regulator NsrR, highlighted residue 8, an Asn in the RirA sequence, as being appropriately positioned to coordinate the cluster. Substitution of Asn8 with Asp, the equivalent, cluster-coordinating residue of NsrR, or with Cys, resulted in proteins that contained a [4Fe-4S] cluster, with N8D RirA exhibiting spectroscopic properties very similar to NsrR. The variant proteins retained the ability to bind RirA-regulated DNA, and could still act as repressors of RirA-regulated genes in vivo. However, they were significantly more stable than wild-type RirA when exposed to O2 and/or low iron. Importantly, they exhibited reduced capacity to respond to cellular iron levels, even abolished in the case of the N8D version, and thus were no longer iron sensing. This work demonstrates the importance of cluster fragility for the iron-sensing function of RirA, and more broadly, how a single residue substitution can alter cluster coordination and functional properties in the Rrf2 superfamily of regulators.