A phage-encoded anti-activator inhibits quorum sensing in Pseudomonas aeruginosa.

The arms race between bacteria and phages has led to the evolution of diverse anti-phage defenses, several of which are controlled by quorum-sensing pathways. In this work, we characterize a quorum-sensing anti-activator protein, Aqs1, found in Pseudomonas phage DMS3. We show that Aqs1 inhibits LasR, the master regulator of quorum sensing, and present the crystal structure of the Aqs1-LasR complex. The 69-residue Aqs1 protein also inhibits PilB, the type IV pilus assembly ATPase protein, which blocks superinfection by phages that require the pilus for infection. This study highlights the remarkable ability of small phage proteins to bind multiple host proteins and disrupt key biological pathways. As quorum sensing influences various anti-phage defenses, Aqs1 provides a mechanism by which infecting phages might simultaneously dampen multiple defenses. Because quorum-sensing systems are broadly distributed across bacteria, this mechanism of phage counter-defense may play an important role in phage-host evolutionary dynamics.

Streptomyces extracellular vesicles are a broad and permissive antimicrobial packaging and delivery system.

Streptomyces are the primary source of bioactive specialized metabolites used in research and medicine, including many antimicrobials. These are presumed to be secreted and function as freely soluble compounds. However, increasing evidence suggests that extracellular vesicles are an alternative secretion system. We assessed environmental and lab-adapted Streptomyces (sporulating filamentous actinomycetes) and found frequent production of antimicrobial vesicles. The molecular cargo included actinomycins, anthracyclines, candicidin, and actinorhodin, reflecting both diverse chemical properties and diverse antibacterial and antifungal activity. The levels of packaged antimicrobials correlated with the level of inhibitory activity of the vesicles, and a strain knocked out for the production of anthracyclines produced vesicles that lacked antimicrobial activity. We demonstrated that antimicrobial containing vesicles achieve direct delivery of the cargo to other microbes. Notably, this delivery via membrane fusion occurred to a broad range of microbes, including pathogenic bacteria and yeast. Vesicle encapsulation offers a broad and permissive packaging and delivery system for antimicrobial specialized metabolites, with important implications for ecology and translation.IMPORTANCEExtracellular vesicle encapsulation changes our picture of how antimicrobial metabolites function in the environment and provides an alternative translational approach for the delivery of antimicrobials. We find many Streptomyces strains are capable of releasing antimicrobial vesicles, and at least four distinct classes of compounds can be packaged, suggesting this is widespread in nature. This is a striking departure from the primary paradigm of the secretion and action of specialized metabolites as soluble compounds. Importantly, the vesicles deliver antimicrobial metabolites directly to other microbes via membrane fusion, including pathogenic bacteria and yeast. This suggests future applications in which lipid-encapsulated natural product antibiotics and antifungals could be used to solve some of the most pressing problems in drug resistance.

A chemical defence against phage infection.

The arms race between bacteria and the phages that infect them drives the continual evolution of diverse anti-phage defences. Previously described anti-phage systems have highly varied defence mechanisms1-11; however, all mechanisms rely on protein components to mediate defence. Here we report a chemical anti-phage defence system that is widespread in Streptomyces. We show that three naturally produced molecules that insert into DNA are able to block phage replication, whereas molecules that target DNA by other mechanisms do not. Because double-stranded DNA phages are the most numerous group in the biosphere and the production of secondary metabolites by bacteria is ubiquitous12, this mechanism of anti-phage defence probably has a major evolutionary role in shaping bacterial communities.

A defect in cell wall recycling confers antibiotic resistance and sensitivity in Staphylococcus aureus.

WalKR is a two-component system that is essential for viability in Gram-positive bacteria that regulates the all-important autolysins in cell wall homeostasis. Further investigation of this essential system is important for identifying ways to address antibiotic resistance. Here, we show that a T101M mutation in walR confers a defect in autolysis, a thickened cell wall, and decreased susceptibility to antibiotics that target lipid II cycle, a phenotype that is reminiscent of the clinical resistance form known as vancomycin intermediate-resistant Staphylococcus aureus. Importantly, this is accompanied by dramatic sensitization to tunicamycin. We demonstrate that this phenotype is due to partial collapse of a pathway consisting of autolysins, AtlA and Sle1, a transmembrane sugar permease, MurP, and GlcNAc recycling enzymes, MupG and MurQ. We suggest that this causes a shortage of substrate for the peptidoglycan biosynthesis enzyme MraY, causing it to be hypersensitive to competitive inhibition by tunicamycin. In conclusion, our results constitute a new molecular model for antibiotic sensitivity in S. aureus and a promising new route for antibiotic discovery.

The ARC2 response in Streptomcyes coelicolor requires the global regulatory genes afsR and afsS.

ARC2 is a synthetic compound, related in structure and mechanism to the antibiotic triclosan, that activates the production of many specialized metabolites in the Streptomyces genus of bacteria. In this work, we demonstrate that the addition of ARC2 to Streptomyces coelicolor cultures results in considerable alterations in overall gene expression including most notably the specialized metabolic genes. Using actinorhodin production as a model system, we show that the effect of ARC2 depends on the pleiotropic regulators afsR and afsS but not afsK. We find that the constitutive expression of afsS can bypass the need for afsR but not the reverse, while the constitutive expression of afsK had no effect on actinorhodin production. These data are consistent with a model in which ARC2 activates a cell stress response that depends on AfsR activating the expression of the afsS gene such that AfsS then triggers the production of actinorhodin.

Dual-PKS Cluster for Biosynthesis of a Light-Induced Secondary Metabolite Found from Genome Sequencing of Hyphodiscus hymeniophilus Fungus.

Filamentous fungi are known producers of important secondary metabolites. In spite of this, the majority of these organisms have not been studied at the genome level, leaving many of the bioactive molecules they produce undiscovered. In this study, we explore the secondary metabolite potential of an understudied fungus, Hyphodiscus hymeniophilus. By sequencing and assembling the first genome from this genus, we show that this fungus has genes for at least 20 natural products and that many of these products are likely novel. One of these metabolites is identified: a new, red-pigmented member of the azaphilone class, hyphodiscorubrin. We show that this metabolite is only produced when the fungus is grown in the light. Furthermore, the biosynthetic gene cluster of hyphodiscorubrin is identified though homology to other known azaphilone producing clusters.

Natural Products Repertoire of the Red Sea.

Marine natural products have achieved great success as an important source of new lead compounds for drug discovery. The Red Sea provides enormous diversity on the biological scale in all domains of life including micro- and macro-organisms. In this review, which covers the literature to the end of 2019, we summarize the diversity of bioactive secondary metabolites derived from Red Sea micro- and macro-organisms, and discuss their biological potential whenever applicable. Moreover, the diversity of the Red Sea organisms is highlighted as well as their genomic potential. This review is a comprehensive study that compares the natural products recovered from the Red Sea in terms of ecological role and pharmacological activities.

Metabolomics analysis and biological investigation of three Malvaceae plants.

INTRODUCTION: Metabolomics is a fast growing technology that has effectively contributed to many plant-related sciences and drug discovery. OBJECTIVE: To use the non-targeted metabolomics approach to investigate the chemical profiles of three Malvaceae plants, namely Hibiscus mutabilis L. (Changing rose), H. schizopetalus (Dyer) Hook.f. (Coral Hibiscus), and Malvaviscus arboreus Cav. (Sleeping Hibiscus), along with evaluating their antioxidant and anti-infective potential. METHODOLOGY: Metabolic profiling was carried out using liquid chromatography coupled with high-resolution electrospray ionisation mass spectrometry (LC-HR-ESI-MS) for dereplication purposes. The chemical composition of the studied plants was further compared by principal component analysis (PCA). The antioxidant and anti-infective properties of their different extracts were correlated to their phytochemical profiles by orthogonal partial least square discriminant analysis (OPLS-DA). RESULTS: A variety of structurally different metabolites, mostly phenolics, were characterized. Comparing the distribution pattern of these tentatively identified metabolites among the studied plant species/fractions revealed the chemical uniqueness of the dichloromethane fraction of M. arboreus. Some extracts and fractions of these plants demonstrated noteworthy antioxidant and antitrypanosomal potential; the latter was partly attributed to their anti-protease activities. The active principles of these plants were pinpointed before any laborious isolation steps, to avoid the redundant isolation of previously known compounds. CONCLUSION: This study highlighted the use of the established procedure in exploring the metabolomes of these species, which could be helpful for chemotaxonomic and authentication purposes, and might expand the basis for their future phytochemical analysis. Coupling the observed biological potential with LC-MS data has also accelerated the tracing of their bioactive principles.

Microbe Profile: Streptomyces coelicolor: a burlesque of pigments and phenotypes.

The streptomycetes are soil-dwelling bacteria that are found in soil everywhere on Earth: the molecule geosmin, which they produce as part of their life cycle, is what gives soil its familiar 'earthy' smell. The species is best known for the production of biologically active small molecules called 'natural products'. These molecules are the source of most of our antibiotics and anti-fungals, as well as many other drugs. The streptomycetes have a filamentous form rather than the more familiar rod-shaped spirochete and coccoid forms. They exhibit a complex life cycle and sporulation mechanism involving several differentiated cell types, each having specific roles in the colony life history. Streptomyces coelicolor is an important model system for this genus - research on this bacterium has provided foundational information for all of these fascinating processes.

Actinorhodin is a redox-active antibiotic with a complex mode of action against Gram-positive cells.

Actinorhodin is a blue-pigmented, redox-active secondary metabolite that is produced by the bacterium Streptomyces coelicolor. Although actinorhodin has been used as a model compound for studying secondary metabolism, its biological activity is not well understood. Indeed, redox-active antibiotics in general have not been widely investigated at the mechanistic level. In this work, we have conducted a comprehensive chemical genetic investigation of actinorhodin's antibacterial effect on target organisms. We find that actinorhodin is a potent, bacteriostatic, pH-responsive antibiotic. Cells activate at least three stress responses in the presence of actinorhodin, including those responsible for managing oxidative damage, protein damage and selected forms of DNA damage. We find that mutations in the Staphylococcus aureus walRKHI operon can confer low-level resistance to actinorhodin, indicating possible targeting of the cell envelope. Our study indicates a complex mechanism of action, involving multiple molecular targets, that is distinct from other antibiotics.

Chromosome level assembly and secondary metabolite potential of the parasitic fungus Cordyceps militaris.

BACKGROUND: Cordyceps militaris is an insect pathogenic fungus that is prized for its use in traditional medicine. This and other entomopathogenic fungi are understudied sources for the discovery of new bioactive molecules. In this study, PacBio SMRT long read sequencing technology was used to sequence the genome of C. militaris with a focus on the genetic potential for secondary metabolite production in the genome assembly of this fungus. RESULTS: This is first chromosome level assembly of a species in the Cordyceps genera. In this seven chromosome assembly of 33.6 Mba there were 9371 genes identified. Cordyceps militaris was determined to have the MAT 1-1-1 and MAT 1-1-2 mating type genes. Secondary metabolite analysis revealed the potential for at least 36 distinct metabolites from a variety of classes. Three of these gene clusters had homology with clusters producing desmethylbassianin, equisetin and emericellamide that had been studied in other fungi. CONCLUSION: Our assembly and analysis has revealed that C. militaris has a wealth of gene clusters for secondary metabolite production distributed among seven chromosomes. The identification of these gene clusters will facilitate the future study and identification of the secondary metabolites produced by this entomopathogenic fungus.

Biosynthetic Genes for the Tetrodecamycin Antibiotics.

UNLABELLED: We recently described 13-deoxytetrodecamycin, a new member of the tetrodecamycin family of antibiotics. A defining feature of these molecules is the presence of a five-membered lactone called a tetronate ring. By sequencing the genome of a producer strain, Streptomyces sp. strain WAC04657, and searching for a gene previously implicated in tetronate ring formation, we identified the biosynthetic genes responsible for producing 13-deoxytetrodecamycin (the ted genes). Using the ted cluster in WAC04657 as a reference, we found related clusters in three other organisms: Streptomyces atroolivaceus ATCC 19725, Streptomyces globisporus NRRL B-2293, and Streptomyces sp. strain LaPpAH-202. Comparing the four clusters allowed us to identify the cluster boundaries. Genetic manipulation of the cluster confirmed the involvement of the ted genes in 13-deoxytetrodecamycin biosynthesis and revealed several additional molecules produced through the ted biosynthetic pathway, including tetrodecamycin, dihydrotetrodecamycin, and another, W5.9, a novel molecule. Comparison of the bioactivities of these four molecules suggests that they may act through the covalent modification of their target(s). IMPORTANCE: The tetrodecamycins are a distinct subgroup of the tetronate family of secondary metabolites. Little is known about their biosynthesis or mechanisms of action, making them an attractive subject for investigation. In this paper we present the biosynthetic gene cluster for 13-deoxytetrodecamycin in Streptomyces sp. strain WAC04657. We identify related clusters in several other organisms and show that they produce related molecules.

Tetrodecamycin: An unusual and interesting tetronate antibiotic.

The tetrodecamycins are a group of secondary metabolites that are characterized by the presence of a tetronate ring in their structure. Originally discovered for their antibiotic activity against Photobacterium damselae ssp. piscicida, the causative agent of pseudotuberculosis in fish, this family of molecules has also been shown to have potent antibiotic activity against methicillin-resistant Staphylococcus aureus. Due to their small size and highly cyclized nature, they represent an unusual member of the much larger group of bioactive molecules called the tetronates. Herein, we review what is known about the mechanism of action of these molecules and also present a hypothesis for their biosynthesis. A deeper understanding of the tetrodecamycins will provide a more holistic view of the tetronate-family, provide new chemical probes of bacterial biology, and may provide therapeutic lead molecules.

David and Goliath: chemical perturbation of eukaryotes by bacteria.

Environmental microbes produce biologically active small molecules that have been mined extensively as antibiotics and a smaller number of drugs that act on eukaryotic cells. It is known that there are additional bioactives to be discovered from this source. While the discovery of new antibiotics is challenged by the frequent discovery of known compounds, we contend that the eukaryote-active compounds may be less saturated. Indeed, despite there being far fewer eukaryotic-active natural products these molecules interact with a far richer diversity of molecular and cellular targets.

Are you talking to me? A possible role for γ-butyrolactones in interspecies signalling.

The secreted γ-butyrolactone signalling molecule SVB1 regulates the biosynthesis of jadomycin in Streptomyces venezuelae. Interestingly, this molecule is identical to SCB3, a secreted regulator of secondary metabolism in Streptomyces coelicolor. This is a departure for this class of signalling molecules as there are no previous reports of identical signalling molecules produced in different species. One implication of this work is that different species of bacteria could use shared extracellular signals to co-ordinate secondary metabolism when and if it is advantageous to do so.

The expression of antibiotic resistance genes in antibiotic-producing bacteria.

Antibiotic-producing bacteria encode antibiotic resistance genes that protect them from the biologically active molecules that they produce. The expression of these genes needs to occur in a timely manner: either in advance of or concomitantly with biosynthesis. It appears that there have been at least two general solutions to this problem. In many cases, the expression of resistance genes is tightly linked to that of antibiotic biosynthetic genes. In others, the resistance genes can be induced by their cognate antibiotics or by intermediate molecules from their biosynthetic pathways. The regulatory mechanisms that couple resistance to antibiotic biosynthesis are mechanistically diverse and potentially relevant to the origins of clinical antibiotic resistance.

Activating secondary metabolism with stress and chemicals.

The available literature on the secondary or nonessential metabolites of the streptomycetes bacteria suggests that there may be poorly expressed or "cryptic" compounds that have yet to be identified and that may have significant medical utility. In addition, it is clear that there is a large and complex regulatory network that controls the production of these molecules in the laboratory and in nature. Two approaches that have been taken to manipulating the yields of secondary metabolites are the use of various stress responses and, more recently, the use of precision chemical probes. Here, we review the status of this work and outline the challenges and opportunities afforded by each of them.

A synthetic, species-specific activator of secondary metabolism and sporulation in Streptomyces coelicolor.

The secondary metabolites produced by bacterial species serve many clinically useful purposes, and Streptomyces have been an abundant source of such compounds. However, a poor understanding of their regulatory cascades leads to an inability to isolate all of the secondary metabolites this genus is capable of producing. This study focuses on comparing synthetic small molecules that were found to alter the production of secondary metabolites in Streptomyces coelicolor. A survey of these molecules suggests that each has a distinct mechanism of action, and hence, could be used as a unique probe of secondary metabolism. A comparative analysis of two of these molecules, ARC2 and ARC6, confirmed that they modulate secondary metabolites in different ways. In a separate study, ARC2 was shown to give rise to a different phenotype through the inhibition of a target in fatty acid biosynthesis. The results of this study suggest that ARC6 does not have the same target, although it might target the same metabolic system. Furthermore, the results demonstrate that ARC2 and ARC6 act through distinct mechanisms and further suggest that chemical probes can be important tools in enhancing our understanding of secondary metabolism and the streptomycete life cycle.