HelR is a helicase-like protein that protects RNA polymerase from rifamycin antibiotics.

Rifamycin antibiotics such as rifampin are potent inhibitors of prokaryotic RNA polymerase (RNAP) used to treat tuberculosis and other bacterial infections. Although resistance arises in the clinic principally through mutations in RNAP, many bacteria possess highly specific enzyme-mediated resistance mechanisms that modify and inactivate rifamycins. The expression of these enzymes is controlled by a 19-bp cis-acting rifamycin-associated element (RAE). Guided by the presence of RAE sequences, we identify a helicase-like protein, HelR, in Streptomyces venezuelae that confers broad-spectrum rifamycin resistance. We show that HelR also promotes tolerance to rifamycins, enabling bacterial evasion of the toxic properties of these antibiotics. HelR forms a complex with RNAP and rescues transcription inhibition by displacing rifamycins from RNAP, thereby providing resistance by target protection . Furthermore, HelRs are broadly distributed in Actinobacteria, including several opportunistic Mycobacterial pathogens, offering yet another challenge for developing new rifamycin antibiotics.

c-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

Streptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

Dissection of 3D chromosome organization in Streptomyces coelicolor A3(2) leads to biosynthetic gene cluster overexpression.

The soil-dwelling filamentous bacteria, Streptomyces, is widely known for its ability to produce numerous bioactive natural products. Despite many efforts toward their overproduction and reconstitution, our limited understanding of the relationship between the host's chromosome three dimension (3D) structure and the yield of the natural products escaped notice. Here, we report the 3D chromosome organization and its dynamics of the model strain, Streptomyces coelicolor, during the different growth phases. The chromosome undergoes a dramatic global structural change from primary to secondary metabolism, while some biosynthetic gene clusters (BGCs) form special local structures when highly expressed. Strikingly, transcription levels of endogenous genes are found to be highly correlated to the local chromosomal interaction frequency as defined by the value of the frequently interacting regions (FIREs). Following the criterion, an exogenous single reporter gene and even complex BGC can achieve a higher expression after being integrated into the chosen loci, which may represent a unique strategy to activate or enhance the production of natural products based on the local chromosomal 3D organization.

Host hydrocarbons protect symbiont transmission from a radical host defense.

Symbioses with microbes play a pivotal role in the evolutionary success of insects, and can lead to intimate host-symbiont associations. However, how the host maintains a stable symbiosis with its beneficial partners while keeping antagonistic microbes in check remains incompletely understood. Here, we uncover a mechanism by which a host protects its symbiont from the host's own broad-range antimicrobial defense during transmission. Beewolves, a group of solitary digger wasps (Hymenoptera: Crabronidae), provide their brood cells with symbiotic Streptomyces bacteria that are later transferred to the cocoon and protect the offspring from opportunistic pathogens by producing antibiotics. In the brood cell, however, the symbiont-containing secretion is exposed to a toxic burst of nitric oxide (NO) released by the beewolf egg, which effectively kills antagonistic microorganisms. How the symbiont survives this lethal NO burst remained unknown. Here, we report that upon NO exposure in vitro, the symbionts mount a global stress response, but this is insufficient to ensure survival at brood cell-level NO concentrations. Instead, in vivo bioassays demonstrate that the host's antennal gland secretion (AGS) surrounding the symbionts in the brood cell provides an effective diffusion barrier against NO. This physicochemical protection can be reconstituted in vitro by beewolf hydrocarbon extracts and synthetic hydrocarbons, indicating that the host-derived long-chain alkenes and alkanes in the AGS are responsible for shielding the symbionts from NO. Our results reveal how host adaptations can protect a symbiont from host-generated oxidative and nitrosative stress during transmission, thereby efficiently balancing pathogen defense and mutualism maintenance.

Cryptic specialized metabolites drive Streptomyces exploration and provide a competitive advantage during growth with other microbes.

Streptomyces bacteria have a complex life cycle that is intricately linked with their remarkable metabolic capabilities. Exploration is a recently discovered developmental innovation of these bacteria, that involves the rapid expansion of a structured colony on solid surfaces. Nutrient availability impacts exploration dynamics, and we have found that glycerol can dramatically increase exploration rates and alter the metabolic output of exploring colonies. We show here that glycerol-mediated growth acceleration is accompanied by distinct transcriptional signatures and by the activation of otherwise cryptic metabolites including the orange-pigmented coproporphyrin, the antibiotic chloramphenicol, and the uncommon, alternative siderophore foroxymithine. Exploring cultures are also known to produce the well-characterized desferrioxamine siderophore. Mutational studies of single and double siderophore mutants revealed functional redundancy when strains were cultured on their own; however, loss of the alternative foroxymithine siderophore imposed a more profound fitness penalty than loss of desferrioxamine during coculture with the yeast Saccharomyces cerevisiae. Notably, the two siderophores displayed distinct localization patterns, with desferrioxamine being confined within the colony area, and foroxymithine diffusing well beyond the colony boundary. The relative fitness advantage conferred by the alternative foroxymithine siderophore was abolished when the siderophore piracy capabilities of S. cerevisiae were eliminated (S. cerevisiae encodes a ferrioxamine-specific transporter). Our work suggests that exploring Streptomyces colonies can engage in nutrient-targeted metabolic arms races, deploying alternative siderophores that allow them to successfully outcompete other microbes for the limited bioavailable iron during coculture.

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.

Elucidation of the ferrichrome siderophore biosynthetic pathway in albomycin-producing Streptomyces sp. ATCC 700974.

Sideromycins are a unique subset of siderophores comprising of a siderophore conjugated to an antimicrobial agent. The "Trojan horse" antibiotic albomycins are unique sideromycins consisting of a ferrichrome-type siderophore conjugated to a peptidyl nucleoside antibiotic. They exhibit potent antibacterial activities against many model bacteria and a number of clinical pathogens. Earlier studies have provided significant insight into the biosynthetic pathway of the peptidyl nucleoside moiety. We herein decipher the biosynthetic pathway of the ferrichrome-type siderophore in Streptomyces sp. ATCC 700974. Our genetic studies suggested that abmA, abmB, and abmQ are involved in the formation of the ferrichrome-type siderophore. Additionally, we performed biochemical studies to demonstrate that a flavin-dependent monooxygenase AbmB and an N-acyltransferase AbmA catalyze sequential modifications of L-ornithine to generate N5-acetyl-N5-hydroxyornithine. Three molecules of N5-acetyl-N5-hydroxyornithine are then assembled to generate the tripeptide ferrichrome through the action of a nonribosomal peptide synthetase AbmQ. Of special note, we found out that orf05026 and orf03299, two genes scattered elsewhere in the chromosome of Streptomyces sp. ATCC 700974, have functional redundancy for abmA and abmB, respectively. Interestingly, both orf05026 and orf03299 are situated within gene clusters encoding putative siderophores. In summary, this study provided new insight into the siderophore moiety of albomycin biosynthesis and shed light on the contingency of multiple siderophores in albomycin-producing Streptomyces sp. ATCC 700974.

Mistranslation of the genetic code by a new family of bacterial transfer RNAs.

The correct coupling of amino acids with transfer RNAs (tRNAs) is vital for translating genetic information into functional proteins. Errors during this process lead to mistranslation, where a codon is translated using the wrong amino acid. While unregulated and prolonged mistranslation is often toxic, growing evidence suggests that organisms, from bacteria to humans, can induce and use mistranslation as a mechanism to overcome unfavorable environmental conditions. Most known cases of mistranslation are caused by translation factors with poor substrate specificity or when substrate discrimination is sensitive to molecular changes such as mutations or posttranslational modifications. Here we report two novel families of tRNAs, encoded by bacteria from the Streptomyces and Kitasatospora genera, that adopted dual identities by integrating the anticodons AUU (for Asn) or AGU (for Thr) into the structure of a distinct proline tRNA. These tRNAs are typically encoded next to a full-length or truncated version of a distinct isoform of bacterial-type prolyl-tRNA synthetase. Using two protein reporters, we showed that these tRNAs translate asparagine and threonine codons with proline. Moreover, when expressed in Escherichia coli, the tRNAs cause varying growth defects due to global Asn-to-Pro and Thr-to-Pro mutations. Yet, proteome-wide substitutions of Asn with Pro induced by tRNA expression increased cell tolerance to the antibiotic carbenicillin, indicating that Pro mistranslation can be beneficial under certain conditions. Collectively, our results significantly expand the catalog of organisms known to possess dedicated mistranslation machinery and support the concept that mistranslation is a mechanism for cellular resiliency against environmental stress.

From nature to cancer therapy: Evaluating the Streptomyces clavuligerus secondary metabolites for potential protein kinase inhibitors.

The study aimed to evaluate the antioxidant, protein kinase inhibitory (PKIs) potential, cytotoxicity activity of Streptomyces clavuligerus extract. DPPH assay revealed a robust free radical scavenging capacity (IC50 28.90 ± 0.24 µg/mL) of organic extract with a maximum inhibition percentage of 61 ± 1.04%. PKIs assay revealed the formation of a whitish bald zone by S. clavuligerus extracts which indicates the presence of PKIs. The cytotoxicity activity of organic fraction of extract through Sulforhodamine B assay on MCF-7, Hop-62, SiHa, and PC-3 cell lines demonstrated the lowest GI50 value against the MCF-7 cell line followed by the PC-3 cell line, showing potent growth inhibitory potential against human breast cancer and human prostate cancer cell line. HR-LCMS analysis identified multiple secondary metabolites from the organic and aqueous extracts of S. clavuligerus when incubated at 30°C under 200 rpm for 3 days. All the secondary metabolites were elucidated for their potential to inhibit RTKs by molecular docking, molecular dynamic simulation, MM/GBSA calculations, and free energy approach. It revealed the superior inhibitory potential of epirubicin (Epi) and dodecaprenyl phosphate-galacturonic acid (DPGA) against fibroblast growth factors receptor (FGFR). Epi also exhibited excellent inhibitory activity against the platelet-derived growth factor receptor (PDGFR), while DPGA effectively inhibited the vascular endothelial growth factor receptor. Additionally, the presence Epi in S. clavuligerus extract was validated through the HPLC technique. Thus, our findings highlight a superior inhibitory potential of Epi against FGFR and PDGFR RTKs than the FDA-approved drug.

Biosynthesis of Arcyriaflavin F from Streptomyces venezuelae ATCC 10712.

Indolocarbazoles are natural products with broad bioactivities. A distinct feature of indolocarbazole biosynthesis is the modification of the indole and maleimide rings by regioselective tailoring enzymes. Here, we study a new indolocarbazole variant, which is encoded by the acfXODCP genes from Streptomyces venezuelae ATCC 10712. First we characterise this pathway by expressing the acfXODCP genes in Streptomyces coelicolor, which led to the production of a C-5/C-5'-dihydroxylated indolocarbazole. We assign as a new product arcyriaflavin F. Second, we demonstrate the flavin-dependent monooxygenase AcfX catalyses the C-5/C-5' dihydroxylation of the unsubstituted arcyriaflavin A into arcyriaflavin F. Interestingly, AcfX shares homology to EspX from erdasporine A biosynthesis, which instead catalyses a single C-6 indolocarbazole hydroxylation. In summary, we report a new indolocarbazole biosynthetic pathway and a regioselective C-5 indole ring tailoring enzyme AcfX.

The Post-Polyketide Synthase Modification Mechanism in Hitachimycin Biosynthesis.

Hitachimycin is a bicyclic macrolactam antibiotic with (S)-ß-phenylalanine (ß-Phe) at the starter position of the polyketide skeleton. While the enzymes that recognize ß-amino acids, modify the aminoacyl groups, and transfer the resultant dipeptide groups to the acyl carrier protein domains of polyketide synthases (PKSs) have been studied extensively, the post-PKS modification mechanism responsible for constructing the unique bicyclic structure of hitachimycin remains elusive. In this study, we first inactivated six genes encoding putative post-PKS modification enzymes, namely hitM1 to hitM6, in Streptomyces scabrisporus to determine their involvement in hitachimycin biosynthesis. The ΔhitM4 strain accumulated an all-trans-2,4,6,8,18-pentaene macrolactam, which was confirmed as a true intermediate in hitachimycin biosynthesis by cellular feeding experiments, and appears to be the initial intermediate in the post-PKS modification pathway. The ΔhitM1 strain accumulated 10-O-demethyl-10-oxohitachimycin (M1-A). In enzymatic experiments, M1-A was reduced by the NAD(P)H-dependent reductase HitM1 in the presence of NADPH. The product of the reaction catalyzed by HitM1 was converted to hitachimycin by the methyltransferase HitM6. We thus propose a plausible post-PKS modification mechanism for the biosynthesis of hitachimycin.

Building a highly efficient Streptomyces super-chassis for secondary metabolite production by reprogramming naturally-evolved multifaceted shifts.

Streptomyces has an extensive array of bioactive secondary metabolites (SMs). Nevertheless, devising a framework for the heterologous production of these SMs remains challenging. We here reprogrammed a versatile plug-and-play Streptomyces super-chassis and established a universal pipeline for production of diverse SMs via understanding of the inherent pleiotropic effects of ethanol shock on jadomycin production in Streptomyces venezuelae. We initially identified and characterized a set of multiplex targets (afsQ1, bldD, bldA, and miaA) that contribute to SM (jadomycin) production when subjected to ethanol shock. Subsequently, we developed an ethanol-induced orthogonal amplification system (EOAS), enabling dynamic and precise control over targets. Ultimately, we integrated these multiplex targets into functional units governed by the EOAS, generating a universal and plug-and-play Streptomyces super-chassis. In addition to achieving the unprecedented titer and yield of jadomycin B, we also evidenced the potential of this super-chassis for production of diverse heterologous SMs, including antibiotic oxytetracycline, anticancer drug doxorubicins, agricultural herbicide thaxtomin A, and plant growth regulator guvermectin, all with the yields of >10 mg/g glucose in a simple mineral medium. Given that the production of SMs all required complexed medium and the cognate yields were usually much lower, our achievement of using a universal super-chassis and engineering pipeline in a simple mineral medium is promising for convenient heterologous production of SMs.

Systems metabolic engineering of the primary and secondary metabolism of Streptomyces albidoflavus enhances production of the reverse antibiotic nybomycin against multi-resistant Staphylococcus aureus.

Nybomycin is an antibiotic compound with proven activity against multi-resistant Staphylococcus aureus, making it an interesting candidate for combating these globally threatening pathogens. For exploring its potential, sufficient amounts of nybomycin and its derivatives must be synthetized to fully study its effectiveness, safety profile, and clinical applications. As native isolates only accumulate low amounts of the compound, superior producers are needed. The heterologous cell factory S. albidoflavus 4N24, previously derived from the cluster-free chassis S. albidoflavus Del14, produced 860 µg L-1 of nybomycin, mainly in the stationary phase. A first round of strain development modulated expression of genes involved in supply of nybomycin precursors under control of the common Perm* promoter in 4N24, but without any effect. Subsequent studies with mCherry reporter strains revealed that Perm* failed to drive expression during the product synthesis phase but that use of two synthetic promoters (PkasOP* and P41) enabled strong constitutive expression during the entire process. Using PkasOP*, several rounds of metabolic engineering successively streamlined expression of genes involved in the pentose phosphate pathway, the shikimic acid pathway, supply of CoA esters, and nybomycin biosynthesis and export, which more than doubled the nybomycin titer to 1.7 mg L-1 in the sixth-generation strain NYB-6B. In addition, we identified the minimal set of nyb genes needed to synthetize the molecule using single-gene-deletion strains. Subsequently, deletion of the regulator nybW enabled nybomycin production to begin during the growth phase, further boosting the titer and productivity. Based on RNA sequencing along the created strain genealogy, we discovered that the nyb gene cluster was unfavorably downregulated in all advanced producers. This inspired removal of a part and the entire set of the four regulatory genes at the 3'-end nyb of the cluster. The corresponding mutants NYB-8 and NYB-9 exhibited marked further improvement in production, and the deregulated cluster was combined with all beneficial targets from primary metabolism. The best strain, S. albidoflavus NYB-11, accumulated up to 12 mg L-1 nybomycin, fifteenfold more than the basic strain. The absence of native gene clusters in the host and use of a lean minimal medium contributed to a selective production process, providing an important next step toward further development of nybomycin.

CslA and GlxA from Streptomyces lividans form a functional cellulose synthase complex.

Filamentous growth of streptomycetes coincides with the synthesis and deposition of an uncharacterized protective glucan at hyphal tips. Synthesis of this glucan depends on the integral membrane protein CslA and the radical copper oxidase GlxA, which are part of a presumably large multiprotein complex operating at growing tips. Here, we show that CslA and GlxA interact by forming a protein complex that is sufficient to synthesize cellulose in vitro. Mass spectrometry analysis revealed that the purified complex produces cellulose chains with a degree of polymerization of at least 80 residues. Truncation analyses demonstrated that the removal of a significant extracellular segment of GlxA had no impact on complex formation, but significantly diminished activity of CslA. Altogether, our work demonstrates that CslA and GlxA form the active core of the cellulose synthase complex and provide molecular insights into a unique cellulose biosynthesis system that is conserved in streptomycetes. IMPORTANCE: Cellulose stands out as the most abundant polysaccharide on Earth. While the synthesis of this polysaccharide has been extensively studied in plants and Gram-negative bacteria, the mechanisms in Gram-positive bacteria have remained largely unknown. Our research unveils a novel cellulose synthase complex formed by the interaction between the cellulose synthase-like protein CslA and the radical copper oxidase GlxA from Streptomyces lividans, a soil-dwelling Gram-positive bacterium. This discovery provides molecular insights into the distinctive cellulose biosynthesis machinery. Beyond expanding our understanding of cellulose biosynthesis, this study also opens avenues for exploring biotechnological applications and ecological roles of cellulose in Gram-positive bacteria, thereby contributing to the broader field of microbial cellulose biosynthesis and biofilm research.

Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways.

Iron is essential to many biological processes but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways in the producing organism. Although the biological role(s) of these new derivatives remain to be elucidated, they may confer advantages in terms of metal chelation in the competitive soil environment due to the additional bidentate hydroxamic functional groups. The metabolites may also find application in various fields including biotechnology, bioremediation, and immuno-PET imaging.IMPORTANCEIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload and in diagnostic imaging, as well as in biotechnology, including as agents for biocontrol of pathogens and bioremediation. In this study, we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens-the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance, 89Zr-based immuno-PET imaging) and other applications of metal chelators.

An atypical two-component system, AtcR/AtcK, simultaneously regulates the biosynthesis of multiple secondary metabolites in Streptomyces bingchenggensis.

Streptomyces bingchenggensis is an industrial producer of milbemycins, which are important anthelmintic and insecticidal agents. Two-component systems (TCSs), which are typically situated in the same operon and are composed of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Here, an atypical TCS, AtcR/AtcK, in which the encoding genes (sbi_06838/sbi_06839) are organized in a head-to-head pair, was demonstrated to be indispensable for the biosynthesis of multiple secondary metabolites in S. bingchenggensis. With the null TCS mutants, the production of milbemycin and yellow compound was abolished but nanchangmycin was overproduced. Transcriptional analysis and electrophoretic mobility shift assays showed that AtcR regulated the biosynthesis of these three secondary metabolites by a MilR3-mediated cascade. First, AtcR was activated by phosphorylation from signal-triggered AtcK. Second, the activated AtcR promoted the transcription of milR3. Third, MilR3 specifically activated the transcription of downstream genes from milbemycin and yellow compound biosynthetic gene clusters (BGCs) and nanR4 from the nanchangmycin BGC. Finally, because NanR4 is a specific repressor in the nanchangmycin BGC, activation of MilR3 downstream genes led to the production of yellow compound and milbemycin but inhibited nanchangmycin production. By rewiring the regulatory cascade, two strains were obtained, the yield of nanchangmycin was improved by 45-fold to 6.08 g/L and the production of milbemycin was increased twofold to 1.34 g/L. This work has broadened our knowledge on atypical TCSs and provided practical strategies to engineer strains for the production of secondary metabolites in Streptomyces.IMPORTANCEStreptomyces bingchenggensis is an important industrial strain that produces milbemycins. Two-component systems (TCSs), which consist of a histidine kinase and a response regulator, are the predominant signal transduction pathways involved in the regulation of secondary metabolism in Streptomyces. Coupled encoding genes of TCSs are typically situated in the same operon. Here, TCSs with encoding genes situated in separate head-to-head neighbor operons were labeled atypical TCSs. It was found that the atypical TCS AtcR/AtcK played an indispensable role in the biosynthesis of milbemycin, yellow compound, and nanchangmycin in S. bingchenggensis. This atypical TCS regulated the biosynthesis of specialized metabolites in a cascade mediated via a cluster-situated regulator, MilR3. Through rewiring the regulatory pathways, strains were successfully engineered to overproduce milbemycin and nanchangmycin. To the best of our knowledge, this is the first report on atypical TCS, in which the encoding genes of RR and HK were situated in separate head-to-head neighbor operons, involved in secondary metabolism. In addition, data mining showed that atypical TCSs were widely distributed in actinobacteria.

Engineering of a TetR family transcriptional regulator BkdR enhances heterologous spinosad production in Streptomyces albus B4 chassis.

Precursor supply plays a significant role in the production of secondary metabolites. In Streptomyces bacteria, propionyl-, malonyl-, and methylmalonyl-CoA are the most common precursors used for polyketide biosynthesis. Although propionyl-CoA synthetases participate in the propionate assimilation pathway and directly convert propionate into propionyl-CoA, malonyl- and methylmalonyl-CoA cannot be formed using common acyl-CoA synthetases. Therefore, both acetyl- and propionyl-CoA carboxylation, catalyzed by acyl-CoA carboxylases, should be considered when engineering a microorganism chassis to increase polyketide production. In this study, we identified a transcriptional regulator of the TetR family, BkdR, in Streptomyces albus B4, which binds directly to the promoter region of the neighboring pccAB operon. This operon encodes acetyl/propionyl-CoA carboxylase and negatively regulates its transcription. In addition to acetate and propionate, the binding of BkdR to pccAB is disrupted by acetyl- and propionyl-CoA ligands. We identified a 16-nucleotide palindromic BkdR-binding motif (GTTAg/CGGTCg/TTAAC) in the intergenic region between pccAB and bkdR. When bkdR was deleted, we found an enhanced supply of malonyl- and methylmalonyl-CoA precursors in S. albus B4. In this study, spinosad production was detected in the recombinant strain after introducing the entire artificial biosynthesized gene cluster into S. albus B4. When supplemented with propionate to provide propionyl-CoA, the novel bkdR-deleted strain produced 29.4% more spinosad than the initial strain in trypticase soy broth (TSB) medium. IMPORTANCE: In this study, we describe a pccAB operon involved in short-chain acyl-CoA carboxylation in S. albus B4 chassis. The TetR family regulator, BkdR, represses this operon. Our results show that BkdR regulates the precursor supply needed for heterologous spinosad biosynthesis by controlling acetyl- and propionyl-CoA assimilation. The deletion of the BkdR-encoding gene exerts an increase in heterologous spinosad yield. Our research reveals a regulatory mechanism in short-chain acyl-CoA metabolism and suggests new possibilities for S. albus chassis engineering to enhance heterologous polyketide yield.

Evolution of genome fragility enables microbial division of labor.

Division of labor can evolve when social groups benefit from the functional specialization of its members. Recently, a novel means of coordinating the division of labor was found in the antibiotic-producing bacterium Streptomyces coelicolor, where specialized cells are generated through large-scale genomic re-organization. We investigate how the evolution of a genome architecture enables such mutation-driven division of labor, using a multiscale computational model of bacterial evolution. In this model, bacterial behavior-antibiotic production or replication-is determined by the structure and composition of their genome, which encodes antibiotics, growth-promoting genes, and fragile genomic loci that can induce chromosomal deletions. We find that a genomic organization evolves, which partitions growth-promoting genes and antibiotic-coding genes into distinct parts of the genome, separated by fragile genomic loci. Mutations caused by these fragile sites mostly delete growth-promoting genes, generating sterile, and antibiotic-producing mutants from weakly-producing progenitors, in agreement with experimental observations. This division of labor enhances the competition between colonies by promoting antibiotic diversity. These results show that genomic organization can co-evolve with genomic instabilities to enable reproductive division of labor.

Exploring the decolorization efficiency and biodegradation mechanisms of different functional textile azo dyes by Streptomyces albidoflavus 3MGH.

Efficiently mitigating and managing environmental pollution caused by the improper disposal of dyes and effluents from the textile industry is of great importance. This study evaluated the effectiveness of Streptomyces albidoflavus 3MGH in decolorizing and degrading three different azo dyes, namely Reactive Orange 122 (RO 122), Direct Blue 15 (DB 15), and Direct Black 38 (DB 38). Various analytical techniques, such as Fourier Transform Infrared (FTIR) spectroscopy, High-Performance Liquid Chromatography (HPLC), and Gas Chromatography-Mass Spectrometry (GC-MS) were used to analyze the degraded byproducts of the dyes. S. albidoflavus 3MGH demonstrated a strong capability to decolorize RO 122, DB 15, and DB 38, achieving up to 60.74%, 61.38%, and 53.43% decolorization within 5 days at a concentration of 0.3 g/L, respectively. The optimal conditions for the maximum decolorization of these azo dyes were found to be a temperature of 35 °C, a pH of 6, sucrose as a carbon source, and beef extract as a nitrogen source. Additionally, after optimization of the decolorization process, treatment with S. albidoflavus 3MGH resulted in significant reductions of 94.4%, 86.3%, and 68.2% in the total organic carbon of RO 122, DB 15, and DB 38, respectively. After the treatment process, we found the specific activity of the laccase enzyme, one of the mediating enzymes of the degradation mechanism, to be 5.96 U/mg. FT-IR spectroscopy analysis of the degraded metabolites showed specific changes and shifts in peaks compared to the control samples. GC-MS analysis revealed the presence of metabolites such as benzene, biphenyl, and naphthalene derivatives. Overall, this study demonstrated the potential of S. albidoflavus 3MGH for the effective decolorization and degradation of different azo dyes. The findings were validated through various analytical techniques, shedding light on the biodegradation mechanism employed by this strain.

Probiotic potential of Streptomyces levis strain HFM-2 isolated from human gut and its antibiofilm properties against pathogenic bacteria.

BACKGROUND: Antimicrobial resistance (AMR) is a serious worldwide public health concern that needs immediate action. Probiotics could be a promising alternative for fighting antibiotic resistance, displaying beneficial effects to the host by combating diseases, improving growth, and stimulating the host immune responses against infection. This study was conducted to evaluate the probiotic, antibacterial, and antibiofilm potential of Streptomyces levis strain HFM-2 isolated from the healthy human gut. RESULTS: In vitro antibacterial activity in the cell-free supernatant of S. levis strain HFM-2 was evaluated against different pathogens viz. K. pneumoniae sub sp. pneumoniae, S. aureus, B. subtilis, VRE, S. typhi, S. epidermidis, MRSA, V. cholerae, M. smegmatis, E. coli, P. aeruginosa and E. aerogenes. Further, the ethyl acetate extract from S. levis strain HFM-2 showed strong biofilm inhibition against S. typhi, K. pneumoniae sub sp. pneumoniae, P. aeruginosa and E. coli. Fluorescence microscopy was used to detect biofilm inhibition properties. MIC and MBC values of EtOAc extract were determined at 500 and 1000 µg/mL, respectively. Further, strain HFM-2 showed high tolerance in gastric juice, pancreatin, bile, and at low pH. It exhibited efficient adhesion properties, displaying auto-aggregation (97.0%), hydrophobicity (95.71%, 88.96%, and 81.15% for ethyl acetate, chloroform and xylene, respectively), and showed 89.75%, 86.53%, 83.06% and 76.13% co-aggregation with S. typhi, MRSA, S. pyogenes and E. coli, respectively after 60 min of incubation. The S. levis strain HFM-2 was susceptible to different antibiotics such as tetracycline, streptomycin, kanamycin, ciprofloxacin, erythromycin, linezolid, meropenem, amikacin, gentamycin, clindamycin, moxifloxacin and vancomycin, but resistant to ampicillin and penicillin G. CONCLUSION: The study shows that S. levis strain HFM-2 has significant probiotic properties such as good viability in bile, gastric juice, pancreatin environment, and at low pH; proficient adhesion properties, and antibiotic susceptibility. Further, the EtOAc extract of Streptomyces levis strain HFM-2 has a potent antibiofilm and antibacterial activity against antibacterial-resistant clinical pathogens.