The role of two major nucleoid-associated proteins in Streptomyces, HupA and HupS, in stress survival and gene expression regulation.

BACKGROUND: Streptomyces are sporulating soil bacteria with enormous potential for secondary metabolites biosynthesis. Regulatory networks governing Streptomyces coelicolor differentiation and secondary metabolites production are complex and composed of numerous regulatory proteins ranging from specific transcriptional regulators to sigma factors. Nucleoid-associated proteins (NAPs) are also believed to contribute to regulation of gene expression. Upon DNA binding, these proteins impact DNA accessibility. Among NAPs, HU proteins are the most widespread and abundant. Unlike other bacteria, the Streptomyces genomes encode two HU homologs: HupA and HupS, which differ in structure and expression profile. However, it remained unclear whether the functions of both homologs overlap. Additionally, although both proteins have been shown to bind the chromosome, their rolesin transcriptional regulation have not been studied so far. RESULTS: In this study, we explore whether HupA and HupS affect S. coelicolor growth under optimal and stressful conditions and how they control global gene expression. By testing both single and double mutants, we address the question of the complementarity of both HU homologs. We show that the lack of both hup genes led to growth and sporulation inhibition, as well as increased spore fragility. We also demonstrate that both HU homologs can be considered global transcriptional regulators, influencing expression of between 2% and 6% genes encoding among others proteins linked to global regulatory networks and secondary metabolite production. CONCLUSIONS: We identify the independent HupA and HupS regulons, as well as genes under the control of both HupA and HupS proteins. Our data indicate a partial overlap between the functions of HupA and HupS during S. coelicolor growth. HupA and HupS play important roles in Streptomyces regulatory network and impact secondary metabolite clusters.

Characterizing and Engineering Rhamnose-Inducible Regulatory Systems for Dynamic Control of Metabolic Pathways in Streptomyces.

Fine-tuning gene expression is of great interest for synthetic biotechnological applications. This is particularly true for the genus Streptomyces, which is well-known as a prolific producer of diverse natural products. Currently, there is an increasing demand to develop effective gene induction systems. In this study, bioinformatic analysis revealed a putative rhamnose catabolic pathway in multiple Streptomyces species, and the removal of the pathway in the model organism Streptomyces coelicolor impaired its growth on minimal media with rhamnose as the sole carbon source. To unravel the regulatory mechanism of RhaR, a LacI family transcriptional regulator of the catabolic pathway, electrophoretic mobility shift assays (EMSAs) were performed to identify potential target promoters. Multiple sequence alignments retrieved a consensus sequence of the RhaR operator (rhaO). A synthetic biology-based strategy was then deployed to build rhamnose-inducible regulatory systems, referred to as rhaRS1 and rhaRS2, by assembling the repressor/operator pair RhaR/rhaO with the well-defined constitutive kasO* promoter. Both rhaRS1 and rhaRS2 exhibited a high level of induced reporter activity, with no leaky expression. rhaRS2 has been proven successful for the programmable production of actinorhodin and violacein in Streptomyces. Our study expanded the toolkit of inducible regulatory systems that will be broadly applicable to many other Streptomyces species.

Genome-Led Discovery of the Antibacterial Cyclic Lipopeptide Kutzneridine A and Its Silent Biosynthetic Gene Cluster from Kutzneria Species.

Genome analysis of Kutzneria sp. CA-103260 revealed a putative lipopeptide-encoding biosynthetic gene cluster (BGC) that was cloned into a bacterial artificial chromosome (BAC) and heterologously expressed in Streptomyces coelicolor M1152. As a result, a novel cyclic lipo-tetrapeptide containing two diaminopropionic acid residues and an exotic N,N-acetonide ring, kutzneridine A (1), was isolated and structurally characterized. Evaluation of the extraction conditions and isotope-labeling chemical modifications showed that the acetonide ring originated from acetone during isolation. The BGC was analyzed in silico and a biosynthetic pathway to 1 was proposed. Kutzneridine A displayed remarkable antibacterial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci.

Combined effect of polyphosphate kinase and lon protease in Streptomyces coelicolor A3(2) antibiotic production.

The bacterial stringent response is a global regulatory process in which polyphosphate kinase (Ppk) and lon protease are important players. Previous studies have shown that overexpression of the lon gene and deletion of the ppk gene significantly increased actinorhodin production in Streptomyces coelicolor (SCO). In this study, a recombinant SCOΔppk-lon cell, expressing the extra lon gene in Δppk cells, was simulated using a modified in silico (computational) model, ecSco-GEM, and the negative effect of Ppk on actinorhodin production was confirmed. In addition, we identified key enzymes that play a positive role in actinorhodin production. Of these, NADH dehydrogenase/complex-I, beta-ketoacyl-[acyl-carrier-protein] synthase III, glycine cleavage system, and superoxide dismutase were identified as the most significant. By confirming these results with experiments, we have shown that GEMs can be a reliable starting point for in vitro (lab-based) studies of Streptomyces..

Characterization of Lomofungin Gene Cluster Enables the Biosynthesis of Related Phenazine Derivatives.

Phenazine-based small molecules are nitrogen-containing heterocyclic compounds with diverse bioactivities and electron transfer properties that exhibit promising applications in pharmaceutical and electrochemical industries. However, the biosynthetic mechanism of highly substituted natural phenazines remains poorly understood. In this study, we report the direct cloning and heterologous expression of the lomofungin biosynthetic gene cluster (BGC) from Streptomyces lomondensis S015. Reconstruction and overexpression of the BGCs in Streptomyces coelicolor M1152 resulted in eight phenazine derivatives including two novel hybrid phenazine metabolites, and the biosynthetic pathway of lomofungin was proposed. Furthermore, gene deletion suggested that NAD(P)H-dependent oxidoreductase gene lomo14 is a nonessential gene in the biosynthesis of lomofungin. Cytotoxicity evaluation of the isolated phenazines and lomofungin was performed. Specifically, lomofungin shows substantial inhibition against two human cancer cells, HCT116 and 5637. These results provide insights into the biosynthetic mechanism of lomofungin, which will be useful for the directed biosynthesis of natural phenazine derivatives.

Consequences of the deletion of the major specialized metabolite biosynthetic pathways of Streptomyces coelicolor on the metabolome and lipidome of this strain.

Chassis strains, derived from Streptomyces coelicolor M145, deleted for one or more of its four main specialized metabolites biosynthetic pathways (CPK, CDA, RED and ACT), in various combinations, were constructed for the heterologous expression of specialized metabolites biosynthetic pathways of various types and origins. To determine consequences of these deletions on the metabolism of the deleted strains comparative lipidomic and metabolomic analyses of these strains and of the original strain were carried out. These studies unexpectedly revealed that the deletion of the peptidic clusters, RED and/or CDA, in a strain deleted for the ACT cluster, resulted into a great increase in the triacylglycerol (TAG) content, whereas the deletion of polyketide clusters, ACT and CPK had no impact on TAG content. Low or high TAG content of the deleted strains was correlated with abundance or paucity in amino acids, respectively, reflecting high or low activity of oxidative metabolism. Hypotheses based on what is known on the bio-activity and the nature of the precursors of these specialized metabolites are proposed to explain the unexpected consequences of the deletion of these pathways on the metabolism of the bacteria and on the efficiency of the deleted strains as chassis strains.

Functional connexion of bacterioferritin in antibiotic production and morphological differentiation in Streptomyces coelicolor.

BACKGROUND: Several two-component systems of Streptomyces coelicolor, a model organism used for studying antibiotic production in Streptomyces, affect the expression of the bfr (SCO2113) gene that encodes a bacterioferritin, a protein involved in iron storage. In this work, we have studied the effect of the deletion mutant ∆bfr in S. coelicolor. RESULTS: The ∆bfr mutant exhibits a delay in morphological differentiation and produces a lesser amount of the two pigmented antibiotics (actinorhodin and undecylprodigiosin) compared to the wild type on complex media. The effect of iron in minimal medium was tested in the wild type and ∆bfr mutant. Consequently, we also observed different levels of production of the two pigmented antibiotics between the two strains, depending on the iron concentration and the medium (solid or liquid) used. Contrary to expectations, no differences in intracellular iron concentration were detected between the wild type and ∆bfr mutant. However, a higher level of reactive oxygen species in the ∆bfr mutant and a higher tolerance to oxidative stress were observed. Proteomic analysis showed no variation in iron response proteins, but there was a lower abundance of proteins related to actinorhodin and ribosomal proteins, as well as others related to secondary metabolite production and differentiation. Additionally, a higher abundance of proteins related to various types of stress, such as respiration and hypoxia among others, was also revealed. Data are available via ProteomeXchange with identifier PXD050869. CONCLUSION: This bacterioferritin in S. coelicolor (Bfr) is a new element in the complex regulation of secondary metabolism in S. coelicolor and, additionally, iron acts as a signal to modulate the biosynthesis of active molecules. Our model proposes an interaction between Bfr and iron-containing regulatory proteins. Thus, identifying these interactions would provide new information for improving antibiotic production in Streptomyces.

ActVI-ORFA directs metabolic flux towards actinorhodin by preventing intermediate degradation.

The biosynthetic pathway of actinorhodin in Streptomyces coelicolor A3(2) has been studied for decades as a model system of type II polyketide biosynthesis. The actinorhodin biosynthetic gene cluster includes a gene, actVI-orfA, that encodes a protein that belongs to the nuclear transport factor-2-like (NTF-2-like) superfamily. The function of this ActVI-ORFA protein has been a long-standing question in this field. Several hypothetical functions, including pyran ring cyclase, enzyme complex stability enhancer, and gene transcription regulator, have been proposed for ActVI-ORFA in previous studies. However, although the recent structural analysis of ActVI-ORFA revealed a solvent-accessible cavity, the protein displayed structural differences to the well-characterized cyclase SnoaL and did not possess a DNA-binding domain. The obtained crystal structure facilitates an inspection of the previous hypotheses regarding the function of ActVI-ORFA. In the present study, we investigated the effects of a series of actVI-orfA test plasmids with different mutations in an established vector/host system. Time-course analysis of dynamic metabolism profiles demonstrated that ActVI-ORFA prevented formation of shunt metabolites and may have a metabolic flux directing function, which shepherds the flux of unstable intermediates towards actinorhodin. The expression studies resulted in the isolation and structure elucidation of two new shunt metabolites from the actinorhodin pathway. Next, we utilized computational modeling to probe the active site of ActVI-ORFA and confirmed the importance of residues R76 and H78 in the flux directing functionality by expression studies. This is the first time such a function has been observed for a member of NTF-2-like superfamily in Streptomyces secondary metabolism.

Polyester degradation by soil bacteria: identification of conserved BHETase enzymes in Streptomyces.

The rising use of plastic results in an appalling amount of waste which is scattered into the environment. One of these plastics is PET which is mainly used for bottles. We have identified and characterized an esterase from Streptomyces, annotated as LipA, which can efficiently degrade the PET-derived oligomer BHET. The Streptomyces coelicolor ScLipA enzyme exhibits varying sequence similarity to several BHETase/PETase enzymes, including IsPETase, TfCut2, LCC, PET40 and PET46. Of 96 Streptomyces strains, 18% were able to degrade BHET via one of three variants of LipA, named ScLipA, S2LipA and S92LipA. SclipA was deleted from S. coelicolor resulting in reduced BHET degradation. Overexpression of all LipA variants significantly enhanced BHET degradation. All variants were expressed in E. coli for purification and biochemical analysis. The optimum conditions were determined as pH 7 and 25 °C for all variants. The activity on BHET and amorphous PET film was investigated. S2LipA efficiently degraded BHET and caused roughening and indents on the surface of PET films, comparable to the activity of previously described TfCut2 under the same conditions. The abundance of the S2LipA variant in Streptomyces suggests an environmental advantage towards the degradation of more polar substrates including these polluting plastics.

Discovery, characterization, and engineering of an advantageous Streptomyces host for heterologous expression of natural product biosynthetic gene clusters.

BACKGROUND: Streptomyces is renowned for its robust biosynthetic capacity in producing medically relevant natural products. However, the majority of natural products biosynthetic gene clusters (BGCs) either yield low amounts of natural products or remain cryptic under standard laboratory conditions. Various heterologous production hosts have been engineered to address these challenges, and yet the successful activation of BGCs has still been limited. In our search for a valuable addition to the heterologous host panel, we identified the strain Streptomyces sp. A4420, which exhibited rapid initial growth and a high metabolic capacity, prompting further exploration of its potential. RESULTS: We engineered a polyketide-focused chassis strain based on Streptomyces sp. A4420 (CH strain) by deleting 9 native polyketide BGCs. The resulting metabolically simplified organism exhibited consistent sporulation and growth, surpassing the performance of most existing Streptomyces based chassis strains in standard liquid growth media. Four distinct polyketide BGCs were chosen and expressed in various heterologous hosts, including the Streptomyces sp. A4420 wild-type and CH strains, alongside Streptomyces coelicolor M1152, Streptomyces lividans TK24, Streptomyces albus J1074, and Streptomyces venezuelae NRRL B-65442. Remarkably, only the Streptomyces sp. A4420 CH strain demonstrated the capability to produce all metabolites under every condition outperforming its parental strain and other tested organisms. To enhance visualization and comparison of the tested strains, we developed a matrix-like analysis involving 15 parameters. This comprehensive analysis unequivocally illustrated the significant potential of the new strain to become a popular heterologous host. CONCLUSION: Our engineered Streptomyces sp. A4420 CH strain exhibits promising attributes for the heterologous expression of natural products with a focus on polyketides, offering an alternative choice in the arsenal of heterologous production strains. As genomics and cloning strategies progress, establishment of a diverse panel of heterologous production hosts will be crucial for expediting the discovery and production of medically relevant natural products derived from Streptomyces.

Streptomyces umbrella toxin particles block hyphal growth of competing species.

Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.

An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor.

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

Impact of C-terminal domains of paralogous single-stranded DNA binding proteins from Streptomyces coelicolor on their biophysical properties and biological functions.

Single-stranded DNA-binding proteins (SSB) are crucial in DNA metabolism. While Escherichia coli SSB is extensively studied, the significance of its C-terminal domain has only recently emerged. This study explored the significance of C-domains of two paralogous Ssb proteins in S. coelicolor. Mutational analyses of C-domains uncovered a novel role of SsbA during sporulation-specific cell division and demonstrated that the C-tip is non-essential for survival. In vitro methods revealed altered biophysical and biochemical properties of Ssb proteins with modified C-domains. Determined hydrodynamic properties suggested that the C-domains of SsbA and SsbB occupy a globular position proposed to mediate cooperative binding. Only SsbA was found to form biomolecular condensates independent of the C-tip. Interestingly, the truncated C-domain of SsbA increased the molar enthalpy of unfolding. Additionally, calorimetric titrations revealed that C-domain mutations affected ssDNA binding. Moreover, this analysis showed that the SsbA C-tip aids binding most likely by regulating the position of the flexible C-domain. It also highlighted ssDNA-induced conformational mobility restrictions of all Ssb variants. Finally, the gel mobility shift assay confirmed that the intrinsically disordered linker is essential for cooperative binding of SsbA. These findings highlight the important role of the C-domain in the functioning of SsbA and SsbB proteins.

Diverse Combinatorial Biosynthesis Strategies for C-H Functionalization of Anthracyclinones.

Streptomyces spp. are "nature's antibiotic factories" that produce valuable bioactive metabolites, such as the cytotoxic anthracycline polyketides. While the anthracyclines have hundreds of natural and chemically synthesized analogues, much of the chemical diversity stems from enzymatic modifications to the saccharide chains and, to a lesser extent, from alterations to the core scaffold. Previous work has resulted in the generation of a BioBricks synthetic biology toolbox in Streptomyces coelicolor M1152ΔmatAB that could produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone. In this work, we extended the platform to generate oxidatively modified analogues via two crucial strategies. (i) We swapped the ketoreductase and first-ring cyclase enzymes for the aromatase cyclase from the mithramycin biosynthetic pathway in our polyketide synthase (PKS) cassettes to generate 2-hydroxylated analogues. (ii) Next, we engineered several multioxygenase cassettes to catalyze 11-hydroxylation, 1-hydroxylation, 10-hydroxylation, 10-decarboxylation, and 4-hydroxyl regioisomerization. We also developed improved plasmid vectors and S. coelicolor M1152ΔmatAB expression hosts to produce anthracyclinones. This work sets the stage for the combinatorial biosynthesis of bespoke anthracyclines using recombinant Streptomyces spp. hosts.

Genomic Insights and Synthetic Biology Applications of Marine Actinomycete Streptomyces griseoincarnatus HNS054.

The marine bacterium Streptomyces sp. HNS054 shows promise as a platform for producing natural products. Isolated from a marine sponge, HNS054 possesses several desirable traits for bioengineering: rapid growth, salt tolerance, and compatibility with genetic tools. Its genome contains 21 potential biosynthetic gene clusters, offering a rich source of natural products. We successfully engineered HNS054 to increase the production of aborycin and actinorhodin by 4.5-fold and 1.2-fold, respectively, compared to S. coelicolor M1346 counterparts. With its unique features and amenability to genetic manipulation, HNS054 emerges as a promising candidate for developing novel marine-derived drugs and other valuable compounds.

Heterologous production of (-)-geosmin in Saccharomyces cerevisiae.

(-)-Geosmin has high demand in perfumes and cosmetic products for its earthy congenial aroma. The current production of (-)-geosmin is either by distillation of sun-baked soil or by inefficient chemical synthesis because of the presence of multiple chiral centers. Fermentation processes are not viable as the titers of the Streptomyces sp. based processes are low. This work presents an alternative route by the heterologous synthesis of (-)-geosmin in Saccharomyces cerevisiae. The enzyme involved is the bifunctional geosmin synthase that catalyzes the conversion of farnesyl diphosphate to germacradienol and germacradienol to geosmin. This study evaluated the activity of many orthologs of geosmin synthase when expressed heterologously in S. cerevisiae. When the well-characterized CAB41566 from Streptomyces coelicolor origin was tested, germacradienol and germacrene D were detected but no geosmin. Bioinformatic analysis based on high/low identities to N-terminal and C-terminal domains of CAB41566 was carried out to identify different orthologs of geosmin synthase proteins from different bacterial and fungal origins. ADO68918 of Stigmatella aurantiaca origin showed the best activity among the tested orthologs, not only in terms of geosmin production but also an order of magnitude higher total abundance of the products of geosmin synthase as compared to CAB41566. This study successfully demonstrated the production of (-)-geosmin in S. cerevisiae and offers an alternative, sustainable and environment-friendly approach to producing (-)-geosmin.

Engineering Site 228 of Streptomyces coelicolor Laccase for Optimizing Catalytic Activity.

Malachite green (MG) poses a formidable threat to ecosystems and human health. Laccase emerges as a promising candidate for MG degradation, prompting an investigation into the catalytic activity modulation of a small laccase (SLAC) from Streptomyces coelicolor, with a focus on amino acid position 228. Through saturation mutagenesis, five mutants with a 50% increase in the specific activity were generated. Characterization revealed notable properties, Km of E228F was 8.8% of the wild type (WT), and E288T exhibited a 133% kcat compared to WT. Structural analyses indicated improved hydrophobicity and electrostatic potential on the mutants' surfaces, with the stable E228F-ABTS complex exhibiting reduced flexibility, possibly contributing to the observed decrease in turnover rate. Mutants demonstrated enhanced MG decolorization, particularly E228G. Site 228 acts as a crucial functional control switch, suggesting its potential role in SLAC engineering. This study provides insights into laccase modulation and offers promising avenues for enzymatic bioremediation applications.

Multiple SigB homologues govern the transcription of the ssgBp promoter in the sporulation-specific ssgB gene in Streptomyces coelicolor A3(2).

Unlike Bacillus subtilis, Streptomyces coelicolor contains nine SigB homologues of the stress-response sigma factor SigB. By using a two-plasmid system, we previously identified promoters recognized by these sigma factors. Almost all promoters were recognized by several SigB homologues. However, no specific sequences of these promoters were found. One of these promoters, ssgBp, was selected to examine this cross-recognition in the native host. It controls the expression of the sporulation-specific gene ssgB. Using a luciferase reporter, the activity of this promoter in S. coelicolor and nine mutant strains lacking individual sigB homologous genes showed that sgBp is dependent on three sigma factors, SigH, SigN, and SigI. To determine which nucleotides in the-10 region are responsible for the selection of a specific SigB homologue, promoters mutated at the last three nucleotide positions were tested in the two-plasmid system. Some mutant promoters were specifically recognized by a distinct set of SigB homologues. Analysis of these mutant promoters in the native host showed the role of these nucleotides. A conserved nucleotide A at position 5 was essential for promoter activity, and two variable nucleotides at positions 4 and 6 were responsible for the partial selectivity of promoter recognition by SigB homologues.

Secretory production and characterization of a highly effective chitosanase from Streptomyces coelicolor A3(2) M145 in Pichia pastoris.

In this study, a glycoside hydrolase family 46 chitosanase from Streptomyces coelicolor A3(2) M145 was firstly cloned and expressed in Pichia pastoris GS115 (P. pastoris GS115). The recombinant enzyme (CsnA) showed maximal activity at pH 6.0 and 65°C. Both thermal stability and pH stability of CsnA expressed in P. pastoris GS115 were significantly increased compared with homologous expression in Streptomyces coelicolor A3(2). A stable chitosanase activity of 725.7 ± 9.58 U mL-1 was obtained in fed-batch fermentation. It's the highest level of CsnA from Streptomyces coelicolor expressed in P. pastoris so far. The hydrolytic process of CsnA showed a time-dependent manner. Chitosan oligosaccharides (COSs) generated by CsnA showed antifungal activity against Fusarium oxysporum sp. cucumerinum (F. oxysporum sp. cucumerinum). The secreted expression and hydrolytic performance make the enzyme a desirable biocatalyst for industrial controllable production of chitooligosaccharides with specific degree of polymerization, which have potential to control fungi that cause important crop diseases.

RIP-seq reveals RNAs that interact with RNA polymerase and primary sigma factors in bacteria.

Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.