Redefining development in Streptomyces venezuelae: integrating exploration into the classical sporulating life cycle.

Two growth modes have been described for the filamentous Streptomyces bacteria. Their classic developmental life cycle culminates in the formation of dormant spores, where movement to new environments is mediated through spore dispersal. In contrast, exploratory growth proceeds as a rapidly expanding vegetative mycelium that leads to extensive surface colonization and is associated with the release of volatile compounds that promote alkalinization (and reduced iron bioavailability) of its surrounding environment. Here, we report that exploratory growth in Streptomyces venezuelae can proceed in tandem with classic sporulating development in response to specific nutritional cues. Sporulating exploration is not accompanied by a rise in environmental pH but has the same iron acquisition requirements as conventional exploration. We found that mutants that were defective in their ability to sporulate were unaffected in exploration, but mutants undergoing precocious sporulation were compromised in their exploratory growth and this appeared to be mediated through premature activation of the developmental regulator WhiI. Cell envelope integrity was also found to be critical for exploration, as mutations in the cell envelope stress-responsive extracytoplasmic function sigma factor SigE led to a failure to explore robustly under all exploration-promoting conditions. Finally, in expanding the known exploration-promoting conditions, we discovered that the model species Streptomyces lividans exhibited exploration capabilities, supporting the proposal that exploration is conserved across diverse streptomycetes. IMPORTANCE: Streptomyces bacteria have evolved diverse developmental and metabolic strategies to thrive in dynamic environmental niches. Here, we report the amalgamation of previously disparate developmental pathways, showing that colony expansion via exploration can proceed in tandem with colony sporulation. This developmental integration extends beyond phenotype to include shared genetic elements, with sporulation-specific repressors being required for successful exploration. Comparing this new exploration mode with previously identified strategies has revealed key differences (e.g., no need for environmental alkalinization), and simultaneously allowed us to define unifying requirements for Streptomyces exploration. The "reproductive exploration" phenomenon reported here represents a unique bet-hedging strategy, with the Streptomyces colony engaging in an aggressive colonization strategy while transporting a protected genetic repository.

Multifactorial genetic control and magnesium levels govern the production of a Streptomyces antibiotic with unusual cell density dependence.

Streptomyces bacteria are renowned both for their antibiotic production capabilities and for their cryptic metabolic potential. Their metabolic repertoire is subject to stringent genetic control, with many of the associated biosynthetic gene clusters being repressed by the conserved nucleoid-associated protein Lsr2. In an effort to stimulate new antibiotic production in wild Streptomyces isolates, we leveraged the activity of an Lsr2 knockdown construct and successfully enhanced antibiotic production in the wild Streptomyces isolate WAC07094. We determined that this new activity stemmed from increased levels of the angucycline-like family member saquayamycin. Saquayamycin has both antibiotic and anti-cancer activities, and intriguingly, beyond Lsr2-mediated repression, we found saquayamycin production was also suppressed at high density on solid or in liquid growth media; its levels were greatest in low-density cultures. This density-dependent control was exerted at the level of the cluster-situated regulatory gene sqnR and was mediated in part through the activity of the PhoRP two-component regulatory system, where deleting phoRP led to both constitutive antibiotic production and sqnR expression. This suggests that PhoP functions to repress the expression of sqnR at high cell density. We further discovered that magnesium supplementation could alleviate this density dependence, although its action was independent of PhoP. Finally, we revealed that the nitrogen-responsive regulators GlnR and AfsQ1 could relieve the repression exerted by Lsr2 and PhoP. Intriguingly, we found that this low density-dependent production of saquayamycin was not unique to WAC07094; saquayamycin production by another wild isolate also exhibited low-density activation, suggesting that this spatial control may serve an important ecological function in their native environments.IMPORTANCEStreptomyces specialized metabolic gene clusters are subject to complex regulation, and their products are frequently not observed under standard laboratory growth conditions. For the wild Streptomyces isolate WAC07094, production of the angucycline-family compound saquayamycin is subject to a unique constellation of control factors. Notably, it is produced primarily at low cell density, in contrast to the high cell density production typical of most antibiotics. This unusual density dependence is conserved in other saquayamycin producers and is driven by the pathway-specific regulator SqnR, whose expression is influenced by both nutritional and genetic elements. Collectively, this work provides new insights into an intricate regulatory system governing antibiotic production and indicates there may be benefits to including low-density cultures in antibiotic screening platforms.

The Best of Both Worlds-Streptomyces coelicolor and Streptomyces venezuelae as Model Species for Studying Antibiotic Production and Bacterial Multicellular Development.

Streptomyces bacteria have been studied for more than 80 years thanks to their ability to produce an incredible array of antibiotics and other specialized metabolites and their unusual fungal-like development. Their antibiotic production capabilities have ensured continual interest from both academic and industrial sectors, while their developmental life cycle has provided investigators with unique opportunities to address fundamental questions relating to bacterial multicellular growth. Much of our understanding of the biology and metabolism of these fascinating bacteria, and many of the tools we use to manipulate these organisms, have stemmed from investigations using the model species Streptomyces coelicolor and Streptomyces venezuelae. Here, we explore the pioneering work in S. coelicolor that established foundational genetic principles relating to specialized metabolism and development, alongside the genomic and cell biology developments that led to the emergence of S. venezuelae as a new model system. We highlight key discoveries that have stemmed from studies of these two systems and discuss opportunities for future investigations that leverage the power and understanding provided by S. coelicolor and S. venezuelae.

Streptomyces behavior and competition in the natural environment.

Streptomyces are ubiquitous terrestrial bacteria that are renowned for their robust metabolic capabilities and their behavioral flexibility. In competing for environmental niches, these bacteria can employ novel growth and dispersal behaviors. They also wield their diverse metabolic repertoire for everything from maximizing nutrient uptake, to preventing phage replication or inhibiting bacterial and fungal growth. Increasingly, they are found to live in association with plants and insects, often conferring protective benefits to their host courtesy of their ability to produce pathogen-inhibitory antimicrobial compounds. Here, we highlight recent advances in understanding the competitive and cooperative interactions between Streptomyces and phage, microbes, and higher organisms in their environment.

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.

Carfentanil Blood Concentrations in Impaired Driving Investigations in Ontario, Canada.

Carfentanil is a potent opioid with no medical use in humans; it presents a serious threat to public health and road safety due to its presence in the illicit drug supply, the potency of the drug and instances of use prior to the operation of a motor vehicle. The identification and quantitation of carfentanil using liquid chromatography with tandem mass spectrometry was performed in blood samples obtained through impaired driving-related investigations from 2017 to 2019. In a series of 66 cases submitted to the Centre of Forensic Sciences in Ontario, Canada, blood concentrations of carfentanil ranged from <0.04 to 2.1 ng/mL in the population studied. Driving behaviors frequently came to the attention of concerned citizens, decreased levels of consciousness were commonly reported and a variety of medical interventions were required, in some cases, to preserve life due to apparent opioid toxicity.

Inducing Global Expression of Actinobacterial Biosynthetic Gene Clusters.

Bacteria produce an impressive array of bioactive specialized metabolites, with Streptomyces (and the actinobacteria more generally) being unusually diverse and prolific producers. However, the biosynthetic potential of these organisms has yet to be fully explored, as many of the biosynthetic gene clusters that direct the synthesis of these natural products are transcriptionally silent under laboratory growth conditions. Here, we describe strategies that can be employed to broadly stimulate the expression of biosynthetic gene clusters in Streptomyces and their relatives, follow the transcription of these genes, and assess the antimicrobial activity of the resulting molecules.

How Streptomyces thrive: Advancing our understanding of classical development and uncovering new behaviors.

Streptomyces are soil- and marine-dwelling microbes that need to survive dramatic fluctuations in nutrient levels and environmental conditions. Here, we explore the advances made in understanding how Streptomyces bacteria can thrive in their natural environments. We examine their classical developmental cycle, and the intricate regulatory cascades that govern it. We discuss alternative growth strategies and behaviors, like the rapid expansion and colonization properties associated with exploratory growth, the release of membrane vesicles and S-cells from hyphal tips, and the acquisition of exogenous DNA along the lateral walls. We further investigate Streptomyces interactions with other organisms through the release of volatile compounds that impact nutrient levels, microbial growth, and insect behavior. Finally, we explore the increasingly diverse strategies employed by Streptomyces species in escaping and thwarting phage infections.

Exploratory Growth in Streptomyces venezuelae Involves a Unique Transcriptional Program, Enhanced Oxidative Stress Response, and Profound Acceleration in Response to Glycerol.

Exploration is a recently discovered mode of growth and behavior exhibited by some Streptomyces species that is distinct from their classical sporulating life cycle. While much has been uncovered regarding initiating environmental conditions and phenotypic outcomes of exploratory growth, how this process is coordinated at a genetic level remains unclear. We used RNA sequencing to survey global changes in the transcriptional profile of exploring cultures over time in the model organism Streptomyces venezuelae. Transcriptomic analyses revealed widespread changes in gene expression impacting diverse cellular functions. Investigations into differentially expressed regulatory elements revealed specific groups of regulatory factors to be impacted, including the expression of several extracytoplasmic function (ECF) sigma factors, second messenger signaling pathways, and members of the whiB-like (wbl) family of transcription factors. Dramatic changes were observed among primary metabolic pathways, especially among respiration-associated genes and the oxidative stress response; enzyme assays confirmed that exploring cultures exhibit an enhanced oxidative stress response compared with classically growing cultures. Changes in the expression of the glycerol catabolic genes in S. venezuelae led to the discovery that glycerol supplementation of the growth medium promotes a dramatic acceleration of exploration. This effect appears to be unique to glycerol as an alternative carbon source, and this response is broadly conserved across other exploration-competent species. IMPORTANCE Exploration represents an alternative growth strategy for Streptomyces bacteria and is initiated in response to other microbes or specific environmental conditions. Here, we show that entry into exploration involves comprehensive transcriptional reprogramming, with an emphasis on changes in primary metabolism and regulatory/signaling functions. Intriguingly, a number of transcription factor classes were downregulated upon entry into exploration. In contrast, respiration-associated genes were strongly induced, and this was accompanied by an enhanced oxidative stress response. Notably, our transcriptional analyses suggested that glycerol may play a role in exploration, and we found that glycerol supplementation dramatically enhanced the exploration response in many streptomycetes. This work sheds new light on the regulatory and metabolic cues that influence a fascinating new microbial behavior.

Global Chromosome Topology and the Two-Component Systems in Concerted Manner Regulate Transcription in Streptomyces.

Bacterial gene expression is controlled at multiple levels, with chromosome supercoiling being one of the most global regulators. Global DNA supercoiling is maintained by the orchestrated action of topoisomerases. In Streptomyces, mycelial soil bacteria with a complex life cycle, topoisomerase I depletion led to elevated chromosome supercoiling, changed expression of a significant fraction of genes, delayed growth, and blocked sporulation. To identify supercoiling-induced sporulation regulators, we searched for Streptomyces coelicolor transposon mutants that were able to restore sporulation despite high chromosome supercoiling. We established that transposon insertion in genes encoding a novel two-component system named SatKR reversed the sporulation blockage resulting from topoisomerase I depletion. Transposition in satKR abolished the transcriptional induction of the genes within the so-called supercoiling-hypersensitive cluster (SHC). Moreover, we found that activated SatR also induced the same set of SHC genes under normal supercoiling conditions. We determined that the expression of genes in this region impacted S. coelicolor growth and sporulation. Interestingly, among the associated products is another two-component system (SitKR), indicating the potential for cascading regulatory effects driven by the SatKR and SitKR two-component systems. Thus, we demonstrated the concerted activity of chromosome supercoiling and a hierarchical two-component signaling system that impacts gene activity governing Streptomyces growth and sporulation. IMPORTANCE Streptomyces microbes, soil bacteria with complex life cycle, are the producers of a broad range of biologically active compounds (e.g., antibiotics). Streptomyces bacteria respond to various environmental signals using a complex transcriptional regulation mechanism. Understanding regulation of their gene expression is crucial for Streptomyces application as industrial organisms. Here, on the basis of the results of extensive transcriptomics analyses, we describe the concerted gene regulation by global DNA supercoiling and novel two-component system. Our data indicate that regulated genes encode growth and sporulation regulators. Thus, we demonstrate that Streptomyces bacteria link the global regulatory strategies to adjust life cycle to unfavorable conditions.

6S-Like scr3559 RNA Affects Development and Antibiotic Production in Streptomyces coelicolor.

Regulatory RNAs control a number of physiological processes in bacterial cells. Here we report on a 6S-like RNA transcript (scr3559) that affects both development and antibiotic production in Streptomyces coelicolor. Its expression is enhanced during the transition to stationary phase. Strains that over-expressed the scr3559 gene region exhibited a shortened exponential growth phase in comparison with a control strain; accelerated aerial mycelium formation and spore maturation; alongside an elevated production of actinorhodin and undecylprodigiosin. These observations were supported by LC-MS analyses of other produced metabolites, including: germicidins, desferrioxamines, and coelimycin. A subsequent microarray differential analysis revealed increased expression of genes associated with the described morphological and physiological changes. Structural and functional similarities between the scr3559 transcript and 6S RNA, and its possible employment in regulating secondary metabolite production are discussed.

Interplay between Nucleoid-Associated Proteins and Transcription Factors in Controlling Specialized Metabolism in Streptomyces.

Lsr2 is a small nucleoid-associated protein found throughout the actinobacteria. Lsr2 functions similarly to the well-studied H-NS, in that it preferentially binds AT-rich sequences and represses gene expression. In Streptomyces venezuelae, Lsr2 represses the expression of many specialized metabolic clusters, including the chloramphenicol antibiotic biosynthetic gene cluster, and deleting lsr2 leads to significant upregulation of chloramphenicol cluster expression. We show here that Lsr2 likely exerts its repressive effects on the chloramphenicol cluster by polymerizing along the chromosome and by bridging sites within and adjacent to the chloramphenicol cluster. CmlR is a known activator of the chloramphenicol cluster, but expression of its associated gene is not upregulated in an lsr2 mutant strain. We demonstrate that CmlR is essential for chloramphenicol production, and further reveal that CmlR functions to "countersilence" Lsr2's repressive effects by recruiting RNA polymerase and enhancing transcription, with RNA polymerase effectively clearing bound Lsr2 from the chloramphenicol cluster DNA. Our results provide insight into the interplay between opposing regulatory proteins that govern antibiotic production in S. venezuelae, which could be exploited to maximize the production of bioactive natural products in other systems. IMPORTANCE Specialized metabolic clusters in Streptomyces are the source of many clinically prescribed antibiotics. However, many clusters are not expressed in the laboratory due to repression by the nucleoid-associated protein Lsr2. Understanding how Lsr2 represses cluster expression, and how repression can be alleviated, is key to accessing the metabolic potential of these bacteria. Using the chloramphenicol biosynthetic cluster from Streptomyces venezuelae as a model, we explored the mechanistic basis underlying Lsr2-mediated repression, and activation by the pathway-specific regulator CmlR. Lsr2 polymerized along the chromosome and bridged binding sites located within and outside the cluster, promoting repression. Conversely, CmlR was essential for chloramphenicol production and further functioned to countersilence Lsr2 repression by recruiting RNA polymerase and promoting transcription, ultimately removing Lsr2 polymers from the chromosome. Manipulating the activity of both regulators led to a >130× increase in chloramphenicol levels, suggesting that combinatorial regulatory strategies can be powerful tools for maximizing natural product yields.

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.

Tactic-specific antimicrobial activity suggests a parental care function for accessory glands in a marine toadfish.

Males of some species possess extra reproductive organs called accessory glands which are outgrowths of the testes or sperm duct. These organs have a well-established role in reproduction; however, they also appear to have other important functions that are less understood. Here, we investigate the function of the highly complex accessory glands of a marine toadfish, Porichthys notatus, a fish with two reproductive male types: large care-providing 'guarder' males and small non-caring 'sneaker' males. While both male types have accessory glands, guarder male accessory glands are much larger relative to their body size. We show that accessory gland fluids strongly inhibit the growth of bacterial genera associated with unhealthy eggs and have no effect on the growth of strains isolated from healthy eggs. This antibacterial effect was particularly pronounced for extracts from guarder males. Furthermore, we demonstrate that both healthy and unhealthy plainfin midshipman eggs have diverse but distinct microbial communities that differ in their composition and abundance. The highly specific inhibitory capacity of accessory gland fluid on bacteria from unhealthy eggs was robust across a wide range of ecologically relevant temperatures and salinities. Collectively, these ecological and molecular observations suggest a care function for the accessory gland mediated by antimicrobial agents.

Specialized and shared functions of diguanylate cyclases and phosphodiesterases in Streptomyces development.

The second messenger bis-3,5-cyclic di-guanosine monophosphate (c-di-GMP) determines when Streptomyces initiate sporulation. c-di-GMP signals are integrated into the genetic differentiation network by the regulator BldD and the sigma factor σWhiG . However, functions of the development-specific diguanylate cyclases (DGCs) CdgB and CdgC, and the c-di-GMP phosphodiesterases (PDEs) RmdA and RmdB, are poorly understood. Here, we provide biochemical evidence that the GGDEF-EAL domain protein RmdB from S. venezuelae is a monofunctional PDE that hydrolyzes c-di-GMP to 5'pGpG. Despite having an equivalent GGDEF-EAL domain arrangement, RmdA cleaves c-di-GMP to GMP and exhibits residual DGC activity. We show that an intact EAL motif is crucial for the in vivo function of both enzymes since strains expressing protein variants with an AAA motif instead of EAL are delayed in development, similar to null mutants. Transcriptome analysis of ∆cdgB, ∆cdgC, ∆rmdA, and ∆rmdB strains revealed that the c-di-GMP specified by these enzymes has a global regulatory role, with about 20% of all S. venezuelae genes being differentially expressed in the cdgC mutant. Our data suggest that the major c-di-GMP-controlled targets determining the timing and mode of sporulation are genes involved in cell division and the production of the hydrophobic sheath that covers Streptomyces aerial hyphae and spores.

Bacterial Volatile Compounds: Functions in Communication, Cooperation, and Competition.

Bacteria produce a multitude of volatile compounds. While the biological functions of these deceptively simple molecules are unknown in many cases, for compounds that have been characterized, it is clear that they serve impressively diverse purposes. Here, we highlight recent studies that are uncovering the volatile repertoire of bacteria, and the functional relevance and impact of these molecules. We present work showing the ability of volatile compounds to modulate nutrient availability in the environment; alter the growth, development, and motility of bacteria and fungi; influence protist and arthropod behavior; and impact plant and animal health. We further discuss the benefits associated with using volatile compounds for communication and competition, alongside the challenges of studying these molecules and their functional roles. Finally, we address the opportunities these compounds present from commercial, clinical, and agricultural perspectives.

Compaction and control-the role of chromosome-organizing proteins in Streptomyces.

Chromosomes are dynamic entities, whose organization and structure depend on the concerted activity of DNA-binding proteins and DNA-processing enzymes. In bacteria, chromosome replication, segregation, compaction and transcription are all occurring simultaneously, and to ensure that these processes are appropriately coordinated, all bacteria employ a mix of well-conserved and species-specific proteins. Unusually, Streptomyces bacteria have large, linear chromosomes and life cycle stages that include multigenomic filamentous hyphae and unigenomic spores. Moreover, their prolific secondary metabolism yields a wealth of bioactive natural products. These different life cycle stages are associated with profound changes in nucleoid structure and chromosome compaction, and require distinct repertoires of architectural-and regulatory-proteins. To date, chromosome organization is best understood during Streptomyces sporulation, when chromosome segregation and condensation are most evident, and these processes are coordinated with synchronous rounds of cell division. Advances are, however, now being made in understanding how chromosome organization is achieved in multigenomic hyphal compartments, in defining the functional and regulatory interplay between different architectural elements, and in appreciating the transcriptional control exerted by these 'structural' proteins.

Roles of LysM and LytM domains in resuscitation-promoting factor (Rpf) activity and Rpf-mediated peptidoglycan cleavage and dormant spore reactivation.

Bacterial dormancy can take many forms, including formation of Bacillus endospores, Streptomyces exospores, and metabolically latent Mycobacterium cells. In the actinobacteria, including the streptomycetes and mycobacteria, the rapid resuscitation from a dormant state requires the activities of a family of cell-wall lytic enzymes called resuscitation-promoting factors (Rpfs). Whether Rpf activity promotes resuscitation by generating peptidoglycan fragments (muropeptides) that function as signaling molecules for spore germination or by simply remodeling the dormant cell wall has been the subject of much debate. Here, to address this question, we used mutagenesis and peptidoglycan binding and cleavage assays to first gain broader insight into the biochemical function of diverse Rpf enzymes. We show that their LysM and LytM domains enhance Rpf enzyme activity; their LytM domain and, in some cases their LysM domain, also promoted peptidoglycan binding. We further demonstrate that the Rpfs function as endo-acting lytic transglycosylases, cleaving within the peptidoglycan backbone. We also found that unlike in other systems, Rpf activity in the streptomycetes is not correlated with peptidoglycan-responsive Ser/Thr kinases for cell signaling, and the germination of rpf mutant strains could not be stimulated by the addition of known germinants. Collectively, these results suggest that in Streptomyces, Rpfs have a structural rather than signaling function during spore germination, and that in the actinobacteria, any signaling function associated with spore resuscitation requires the activity of additional yet to be identified enzymes.

Streptomyces IHF uses multiple interfaces to bind DNA.

BACKGROUND: Nucleoid associated proteins (NAPs) are essential for chromosome condensation in bacterial cells. Despite being a diverse group, NAPs share two common traits: they are small, oligomeric proteins and their oligomeric state is critical for DNA condensation. Streptomyces coelicolor IHF (sIHF) is an actinobacterial-specific nucleoid-associated protein that despite its name, shares neither sequence nor structural homology with the well-characterized Escherichia coli IHF. Like E. coli IHF, sIHF is needed for efficient nucleoid condensation, morphological development and antibiotic production in S. coelicolor. METHODS: Using a combination of crystallography, small-angle X-ray scattering, electron microscopy and structure-guided functional assays, we characterized how sIHF binds and remodels DNA. RESULTS: The structure of sIHF bound to DNA revealed two DNA-binding elements on opposite surfaces of the helix bundle. Using structure-guided functional assays, we identified an additional surface that drives DNA binding in solution. Binding by each element is necessary for both normal development and antibiotic production in vivo, while in vitro, they act collectively to restrain negative supercoils. CONCLUSIONS: The cleft defined by the N-terminal and the helix bundle of sIHF drives DNA binding, but the two additional surfaces identified on the crystal structure are necessary to stabilize binding, remodel DNA and maintain wild-type levels of antibiotic production. We propose a model describing how the multiple DNA-binding elements enable oligomerization-independent nucleoid condensation. GENERAL SIGNIFICANCE: This work provides a new dimension to the mechanistic repertoire ascribed to bacterial NAPs and highlights the power of combining structural biology techniques to study sequence unspecific protein-DNA interactions.

Transcriptional Response of Streptomyces coelicolor to Rapid Chromosome Relaxation or Long-Term Supercoiling Imbalance.

Negative DNA supercoiling allows chromosome condensation and facilitates DNA unwinding, which is required for the occurrence of DNA transaction processes, i.e., DNA replication, transcription and recombination. In bacteria, changes in chromosome supercoiling impact global gene expression; however, the limited studies on the global transcriptional response have focused mostly on pathogenic species and have reported various fractions of affected genes. Furthermore, the transcriptional response to long-term supercoiling imbalance is still poorly understood. Here, we address the transcriptional response to both novobiocin-induced rapid chromosome relaxation or long-term topological imbalance, both increased and decreased supercoiling, in environmental antibiotic-producing bacteria belonging to the Streptomyces genus. During the Streptomyces complex developmental cycle, multiple copies of GC-rich linear chromosomes present in hyphal cells undergo profound topological changes, from being loosely condensed in vegetative hyphae, to being highly compacted in spores. Moreover, changes in chromosomal supercoiling have been suggested to be associated with the control of antibiotic production and environmental stress response. Remarkably, in S. coelicolor, a model Streptomyces species, topoisomerase I (TopA) is solely responsible for the removal of negative DNA supercoils. Using a S. coelicolor strain in which topA transcription is under the control of an inducible promoter, we identified genes involved in the transcriptional response to long-term supercoiling imbalance. The affected genes are preferentially organized in several clusters, and a supercoiling-hypersensitive cluster (SHC) was found to be located in the core of the S. coelicolor chromosome. The transcripts affected by long-term topological imbalance encompassed genes encoding nucleoid-associated proteins, DNA repair proteins and transcriptional regulators, including multiple developmental regulators. Moreover, using a gyrase inhibitor, we identified those genes that were directly affected by novobiocin, and found this was correlated with increased AT content in their promoter regions. In contrast to the genes affected by long-term supercoiling changes, among the novobiocin-sensitive genes, a significant fraction encoded for proteins associated with membrane transport or secondary metabolite synthesis. Collectively, our results show that long-term supercoiling imbalance globally regulates gene transcription and has the potential to impact development, secondary metabolism and DNA repair, amongst others.