The stringent response is strongly activated in the antibiotic producing strain, Streptomyces coelicolor.

S. lividans and S. coelicolor are phylogenetically closely related strains with different abilities to produce the same specialized metabolites. Previous studies revealed that the strong antibiotic producer, S. coelicolor, had a lower ability to assimilate nitrogen and phosphate than the weak producer, Streptomyces lividans, and this resulted into a lower growth rate. A comparative proteomic dataset was used to establish the consequences of these nutritional stresses on the abundance of proteins of the translational apparatus of these strains, grown in low and high phosphate availability. Our study revealed that most proteins of the translational apparatus were less abundant in S. coelicolor than in S. lividans whereas it was the opposite for ET-Tu 3 and a TrmA-like methyltransferase. The expression of the latter being known to be under the positive control of the stringent response whereas that of the other ribosomal proteins is under its negative control, this indicated the occurrence of a strong activation of the stringent response in S. coelicolor. Furthermore, in S. lividans, ribosomal proteins were more abundant in phosphate proficiency than in phosphate limitation suggesting that a limitation in phosphate, that was also shown to trigger RelA expression, contributes to the induction of the stringent response.

Cross-Recognition of Promoters by the Nine SigB Homologues Present in Streptomyces coelicolor A3(2).

In contrast to Bacillus subtilis, Streptomyces coelicolor A3(2) contains nine homologues of stress response sigma factor SigB with a major role in differentiation and osmotic stress response. The aim of this study was to further characterize these SigB homologues. We previously established a two-plasmid system to identify promoters recognized by sigma factors and used it to identify promoters recognized by the three SigB homologues, SigF, SigG, and SigH from S. coelicolor A3(2). Here, we used this system to identify 14 promoters recognized by SigB. The promoters were verified in vivo in S. coelicolor A3(2) under osmotic stress conditions in sigB and sigH operon mutants, indicating some cross-recognition of these promoters by these two SigB homologues. This two-plasmid system was used to examine the recognition of all identified SigB-, SigF-, SigG-, and SigH-dependent promoters with all nine SigB homologues. The results confirmed this cross-recognition. Almost all 24 investigated promoters were recognized by two or more SigB homologues and data suggested some distinguishing groups of promoters recognized by these sigma factors. However, analysis of the promoters did not reveal any specific sequence characteristics for these recognition groups. All promoters showed high similarity in the -35 and -10 regions. Immunoblot analysis revealed the presence of SigB under osmotic stress conditions and SigH during morphological differentiation. Together with the phenotypic analysis of sigB and sigH operon mutants in S. coelicolor A3(2), the results suggest a dominant role for SigB in the osmotic stress response and a dual role for SigH in the osmotic stress response and morphological differentiation. These data suggest a complex regulation of the osmotic stress response in relation to morphological differentiation in S. coelicolor A3(2).

Heterologous expression of 8-demethyl-tetracenomycin (8-dmtc) affected Streptomyces coelicolor life cycle.

Heterologous hosts are highly important to detect the expression of biosynthetic gene clusters that are cryptic or poorly expressed in their natural hosts. To investigate whether actinorhodin-overproducer Streptomyces coelicolor ∆ppk mutant strain could be a possible prototype as a heterologous expression host, a cosmid containing most of the elm gene cluster of Streptomyces olivaceus Tü2353 was integrated into chromosomes of both S. coelicolor A3(2) and ∆ppk strains. Interestingly, it was found that the production of tetracyclic polyketide 8-demethyl-tetracenomycin (8-DMTC) by recombinant strains caused significant changes in the morphology of cells. All the pellets and clumps were disentangled and mycelia were fragmented in the recombinant strains. Moreover, they produce neither pigmented antibiotics nor agarase and did not sporulate. By eliminating the elm biosynthesis genes from the cosmid, we showed that the morphological properties of recombinants were caused by the production of 8-DMTC. Extracellular application of 8-DMTC on S. coelicolor wild-type cells caused a similar phenotype with the 8-DMTC-producing recombinant strains. The results of this study may contribute to the understanding of the effect of 8-DMTC in Streptomyces since the morphological changes that we have observed have not been reported before. It is also valuable in that it provides useful information about the use of Streptomyces as hosts for the heterologous expression of 8-DMTC.

Teichoic acids anchor distinct cell wall lamellae in an apically growing bacterium.

The bacterial cell wall is a multicomponent structure that provides structural support and protection. In monoderm species, the cell wall is made up predominantly of peptidoglycan, teichoic acids and capsular glycans. Filamentous monoderm Actinobacteria incorporate new cell-wall material at their tips. Here we use cryo-electron tomography to reveal the architecture of the actinobacterial cell wall of Streptomyces coelicolor. Our data shows a density difference between the apex and subapical regions. Removal of teichoic acids results in a patchy cell wall and distinct lamellae. Knock-down of tagO expression using CRISPR-dCas9 interference leads to growth retardation, presumably because build-in of teichoic acids had become rate-limiting. Absence of extracellular glycans produced by MatAB and CslA proteins results in a thinner wall lacking lamellae and patches. We propose that the Streptomyces cell wall is composed of layers of peptidoglycan and extracellular polymers that are structurally supported by teichoic acids.

Expression of genes of the Pho regulon is altered in Streptomyces coelicolor.

Most currently used antibiotics originate from Streptomycetes and phosphate limitation is an important trigger of their biosynthesis. Understanding the molecular processes underpinning such regulation is of crucial importance to exploit the great metabolic diversity of these bacteria and get a better understanding of the role of these molecules in the physiology of the producing bacteria. To contribute to this field, a comparative proteomic analysis of two closely related model strains, Streptomyces lividans and Streptomyces coelicolor was carried out. These strains possess identical biosynthetic pathways directing the synthesis of three well-characterized antibiotics (CDA, RED and ACT) but only S. coelicolor expresses them at a high level. Previous studies established that the antibiotic producer, S. coelicolor, is characterized by an oxidative metabolism and a reduced triacylglycerol content compared to the none producer, S. lividans, characterized by a glycolytic metabolism. Our proteomic data support these findings and reveal that these drastically different metabolic features could, at least in part, due to the weaker abundance of proteins of the two component system PhoR/PhoP in S. coelicolor compared to S. lividans. In condition of phosphate limitation, PhoR/PhoP is known to control positively and negatively, respectively, phosphate and nitrogen assimilation and our study revealed that it might also control the expression of some genes of central carbon metabolism. The tuning down of the regulatory role of PhoR/PhoP in S. coelicolor is thus expected to be correlated with low and high phosphate and nitrogen availability, respectively and with changes in central carbon metabolic features. These changes are likely to be responsible for the observed differences between S. coelicolor and S. lividans concerning energetic metabolism, triacylglycerol biosynthesis and antibiotic production. Furthermore, a novel view of the contribution of the bio-active molecules produced in this context, to the regulation of the energetic metabolism of the producing bacteria, is proposed and discussed.

A Comparison of Protein and mRNA Expression during Development of the Soil Dwelling Prokaryote (S. coelicolor).

In this paper, correlation analysis of protein and mRNA levels in the soil dwelling bacteria Streptomyces coelicolor (S. coelicolor M145) is presented during development of the population as it grew in liquid medium using three biological and two technical replicates, measured during exponential growth, and its entry into the stationary phase. The proteome synthesis time series are compared with the gene expression time series measured previously under identical experimental conditions. Results reveal that about one third of protein/mRNA synthesis profiles are well correlated while another third are correlated negatively. Functional analysis of the highly correlated groups is presented. Based on numerical simulation, the negative correlation between protein and mRNA is shown to be caused by the difference between the rate of translation and protein degradation.

The Potential for Redox-Active Metabolites To Enhance or Unlock Anaerobic Survival Metabolisms in Aerobes.

Classifying microorganisms as "obligate" aerobes has colloquially implied death without air, leading to the erroneous assumption that, without oxygen, they are unable to survive. However, over the past few decades, more than a few obligate aerobes have been found to possess anaerobic energy conservation strategies that sustain metabolic activity in the absence of growth or at very low growth rates. Similarly, studies emphasizing the aerobic prowess of certain facultative aerobes have sometimes led to underrecognition of their anaerobic capabilities. Yet an inescapable consequence of the affinity both obligate and facultative aerobes have for oxygen is that the metabolism of these organisms may drive this substrate to scarcity, making anoxic survival an essential skill. To illustrate this, we highlight the importance of anaerobic survival strategies for Pseudomonas aeruginosa and Streptomyces coelicolor, representative facultative and obligate aerobes, respectively. Included among these strategies, we describe a role for redox-active secondary metabolites (RAMs), such as phenazines made by P. aeruginosa, in enhancing substrate-level phosphorylation. Importantly, RAMs are made by diverse bacteria, often during stationary phase in the absence of oxygen, and can sustain anoxic survival. We present a hypothesis for how RAMs may enhance or even unlock energy conservation pathways that facilitate the anaerobic survival of both RAM producers and nonproducers.

High Spatial Resolution Imaging Mass Spectrometry Reveals Chemical Heterogeneity Across Bacterial Microcolonies.

Microbes interact with the world around them at the chemical level. However, directly examining the chemical exchange between microbes and microbes and their environment, at ecological scales, i.e., the scale of a single bacterial cell or small groups of cells, remains a key challenge. Here we address this obstacle by presenting a methodology that enables matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) of bacterial microcolonies. By combining optimized sample preparation with subatmospheric pressure MALDI, we demonstrate that chemical output from groups of as few as ∼50 cells can be visualized with MALDI-IMS. Application of this methodology to Bacillus subtilis and Streptomyces coelicolor revealed heterogeneity in chemical output across microcolonies and asymmetrical metabolite production when cells grew within physiological gradients produced by Medicago sativa roots. Taken together, these results indicate that MALDI-IMS can readily visualize metabolites made by very small assemblages of bacterial cells and that even these small groups of cells can differentially produce metabolites in response to local chemical gradients.

Reconciling DNA replication and transcription in a hyphal organism: visualizing transcription complexes in live Streptomyces coelicolor.

Reconciling transcription and DNA replication in the growing hyphae of the filamentous bacterium Streptomyces presents several physical constraints on growth due to their apically extending and branching, multigenomic cells and chromosome replication being independent of cell division. Using a GFP translational fusion to the ß'-subunit of RNA polymerase (rpoC-egfp), in its native chromosomal location, we observed growing Streptomyces hyphae using time-lapse microscopy throughout the lifecycle and under different growth conditions. The RpoC-eGFP fusion co-localized with DNA around 1.8 µm behind the extending tip, whereas replisomes localize around 4-5 µm behind the tip, indicating that at the growing tip, transcription and chromosome replication are to some degree spatially separated. Dual-labelled RpoC-egfp/DnaN-mCherry strains also indicate that there is limited co-localization of transcription and chromosome replication at the extending hyphal tip. This likely facilitates the use of the same DNA molecule for active transcription and chromosome replication in growing cells, independent of cell division. This represents a novel, but hitherto unknown mechanism for reconciling two fundamental processes that utilize the same macromolecular template that allows for rapid growth without compromising chromosome replication in filamentous bacteria and may have implications for evolution of filamentous growth in micro-organisms, where uncoupling of DNA replication from cell division is required.

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

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

Anaerobic nitrate respiration in the aerobe Streptomyces coelicolor A3(2): helping maintain a proton gradient during dormancy.

Respiratory nitrate reductases (Nar) catalyse the reduction of nitrate to nitrite, coupling this process to energy conservation. The obligate aerobic actinobacterium Streptomyces coelicolor synthesizes three Nar enzymes that contribute to maintenance of a membrane potential when either the mycelium or the spores become hypoxic or anoxic. No growth occurs under such conditions but the bacterium survives the lack of O2 by remaining metabolically active; reducing nitrate is one means whereby this process is aided. Nar1 is exclusive to spores, Nar2 to vegetative mycelium and Nar3 to stationary-phase mycelium, each making a distinct contribution to energy conservation. While Nar2 and Nar3 appear to function like conventional menaquinol oxidases, unusually, Nar1 is completely dependent for its activity on a cytochrome bcc-aa 3 oxidase supercomplex. This suggest that electrons within this supercomplex are diverted to Nar1 during O2 limitation. Receiving electrons from this supercomplex potentially allows nitrate reduction to be coupled to the Q-cycle of the cytochrome bcc complex. This modification likely improves the efficiency of energy conservation, extending longevity of spores under O2 limitation. Knowledge gained on the bioenergetics of NO3 - respiration in the actinobacteria will aid our understanding of how many microorganisms survive under conditions of extreme nutrient and energy restriction.

One Extra Copy of lon Gene Causes a Dramatic Increase in Actinorhodin Production by Streptomyces coelicolor A3(2).

ATP-dependent Lon protease plays important roles in different physiological processes, including cellular differentiation of the bacteria and is a part of an important stress response regulon (HspR/HAIR). In Streptomyces, biosynthesis of secondary metabolites starts with cellular differentiation and stress is one of the factor that affect metabolite production. To clarify the effect of Lon protease on secondary metabolite production, we constructed a recombinant strain of Streptomyces coelicolor A3(2) that has one extra copy of lon gene with its own promoter and transcriptional terminator in its genome. Expression of lon gene in the recombinant strain was determined by quantitative real time (RT-qPCR). Actinorhodin and undecylprodigiosin production of the recombinant cell was measured in liquid R2YE and it was found to produce about 34 times more actinorhodin and 9 times more undecylprodigiosin than the wild-type at 168 h of growth. Development of stable Streptomyces strains capable of producing high amounts of secondary metabolites is valuable for biotechnology industry. One extra copy of lon gene is enough to boost antibiotic production by S. coelicolor A3(2) and this change do not cause any metabolic burden in the cell.

Initial Metabolic Step of a Novel Ethanolamine Utilization Pathway and Its Regulation in Streptomyces coelicolor M145.

Streptomyces coelicolor is a Gram-positive soil bacterium with a high metabolic and adaptive potential that is able to utilize a variety of nitrogen sources. However, little is known about the utilization of the alternative nitrogen source ethanolamine. Our study revealed that S. coelicolor can utilize ethanolamine as a sole nitrogen or carbon (N/C) source, although it grows poorly on this nitrogen source due to the absence of a specific ethanolamine permease. Heterologous expression of a putative ethanolamine permease (SPRI_5940) from Streptomycespristinaespiralis positively influenced the biomass accumulation of the overexpression strain grown in defined medium with ethanolamine. In this study, we demonstrated that a glutamine synthetase-like protein, GlnA4 (SCO1613), is involved in the initial metabolic step of a novel ethanolamine utilization pathway in S. coelicolor M145. GlnA4 acts as a gamma-glutamylethanolamide synthetase. Transcriptional analysis revealed that expression of glnA4 was induced by ethanolamine and repressed in the presence of ammonium. Regulation of glnA4 is governed by the transcriptional repressor EpuRI (SCO1614). The ΔglnA4 mutant strain was unable to grow on defined liquid Evans medium supplemented with ethanolamine. High-performance liquid chromatography (HPLC) analysis demonstrated that strain ΔglnA4 is unable to utilize ethanolamine. GlnA4-catalyzed glutamylation of ethanolamine was confirmed in an enzymatic in vitro assay, and the GlnA4 reaction product, gamma-glutamylethanolamide, was detected by HPLC/electrospray ionization-mass spectrometry (HPLC/ESI-MS). In this work, the first step of ethanolamine utilization in S. coelicolor M145 was elucidated, and a putative ethanolamine utilization pathway was deduced based on the sequence similarity and genomic localization of homologous genes.IMPORTANCE Until now, knowledge of the utilization of ethanolamine in Streptomyces was limited. Our work represents the first attempt to reveal a novel ethanolamine utilization pathway in the actinobacterial model organism S. coelicolor through the characterization of the key enzyme gamma-glutamylethanolamide synthetase GlnA4, which is absolutely required for growth in the presence of ethanolamine. The novel ethanolamine utilization pathway is dissimilar to the currently known ethanolamine utilization pathway, which occurs in metabolome. The novel ethanolamine utilization pathway does not result in the production of toxic by-products (such as acetaldehyde); thus, it is not encapsulated. We believe that this contribution is a milestone in understanding the ecology of Streptomyces and the utilization of alternative nitrogen sources. Our report provides new insight into bacterial primary metabolism, which remains complex and partially unexplored.

Novel Two-Component System MacRS Is a Pleiotropic Regulator That Controls Multiple Morphogenic Membrane Protein Genes in Streptomyces coelicolor.

As with most annotated two-component systems (TCSs) of Streptomyces coelicolor, the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS, for morphogenesis and actinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using an S. coelicolor strain expressing MacR-Flag fusion protein, identified in vivo targets of MacR, and DNase I footprinting of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNase I footprinting and electrophoretic mobility shift assays in vitro and by ChIP-quantitative PCR in vivo, and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes, sco6728, sco4924, and sco4011, markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein A (MmpA; SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS from S. avermitilis and S. venezuelae, respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism in StreptomycesIMPORTANCE TCSs help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in the Streptomyces model strain S. coelicolor are unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that other Streptomyces species have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems in Streptomyces.

Non-targeted metabolomics unravels a media-dependent prodiginines production pathway in Streptomyces coelicolor A3(2).

The genus Streptomyces is the best-known source of therapeutic secondary metabolites, especially antibiotics with pharmaceutical applications. Here, we performed a comparative study based on the time-resolved metabolic disparity in S. coelicolor A3(2) subjected to fermentative cultivation in two different types of media (R2YE and RSM3) in order to investigate secondary metabolite production pathways. The relative abundance of secondary metabolites, such as prodiginines, indoles, germicidins, and selected diketopiperazines, was increased in S. coelicolor A3(2) cultivated in R2YE medium compared to that in RSM3 medium, variably at the late-log and stationary phases of fermentative growth. Correlation analysis indicated that "antibiotic prodiginines" contributed maximally to the absorption maxima (A530) of culture supernatants, indicating their optimal production at 96 hours in R2YE medium. A higher abundance of L-proline (48-72 hours) followed by prodiginines (96 hours) was evident, substantiating the intertwined links between precursor and activated prodiginines pathway. Similarly, the higher abundance of indoles was concurrent with tryptophan levels in the shikimate pathway, whereas diketopiperazines were synchronously abundant along with the levels of phenylalanine, leucine, and proline. Additionally, acetyl-CoA induced the acetate pathway, resulting in the production of germicidins. Thus, our results demonstrate that S. coelicolor A3(2) produces specific secondary metabolites by enhancing the dedicated metabolic pathway responsible for their production. In conclusion, our results from this study provide insight into the metabolic pathways of S. coelicolor A3(2), and can be applied to further optimize the production of prodiginines.

Amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen.

The rapid emergence of antibiotic-resistant pathogenic bacteria has accelerated the search for new antibiotics. Many clinically used antibacterials were discovered through culturing a single microbial species under nutrient-rich conditions, but in the environment, bacteria constantly encounter poor nutrient conditions and interact with neighboring microbial species. In an effort to recapitulate this environment, we generated a nine-strain actinomycete community and used 16S rDNA sequencing to deconvolute the stochastic production of antimicrobial activity that was not observed from any of the axenic cultures. We subsequently simplified the community to just two strains and identified Amycolatopsis sp. AA4 as the producing strain and Streptomyces coelicolor M145 as an inducing strain. Bioassay-guided isolation identified amycomicin (AMY), a highly modified fatty acid containing an epoxide isonitrile warhead as a potent and specific inhibitor of Staphylococcus aureus Amycomicin targets an essential enzyme (FabH) in fatty acid biosynthesis and reduces S. aureus infection in a mouse skin-infection model. The discovery of AMY demonstrates the utility of screening complex communities against specific targets to discover small-molecule antibiotics.

Development and validation of an updated computational model of Streptomyces coelicolor primary and secondary metabolism.

BACKGROUND: Streptomyces species produce a vast diversity of secondary metabolites of clinical and biotechnological importance, in particular antibiotics. Recent developments in metabolic engineering, synthetic and systems biology have opened new opportunities to exploit Streptomyces secondary metabolism, but achieving industry-level production without time-consuming optimization has remained challenging. Genome-scale metabolic modelling has been shown to be a powerful tool to guide metabolic engineering strategies for accelerated strain optimization, and several generations of models of Streptomyces metabolism have been developed for this purpose. RESULTS: Here, we present the most recent update of a genome-scale stoichiometric constraint-based model of the metabolism of Streptomyces coelicolor, the major model organism for the production of antibiotics in the genus. We show that the updated model enables better metabolic flux and biomass predictions and facilitates the integrative analysis of multi-omics data such as transcriptomics, proteomics and metabolomics. CONCLUSIONS: The updated model presented here provides an enhanced basis for the next generation of metabolic engineering attempts in Streptomyces.

CRISPR/dCas9-Mediated Multiplex Gene Repression in Streptomyces.

Streptomycetes are Gram-positive bacteria with the capacity to produce copious bioactive secondary metabolites, which are the main source of medically and industrially relevant drugs. However, genetic manipulation of Streptomyces strains is much more difficult than other model microorganisms like Escherichia coli and Saccharomyces cerevisiae. Recently, CRISPR/Cas9 or dCas9-mediated genetic manipulation tools have been developed and facilitated Streptomyces genome editing. However, till now, CRISPR/dCas9-based interference system (CRISPRi) is only designed to repress single gene expression. Herein, the authors developed a novel CRISPRi system for multiplex gene repression in the model strain Streptomyces coelicolor. In this system, the integrative plasmid pSET152 is used as the backbone for the expression of the dCas9/sgRNA complex and both dCas9 and sgRNAs are designed to be under the control of constitutive promoters. Using the integrative CRISPRi system, the authors achieved efficient repression of multiple genes simultaneously; the mRNA levels of four targets are reduced to 2-32% of the control. Furthermore, it is successfully employed for functional gene screening, and an orphan response regulator (RR) (encoded by SCO2013) containing an RNA-binding ANTAR domain is identified being involved in bacterial growth. Collectively, this integrative CRISPRi system is very effective for multiplex gene repression in S. coelicolor, which could be extended to other Streptomyces strains for functional gene screening as well as for metabolic engineering.

SCO5351 is a pleiotropic factor that impacts secondary metabolism and morphological development in Streptomyces coelicolor.

The genome of Streptomyces coelicolor encodes hundreds of putative regulatory proteins, most of which are of unknown function, including SCO5351. In this study, we determined that deletion of sco5351 largely abrogates production of actinorhodin (ACT) and reduces production of the calcium-dependent antibiotic (CDA). Comprehensive transcriptional analyses indicated that transcription of genes of the ACT pathway, including the pathway-specific regulator actII-orf4 and those involved in the building of the chemical compound, was markedly lower in Δsco5351 in the late growth phase. However, transcription of genes in the CDA cluster was notably reduced in Δsco5351 only in the early growth phase, suggesting that SCO5351 has a regulatory role throughout growth. Similar to the observations with Δsco5351, ACT production was blocked by mutagenesis of three conserved amino acids potentially involved in dimerization of SCO5351, indicating that protein dimerization is critical to the function of SCO5351. In addition, disruption of sco5351 delayed the formation of aerial mycelium and spores under the conditions tested and, consistent with this, transcription of developmental genes associated with spore formation was reduced in Δsco5351, implying that SCO5351 is involved in developmental control. Our findings reveal SCO5351 as a pleiotropic regulator with roles in both secondary metabolism and morphological development in S. coelicolor.

Tricarboxylic acid cycle without malate dehydrogenase in Streptomyces coelicolor M-145.

The oxidation of malate to oxaloacetate is catalysed only by a nicotinamide adenine dinucleotide-dependent malate dehydrogenase encoded by SCO4827 in Streptomyces coelicolor. A mutant lacking the malate dehydrogenase gene was isolated and no enzymatic activity was detected. As expected, the ∆mdh mutant was unable to grow on malate as the sole carbon source. However, the mutant grew less in minimal medium with glucose and there was a delay of 36 h. The same behaviour was observed when the mutant was grown on minimal medium with casamino acids or glycerol. For unknown reasons, the mutant was not able to grow in YEME medium with glucose. The deficiency of malate dehydrogenase affected the expression of the isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase genes, decreasing the expression of both genes by approximately two- to threefold.