Differentiation In Streptomyces Research Articles

Nitric Oxide Signaling for Aerial Mycelium Formation in Streptomyces coelicolor A3(2) M145.

Nitric oxide (NO) is a well-known signaling molecule in various organisms. Streptomyces undergoes complex morphological differentiation, similar to that of fungi. A recent study revealed a nitrogen oxide metabolic cycle that forms NO in Streptomyces coelicolor A3(2) M145. Further, endogenously produced NO serves as a signaling molecule. Here, we report that endogenously produced NO regulates cyclic 3',5'-diguanylate (c-di-GMP) levels and controls aerial mycelium formation through the c-di-GMP-binding transcriptional regulator BldD in S. coelicolor A3(2) M145. These observations provide important insights into the mechanisms regulating morphological differentiation. This is the first study to demonstrate a link between NO and c-di-GMP in S. coelicolor A3(2) M145. Morphological differentiation is closely linked to the initiation of secondary metabolism in actinomycetes. Thus, the NO signaling-based regulation of aerial mycelium formation has potential applications in the fermentation industry employing useful actinomycetes. IMPORTANCE Eukaryotic and prokaryotic cells utilize nitric oxide (NO) to regulate physiological functions. Besides its role as a producer of different bioactive substances, Streptomyces is suggested to be involved in mycelial development regulated by endogenously produced NO. However, the regulatory mechanisms are unclear. In this study, we proposed that NO signaling is involved in aerial mycelium formation in S. coelicolor A3(2) M145. NO serves as a signaling molecule for the regulation of intracellular cyclic 3',5'-diguanylate (c-di-GMP) levels, resulting in aerial mycelium formation controlled by a c-di-GMP receptor, BldD. As the abundant production of valuable secondary metabolites is closely related to the initiation of morphological differentiation in Streptomyces, NO may provide value for application in industrial fermentation by serving as a tool for regulating secondary metabolism.

Quantitative phosphoproteome analysis of Streptomyces coelicolor by immobilized zirconium (IV) affinity chromatography and mass spectrometry reveals novel regulated protein phosphorylation sites and sequence motifs

Streptomycetes are multicellular gram-positive bacteria that produce many bioactive compounds, including antibiotics, antitumorals and immunosuppressors. The Streptomyces phosphoproteome remains largely uncharted even though protein phosphorylation at Ser/Thr/Tyr is known to modulate morphological differentiation and specialized metabolic processes. We here expand the S. coelicolor phosphoproteome by optimised immobilized zirconium (IV) affinity chromatography and mass spectrometry to identify phosphoproteins at the vegetative and sporulating stages. We mapped 361 phosphorylation sites (41% pSer, 56.2% pThr, 2.8% pTyr) and discovered four novel Thr phosphorylation motifs (“Kxxxx(pT)xxxxK”, “DxE(pT)”, “D(pT)” and “Exxxxx(pT)”) in 351 phosphopeptides derived from 187 phosphoproteins. We identified 154 novel phosphoproteins, thereby almost doubling the number of experimentally verified Streptomyces phosphoproteins. Novel phosphoproteins included cell division proteins (FtsK, CrgA) and specialized metabolism regulators (ArgR, AfsR, CutR and HrcA) that were differentially phosphorylated in the vegetative and in the antibiotic producing sporulating stages. Phosphoproteins involved in primary metabolism included 27 novel ribosomal proteins that were phosphorylated during the vegetative stage. Phosphorylation of these proteins likely participate in the intricate and incompletely understood regulation of Streptomyces development and secondary metabolism. We conclude that Zr(IV)-IMAC is an efficient and sensitive method to study protein phosphorylation and regulation in bacteria and enhance our understanding of bacterial signalling. SignificanceTwo thirds of the secondary metabolites used in clinic, especially antibiotics, were discovered in Streptomyces strains. Antibiotic resistance became one of the major challenges in clinic, and new antibiotics are urgently required in clinic. Next-generation sequencing analyses revealed that streptomycetes harbour many cryptic secondary metabolite pathways, i.e. pathways not expressed in the laboratory. Secondary metabolism is tightly connected with hypha differentiation and sporulation, and understanding Streptomyces differentiation is one of the main challenges in industrial microbiology, in order to activate the expression of cryptic pathways in the laboratory.Protein phosphorylation at Ser/Thr/Tyr modulates development and secondary metabolism, but the Streptomyces phosphoproteome is still largely uncharted. Previous S. coelicolor phosphoproteomic studies used TiO2 affinity enrichment and LC-MS/MS identifying a total of 184 Streptomyces phosphoproteins. Here, we used by first time zirconium (IV) affinity chromatography and mass spectrometry, identifying 186 S. coelicolor phosphoproteins. Most of these phosphoproteins (154) were not identified in previous phosphoproteomic studies using TiO2 affinity enrichment. Thereby we almost doubling the number of experimentally verified Streptomyces phosphoproteins. Zr(IV)-IMAC affinity chromatography also worked in E. coli, allowing the identification of phosphoproteins that were not identified by TiO2 affinity chromatography.We conclude that Zr(IV)-IMAC is an efficient and sensitive method for studies of protein phosphorylation and regulation in bacteria to enhance our understanding of bacterial signalling networks. Moreover, the new Streptomyces phosphoproteins identified will contribute to design further works to understand and modulate Streptomyces secondary metabolism activation.

Involvement of BldC in the Formation of Physiologically Mature Sporangium in Actinoplanes missouriensis.

AmBldD is a global transcriptional regulator that represses the transcription of several genes required for sporangium formation in Actinoplanes missouriensis. Here, we characterized one of the AmBldD regulons: AMIS_1980, encoding an ortholog of BldC, which is a transcriptional regulator involved in the morphological development of Streptomyces. We determined the transcriptional start point of the bldC ortholog by high-resolution S1 nuclease mapping and found an AmBldD box in its 5'-untranslated region. Reverse transcription-quantitative PCR analysis revealed that the transcription of bldC is activated during sporangium formation. A bldC null mutant (ΔbldC) strain formed normally shaped sporangia, but they exhibited defective sporangium dehiscence; under a dehiscence-inducing condition, the number of spores released from the sporangia of the ΔbldC strain was 2 orders of magnitude lower than that from the sporangia of the wild-type strain. RNA sequencing analysis indicated that BldC functions as a transcriptional activator of several developmental genes, including tcrA, which encodes a key transcriptional activator that regulates sporangium formation, sporangium dehiscence, and spore dormancy. Using electrophoretic mobility shift assay (EMSA), we showed that a recombinant BldC protein directly binds to upstream regions of at least 18 genes, the transcription of which is downregulated in the ΔbldC strain. Furthermore, using DNase I footprinting and EMSA, we demonstrated that BldC binds to the direct repeat sequences containing an AT-rich motif. Thus, BldC is a global regulator that activates the transcription of several genes, some of which are likely to be required for sporangium dehiscence. IMPORTANCE BldC is a global transcriptional regulator that acts as a "brake" in the morphological differentiation of Streptomyces. BldC-like proteins are widely distributed throughout eubacteria, but their orthologs have not been studied outside streptomycetes. Here, we revealed that the BldC ortholog in Actinoplanes missouriensis is essential for sporangium dehiscence and that its regulon is different from the BldC regulon in Streptomyces venezuelae, suggesting that BldC has evolved to play different roles in morphological differentiation between the two genera of filamentous actinomycetes.

AdpA, a developmental regulator, promotes\xa0\u03b5-poly-l-lysine biosynthesis in Streptomycesalbulus

BackgroundAdpA is a global regulator of morphological differentiation and secondary metabolism in Streptomyces, but the regulatory roles of the Streptomyces AdpA family on the biosynthesis of the natural product ε-poly-l-lysine (ε-PL) remain unidentified, and few studies have focused on increasing the production of ε-PL by manipulating transcription factors in Streptomyces.ResultsIn this study, we revealed the regulatory roles of different AdpA homologs in ε-PL biosynthesis and morphological differentiation and effectively promoted ε-PL production and sporulation in Streptomycesalbulus NK660 by heterologously expressing adpA from S.neyagawaensis NRRLB-3092 (adpASn). First, we identified a novel AdpA homolog named AdpASa in S.albulus NK660 and characterized its function as an activator of ε-PL biosynthesis and morphological differentiation. Subsequently, four heterologous AdpA homologs were selected to investigate their phylogenetic relationships and regulatory roles in S.albulus, and AdpASn was demonstrated to have the strongest ability to promote both ε-PL production and sporulation among these five AdpA proteins. The ε-PL yield of S.albulus heterologously expressing adpASn was approximately 3.6-fold higher than that of the control strain. Finally, we clarified the mechanism of AdpASn in enhancing ε-PL biosynthesis and its effect on ε-PL polymerization degree using real-time quantitative PCR, microscale thermophoresis and MALDI-TOF–MS. AdpASn was purified, and its seven direct targets, zwf, tal, pyk2, pta, ack, pepc and a transketolase gene (DC74_2409), were identified, suggesting that AdpASn may cause the redistribution of metabolic flux in central metabolism pathways, which subsequently provides more carbon skeletons and ATP for ε-PL biosynthesis in S.albulus.ConclusionsHere, we characterized the positive regulatory roles of Streptomyces AdpA homologs in ε-PL biosynthesis and their effects on morphological differentiation and reported for the first time that AdpASn promotes ε-PL biosynthesis by affecting the transcription of its target genes in central metabolism pathways. These findings supply valuable insights into the regulatory roles of the Streptomyces AdpA family on ε-PL biosynthesis and morphological differentiation and suggest that AdpASn may be an effective global regulator for enhanced production of ε-PL and other valuable secondary metabolites in Streptomyces.

C-di-AMP hydrolysis by the phosphodiesterase AtaC promotes differentiation of multicellular bacteria

Antibiotic-producing Streptomyces use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as Streptococcus pneumoniae and Mycobacterium tuberculosis AtaC is monomeric in solution and binds Mn2+ to specifically hydrolyze c-di-AMP to AMP via the intermediate 5'-pApA. As an effector of c-di-AMP signaling, we characterize the RCK_C domain protein CpeA. c-di-AMP promotes interaction between CpeA and the predicted cation/proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in Actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in Streptomyces, connecting ionic stress to development. Thus, we present the discovery of two components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for ion balance and coordinated development in Streptomyces.

Growth, Differentiation and Secondary Metabolism in Streptomyces

Growth, Differentiation and Secondary Metabolism in Streptomyces

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

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

BldC Delays Entry into Development To Produce a Sustained Period of Vegetative Growth in Streptomyces venezuelae.

Streptomycetes are filamentous bacteria that differentiate by producing spore-bearing reproductive structures called aerial hyphae. The transition from vegetative to reproductive growth is controlled by the bld (bald) loci, and mutations in bld genes prevent the formation of aerial hyphae, either by blocking entry into development (typically mutations in activators) or by inducing precocious sporulation in the vegetative mycelium (typically mutations in repressors). One of the bld genes, bldC, encodes a 68-residue DNA-binding protein related to the DNA-binding domain of MerR-family transcription factors. Recent work has shown that BldC binds DNA by a novel mechanism, but there is less insight into its impact on Streptomyces development. Here we used ChIP-seq coupled with RNA-seq to define the BldC regulon in the model species Streptomyces venezuelae, showing that BldC can function both as a repressor and as an activator of transcription. Using electron microscopy and time-lapse imaging, we show that bldC mutants are bald because they initiate development prematurely, bypassing the formation of aerial hyphae. This is consistent with the premature expression of BldC target genes encoding proteins with key roles in development (e.g., whiD, whiI, sigF), chromosome condensation and segregation (e.g., smeA-sffA, hupS), and sporulation-specific cell division (e.g., dynAB), suggesting that BldC-mediated repression is critical to maintain a sustained period of vegetative growth prior to sporulation. We discuss the possible significance of BldC as an evolutionary link between MerR family transcription factors and DNA architectural proteins.IMPORTANCE Understanding the mechanisms that drive bacterial morphogenesis depends on the dissection of the regulatory networks that underpin the cell biological processes involved. Recently, Streptomyces venezuelae has emerged as an attractive model system for the study of morphological differentiation in Streptomyces This has led to significant progress in identifying the genes controlled by the transcription factors that regulate aerial mycelium formation (Bld regulators) and sporulation (Whi regulators). Taking advantage of S. venezuelae, we used ChIP-seq coupled with RNA-seq to identify the genes directly under the control of BldC. Because S. venezuelae sporulates in liquid culture, the complete spore-to-spore life cycle can be examined using time-lapse microscopy, and we applied this technique to the bldC mutant. These combined approaches reveal BldC to be a member of an emerging class of Bld regulators that function principally to repress key sporulation genes, thereby extending vegetative growth and blocking the onset of morphological differentiation.

Streptomyces Differentiation in Liquid Cultures as a Trigger of Secondary Metabolism.

Streptomyces is a diverse group of gram-positive microorganisms characterised by a complex developmental cycle. Streptomycetes produce a number of antibiotics and other bioactive compounds used in the clinic. Most screening campaigns looking for new bioactive molecules from actinomycetes have been performed empirically, e.g., without considering whether the bacteria are growing under the best developmental conditions for secondary metabolite production. These screening campaigns were extremely productive and discovered a number of new bioactive compounds during the so-called “golden age of antibiotics” (until the 1980s). However, at present, there is a worrying bottleneck in drug discovery, and new experimental approaches are needed to improve the screening of natural actinomycetes. Streptomycetes are still the most important natural source of antibiotics and other bioactive compounds. They harbour many cryptic secondary metabolite pathways not expressed under classical laboratory cultures. Here, we review the new strategies that are being explored to overcome current challenges in drug discovery. In particular, we focus on those aimed at improving the differentiation of the antibiotic-producing mycelium stage in the laboratory.

Genomic Insights into Evolution of AdpA Family Master Regulators of Morphological Differentiation and Secondary Metabolism in Streptomyces.

The AdpA protein from a streptomycin producer Streptomyces griseus is a founding member of the AdpA family of pleiotropic regulators, known to be ubiquitously present in streptomycetes. Functional genomic approaches revealed a huge number of AdpA targets, leading to the claim that the AdpA regulon is the largest one in bacteria. The expression of adpA is limited at the level of translation of the rare leucyl UUA codon. All known properties of AdpA regulators were discovered on a few streptomycete strains. There are open questions about the true abundance and diversity of AdpA across actinobacterial taxa (and beyond) and about the possible evolutionary forces that shape the AdpA orthologous group in Streptomyces. Here we show that, with respect to the TTA codon, streptomycete adpA is more diverse than has been previously thought, as the genes differ in presence/position of this codon. Reciprocal best hits to AdpA can be found in many actinobacterial orders, with a domain organization resembling that of the prototypical AdpA, but other configurations also exist. Diversifying positive selection was detected within the DNA-binding (AraC) domain in adpA of Streptomyces origin, most likely affecting residues enabling AdpA to recognize a degenerate operator. Sequence coding for putative glutamine amidotransferase (GATase-1) domain also shows signs of positive selection. The two-domain organization of AdpA most likely arose from a fusion of genes encoding separate GATase-1 and AraC domains. Indeed, we show that the AraC domain retains a biological function in the absence of the GATase-1 part. We suggest that acquisition of the regulatory role by TTA codon is a relatively recent event in the evolution of AdpA, which coincided with the rise of the Streptomycetales clade and, at present, is under relaxed selective constraints. Further experimental scrutiny of our findings is invited, which should provide new insights into the evolution and prospects for engineering of an AdpA-centered regulatory network.

In conditions of over-expression, WblI, a WhiB-like transcriptional regulator, has a positive impact on the weak antibiotic production of Streptomyces lividans TK24.

Regulators of the WhiB-like (wbl) family are playing important role in the complex regulation of metabolic and morphological differentiation in Streptomyces. In this study, we investigated the role of wblI, a member of this family, in the regulation of secondary metabolite production in Streptomyces lividans. The over-expression of wblI was correlated with an enhanced biosynthesis of undecylprodigiosin and actinorhodin and with a reduction of the biosynthesis of yCPK and of the grey spore pigment encoded by the whiE locus. Five regulatory targets of WblI were identified using in vitro formaldehyde crosslinking and confirmed by EMSA and qRT-PCR. These included the promoter regions of wblI itself, two genes of the ACT cluster (actVA3 and the intergenic region between the divergently orientated genes actII-1 and actII-2) and that of wblA, another member of the Wbl family. Quantitative RT-PCR analysis indicated that the expression of actVA3 encoding a protein of unknown function as well as that of actII-1, a TetR regulator repressing the expression of actII-2, encoding the ACT transporter, were down regulated in the WblI over-expressing strain. Consistently the expression of the transporter actII-2 was up-regulated. The expression of WblA, that is known to have a negative impact on ACT biosynthesis, was strongly down regulated in the WblI over-expressing strain. These data are consistent with the positive impact that WblI over-expression has on ACT biosynthesis. The latter might result from direct activation of ACT biosynthesis and export and from repression of the expression of WblA, a likely indirect, repressor of ACT biosynthesis.

Multi-layered inhibition of Streptomyces development: BldO is a dedicated repressor of whiB.

SummaryBldD‐(c‐di‐GMP) sits on top of the regulatory network that controls differentiation in Streptomyces, repressing a large regulon of developmental genes when the bacteria are growing vegetatively. In this way, BldD functions as an inhibitor that blocks the initiation of sporulation. Here, we report the identification and characterisation of BldO, an additional developmental repressor that acts to sustain vegetative growth and prevent entry into sporulation. However, unlike the pleiotropic regulator BldD, we show that BldO functions as the dedicated repressor of a single key target gene, whiB, and that deletion of bldO or constitutive expression of whiB is sufficient to induce precocious hypersporulation.

Polydiglycosylphosphate Transferase PdtA (SCO2578) of Streptomyces coelicolor A3(2) Is Crucial for Proper Sporulation and Apical Tip Extension under Stress Conditions.

Although anionic glycopolymers are crucial components of the Gram-positive cell envelope, the relevance of anionic glycopolymers for vegetative growth and morphological differentiation of Streptomyces coelicolor A3(2) is unknown. Here, we show that the LytR-CpsA-Psr (LCP) protein PdtA (SCO2578), a TagV-like glycopolymer transferase, has a dual function in the S. coelicolor A3(2) life cycle. Despite the presence of 10 additional LCP homologs, PdtA is crucial for proper sporulation. The integrity of the spore envelope was severely affected in a pdtA deletion mutant, resulting in 34% nonviable spores. pdtA deletion caused a significant reduction in the polydiglycosylphosphate content of the spore envelope. Beyond that, apical tip extension and normal branching of vegetative mycelium were severely impaired on high-salt medium. This growth defect coincided with the mislocalization of peptidoglycan synthesis. Thus, PdtA itself or the polydiglycosylphosphate attached to the peptidoglycan by the glycopolymer transferase PdtA also has a crucial function in apical tip extension of vegetative hyphae under stress conditions. Anionic glycopolymers are underappreciated components of the Gram-positive cell envelope. They provide rigidity to the cell wall and position extracellular enzymes involved in peptidoglycan remodeling. Although Streptomyces coelicolor A3(2), the model organism for bacterial antibiotic production, is known to produce two distinct cell wall-linked glycopolymers, teichulosonic acid and polydiglycosylphosphate, the role of these glycopolymers in the S. coelicolor A3(2) life cycle has not been addressed so far. This study reveals a crucial function of the anionic glycopolymer polydiglycosylphosphate for the growth and morphological differentiation of S. coelicolor A3(2). Polydiglycosylphosphate is attached to the spore wall by the LytR-CpsA-Psr protein PdtA (SCO2578), a component of the Streptomyces spore wall-synthesizing complex (SSSC), to ensure the integrity of the spore envelope. Surprisingly, PdtA also has a crucial role in vegetative growth under stress conditions and is required for proper peptidoglycan incorporation during apical tip extension.

Carbon Catabolite Regulation of Secondary Metabolite Formation and Morphological Differentiation in Streptomyces coelicolor.

In the genus Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. However, there is little information about the mechanism involved in these effects. In the present work, it was found that glucose exerted a suppressive effect on the Streptomyces coelicolor actinorhodin (Act) and undecylprodigiosin (Red) production, as well as in its morphological differentiation. Accordingly, using a high-density microarray approach in S. coelicolor grown under glucose repression, at early growth stages, a negative effect was exerted on the transcription of genes involved in Act and Red production, when compared with non-repressive conditions. Seven genes of Act and at least ten genes of Red production were down-regulated by glucose. Stronger repression was observed on the initial steps of antibiotics formation. On the contrary, the coelimycin P1 cluster was up-regulated by glucose. Regarding differentiation, no sporulation was observed in the presence of glucose and expression of a set of genes of the bld cascade was repressed as well as chaplins and rodlins genes. Finally, a series of transcriptional regulators involved in both processes were up- or down-regulated by glucose. This is the first global transcriptomic approach performed to understand the molecular basis of the glucose effect on the synthesis of secondary metabolism and differentiation in the genus Streptomyces. The results of this study are opening new avenues for further exploration.

Response regulator heterodimer formation controls a key stage in Streptomyces development.

The orphan, atypical response regulators BldM and WhiI each play critical roles in Streptomyces differentiation. BldM is required for the formation of aerial hyphae, and WhiI is required for the differentiation of these reproductive structures into mature spores. To gain insight into BldM function, we defined the genome-wide BldM regulon using ChIP-Seq and transcriptional profiling. BldM target genes clustered into two groups based on their whi gene dependency. Expression of Group I genes depended on bldM but was independent of all the whi genes, and biochemical experiments showed that Group I promoters were controlled by a BldM homodimer. In contrast, Group II genes were expressed later than Group I genes and their expression depended not only on bldM but also on whiI and whiG (encoding the sigma factor that activates whiI). Additional ChIP-Seq analysis showed that BldM Group II genes were also direct targets of WhiI and that in vivo binding of WhiI to these promoters depended on BldM and vice versa. We go on to demonstrate that BldM and WhiI form a functional heterodimer that controls Group II promoters, serving to integrate signals from two distinct developmental pathways. The BldM-WhiI system thus exemplifies the potential of response regulator heterodimer formation as a mechanism to expand the signaling capabilities of bacterial cells.

Transcriptomic Analysis of Liquid Non-Sporulating Streptomyces coelicolor Cultures Demonstrates the Existence of a Complex Differentiation Comparable to That Occurring in Solid Sporulating Cultures

Streptomyces species produce many clinically relevant secondary metabolites and exhibit a complex development that includes hyphal differentiation and sporulation in solid cultures. Industrial fermentations are usually performed in liquid cultures, conditions in which Streptomyces strains generally do not sporulate, and it was traditionally assumed that no differentiation took place. The aim of this work was to compare the transcriptomes of S. coelicolor growing in liquid and solid cultures, deepening the knowledge of Streptomyces differentiation. Microarrays demonstrated that gene expression in liquid and solid cultures were comparable and data indicated that physiological differentiation was similar for both conditions. Eighty-six percent of all transcripts showed similar abundances in liquid and solid cultures, such as those involved in the biosynthesis of actinorhodin (actVA, actII-4) and undecylprodigiosin (redF); activation of secondary metabolism (absR1, ndsA); genes regulating hydrophobic cover formation (aerial mycelium) (bldB, bldC, bldM, bldN, sapA, chpC, chpD, chpE, chpH, ramA, ramC, ramS); and even some genes regulating early stages of sporulation (wblA, whiG, whiH, whiJ). The two most important differences between transcriptomes from liquid and solid cultures were: first, genes related to secondary metabolite biosynthesis (CDA, CPK, coelichelin, desferrioxamine clusters) were highly up-regulated in liquid but not in solid cultures; and second, genes involved in the final stages of hydrophobic cover/spore maturation (chpF, rdlA, whiE, sfr) were up-regulated in solid but not in liquid cultures. New information was also provided for several non-characterized genes differentially expressed in liquid and solid cultures which might be regulating, at least in part, the metabolic and developmental differences observed between liquid and solid cultures.

Regulation of the clpP1clpP2 operon by the pleiotropic regulator AdpA in Streptomyces lividans

Insertion of an apramycin resistance cassette in the clpP1clpP2 operon (encoding the ClpP1 and ClpP2 peptidase subunits) affects morphological and physiological differentiation of Streptomyces lividans. Another key factor controlling Streptomyces differentiation is the pleiotropic transcriptional regulator AdpA. We have identified a spontaneous missense mutation (-1 frameshift) in the adpA (bldH) open reading frame in a clpP1clpP2 mutant that led to the synthesis of a non-functional AdpA protein. Electrophoretic mobility shift assays showed that AdpA bound directly to clpP1clpP2 promoter region. Quantitative real-time PCR analysis showed that AdpA regulated the clpP1clpP2 operon expression at specific growth times. In vitro, AdpA and ClgR, a transcriptional activator of clpP1clpP2 operon and other genes, were able to bind simultaneously to clpP1 promoter, which suggests that AdpA binding to clpP1 promoter did not affect that of ClgR. This study allowed to uncover an interplay between the ClpP peptidases and AdpA in S. lividans.

Mycelium differentiation and development of Streptomyces coelicolor in lab-scale bioreactors: programmed cell death, differentiation, and lysis are closely linked to undecylprodigiosin and actinorhodin production.

Streptomycetes are mycelium-forming bacteria that produce two thirds of clinically relevant secondary metabolites. Secondary metabolite production is activated at specific developmental stages of Streptomyces life cycle. Despite this, Streptomyces differentiation in industrial bioreactors tends to be underestimated and the most important parameters managed are only indirectly related to differentiation: modifications to the culture media, optimization of productive strains by random or directed mutagenesis, analysis of biophysical parameters, etc. In this work the relationship between differentiation and antibiotic production in lab-scale bioreactors was defined. Streptomyces coelicolor was used as a model strain. Morphological differentiation was comparable to that occurring during pre-sporulation stages in solid cultures: an initial compartmentalized mycelium suffers a programmed cell death, and remaining viable segments then differentiate to a second multinucleated antibiotic-producing mycelium. Differentiation was demonstrated to be one of the keys to interpreting biophysical fermentation parameters and to rationalizing the optimization of secondary metabolite production in bioreactors.

Dual Positive Feedback Regulation of Protein Degradation of an Extra-cytoplasmic Function σ Factor for Cell Differentiation in Streptomyces coelicolor

Here we report that in Streptomyces coelicolor, the protein stability of an ECF σ factor SigT, which is involved in the negative regulation of cell differentiation, was completely dependent on its cognate anti-σ factor RstA. The degradation of RstA caused a ClpP/SsrA-dependent degradation of SigT during cell differentiation. This was consistent with the delayed morphological development or secondary metabolism in the ΔclpP background after rstA deletion or sigT overexpression. Meanwhile, SigT negatively regulated clpP/ssrA expression by directly binding to the clpP promoter (clpPp). The SigT-clpPp interaction could be disrupted by secondary metabolites, giving rise to the stabilized SigT protein and retarded morphological development in a non-antibiotic-producing mutant. Thus a novel regulatory mechanism was revealed that the protein degradation of the ECF σ factor was initiated by the degradation of its anti-σ factor, and was accelerated in a dual positive feedback manner, through regulation by secondary metabolites, to promote rapid and irreversible development of the secondary metabolism. This ingenious cooperation of intracellular components can ensure economical and exquisite control of the ECF σ factor protein level for the proper cell differentiation in Streptomyces.

Pre-sporulation stages ofStreptomycesdifferentiation: state-of-the-art and future perspectives

Streptomycetes comprise very important industrial bacteria, producing two-thirds of all clinically relevant secondary metabolites. They are mycelial microorganisms with complex developmental cycles that include programmed cell death (PCD) and sporulation. Industrial fermentations are usually performed in liquid cultures (large bioreactors), conditions in which Streptomyces strains generally do not sporulate, and it was traditionally assumed that there was no differentiation. In this work, we review the current knowledge on Streptomyces pre-sporulation stages of Streptomyces differentiation.