SVEN_5003 is a Major Developmental Regulator in Streptomyces venezuelae.

Many regulatory genes that affect cellular development in Streptomyces, such as the canonical bld genes, have already been identified. However, in this study, we identified sven_5003 in Streptomyces venezuelae as a major new developmental regulatory gene, the deletion of which leads to a bald phenotype, typical of bld mutants, under multiple growth conditions. Our data indicated that disruption of sven_5003 also has a differential impact on the production of the two antibiotics jadomycin and chloramphenicol. Enhanced production of jadomycin but reduced production of chloramphenicol were detected in our sven_5003 mutant strain (S. venezuelae D5003). RNA-Seq analysis indicated that SVEN_5003 impacts expression of hundreds of genes, including genes involved in development, primary and secondary metabolism, and genes of unknown function, a finding confirmed by real-time PCR analysis. Transcriptional analysis indicated that sven_5003 is an auto-regulatory gene, repressing its own expression. Despite the evidence indicating that SVEN_5003 is a regulatory factor, a putative DNA-binding domain was not predicted from its primary amino acid sequence, implying an unknown regulatory mechanism by SVEN_5003. Our findings revealed that SVEN_5003 is a pleiotropic regulator with a critical role in morphological development in S. venezuelae.

The regulatory gene wblA is a target of the orphan response regulator OrrA in Streptomyces coelicolor.

Our previous study using transposon mutagenesis indicated that disruption of the putative response regulator gene orrA impacted antibiotic production in Streptomyces coelicolor. In this study, the role of OrrA was further characterized by comparing the phenotypes and transcriptomic profiles of the wild-type S. coelicolor strain M145 and ΔorrA, a strain with an inactivated orrA gene. Chromatin immunoprecipitation using a strain expressing OrrA fused with FLAG showed that OrrA binds the promoter of wblA, whose expression was downregulated in ΔorrA. The interaction of OrrA with the wblA promoter was further validated by a pull-down assay. Similar to ΔorrA, the deletion mutant of wblA (ΔwblA) was defective in development, and developmental genes were expressed at similar levels in ΔorrA and ΔwblA. Although both OrrA and WblA downregulated actinorhodin and undecylprodigiosin, their roles in regulation of the calcium-dependent antibiotic and yellow-pigmented type I polyketide differed. sco1375, a gene of unknown function, was identified as another OrrA target, and overexpression of either sco1375 or wblA in ΔorrA partially restored the wild-type phenotype, indicating that these genes mediate some of the effects of OrrA. This study revealed targets of OrrA and provided more insights into the role of the orphan response regulator OrrA in Streptomyces.

Mutation of MtrA at the Predicted Phosphorylation Site Abrogates Its Role as a Global Regulator in Streptomyces venezuelae.

The global regulator MtrA controls development and primary and secondary metabolism in Streptomyces species. However, residues critical for its function have not yet been characterized. In this study, we identified residue D53 as the potential phosphorylation site of MtrA from Streptomyces venezuelae, a model Streptomyces strain. MtrA variants with amino acid substitutions at the D53 site were generated, and the effects of these substitutions were evaluated in vitro and in vivo. We showed that, although substitutions at D53 did not alter MtrA's secondary structure, the MtrA D53 protein variants lost the ability to bind known MtrA recognition sequences (MtrA sites) in electrophoretic mobility shift assays. Complementation of the ΔmtrA strain with MtrA D53 protein variants did not affect overall strain growth. However, in comparison to the wild-type strain, chloramphenicol and jadomycin production were aberrant in the D53 variant strains, with levels similar to the levels in the ΔmtrA strain. Transcriptional analysis showed that the expression patterns of genes were also similar in the ΔmtrA strain and the D53 variant strains. Although the D53 protein variants and wild-type MtrA were produced at similar levels in S. venezuelae, chromatin immunoprecipitation-quantitative PCR results indicated that replacing the D53 residue rendered the altered proteins unable to bind MtrA sites in vivo, including MtrA sites that regulate genes involved in nitrogen metabolism and in chloramphenicol and jadomycin biosynthesis. In conclusion, our study demonstrates that the predicted phosphorylation site D53 is critical for the role of MtrA in regulation and suggests that MtrA functions in a phosphorylated form in the genus Streptomyces. IMPORTANCE Although phosphorylation has been shown to be essential for the activation of many response regulator proteins of two-component systems, the role of the phosphorylation site in the function of the global regulator MtrA in the genus Streptomyces has not been reported. In this study, we generated Streptomyces mutants that had amino acid substitutions at the predicted phosphorylation site of MtrA, and the effects of the substitutions were investigated by comparing the phenotypes of the resulting strains and their gene expression patterns with those of the wild-type strain and an MtrA deletion mutant. The ability of the altered proteins to bind known promoter targets in vitro was also evaluated. Our analyses showed that the predicted phosphorylation site D53 is critical for MtrA binding in vitro and for the normal functioning of MtrA in vivo. These studies further demonstrate the importance of MtrA as a global regulator in the genus Streptomyces.

Effects of dual deletion of glnR and mtrA on expression of nitrogen metabolism genes in Streptomyces venezuelae.

GlnR activates nitrogen metabolism genes under nitrogen-limited conditions, whereas MtrA represses these genes under nutrient-rich conditions in Streptomyces. In this study, we compared the transcription patterns of nitrogen metabolism genes in a double deletion mutant (ΔmtrA-glnR) lacking both mtrA and glnR and in mutants lacking either mtrA (ΔmtrA) or glnR (ΔglnR). The nitrogen metabolism genes were expressed similarly in ΔmtrA-glnR and ΔglnR under both nitrogen-limited and nutrient-rich conditions, with patterns distinctly different from that of ΔmtrA, suggesting a decisive role for GlnR in the control of nitrogen metabolism genes and further suggesting that regulation of these genes by MtrA is GlnR-dependent. MtrA and GlnR utilize the same binding sites upstream of nitrogen metabolism genes, and we showed stronger in vivo binding of MtrA to these sites under nutrient-rich conditions and of GlnR under nitrogen-limited conditions, consistent with the higher levels of MtrA or GlnR under those respective conditions. In addition, we showed that both mtrA and glnR are self-regulated. Our study provides new insights into the regulation of nitrogen metabolism genes in Streptomyces.

The Response Regulator MacR and its Potential in Improvement of Antibiotic Production in Streptomyces coelicolor.

We previously reported that the two-component system MacRS regulates morphogenesis and production of the blue-pigmented antibiotic actinorhodin (ACT) in Streptomyces coelicolor. In this study, the role of MacRS was further extended to include control of the production of the red-pigmented antibiotic undecylprodigiosin (RED) and the calcium-dependent antibiotic (CDA), and control of other important cellular activities. Our data indicated that disruption of the MacRS TCS reduced production not only of ACT but also of RED and CDA. RNA-Seq analysis revealed that genes involved in both secondary metabolism and primary metabolism are differentially expressed in the MacRS deletion mutant ΔmacRS. Moreover, we found that genes of the Zur regulon are also markedly downregulated in ΔmacRS, suggesting a role for macRS in zinc homeostasis. In addition to previously identified MacR sites with strong matches to the MacR consensus recognition sequence, a genome-wide search revealed over one hundred less-stringent matches, including potential sites upstream of absR1, crgA, and smeA. Electrophoretic mobility shift assays demonstrated that MacR binds some of these sites in vitro. Although there is no strong MacR site upstream of the ACT regulatory gene actII-orf4 (sco5085), we showed that an engineered MacR site enhanced ACT production, providing an approach for modulating production of useful compounds. Altogether, our work suggests an important role for MacRS in a range of cellular activities in Streptomyces and its potential application in strain engineering.

Impact of MtrA on phosphate metabolism genes and the response to altered phosphate conditions in Streptomyces.

Phosphate metabolism is known to be regulated by the PhoPR regulatory system in Streptomyces and some other bacteria. In this study, we report that MtrA also regulates phosphate metabolism in Streptomyces. Our data showed that, in Streptomyces coelicolor, MtrA regulates not only phosphate metabolism genes such as phoA but also phoP under different phosphate conditions, including growth on rich complex media without added inorganic phosphate and on defined media with low or high concentrations of inorganic phosphate. Cross-regulation was also observed among mtrA, phoP and glnR under these conditions. We demonstrated both in vitro and in vivo binding of MtrA to the promoter regions of genes associated with phosphate metabolism and to the intergenic region between phoR and phoU, indicating that these phosphate metabolism genes are targets of MtrA. We further showed that MtrA in S. lividans and S. venezuelae has detectable regulatory effects on expression of phosphate metabolism genes. Additionally, the MtrA homologue from Corynebacterium glutamicum bound predicted MtrA sites of multiple phosphate metabolism genes, implying its potential for regulating phosphate metabolism in this species. Overall, our findings support MtrA as a major regulator for phosphate metabolism in Streptomyces and also potentially in other actinobacteria.

Engineering the Erythromycin-Producing Strain Saccharopolyspora erythraea HOE107 for the Heterologous Production of Polyketide Antibiotics.

Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.

A novel XRE family regulator that controls antibiotic production and development in Streptomyces coelicolor.

Although the genome of the Streptomyces model strain S. coelicolor was sequenced nearly two decades ago, the function of many annotated genes has not been verified, including that of gene sco1979, which was predicted to encode a transcriptional regulator of the xenobiotic response element (XRE) family. In this study, we showed that SCO1979 represses its own transcription and that deletion of sco1979 from S. coelicolor markedly enhanced production of three antibiotics, which are actinorhodin (ACT), undecylprodigiosin (RED), and calcium-dependent antibiotic (CDA), suggesting that SCO1979 represses their biosynthesis. We demonstrated that transcription of genes in the ACT, RED, and CDA pathways was generally increased in the mutant strain Δ1979 compared with levels in the wild-type strain M145. Additionally, purified recombinant SCO1979 interacted with DNA sequences upstream of sco1979 and actII-orf4, redZ, and cdaR, the pathway-specific regulators for the three pathways, implying that SCO1979 potentially regulates the ACT, RED, and CDA pathways via their specific regulators. In addition, disruption of sco1979 led to the notably delayed formation of aerial mycelium and spores, and consistent with this, transcription of genes associated with aerial hyphae and spore formation, such as chp and rdl, and ram, was reduced in Δ1979, implying the involvement of SCO1979 in cellular development control as well. In summary, our findings demonstrated that SCO1979 is a pleiotropic regulator with roles in both secondary metabolism and morphological development in S. coelicolor. KEY POINTS: • SCO1979 is a novel Streptomyces regulator of the XRE family. • SCO1979 regulates its own transcription. • SCO1979 regulates antibiotic production and cellular development.

Impact on Multiple Antibiotic Pathways Reveals MtrA as a Master Regulator of Antibiotic Production in Streptomyces spp. and Potentially in Other Actinobacteria.

Regulation of antibiotic production by Streptomyces is complex. We report that the response regulator MtrA is a master regulator for antibiotic production in Streptomyces Deletion of MtrA altered production of actinorhodin, undecylprodigiosin, calcium-dependent antibiotic, and the yellow-pigmented type I polyketide and resulted in altered expression of the corresponding gene clusters in S. coelicolor Integrated in vitro and in vivo analyses identified MtrA binding sites upstream of cdaR, actII-orf4, and redZ and between cpkA and cpkD MtrA disruption also led to marked changes in chloramphenicol and jadomycin production and in transcription of their biosynthetic gene clusters (cml and jad, respectively) in S. venezuelae, and MtrA sites were identified within cml and jad MtrA also recognized predicted sites within the avermectin and oligomycin pathways in S. avermitilis and in the validamycin gene cluster of S. hygroscopicus The regulator GlnR competed for several MtrA sites and impacted production of some antibiotics, but its effects were generally less dramatic than those of MtrA. Additional potential MtrA sites were identified in a range of other antibiotic biosynthetic gene clusters in Streptomyces species and other actinobacteria. Overall, our study suggests a universal role for MtrA in antibiotic production in Streptomyces and potentially other actinobacteria.IMPORTANCE In natural environments, the ability to produce antibiotics helps the producing host to compete with surrounding microbes. In Streptomyces, increasing evidence suggests that the regulation of antibiotic production is complex, involving multiple regulatory factors. The regulatory factor MtrA is known to have additional roles beyond controlling development, and using bioassays, transcriptional studies, and DNA-binding assays, our study identified MtrA recognition sequences within multiple antibiotic pathways and indicated that MtrA directly controls the production of multiple antibiotics. Our analyses further suggest that this role of MtrA is evolutionarily conserved in Streptomyces species, as well as in other actinobacterial species, and also suggest that MtrA is a major regulatory factor in antibiotic production and in the survival of actinobacteria in nature.

Sulfane sulfur-activated actinorhodin production and sporulation is maintained by a natural gene circuit in Streptomyces coelicolor.

Sulfane sulfur, including polysulfide and persulfide, is a newly identified cellular component present in microorganisms; however, its physiological functions are unclear. Streptomyces coelicolor M145 is a model strain of actinomycetes, which produces several polyketides, including actinorhodin. Herein, we found that both exogenously added and endogenously generated sulfane sulfur increased the actinorhodin production and accelerated spore formation of S. coelicolor M145. This bacterial species carries a natural gene circuit containing four genes that encode a CsoR-like transcription factor (ScCsoR), persulfide dioxygenase (ScPDO), rhodanese and a sulfite transporter, which were shown to be responsible for sensing and removal of excessive sulfane sulfur. ScCsoR was observed to bind to the promoters of the four genes, thus repressing their transcription. Sulfane sulfur modified Cys37 of ScCsoR, and the modified ScCSoR did not bind to the promoters, thereby activating the transcription of ScPDO. The deletion of ScCsoR decreased cellular sulfane sulfur, while the deletion of ScPDO increased its levels. The increased sulfane sulfur promoted actinorhodin production and sporulation. This study unveiled a natural gene circuit for maintaining sulfane sulfur homeostasis in bacteria. Further, we identified the trigger effect of sulfane sulfur on actinorhodin production, presenting a new approach for activating polyketide gene clusters in actinomycetes.

A Hierarchical Network of Four Regulatory Genes Controlling Production of the Polyene Antibiotic Candicidin in Streptomyces sp. Strain FR-008.

The four regulatory genes fscR1 to fscR4 in Streptomyces sp. strain FR-008 form a genetic arrangement that is widely distributed in macrolide-producing bacteria. Our previous work has demonstrated that fscR1 and fscR4 are critical for production of the polyene antibiotic candicidin. In this study, we further characterized the roles of the other two regulatory genes, fscR2 and fscR3, focusing on the relationship between these four regulatory genes. Disruption of a single or multiple regulatory genes did not affect bacterial growth, but transcription of genes in the candicidin biosynthetic gene cluster decreased, and candicidin production was abolished, indicating a critical role for each of the four regulatory genes, including fscR2 and fscR3, in candicidin biosynthesis. We found that fscR1 to fscR4, although differentially expressed throughout the growth phase, displayed similar temporal expression patterns, with an abrupt increase in the early exponential phase, coincident with initial detection of antibiotic production in the same phase. Our data suggest that the four regulatory genes fscR1 to fscR4 have various degrees of control over structural genes in the biosynthetic cluster under the conditions examined. Extensive transcriptional analysis indicated that complex regulation exists between these four regulatory genes, forming a regulatory network, with fscR1 and fscR4 functioning at a lower level. Comprehensive cross-complementation analysis indicates that functional complementation is restricted among the four regulators and unidirectional, with fscR1 complementing the loss of fscR3 or -4 and fscR4 complementing loss of fscR2 Our study provides more insights into the roles of, and the regulatory network formed by, these four regulatory genes controlling production of an important pharmaceutical compound.IMPORTANCE The regulation of antibiotic biosynthesis by Streptomyces species is complex, especially for biosynthetic gene clusters with multiple regulatory genes. The biosynthetic gene cluster for the polyene antibiotic candicidin contains four consecutive regulatory genes, which encode regulatory proteins from different families and which form a subcluster within the larger biosynthetic gene cluster in Streptomyces sp. FR-008. Syntenic arrangements of these regulatory genes are widely distributed in polyene gene clusters, such as the amphotericin and nystatin gene clusters, suggesting a conserved regulatory mechanism controlling production of these clinically important medicines. However, the relationships between these multiple regulatory genes are unknown. In this study, we determined that each of these four regulatory genes is critical for candicidin production. Additionally, using transcriptional analyses, bioassays, high-performance liquid chromatography (HPLC) analysis, and genetic cross-complementation, we showed that FscR1 to FscR4 comprise a hierarchical regulatory network that controls candicidin production and is likely representative of how expression of other polyene biosynthetic gene clusters is controlled.

The developmental regulator MtrA binds GlnR boxes and represses nitrogen metabolism genes in Streptomyces coelicolor.

In Streptomyces, GlnR is an activator protein that activates nitrogen-assimilation genes under nitrogen-limiting conditions. However, less is known regarding the regulation of these genes under nitrogen-rich conditions. We determined that the developmental regulator MtrA represses nitrogen-assimilation genes in nitrogen-rich media and that it competes with GlnR for binding to GlnR boxes. The GlnR boxes upstream of multiple nitrogen genes, such as amtB, were confirmed as MtrA binding sites in vitro by electrophoretic mobility shift assays and in vivo by ChIP-qPCR analysis. Transcriptional analysis indicated that, on nutrient-rich medium, MtrA profoundly repressed expression of nitrogen-associated genes, indicating opposing roles for MtrA and GlnR in the control of nitrogen metabolism. Using in vitro and in vivo analysis, we also showed that glnR is itself a direct target of MtrA and that MtrA represses glnR transcription. We further demonstrated functional conservation of MtrA homologues in the recognition of GlnR boxes upstream of nitrogen genes from different actinobacterial species. As mtrA and glnR are widespread among actinomycetes, this mechanism of potential competitive control over nitrogen metabolism genes may be common in this group, adding a major new layer of complexity to the known regulatory network for nitrogen metabolism in Streptomyces and related species.

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.

Novel MprA binding motifs in the phoP regulatory region in Mycobacterium tuberculosis.

MprAB and PhoPR are important two-component systems (TCSs) in Mycobacterium tuberculosis, and both regulate EspR, a key regulator of the ESX-1 secretion system. Although previous studies suggest that the response regulator PhoP does not directly regulate mprA, the interplay between MprAB and PhoPR remains unclear. In this study, we found that the response regulator MprA can bind to the phoP promoter. Four repeat motifs, D1-D4, constituting two predicted binding sites, were located in the region protected by MprA in DNA footprinting. D1-D4 lack the reported conserved MprA binding sequences, indicating that MprA can recognize a greater range of target sites. Interestingly, D1-D2 overlap a previously reported PhoP binding site, and mutation of D1-D2 inhibited PhoP binding, whereas the D3-D4 site, but not the D1-D2 site, was required for MprA binding. EMSA assays also suggest that MprA and PhoP compete to bind to the phoP promoter. The results of the transcriptional and western blot assays are consistent with a model in which MprA positively controls the phoP expression, which in turn upregulates the expression of espR. These findings reveal complex regulation of a major mycobacterial TCS by dual TCSs.

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.

Deletion of MtrA Inhibits Cellular Development of Streptomyces coelicolor and Alters Expression of Developmental Regulatory Genes.

The developmental life cycle of Streptomyces species includes aerial hyphae formation and spore maturation, two distinct developmental processes that are controlled, respectively, by two families of developmental regulatory genes, bld and whi. In this study, we show that the response regulator MtrA (SCO3013) is critical for normal development of aerial hyphae in S. coelicolor and related species. ΔmtrA, a deletion mutant of the response regulator gene mtrA, exhibited the bald phenotype typical of bld mutants defective in aerial mycelium formation, with formation either much delayed or absent depending on the culture medium. Transcriptional analysis indicated that MtrA activates multiple genes involved in formation of aerial mycelium, including chp, rdl, and ram genes, as well as developmental regulatory genes of the bld and whi families. However, the major regulatory gene bldD showed enhanced expression in ΔmtrA, suggesting it is repressed by MtrA. electrophoretic mobility shift assays indicated that MtrA binds upstream of several genes with altered expression in ΔmtrA, including bldD and whiI, and sequences similar to the consensus binding sequence for MtrA of another actinomycete, Mycobacterium tuberculosis, were found in the bound sites. A loosely conserved recognition sequence containing two short, direct repeats was identified for MtrA of S. coelicolor and was validated using mutational analysis. MtrA homologs are widely distributed among Streptomyces species, and as with S. coelicolor, deletion of the mtrA homologs sve_2757 from S. venezuelae and sli_3357 from S. lividans resulted in conditional bald morphology. Our study suggests a critical and conserved role for MtrA in Streptomyces development.

XdhR negatively regulates actinorhodin biosynthesis in Streptomyces coelicolor M145.

The xdhR gene encodes a TetR-family regulator in Streptomyces coelicolor. However, little is known about the function of XdhR in regulating actinorhodin production. Here, we report that XdhR negatively regulates actinorhodin biosynthesis in S. coelicolor. Deletion of xdhR resulted in overproduction of actinorhodin by approximately 2.5-fold compared to the wild-type strain. Complementation of the xdhR deletion strain restored actinorhodin production to normal levels. In addition, the relative expression levels of actinorhodin cluster genes were all significantly increased in the xdhR deletion strain compared to the wild-type strain. XdhR can specifically bind the promoters of actII-4 and actII-1, two pathway-specific regulators of actinorhodin biosynthesis. These results suggest that xdhR negatively controls actinorhodin biosynthesis by directly regulating actII-4 and actII-1 in S. coelicolor.

Improvement of clavulanic acid production in Streptomyces clavuligerus F613-1 by using a claR-neo reporter strategy

Background: Streptomyces clavuligerus was the producer of clavulanic acid, claR, a pathway-specific transcriptional regulator in S. clavuligerus, positively regulates clavulanic acid biosynthesis. In this study, the promoter-less kanamycin resistance gene neo was fused with claR to obtain strain NEO from S. clavuligerus F613-1. The claR-neo fusion strain NEO was mutated using physical and chemical mutagens and then screened under high concentrations of kanamycin for high-yield producers of clavulanic acid. Results: The reporter gene neo was fused downstream of claR and used as an indicator for expression levels of claR in strain NEO. After three rounds of continuous treatment and screening, the high-yield clavulanic acid-producing strain M3-19 was obtained. In the shaking flask model, the clavulanic acid titer of M3-19 reached 4.33 g/L, which is an increase of 33% over the titer of 3.26 g/L for the starting strains S. clavuligerus F613-1 and NEO. Conclusions: Our results indicate that neo can be effectively used as a reporter for the expression of late-stage biosynthetic genes when screening for high-yield strains and that this approach has strong potential for improving Streptomyces strains of industrial value.

Gene Overexpression in Streptomyces hygroscopicus Associated with DNA Amplification.

The genetics of the Streptomyces hygroscopicus strain 10-22 is of interest due to the ability of this strain to produce antifungal compounds. Strain T110 was obtained through insertional mutagenesis of strain 10-22 and was found to have undergone DNA amplification, as determined by both conventional and pulsed-field gel electrophoresis (PFGE). pIJ702, the vector used for insertional mutagenesis, was shown to have integrated into and co-amplified with the chromosomal DNA sequence of T110, as pIJ702 hybridized predominantly with two of the three amplified BamHI fragments. The amplified DNA sequence in T110 is 10.8 kb in length and consists of 5.18 kb of Streptomyces chromosomal DNA and the entire 5.62 kb pIJ702 sequence. Sequence analysis of the 5.18 kb chromosomal sequence revealed two open reading frames, one encoding a putative IS5 family transposase and the other encoding a putative dihydroxy-acid dehydratase. Real-time PCR analysis showed that expression of the putative dehydratase gene in T110 is about 50 times greater than in the wild-type strain, consistent with the high level of amplification of this DNA region, and therefore this system has the potential for producing economically or clinically important molecules.

Novel Molecular Insights into the Catalytic Mechanism of Marine Bacterial Alginate Lyase AlyGC from Polysaccharide Lyase Family 6.

Alginate lyases that degrade alginate via a ß-elimination reaction fall into seven polysaccharide lyase (PL) families. Although the structures and catalytic mechanisms of alginate lyases in the other PL families have been clarified, those in family PL6 have yet to be revealed. Here, the crystal structure of AlyGC, a PL6 alginate lyase from marine bacterium Glaciecola chathamensis S18K6T, was solved, and its catalytic mechanism was illustrated. AlyGC is a homodimeric enzyme and adopts a structure distinct from other alginate lyases. Each monomer contains a catalytic N-terminal domain and a functionally unknown C-terminal domain. A combined structural and mutational analysis using the structures of AlyGC and of an inactive mutant R241A in complex with an alginate tetrasaccharide indicates that conformational changes occur in AlyGC when a substrate is bound and that the two active centers in AlyGC may not bind substrates simultaneously. The C-terminal domain is shown to be essential for the dimerization and the catalytic activity of AlyGC. Residues Tyr130, Arg187, His242, Arg265, and Tyr304 in the active center are also important for the activity of AlyGC. In catalysis, Lys220 and Arg241 function as the Brønsted base and acid, respectively, and a Ca2+ in the active center neutralizes the negative charge of the C5 carboxyl group of the substrate. Finally, based on our data, we propose a metal ion-assisted catalytic mechanism of AlyGC for alginate cleavage with a state change mode, which provides a better understanding for polysaccharide lyases and alginate degradation.