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.

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.

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.

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.

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.

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.

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.

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 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.

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.

Functional Genome Mining for Metabolites Encoded by Large Gene Clusters through Heterologous Expression of a Whole-Genome Bacterial Artificial Chromosome Library in Streptomyces spp.

UNLABELLED: Genome sequencing projects in the last decade revealed numerous cryptic biosynthetic pathways for unknown secondary metabolites in microbes, revitalizing drug discovery from microbial metabolites by approaches called genome mining. In this work, we developed a heterologous expression and functional screening approach for genome mining from genomic bacterial artificial chromosome (BAC) libraries in Streptomyces spp. We demonstrate mining from a strain of Streptomyces rochei, which is known to produce streptothricins and borrelidin, by expressing its BAC library in the surrogate host Streptomyces lividans SBT5, and screening for antimicrobial activity. In addition to the successful capture of the streptothricin and borrelidin biosynthetic gene clusters, we discovered two novel linear lipopeptides and their corresponding biosynthetic gene cluster, as well as a novel cryptic gene cluster for an unknown antibiotic from S. rochei This high-throughput functional genome mining approach can be easily applied to other streptomycetes, and it is very suitable for the large-scale screening of genomic BAC libraries for bioactive natural products and the corresponding biosynthetic pathways. IMPORTANCE: Microbial genomes encode numerous cryptic biosynthetic gene clusters for unknown small metabolites with potential biological activities. Several genome mining approaches have been developed to activate and bring these cryptic metabolites to biological tests for future drug discovery. Previous sequence-guided procedures relied on bioinformatic analysis to predict potentially interesting biosynthetic gene clusters. In this study, we describe an efficient approach based on heterologous expression and functional screening of a whole-genome library for the mining of bioactive metabolites from Streptomyces The usefulness of this function-driven approach was demonstrated by the capture of four large biosynthetic gene clusters for metabolites of various chemical types, including streptothricins, borrelidin, two novel lipopeptides, and one unknown antibiotic from Streptomyces rochei Sal35. The transfer, expression, and screening of the library were all performed in a high-throughput way, so that this approach is scalable and adaptable to industrial automation for next-generation antibiotic discovery.

Characterization of a novel subtilisin-like protease myroicolsin from deep sea bacterium Myroides profundi D25 and molecular insight into its collagenolytic mechanism.

Collagen is an insoluble protein that widely distributes in the extracellular matrix of marine animals. Collagen degradation is an important step in the marine nitrogen cycle. However, the mechanism of marine collagen degradation is still largely unknown. Here, a novel subtilisin-like collagenolytic protease, myroicolsin, which is secreted by the deep sea bacterium Myroides profundi D25, was purified and characterized, and its collagenolytic mechanism was studied. Myroicolsin displays low identity (<30%) to previously characterized subtilisin-like proteases, and it contains a novel domain structure. Protein truncation indicated that the Pro secretion system C-terminal sorting domain in the precursor protein is involved in the cleavage of the N-propeptide, and the linker is required for protein folding during myroicolsin maturation. The C-terminal ß-jelly roll domain did not bind insoluble collagen fiber, suggesting that myroicolsin may degrade collagen without the assistance of a collagen-binding domain. Myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. The favored residue at the P1 site was basic arginine. Scanning electron microscopy and atomic force microscopy, together with biochemical analyses, confirmed that collagen fiber degradation by myroicolsin begins with the hydrolysis of proteoglycans and telopeptides in collagen fibers and fibrils. Myroicolsin showed strikingly different cleavage patterns between native and denatured collagens. A collagen degradation model of myroicolsin was proposed based on our results. Our study provides molecular insight into the collagen degradation mechanism and structural characterization of a subtilisin-like collagenolytic protease secreted by a deep sea bacterium, shedding light on the degradation mechanism of deep sea sedimentary organic nitrogen.

Molecular insight into the role of the N-terminal extension in the maturation, substrate recognition, and catalysis of a bacterial alginate lyase from polysaccharide lyase family 18.

Bacterial alginate lyases, which are members of several polysaccharide lyase (PL) families, have important biological roles and biotechnological applications. The mechanisms for maturation, substrate recognition, and catalysis of PL18 alginate lyases are still largely unknown. A PL18 alginate lyase, aly-SJ02, from Pseudoalteromonas sp. 0524 displays a ß-jelly roll scaffold. Structural and biochemical analyses indicated that the N-terminal extension in the aly-SJ02 precursor may act as an intramolecular chaperone to mediate the correct folding of the catalytic domain. Molecular dynamics simulations and mutational assays suggested that the lid loops over the aly-SJ02 active center serve as a gate for substrate entry. Molecular docking and site-directed mutations revealed that certain conserved residues at the active center, especially those at subsites +1 and +2, are crucial for substrate recognition. Tyr(353) may function as both a catalytic base and acid. Based on our results, a model for the catalysis of aly-SJ02 in alginate depolymerization is proposed. Moreover, although bacterial alginate lyases from families PL5, 7, 15, and 18 adopt distinct scaffolds, they share the same conformation of catalytic residues, reflecting their convergent evolution. Our results provide the foremost insight into the mechanisms of maturation, substrate recognition, and catalysis of a PL18 alginate lyase.

Physiological and genetic analyses reveal a mechanistic insight into the multifaceted lifestyles of Pseudoalteromonas sp. SM9913 adapted to the deep-sea sediment.

Although bacteriobenthos play a major role in the degradation of particulate organic matter in marine sediment, knowledge of the sediment-adapted lifestyles of bacteriobenthos is still scarce. Here, the particle-associated, swimming and swarming lifestyles of the benthonic bacterium Pseudoalteromonas sp. SM9913 (SM9913) were illustrated. SM9913 had a clay particle-associated lifestyle, and its exopolysaccharide played an important role in this lifestyle. SM9913 also had swimming and swarming motilities, indicating that it may have swimming and swarming lifestyles in the sediment. The lateral flagella were responsible for the swarming motility, and the polar flagella were responsible for the swimming motility. Iron limitation was an indispensable inductive signal of the swarming motility. An analysis of the motilities of SM9913 and its mutants in clay demonstrated that SM9913 moved in clay by both swimming and swarming motilities. Genomic analysis suggests that having two flagella systems is most likely a common adaptation of some bacteriobenthos to the sediment environment. Our results reveal the lifestyles of benthonic SM9913, providing a better understanding of the environmental adaptation of benthonic bacteria.

EspR, a regulator of the ESX-1 secretion system in Mycobacterium tuberculosis, is directly regulated by the two-component systems MprAB and PhoPR.

The regulatory mechanisms that control the ESX-1 secretion system, a key player in the pathogenesis of Mycobacterium tuberculosis, have not been fully elucidated. However, factors that regulate the ESX-1 substrate EspA usually affect ESX-1 function. Previous studies showed that espA is directly regulated by the nucleoid-associated protein EspR and the two-component system (TCS) MprAB. The PhoPR TCS also activates espA, but the direct target of PhoP was unknown. In this report, we reveal that EspR is directly regulated by MprA and PhoP-Rv, but not by PhoP-Ra. PhoP-Rv and MprA binding sites in the espR promoter were determined by gel-shift and DNase I footprinting assays, which identified a PhoP-protected region centred approximately 205 bp before the espR start codon and that encompasses MprA Region-1, one of two MprA-protected regions. MprA Region-2 is located approximately 60 bp downstream of MprA Region-1 and overlaps a known EspR binding site. Nucleotides essential for the binding of PhoP and/or MprA were identified through site-directed DNA mutagenesis. Our studies also indicate that MprA Region-2, but not MprA Region-1/PhoP region, is required for the full expression of espR. Recombinant strains carrying mutations at MprA Region-2 exhibited lower transcription levels for espR, espA and espD, and had reduced EspR and EspA levels in cell lysates. These findings indicate that EspR may mediate the regulatory effect of PhoPR and MprAB, and provide more insight into the mechanisms underlying ESX-1 control.

Production of the antibiotic FR-008/candicidin in Streptomyces sp. FR-008 is co-regulated by two regulators, FscRI and FscRIV, from different transcription factor families.

In Streptomyces sp. FR-008, the biosynthetic gene cluster of the polyene antibiotic FR-008, also known as candicidin, consists of 21 genes, including four regulatory genes, fscRI-fscRIV. Our bioinformatics analyses indicate that FscRI has an N-terminal PAS domain, whereas the other three regulators have N-terminal AAA domains and are members of the LAL (large ATP-binding regulators of the LuxR type) family. Deletion of fscRI abolished the production of FR-008, with production restored in the complemented strain, supporting a critical role for FscRI in FR-008 biosynthesis. Consistent with these findings, transcription of genes involved in the biosynthesis and efflux of FR-008 was greatly downregulated in a ΔfscRI mutant. Interestingly, the regulatory gene fscRIV was also downregulated in the ΔfscRI mutant. Production of FR-008 was reduced, but not abrogated, in an fscRIV deletion mutant, and although structural genes were downregulated in ΔfscRIV, the changes were much less dramatic than in ΔfscRI, suggesting a stronger regulatory role for FscRI. Remarkably, transcription of fscRI was also decreased in ΔfscRIV. Expression of fscRI restored antibiotic production in a ΔfscRIV mutant, but not vice versa. Putative binding sequences for FscRI were identified upstream of fscRIV and the three structural genes fscA, fscB and fscD, which encode large modular polyketide synthases. Our findings suggest that fscRI and fscRIV are interregulatory, whereas expression of fscRII and fscRIII appears to be independent of fscRI and fscRIV. This study demonstrates that the regulation of polyene antibiotic synthesis can involve mutually regulated transcriptional activators that belong to different families.

Regulation of the biosynthesis of thiopeptide antibiotic cyclothiazomycin by the transcriptional regulator SHJG8833 in Streptomyces hygroscopicus 5008.

Cyclothiazomycin is a member of the thiopeptide antibiotics, which are usually complicated derivatives of ribosomally synthesized peptides. A gene cluster containing 12 ORFs identical to the clt cluster encoding cyclothiazomycin from Streptomyces hygroscopicus 10-22 was revealed by genome sequencing in S. hygroscopicus 5008. Genes SHJG8833 and SHJG8837 of the cluster and flanking gene SHJG8838 were predicted to encode regulatory proteins from different families. In this study, we showed that the newly identified cluster is functional and we investigated the roles of these regulatory genes in the regulation of cyclothiazomycin biosynthesis. We determined that SHJG8833, but not SHJG8837 or SHJG8838, is critical for cyclothiazomycin biosynthesis. The transcriptional start point of SHJG8833 was located to a thymidine 54 nt upstream of the start codon. Inactivation of SHJG8833 abrogated the production of cyclothiazomycin, and synthesis could be restored by reintroducing SHJG8833 into the mutant strain. Gene expression analyses indicated that SHJG8833 regulates a consecutive set of seven genes from SHJG8826 to SHJG8832, whose products are predicted to be involved in different steps in the construction of the main framework of cyclothiazomycin. Transcriptional analysis indicated that these seven genes may form two operons, SHJG8826-27 and SHJG8828-32. Gel-shift analysis demonstrated that the DNA-binding domain of SHJG8833 binds the promoters of SHJG8826 and SHJG8828 and sequences internal to SHJG8826 and SHJG8829, and a conserved binding sequence was deduced. These results indicate that SHJG8833 is a positive regulator that controls cyclothiazomycin biosynthesis by activating structural genes in the clt cluster.

Development of a genetic system for the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913.

BACKGROUND: Pseudoalteromonas species are a group of marine gammaproteobacteria frequently found in deep-sea sediments, which may play important roles in deep-sea sediment ecosystem. Although genome sequence analysis of Pseudoalteromonas has revealed some specific features associated with adaptation to the extreme deep-sea environment, it is still difficult to study how Pseudoalteromonas adapt to the deep-sea environment due to the lack of a genetic manipulation system. The aim of this study is to develop a genetic system in the deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913, making it possible to perform gene mutation by homologous recombination. RESULTS: The sensitivity of Pseudoalteromonas sp. SM9913 to antibiotic was investigated and the erythromycin resistance gene was chosen as the selective marker. A shuttle vector pOriT-4Em was constructed and transferred into Pseudoalteromonas sp. SM9913 through intergeneric conjugation with an efficiency of 1.8 × 10-3, which is high enough to perform the gene knockout assay. A suicide vector pMT was constructed using pOriT-4Em as the bone vector and sacB gene as the counterselective marker. The epsT gene encoding the UDP-glucose lipid carrier transferase was selected as the target gene for inactivation by in-frame deletion. The epsT was in-frame deleted using a two-step integration-segregation strategy after transferring the suicide vector pMT into Pseudoalteromonas sp. SM9913. The ΔepsT mutant showed approximately 73% decrease in the yield of exopolysaccharides, indicating that epsT is an important gene involved in the EPS production of SM9913. CONCLUSIONS: A conjugal transfer system was constructed in Pseudoalteromonas sp. SM9913 with a wide temperature range for selection and a high transfer efficiency, which will lay the foundation of genetic manipulation in this strain. The epsT gene of SM9913 was successfully deleted with no selective marker left in the chromosome of the host, which thus make it possible to knock out other genes in the same host. The construction of a gene knockout system for Pseudoalteromonas sp. SM9913 will contribute to the understanding of the molecular mechanism of how Pseudoalteromonas adapt to the deep-sea environment.

A novel exopolysaccharide from deep-sea bacterium Zunongwangia profunda SM-A87: low-cost fermentation, moisture retention, and antioxidant activities.

Many marine microorganisms can secrete exopolysaccharides (EPSs) which have important applications in biotechnology. We have purified a novel EPS from deep-sea bacterium Zunongwangia profunda SM-A87, identified its glycosyl composition and linkage, and optimized its production to 8.9 g/l in previous studies. To reduce the fermentation cost, an economical fermentation medium containing 60.9 % whey, 10 g/l soybean meal, and 2.9 % NaCl was developed. The EPS yield of batch fermentation in this medium reached 12.1 ± 0.3 g/l. Fed-batch fermentation was conducted and led to an EPS yield of 17.2 ± 0.4 g/l, which represents the highest EPS yield ever reported for a marine bacterium. The EPS was extracted and it displayed good rheological properties, moisture-retention ability, and antioxidant activity. Particularly, its moisture-retention ability is superior to that of other marine bacterial EPSs reported to date. SM-A87 EPS also showed high antioxidant activity. These results suggest that SM-A87 EPS has promising potentials in biotechnology.