A stable vector for efficient production of heterologous proteins and secondary metabolites in streptomycetes.

The bacteria of the genus Streptomyces are important producers of a large number of biologically active natural products. Examination of their genomes has revealed great biosynthetic potential for the production of new products, but many of them are silent under laboratory conditions. One of the promising avenues for harnessing this biosynthetic potential is the refactoring and heterologous expression of relevant biosynthetic gene clusters (BGCs) in suitable optimized chassis strains. Although several Streptomyces strains have been used for this purpose, the efficacy is relatively low, and some BGCs have not been expressed. In this study, we optimized our long-term genetically studied Streptomyces lavendulae subsp. lavendulae CCM 3239 strain as a potential host for heterologous expression along with its stable large linear plasmid pSA3239 as a vector system. Two reporter genes, mCherry and gusA under the control of ermEp* promoter, were successfully integrated into pSA3239. The activity of GUS reporter was four-fold higher in pSA3239 than in a single site in S. lavendulae subsp. lavendulae CCM 3239 chromosome, consistent with a higher copy number of pSA3239 (4 copies per chromosome). In addition, the two Att/Int systems (based on PhiC31 and pSAM2) were able to integrate into the corresponding individual attB sites in the chromosome. The BGC for actinorhodin was successfully integrated into pSA3239. However, the resulting strain produced very low amounts of actinorhodin. Its level increased dramatically after integration of the actII-ORF4 gene for the positive regulator under the control of the kasOp* promoter into this strain using the PhiC31 phage integration system. KEY POINTS: • New Streptomyces chassis for heterologous expression of genes and BGCs • Optimized strategy for insertion of heterologous genes into linear plasmid pSA3239 • Efficient heterologous production of actinorhodin after induction of its regulator.

A New Family of Transcriptional Regulators Activating Biosynthetic Gene Clusters for Secondary Metabolites.

We previously identified the aur1 biosynthetic gene cluster (BGC) in Streptomyceslavendulae subsp. lavendulae CCM 3239 (formerly Streptomycesaureofaciens CCM 3239), which is responsible for the production of the unusual angucycline-like antibiotic auricin. Auricin is produced in a narrow interval of the growth phase after entering the stationary phase, after which it is degraded due to its instability at the high pH values reached after the production phase. The complex regulation of auricin BGC is responsible for this specific production by several regulators, including the key activator Aur1P, which belongs to the family of atypical response regulators. The aur1P gene forms an operon with the downstream aur1O gene, which encodes an unknown protein without any conserved domain. Homologous aur1O genes have been found in several BGCs, which are mainly responsible for the production of angucycline antibiotics. Deletion of the aur1O gene led to a dramatic reduction in auricin production. Transcription from the previously characterized Aur1P-dependent biosynthetic aur1Ap promoter was similarly reduced in the S. lavendulaeaur1O mutant strain. The aur1O-specific coactivation of the aur1Ap promoter was demonstrated in a heterologous system using a luciferase reporter gene. In addition, the interaction between Aur1O and Aur1P has been demonstrated by a bacterial two-hybrid system. These results suggest that Aur1O is a specific coactivator of this key auricin-specific positive regulator Aur1P. Bioinformatics analysis of Aur1O and its homologues in other BGCs revealed that they represent a new family of transcriptional coactivators involved in the regulation of secondary metabolite biosynthesis. However, they are divided into two distinct sequence-specific subclasses, each of which is likely to interact with a different family of positive regulators.

An efficient system for stable markerless integration of large biosynthetic gene clusters into Streptomyces chromosomes.

The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties. We have recently developed an efficient markerless genome modification system for streptomycetes based on positive blue/white selection of double crossovers using the bpsA gene from indigoidine biosynthesis, which has been successfully applied for markerless deletions of genes and BGCs. In the present study, we optimized this system for markerless insertion of large BGCs. In a pilot test experiment, we successfully inserted a part of the landomycin BGC (lanFABCDL) under the control of the ermEp* promoter in place of the actinorhodin BGC (act) of Streptomyces lividans TK24 and RedStrep 1.3. The resulting strains correctly produced UWM6 and rabelomycin in twice the yield compared to S. lividans strains with the same construct inserted using the PhiBT1 phage-based integration vector system. Moreover, the system was more stable. Subsequently, using the same strategy, we effectively inserted the entire BGC for mithramycin (MTM) in place of the calcium-dependent antibiotic BGC (cda) of S. lividans RedStrep 1.3 without antibiotic-resistant markers. The resulting strain produced similar levels of MTM when compared to the previously described S. lividans RedStrep 1.3 strain with the VWB phage-based integration plasmid pMTMF. The system was also more stable. KEY POINTS: • Optimized genome editing system for markerless insertion of BGCs into Streptomyces genomes • Efficient heterologous production of MTM in the stable engineered S. lividans strain.

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

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

The antitumor antibiotic mithramycin: new advanced approaches in modification and production.

The aureolic acid-type polyketide mithramycin (MTM) has a remarkable cytotoxicity against a variety of human tumors and has been used for the treatment of several types of cancer, including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia, and Paget's disease. However, its clinical use is quite limited due to its toxicity. Recently, interest in MTM has been renewed after its identification as a top candidate for the inhibition of the aberrant fusion transcription factor EWS-FLI1, associated with malignant transformation and progression of Ewing sarcoma tumor family. The mechanism of MTM inhibition involves its reversible non-intercalative interaction with GC-rich DNA regions. As a result of this binding, MTM blocks binding of transcription factors (such as Sp1) to their GC-rich promoters and inhibits transcription of several proto-oncogenes and thus suppresses various types of cancer. Knowledge of the biosynthesis of MTM and its gene cluster has enabled genetic modifications of the gene cluster and combinatorial biosynthesis to produce new modified MTM molecules ("mithralogues") with improved efficacy and lower toxicity, which has also renewed interest in the clinical development of MTM. However, production yields of MTM and its analogues are low in the natural production strains. Recent developments in genetic engineering approaches have made it possible to increase MTM production through more rational strategies based on genetic manipulations and heterologous expression in optimized chassis. Recent construction of various genetically modified strains of Streptomyces lividans has shown their use for efficient heterologous production of various biologically active secondary metabolites including MTM. KEY POINTS: • Discovery a novel bifunctional glycosyl hydrolase from uncultured microorganism. • Heterologous production of MTM in engineered S. lividans strains is efficient.

Recent achievements in the generation of stable genome alterations/mutations in species of the genus Streptomyces.

The bacteria of the genus Streptomyces are the most valuable source of natural products of industrial and medical importance. A recent explosion of Streptomyces genome sequence data has revealed the enormous genetic potential of new biologically active compounds, although many of them are silent under laboratory conditions. Efficient and stable manipulation of the genome is necessary to induce their production. Comprehensive studies in the past have led to a large and versatile collection of molecular biology tools for gene manipulation of Streptomyces, including various replicative plasmids. However, biotechnological applications of these bacteria require stable genome alterations/mutations. To accomplish such stable genome editing, two major strategies for streptomycetes have been developed: (1) integration into the chromosome through Att/Int site-specific integration systems based on Streptomyces actinophages (ΦC31, ΦBT1, VWB, TG1, SV1, R4, ΦJoe, µ1/6) or pSAM2 integrative plasmid; (2) integration by homologous recombination using suicidal non-replicating vectors. The present review is an attempt to provide a comprehensive summary of both approaches for stable genomic engineering and to outline recent advances in these strategies, such as CRISPR/Cas9, which have successfully manipulated Streptomyces strains to improve their biotechnological properties and increase production of natural or new gene-manipulated biologically active compounds.

An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA.

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.

Correction to: Increased heterologous production of the antitumoral polyketide mithramycin a by engineered Streptomyces lividans TK24 strains.

The original publication contains error error in the Materials and Methods section and in the acknowledgement section.

Increased heterologous production of the antitumoral polyketide mithramycin A by engineered Streptomyces lividans TK24 strains.

Mithramycin A is an antitumor compound used for treatment of several types of cancer including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia and Paget's disease. Selective modifications of this molecule by combinatorial biosynthesis and biocatalysis opened the possibility to produce mithramycin analogues with improved properties that are currently under preclinical development. The mithramycin A biosynthetic gene cluster from Streptomyces argillaceus ATCC12956 was cloned by transformation assisted recombination in Saccharomyces cerevisiae and heterologous expression in Streptomyces lividans TK24 was evaluated. Mithramycin A was efficiently produced by S. lividans TK24 under standard fermentation conditions. To improve the yield of heterologously produced mithramycin A, a collection of derivative strains of S. lividans TK24 were constructed by sequential deletion of known potentially interfering secondary metabolite gene clusters using a protocol based on the positive selection of double crossover events with blue pigment indigoidine-producing gene. Mithramycin A production was evaluated in these S. lividans strains and substantially improved mithramycin A production was observed depending on the deleted gene clusters. A collection of S. lividans strains suitable for heterologous expression of actinomycetes secondary metabolites were generated and efficient production of mithramycin A with yields close to 3 g/L, under the tested fermentation conditions was achieved using these optimized collection of strains.

Characterisation of the genes involved in the biosynthesis and attachment of the aminodeoxysugar D-forosamine in the auricin gene cluster of Streptomyces aureofaciens CCM3239.

We previously identified the aur1 gene cluster which produces the angucycline antibiotic auricin. Preliminary characterisation of auricin revealed that it is modified by a single aminodeoxysugar, D-forosamine. Here we characterise the D-forosamine-specific genes. The four close tandem genes, aur1TQSV, encoding enzymes involved in the initial steps of the deoxysugar biosynthesis, were located on a large operon with other core auricin biosynthetic genes. Deleting these genes resulted in the absence of auricin and the production of deglycosylated auricin intermediates. The two final D-forosamine biosynthetic genes, sa59, an NDP-hexose aminotransferase, and sa52, an NDP-aminohexose N-dimethyltransferase, are located in a region rather distant from the core auricin genes. A deletion analysis of these genes confirmed their role in D-forosamine biosynthesis. The Δsa59 mutant had a phenotype similar to that of the cluster deletion mutant, while the Δsa52 mutant produced an auricin with a demethylated D-forosamine. Although auricin contains a single deoxyhexose, two glycosyltransferase genes were found to participate in the attachment of D-forosamine to the auricin aglycon. An analysis of the expression of the D-forosamine biosynthesis genes revealed that the initial D-forosamine biosynthetic genes aur1TQSV are regulated together with the other auricin core genes by the aur1Ap promoter under the control of the auricin-specific activator Aur1P. The expression of the other D-forosamine genes, however, is governed by promoters differentially dependent upon the two SARP family auricin-specific activators Aur1PR3 and Aur1PR4. These promoters contain direct repeats similar to the SARP consensus sequence and are involved in the interaction with both regulators.

A γ-butyrolactone autoregulator-receptor system involved in the regulation of auricin production in Streptomyces aureofaciens CCM 3239.

The γ-butyrolactone (GBL) autoregulator-receptor systems play a role in controlling secondary metabolism and/or morphological differentiation in many Streptomyces species. We previously identified the aur1 gene cluster, located on the Streptomyces aureofaciens CCM 3239 large linear plasmid pSA3239, which is responsible for the production of the angucycline antibiotic auricin. Here, we describe the characterisation of two genes, sagA and sagR, encoding GBL autoregulatory signalling homologues, which lie in the upstream part of the aur1 cluster. SagA was similar to GBL synthases and SagR to GBL receptors. The expression of each gene is directed by its own promoter, sagAp for sagA and sagRp for sagR. Both genes were active mainly during the exponential phase, and their transcription was interdependent. The disruption of sagA abolished auricin production, while the disruption of sagR resulted in precocious but dramatically reduced auricin production. Transcription from the aur1Pp and aur1Rp promoters, which direct the expression of auricin-specific cluster-situated regulators (CSRs), was also precocious and increased in the sagR mutant strain. In addition, SagR was also shown to specifically bind both promoters in vitro. These results indicated that the SagA-SagR GBL system regulates auricin production. Unlike many other GBL receptors, SagR does not bind its own promoter, but Aur1R, an auricin-specific repressor from the family of pseudo GBL receptors, does bind both sagAp and sagRp promoters. Moreover, the expression of both promoters was deregulated in an aur1R mutant, indicating that the SagA-SagR GBL system is regulated by a feedback mechanism involving the auricin-specific CSR Aur1R, which regulates downstream.

Intriguing properties of the angucycline antibiotic auricin and complex regulation of its biosynthesis.

Streptomyces bacteria are major producers of bioactive natural products, including many antibiotics. We identified a gene cluster, aur1, in a large linear plasmid of Streptomyces aureofaciens CCM3239. The cluster is responsible for the production of a new angucycline polyketide antibiotic auricin. Several tailoring biosynthetic genes were scatted in rather distant aur1 flanking regions. Auricin was produced in a very narrow growth phase interval of several hours after entry into stationary phase, after which it was degraded to non-active metabolites because of its instability at the high pH values reached after the production stage. Strict transcriptional regulation of the auricin biosynthetic gene cluster has been demonstrated, including feed-forward and feedback control by auricin intermediates via several of the huge number of regulatory genes present in the aur1 cluster. The complex mechanism may ensure strict confinement of auricin production to a specific growth stage.

Multiple SigB homologues govern the transcription of the ssgBp promoter in the sporulation-specific ssgB gene in Streptomyces coelicolor A3(2).

Unlike Bacillus subtilis, Streptomyces coelicolor contains nine SigB homologues of the stress-response sigma factor SigB. By using a two-plasmid system, we previously identified promoters recognized by these sigma factors. Almost all promoters were recognized by several SigB homologues. However, no specific sequences of these promoters were found. One of these promoters, ssgBp, was selected to examine this cross-recognition in the native host. It controls the expression of the sporulation-specific gene ssgB. Using a luciferase reporter, the activity of this promoter in S. coelicolor and nine mutant strains lacking individual sigB homologous genes showed that sgBp is dependent on three sigma factors, SigH, SigN, and SigI. To determine which nucleotides in the-10 region are responsible for the selection of a specific SigB homologue, promoters mutated at the last three nucleotide positions were tested in the two-plasmid system. Some mutant promoters were specifically recognized by a distinct set of SigB homologues. Analysis of these mutant promoters in the native host showed the role of these nucleotides. A conserved nucleotide A at position 5 was essential for promoter activity, and two variable nucleotides at positions 4 and 6 were responsible for the partial selectivity of promoter recognition by SigB homologues.

Phenotypic analysis of Salmonella enterica serovar Typhimurium rpoE mutants encoding RNA polymerase extracytoplasmic stress response sigma factors σ(E) with altered promoter specificity.

We previously identified mutants in the rpoE gene of Salmonella enterica serovar Typhimurium (S. Typhimurium) encoding RNA polymerase extracytoplasmic stress response sigma factors σ(E) with altered promoter specificity. The replacement of the conserved R171 residue in the conserved region 4.2 of σ(E) by different amino acid residues exhibited different phenotypes. While R171A almost completely abolished sigma factor activity, R171G and R171C mutant changes imparted a relaxed recognition phenotype to the sigma factor. In the present study, we introduced these mutations into the S. Typhimurium chromosome to investigate their phenotype during ethanol stress and in promoter recognition. Both relaxed sigma factors were found to initiate transcription from a high number of artificial promoters in the S. Typhimurium genome. Both mutants had substantially decreased activity under stress conditions. However, this decreased activity and also the recognition of atypical promoters had no significant effect upon growth, even in stressful conditions.

Strict control of auricin production in Streptomyces aureofaciens CCM 3239 involves a feedback mechanism.

The polyketide gene cluster aur1 is responsible for the production of the angucycline antibiotic auricin in Streptomyces aureofaciens CCM 3239. Auricin production is regulated in a complex manner involving several regulators, including a key pathway-specific positive regulator Aur1P that belongs to the family of 'atypical' response regulators. Production of auricin is induced after entry into stationary phase. However, auricin was produced in only a short time interval of several hours. We found that the decrease of auricin production was due to a strict regulation of auricin biosynthetic genes at the transcriptional level by a feedback mechanism; auricin and/or its intermediate(s) inhibited binding of Aur1P to its cognate biosynthetic promoter aur1Ap and consequently stopped its activation. In addition, we also determined that synthesised auricin is unstable during growth of S. aureofaciens CCM3239 in the production medium even though purified auricin is stable for days in various organic solvents. The critical parameter affecting its stability was pH. Auricin is stable at acid pH and unstable at neutral and alkaline pH. The drop in auricin concentration was due to an increase of pH shortly after induction of auricin production during cultivation of S. aureofaciens CCM3239.

Investigating the initial steps of auricin biosynthesis using synthetic biology.

Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239) contains a type II polyketide synthase (PKS) biosynthetic gene cluster (BGC) aur1 whose genes were highly similar to angucycline BGCs. However, its product auricin is structurally different from all known angucyclines. It contains a spiroketal pyranonaphthoquinone aglycone similar to griseusins and is modified with D-forosamine. Here, we describe the characterization of the initial steps in auricin biosynthesis using a synthetic-biology-based approach. We have created a plasmid system based on the strong kasOp* promoter, RBS and phage PhiBT1-based integration vector, where each gene in the artificial operon can be easily replaced by another gene using unique restriction sites surrounding each gene in the operon. The system was validated with the initial landomycin biosynthetic genes lanABCFDLE, leading to the production of rabelomycin after its integration into Streptomyces coelicolor M1146. However, the aur1DEFCGHA homologous genes from the auricin aur1 BGC failed to produce rabelomycin in this system. The cause of this failure was inactive aur1DE genes encoding ketosynthases α and ß (KSα, KSß). Their replacement with homologous aur2AB genes from the adjacent aur2 BGC resulted in rabelomycin production that was even higher after the insertion of two genes from the aur1 BGC, aur1L encoding 4-phosphopantetheinyl transferase (PPTase) and aur1M encoding malonyl-CoA:ACP transacylase (MCAT), suggesting that Aur1L PPTase is essential for the activation of the acyl carrier protein Aur1F. These results suggest an interesting communication of two BGCs, aur1 and aur2, in the biosynthesis of the initial structure of auricin aglycone.

The role of two SARP family transcriptional regulators in regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239.

Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.

The linear plasmid pSA3239 is essential for the replication of the Streptomyces lavendulae subsp. lavendulae CCM 3239 chromosome.

We previously reported the complete genome of Streptomyces lavendulae subsp. lavendulae CCM 3239, containing the linear chromosome and the large linear plasmid pSA3239. Although the chromosome exhibited replication features characteristic for the archetypal end-patching replication, it lacked the tap/tpg gene pair for two proteins essential for this process. However, this archetypal tpgSa-tapSa operon is present in pSA3239. Complete genomic sequence of the S. lavendulae Del-LP strain lacking this plasmid revealed the circularization of its chromosome with a large deletion of both arms. These results suggest an essential role of pSA3239-encoded TapSa/TpgSa in the end-patching replication of the chromosome.

The role of the TetR-family transcriptional regulator Aur1R in negative regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239.

Two regulatory genes, aur1P and aur1R, have been previously identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. The aur1P gene encodes a protein similar to the response regulators of bacterial two-component signal transduction systems and has been shown to specifically activate expression of the auricin biosynthetic genes. The aur1R gene encodes a protein homologous to transcriptional repressors of the TetR family. Here we describe the characterization of the aur1R gene. Expression of the gene is directed by a single promoter, aur1Rp, which is induced just before stationary phase. Disruption of aur1R in S. aureofaciens CCM 3239 had no effect on growth and differentiation. However, the disrupted strain produced more auricin than its parental wild-type S. aureofaciens CCM 3239 strain. Transcription from the aur1Ap and aur1Pp promoters, directing expression of the first biosynthetic gene in the auricin gene cluster and the pathway-specific transcriptional activator, respectively, was increased in the S. aureofaciens CCM 3239 aur1R mutant strain. However, Aur1R was shown to bind specifically only to the aur1Pp promoter in vitro. This binding was abolished by the addition of auricin and/or its intermediates. The results indicate that the Aur1R regulator specifically represses expression of the aur1P gene, which encodes a pathway-specific activator of the auricin biosynthetic gene cluster in S. aureofaciens CCM 3239, and that this repression is relieved by auricin or its intermediates.

Pleiotropic anti-anti-sigma factor BldG is phosphorylated by several anti-sigma factor kinases in the process of activating multiple sigma factors in Streptomyces coelicolor A3(2).

The anti-anti-sigma factor BldG has a pleiotropic function in Streptomyces coelicolor A3(2), regulating both morphological and physiological differentiation. Together with the anti-sigma factor UshX, it participates in a partner-switching activation of the sigma factor σH, which has a dual role in the osmotic stress response and morphological differentiation in S. coelicolor A3(2). In addition to UshX, BldG also interacts with the anti-sigma factor ApgA, although no target sigma factor has yet been identified. However, neither UshX nor ApgA phosphorylates BldG. This phosphorylation is provided by the anti-sigma factor RsfA, which is specific for the late developmental sigma factor σF. However, BldG is phosphorylated in the rsfA mutant, suggesting that some other anti-sigma factors containing HATPase_c kinase domain are capable to phosphorylate BldG in vivo. Bacterial two-hybrid system (BACTH) was therefore used to investigate the interactions of all suitable anti-sigma factors of S. coelicolor A3(2) with BldG. At least 15 anti-sigma factors were found to interact with BldG. These interactions were confirmed by native PAGE. In addition to RsfA, BldG is specifically phosphorylated on the conserved phosphorylation Ser57 residue by at least seven additional anti-sigma factors. However, only one of them, SCO7328, has been shown to interact with three sigma factors, σG, σK and σM. A mutant with deleted SCO7328 gene was prepared in S. coelicolor A3(2), however, no specific function of SCO7328 in growth, differentiation or stress response could be attributed to this anti-sigma factor. These results suggest that BldG is specifically phosphorylated by several anti-sigma factors and it plays a role in the regulation of several sigma factors in S. coelicolor A3(2). This suggests a complex regulation of the stress response and differentiation in S. coelicolor A3(2) through this pleiotropic anti-sigma factor.