Actinorhodin Production Research Articles

Gene-expression analysis of acidic pH shock effects on two-component systems in Streptomyces coelicolor

In this study, transcriptional analyses by using reverse transcription-polymerase chain reaction (RT-PCR), DNA chip, and quantitative real time-PCR were performed to investigate effects of acidic pH shock on two-component systems in Streptomyces coelicolor A3(2). Two-component systems of cseC/cseB and vanS/vanR known to be closely linked with self-protection against cell wall hydrolysis caused by external stimuli were upregulated by the pH shock. The chiS/chiR, afsQ2/afsQ1, ecrA2/ecrA1, bldM, ramC/ramR, and ragK/ragR known to be positively associated with the initiation of secondary metabolism were also upregulated. The cutS/cutR known to be negatively related to the secondary metabolism was, however, slightly downregulated. Upregulation or downregulation by the acidic pH shock of these two-component regulator systems might have contributed in a concerted manner to the enhancement of secondary metabolite production, at least, in this particular case of actinorhodin production in S. coelicolor A3(2).

Cross-talk between an orphan response regulator and a noncognate histidine kinase inStreptomyces coelicolor

Two-component systems (TCSs), typically consisting of a histidine kinase (HK) and a cognate response regulator (RR), are the most common signaling systems in bacteria. Besides paired genes encoding TCSs, there also exists unpaired HKs and orphan RRs. In Streptomyces coelicolor, 13 orphan RRs have been annotated. Because of lack of cognate HKs, little is known as yet about the regulation of orphan RRs. Bioinformatic analysis revealed that several orphan RRs had high amino acid sequence identities with RRs from typical TCSs in S. coelicolor. Among them, the orphan RR SCO3818 and RR SCO0204, which paired with HK SCO0203, showed the highest identity (65%), suggesting that the two RRs might both be under the regulation of SCO0203. Following studies showed that SCO0203 could phosphorylate not only SCO0204 but also SCO3818. Deletion of either sco0203 or sco3818 led to enhanced production of blue-pigmented antibiotic actinorhodin, which indicated a functional correlation between SCO0203 and SCO3818. These results suggested that SCO3818 might be regulated by SCO0203. This is the first report describing the regulation of an orphan RR by an HK. Moreover, this is also the first identification of cross-talk between different TCS components in S. coelicolor.

NdgR, an IclR-like regulator involved in amino-acid-dependent growth, quorum sensing, and antibiotic production in Streptomyces coelicolor

NdgR (regulator for nitrogen source-dependent growth and antibiotic production), an IclR-like regulator, has been initially identified as a binding protein to the promoters of doxorubicin biosynthetic genes in Streptomyces peucetius by DNA affinity capture assay method. NdgR is well conserved throughout the Streptomyces species and many other bacteria such as Mycobacteria and Corynebacteria. In Streptomyces coelicolor, ndgR deletion mutant showed slow cell growth and defects in differentiation and enhances the production of actinorhodin (ACT) in minimal media containing certain amino acids where wild-type strain could not produce ACT. Although deletion mutant of ndgR showed different antibiotic production in minimal media containing Leu or Gln, it only showed reduced mRNA expression levels of the genes involved in leucine metabolism. Neither NdgR-dependent expression of glnA nor direct binding of NdgR protein to glnA, glnII, and glnR promoters was observed. However, ScbR, which is governed by NdgR shown by gel mobility shift assay, binds to promoter of glnR, suggesting indirect regulation of glutamine metabolism by NdgR. NdgR protein binds to intergenic region of ndgR-leuC, and scbR-scbA involved in gamma-butyrolactone. Two-dimensional gel analysis has shown a global effect of ndgR deletion in protein expression, including up-regulated proteins involved in ACT synthesis and down-regulation of chaperones such as GroEL, GroES, and DnaK. These results suggest a global regulatory role for NdgR in amino acid metabolisms, quorum sensing, morphological changes, antibiotic production, and expression of chaperonines in S. coelicolor.

Functional Expression of SAV3818, a Putative TetR-Family Transcriptional Regulatory Gene from Streptomyces avermitilis, Stimulates Antibiotic Production in Streptomyces Species

Avermectin and its analogs are major commercial antiparasitic agents in the fields of animal health, agriculture, and human infections. Previously, comparative transcriptome analysis between the low-producer S. avermitilis ATCC31267 and the high-producer S. avermitilis ATCC31780 using a S. avermitilis whole genome chip revealed that 50 genes were overexpressed at least two-fold higher in S. avermitilis ATCC31780. To verify the biological significance of some of the transcriptomics-guided targets, five putative regulatory genes were individually cloned under the strong-andconstitutive promoter of the Streptomyces expression vector pSE34, followed by the transformation into the lowproducer S. avermitilis ATCC31267. Among the putative genes tested, three regulatory genes including SAV213, SAV3818, and SAV4023 exhibited stimulatory effects on avermectin production in S. avermitilis ATCC31267. Moreover, overexpression of SAV3818 also stimulated actinorhodin production in both S. coelicolor M145 and S. lividans TK21, implying that the SAV3818, a putative TetR-family transcriptional regulator, could be a global upregulator acting in antibiotic production in Streptomyces species.

AfsQ1-Q2-sigQ is a pleiotropic but conditionally required signal transduction system for both secondary metabolism and morphological development in Streptomyces coelicolor

Two-component system AfsQ1-Q2 of Streptomyces coelicolor was identified previously for its ability to stimulate actinorhodin (ACT) and undecylprodigiosin (RED) production in Streptomyces lividans. However, disruption of either afsQ1 or afsQ2 in S. coelicolor led to no detectable changes in secondary metabolite formation or morphogenesis. In this study, we reported that, when cultivated on defined minimal medium (MM) with glutamate as the sole nitrogen source, the afsQ mutant exhibited significantly decreased ACT, RED, and calcium-dependent antibiotic (CDA) production and rapid growth of aerial mycelium. In addition, we also found that deletion of sigQ, which is located upstream of afsQ1-Q2 and encodes a putative sigma factor, led to the precocious hyperproduction of these antibiotics and delayed formation of sporulating aerial mycelium in the same glutamate-based defined MM. Reverse-transcription polymerase chain reaction and egfp fusion analyses showed that the expression of sigQ was under control by afsQ. In addition, deletion of both afsQ-sigQ resulted in the phenotype identical to that of afsQ mutant. The results suggested that afsQ1-Q2 and sigQ worked together in the regulation of both antibiotic biosynthesis and morphological development, and sigQ might be responsible for antagonizing the function of AfsQ1-Q2 in S. coelicolor, however, in a medium-dependent manner. Moreover, the study showed that the medium-dependent regulation of antibiotic biosynthesis by AfsQ1-Q2-SigQ was through pathway-specific activator genes actII-ORF4, redD, and cdaR. The study provides new insights on regulation of antibiotic biosynthesis and morphological development in S. coelicolor.

Enhanced Production of the Fluorinated Nucleoside Antibiotic Nucleocidin by a rifR-Resistant Mutant of Streptomyces calvus IFO13200

Nucleocidin, a fairly broad antibacterial and trypanocidal agent produced by Streptomyces calvus, has the unique structure of 4′-α-fluoro-5′-O-sulfamoyl adenosine. This nucleoside antibiotic has been a target for organic synthesis in past decades; however, microbial large-scale production has not been established due to low yield and poor reproducibility. To activate the dormant secondary metabolism of S. calvus, we examined the effect of an rpoB mutation that was induced by ultraviolet light irradiation. The resulting rifampicin-resistant strains showed remarkably improved antibiotic activity, which was extracted by n-butanol and identified by Electron-Spray-Ionization Mass Spectrometry. DNA sequencing identified double mutations, C1309A and C1318A, in the rpoB gene (according to the numbering of Streptomyces coelicolor rpoB). The resulting amino acid substitutions, H437N and R440S, corresponded to two of the previously reported amino acid substitutions that allowed the activation of dormant actinorhodin production in S. lividans 66.

A Novel Insertion Mutation in Streptomyces coelicolor Ribosomal S12 Protein Results in Paromomycin Resistance and Antibiotic Overproduction

We identified a novel paromomycin resistance-associated mutation in rpsL, caused by the insertion of a glycine residue at position 92, in Streptomyces coelicolor ribosomal protein S12. This insertion mutation (GI92) resulted in a 20-fold increase in the paromomycin resistance level. In combination with another S12 mutation, K88E, the GI92 mutation markedly enhanced the production of the blue-colored polyketide antibiotic actinorhodin and the red-colored antibiotic undecylprodigiosin. The gene replacement experiments demonstrated that the K88E-GI92 double mutation in the rpsL gene was responsible for the marked enhancement of antibiotic production observed. Ribosomes with the K88E-GI92 double mutation were characterized by error restrictiveness (i.e., hyperaccuracy). Using a cell-free translation system, we found that mutant ribosomes harboring the K88E-GI92 double mutation but not ribosomes harboring the GI92 mutation alone displayed sixfold greater translation activity relative to that of the wild-type ribosomes at late growth phase. This resulted in the overproduction of actinorhodin, caused by the transcriptional activation of the pathway-specific regulatory gene actII-orf4, possibly due to the increased translation of transcripts encoding activators of actII-orf4. The mutant with the K88E-GI92 double mutation accumulated a high level of ribosome recycling factor at late stationary phase, underlying the high level of protein synthesis activity observed.

A putative secreted solute binding protein, SCO6569 is a possible AfsR2-dependent down-regulator of actinorhodin biosynthesis in Streptomyces coelicolor

AfsR2 is a global regulatory protein involved in stimulating the biosynthesis of antibiotics in various Streptomyces species. Using 2-D gel electrophoresis and MALDI-TOF analyses, several putative AfsR2-target proteins were previously identified. In order to verify the biological significance of the proteomics-guided targets, the sixteen putative AfsR2 target genes were individually cloned under the strong-and-constitutive promoter (PermE *) of the Streptomyces expression vector, pSE34, followed by the transformation into both Streptomyces lividans and Streptomyces coelicolor. Among the putative AfsR2-target genes tested, the expression of a putative secreted solute binding protein, SCO6569 significantly repressed biosynthesis of actinorhodin in S. coelicolor, and the transcripts encoded by the pathway-specific activator, actII-ORF4 for actinorhodin were also reduced in SCO6569-overexpressing S. coelicolor. Moreover, SCO6569 gene-disruption in S. coelicolor further enhanced actinorhodin production, suggesting that the SCO6569 could be a possible AfsR2-dependent down-regulator acting in actinorhodin biosynthesis in S. coelicolor.

Acidic pH shock induces the expressions of a wide range of stress-response genes

BackgroundEnvironmental signals usually enhance secondary metabolite production in Streptomycetes by initiating complex signal transduction system. It is known that different sigma factors respond to different types of stresses, respectively in Streptomyces strains, which have a number of unique signal transduction mechanisms depending on the types of environmental shock. In this study, we wanted to know how a pH shock would affect the expression of various sigma factors and shock-related proteins in S. coelicolor A3(2).ResultsAccording to the results of transcriptional and proteomic analyses, the major number of sigma factor genes were upregulated by an acidic pH shock. Well-studied sigma factor genes of sigH (heat shock), sigR (oxidative stress), sigB (osmotic shock), and hrdD that play a major role in the secondary metabolism, were all strongly upregulated by the pH shock. A number of heat shock proteins including the DnaK family and chaperones such as GroEL2 were also observed to be upregulated by the pH shock, while their repressor of hspR was strongly downregulated. Oxidative stress-related proteins such as thioredoxin, catalase, superoxide dismutase, peroxidase, and osmotic shock-related protein such as vesicle synthases were also upregulated in overall.ConclusionFrom these observations, an acidic pH shock was considered to be one of the strongest stresses to influence a wide range of sigma factors and shock-related proteins including general stress response proteins. The upregulation of the sigma factors and shock proteins already found to be related to actinorhodin biosynthesis was considered to have contributed to enhanced actinorhodin productivity by mediating the pH shock signal to regulators or biosynthesis genes for actinorhodin production.

SarA influences the sporulation and secondary metabolism inStreptomyces coelicolorM145

The filamentous bacteria Streptomyces exhibit a complex life cycle involving morphological differentiation and secondary metabolism. A putative membrane protein gene sarA (sco4069), sporulation and antibiotic production related gene A, was partially characterized in Streptomyces coelicolor M145. The gene product had no characterized functional domains and was highly conserved in Streptomyces. Compared with the wild-type M145, the sarA mutant accelerated sporulation and dramatically decreased the production of actinorhodin and undecylprodigiosin. Reverse transcription-polymerase chain reaction analysis showed that SarA influenced antibiotic production by controlling the abundance of actII-orf4 and redZ messenger RNA.

SarA influences the sporulation and secondary metabolism in <italic>Streptomyces coelicolor</italic> M145

The filamentous bacteria Streptomyces exhibit a complex life cycle involving morphological differentiation and secondary metabolism. A putative membrane protein gene sarA (sco4069), sporulation and antibiotic production related gene A, was partially characterized in Streptomyces coelicolor M145. The gene product had no characterized functional domains and was highly conserved in Streptomyces. Compared with the wild-type M145, the sarA mutant accelerated sporulation and dramatically decreased the production of actinorhodin and undecylprodigiosin. Reverse transcription-polymerase chain reaction analysis showed that SarA influenced antibiotic production by controlling the abundance of actII-orf4 and redZ messenger RNA.

Actinorhodin biosynthesis is negatively regulated by a putative AfsR2-dependent secreted solute binding protein, SCO6569 in Streptomyces coelicolor

Deletion of either of the genes (zwf), coding for glucose-6-phosphate dehydrogenase isoforms in Streptomyces coelicolor, resulted in an increase in in vitro determined glucose-6-phosphate dehydrogenase activity in the surviving isoform. Mutants carrying either one of these deletions exhibited significantly increased actinorhodin biosynthesis, an effect that was not predicted by simple flux balance simulation. The flux variability distribution, obtained using experimental measured variables as constraints, was examined. The reactions whose flux variabilities were most significantly affected, as a result of the increased actinorhodin production and changes in nutrient uptake rates, included reactions from a number of different pathways. These could be connected together to form an actinorhodin-generating network. They included glutamate synthesising and catabolising reactions. Subsequent experiments, in which glutamate was fed to steady state cultures, resulted in increased actinorhodin production and the in silico model predicted the importance of the same reactions that were amplified in the zwf model. Introduction of a constitutively expressed copy of actII-orf4, the pathway-specific activator of the actinorhodin pathway genes, on the integrating plasmid pIJ8714 resulted in a switch from growth dissociated (typical secondary metabolite) to growth associated antibiotic production.

Antibiotic Overproduction in Streptomyces coelicolor A3(2) Mediated by Phosphofructokinase Deletion

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by (13)C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

Role of Phosphopantetheinyl Transferase Genes in Antibiotic Production by Streptomyces coelicolor

The phosphopantetheinyl transferase genes SCO5883 (redU) and SCO6673 were disrupted in Streptomyces coelicolor. The redU mutants did not synthesize undecylprodigiosin, while SCO6673 mutants failed to produce calcium-dependent antibiotic. Neither gene was essential for actinorhodin production or morphological development in S. coelicolor, although their mutation could influence these processes.

Characterization of a negative regulator AveI for avermectin biosynthesis in Streptomyces avermitilis NRRL8165

A transcriptional activator for actinorhodin biosynthesis, AtrA, was previously characterized in Streptomyces coelicolor A3(2), and an orthologue of atrA, named aveI, is identified in the Streptomyces avermitilis NRRL8165 genome (Uguru et al., Mol Microbiol, 58:131-150, 2005). In this study, genetic and functional characterization of aveI gene was reported. Deletion of aveI gene led to increased biosynthesis of avermectin B1a by about 16-fold. The increased synthesis of avermectin B1a was suppressed by complementation with either aveI gene or its orthologue gene atrA from S. coelicolor, suggesting AveI and AtrA shared the similar functionality and were negative regulators for avermectin biosynthesis in S. avermitilis. However, when aveI was introduced into S. coelicolor on a multi-copy plasmid, the production of actinorhodin was significantly increased, indicating that aveI had a positive effect on actinorhodin biosynthesis in S. coelicolor, the same as its orthologue atrA. Electrophoretic mobility shift assays revealed AveI can bind specifically to the promoter region of actII-ORF4 in vitro but not that of aveR. Although its mechanism still needs to be defined, the species-differential regulation by the same regulator may represent an example of the evolutional strategy that enables bacteria to adapt the existing molecular machinery to a variety of functionalities for growth and survival.

Effects of extracellular ATP on the physiology ofStreptomyces coelicolorA3(2)

Because ATP is an extracellular effector in animal and plant systems and derivatives of ATP, such as S-adenosylmethionine and cAMP, can control antibiotic production and morphological differentiation in Streptomyces, we hypothesized that extracellular ATP (exATP) can also affect physiologies of Streptomyces. We found that the addition of 10 microM exATP to Streptomyces coelicolor A3(2) cultures resulted in enhanced actinorhodin and undecylprodigiosin production and morphological differentiation on solid medium. However, these phenotypes were reduced by the addition of a 10-fold higher concentration of exATP (100 microM). Intracellular ATP concentrations were also modulated in response to changes in exATP. ATP analogs, added at a 100-fold lower concentration, affected Streptomyces similarly to that seen for 10 microM exATP. The enhanced promoter activity of actII-orf4 indicated that 10 microM exATP affect the transcriptional level for actinorhodin production. Results from this study suggest that exATP is an effector for the physiology of S. coelicolor and careful manipulation of exATP may significantly enhance the high-yield production of antibiotics by S. coelicolor.

Selection of objective function in genome scale flux balance analysis for process feed development in antibiotic production

Using flux variability analysis of a genome scale metabolic network of Streptomyces coelicolor, a series of reactions were identified, from disparate pathways that could be combined into an actinorhodin-generating mini-network. Candidate process feed nutrients that might be expected to influence this network were used in process simulations and in silico predictions compared to experimental findings. Ranking potential process feeds by flux balance analysis optimisation, using either growth or antibiotic production as objective function, did not correlate with experimental actinorhodin yields in fed processes. However, the effect of the feeds on glucose assimilation rate (using glucose uptake as objective function) ranked them in the same order as in vivo antibiotic production efficiency, consistent with results of a robustness analysis of the effect of glucose assimilation on actinorhodin production.

Expression and Characterization of theStreptomyces coelicolorSerine/Threonine Protein Kinase PkaD

We identified and characterized the gene encoding a new eukaryotic-type protein kinase from Streptomyces coelicolor A3(2) M145. PkaD, consisting of 598 amino acid residues, contained the catalytic domain of eukaryotic protein kinases in the N-terminal region. A hydrophobicity plot indicated the presence of a putative transmembrane spanning sequence downstream of the catalytic domain, suggesting that PkaD is a transmembrane protein kinase. The recombinant PkaD was found to be phosphorylated at the threonine and tyrosine residues. In S. coelicolor A3(2), pkaD was transcribed as a monocistronic mRNA, and it was expressed constitutively throughout the life cycle. Disruption of chromosomal pkaD resulted in a significant loss of actinorhodin production. This result implies the involvement of pkaD in the regulation of secondary metabolism.

HmgA, transcriptionally activated by HpdA, influences the biosynthesis of actinorhodin in Streptomyces coelicolor

Homogentisate 1,2-dioxygenase (HmgA) converts homogentisate to maleylacetoacetate in the pathway of tyrosine catabolism in living organisms. Studies published to date have not elucidated the transcriptional regulation of hmgA and its biological function in Streptomyces spp. Here it is shown that the transcription of hmgA in Streptomyces coelicolor was activated by HpdA, an AsnC-type activator, in the presence of tyrosine. Disruption of hmgA led to enhanced production of diffusible brown pigment, suggesting that hmgA is required for tyrosine catabolism in Streptomyces coelicolor. The production of actinorhodin in the hmgA disruption mutant was several fold higher than that in the wild-type strain (M145), indicating that hmgA of tyrosine catabolism correlates with actinorhodin biosynthesis. Furthermore, S1 mapping showed that the transcription of actII-ORF4, a pathway-specific activator gene of actinorhodin biosynthesis, was increased in the hmgA disruption mutant. These results reveal the transcriptional regulation of hmgA and the positive contribution of hmgA disruption to the actinorhodin biosynthesis in Streptomyces coelicolor.

Genome-wide transcriptome analysis reveals that a pleiotropic antibiotic regulator, AfsS, modulates nutritional stress response in Streptomyces coelicolor A3(2)

BackgroundA small "sigma-like" protein, AfsS, pleiotropically regulates antibiotic biosynthesis in Streptomyces coelicolor. Overexpression of afsS in S. coelicolor and certain related species causes antibiotic stimulatory effects in the host organism. Although recent studies have uncovered some of the upstream events activating this gene, the mechanisms through which this signal is relayed downstream leading to the eventual induction of antibiotic pathways remain unclear.ResultsIn this study, we employed whole-genome DNA microarrays and quantitative PCRs to examine the transcriptome of an afsS disruption mutant that is completely deficient in the production of actinorhodin, a major S. coelicolor antibiotic. The production of undecylprodigiosin, another prominent antibiotic, was, however, perturbed only marginally in the mutant. Principal component analysis of temporal gene expression profiles identified two major gene classes each exhibiting a distinct coordinate differential expression pattern. Surprisingly, nearly 70% of the >117 differentially expressed genes were conspicuously associated with nutrient starvation response, particularly those of phosphate, nitrogen and sulfate. Furthermore, expression profiles of some transcriptional regulators including at least two sigma factors were perturbed in the mutant. In almost every case, the effect of afsS disruption was not observed until the onset of stationary phase.ConclusionOur data suggests a comprehensive role for S. coelicolor AfsS as a master regulator of both antibiotic synthesis and nutritional stress response, reminiscent of alternative sigma factors found in several bacteria.