Actinorhodin Production Research Articles

SsgA Is Essential for Sporulation of Streptomyces coelicolor A3(2) and Affects Hyphal Development by Stimulating Septum Formation

The role of ssgA in cell division and development of streptomycetes was analyzed. An ssgA null mutant of Streptomyces coelicolor produced aerial hyphae but failed to sporulate, and ssgA can therefore be regarded as a novel whi gene. In addition to the morphological changes, antibiotic production was also disturbed, with strongly reduced actinorhodin production. These defects could be complemented by plasmid-borne ssgA. In the wild-type strain, transcription of ssgA was induced by nutritional shift-down and was shown to be linked to that of the upstream-located gene ssgR, which belongs to the family of iclR-type transcriptional regulator genes. Analysis of mycelium harvested from liquid-grown cultures by transmission electron microscopy showed that septum formation had strongly increased in ssgA-overexpressing strains in comparison to wild-type S. coelicolor and that spore-like compartments were produced at high frequency. Furthermore, the hyphae were significantly wider and contained irregular and often extremely thick septa. These data underline the important role for ssgA in Streptomyces cell division.

Cloning of a two-component regulatory system probably involved in the regulation of chitinase in Streptomyces coelicolor A3(2).

Using the method for the identification of promoters recognized by the sporulation specific sigma factor (sigma F), we identified a positive 950 bp Sau3AI DNA fragment in Streptomyces coelicolor A3(2). High-resolution S1-nuclease mapping identified a potential promoter, PF35, in the E. coli two-plasmid system similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor (sigma B). However, the putative sigF-dependent promoter, PF35, was inactive in S. coelicolor in the course of differentiation, and it was located divergently in the promoter region directing expression of the chiC gene encoding chitinase. Sequence analysis of the region potentially governed by PF35 revealed two translationally coupled genes encoding proteins similar to bacterial two-component regulatory systems, and with the highest similarity to the two-component system chiS, chiR, regulating chitinase activity in Streptomyces thermoviolaceus. However, the genes had a divergent orientation with respect to the PF35 promoter. Disruption of the S. coelicolor chiR gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin. Moreover, the chiR disruption did not affect the overall chitinase activity.

Purification and Structural Determination of SCB1, a γ-Butyrolactone That Elicits Antibiotic Production inStreptomyces coelicolor A3(2)

Early stationary phase culture supernatants of Streptomyces coelicolor A3(2) contained at least four small diffusible signaling molecules that could elicit precocious antibiotic synthesis in the producing strain. The compounds were not detected in exponentially growing cultures. One of these compounds, SCB1, was purified to homogeneity and shown to be a gamma-butyrolactone of structure (2R, 3R,1'R)-2-(1'-hydroxy-6-methylheptyl)-3-hydroxymethylbutanolide . Bioassays of chemically synthesized SCB1, and of its purified stereoisomers, suggest that SCB1 acts in a highly specific manner to elicit the production of both actinorhodin and undecylprodigiosin, the two pigmented antibiotics made by S. coelicolor.

Metabolic Flux Analysis in Streptomyces coelicolor under Various Nutrient Limitations

Metabolic flux analysis was applied to Streptomyces coelicolor continuous culture data obtained under nitrogen, phosphate, sulfate, and potassium limitations. The metabolic reaction network involved more than 200 reactions describing the major pathways as well as the secondary metabolism for the production of actinorhodin and excretion of certain metabolites. Linear programming was used for the optimization of specific growth rates and energy requirements. Two types of specific growth rates, stoichiometric and theoretical, were defined, maximized, and compared in order to investigate the microbial potential. Potassium limitation led to the largest and nitrogen limitation to the smallest difference between the stoichiometric and theoretical specific growth rates. Although the value of the maximum theoretical specific growth rate was close to that of the experimental specific growth rate with potassium limitation, this difference was the largest in the case of nitrogen limitation. Energy requirements during different nutrient limitations were also investigated. The model indicated that although the highest actinorhodin production rate was with nitrogen limitation, this was accompanied with the undesired excretion of certain metabolites.

The Streptomyces coelicolor A3(2) lipAR operon encodes an extracellular lipase and a new type of transcriptional regulator.

A region of the Streptomyces coelicolor A3(2) chromosome was identified and cloned by using as a probe the lipase gene from Streptomyces exfoliatus M11. The cloned region consisted of 6286 bp, and carried a complete lipase gene, lipA, as well as a gene encoding a transcriptional activator (lipR). The S. coelicolor A3(2) lipA gene encodes a functional extracellular lipase 82% identical to the S. exfoliatus M11 lipase; the partially purified S. coelicolor enzyme showed a preference for substrates of short to medium chain length. Transcription of lipA was completely dependent on the presence of lipR, and occurred from a single promoter similar to the lipA promoters of S. exfoliatus M11 and Streptomyces albus G. These three Streptomyces lipA promoters have well-conserved -10 and -35 regions, as well as additional conserved sequences upstream of the -35 region, which could function as targets for transcriptional activation by the cognate LipR regulators. The Streptomyces LipR activators are related to other bacterial regulators of a similar size, constituting a previously unidentified family of proteins that includes MalT, AcoK, AlkS, AfsR, five mycobacterial proteins of unknown function and some Streptomyces regulators in antibiotic synthesis clusters. A lipase-deficient strain of S. coelicolor was constructed and found to be slightly affected in production of the polyketide antibiotic actinorhodin.

A kinetic model for actinorhodin production by Streptomyces coelicolor A3(2)

The fermentation kinetics of an extracellular antibiotic, actinorhodin, by Streptomyces coelicolor were studied in a batch system. A simple model was proposed using the Logistic equation for growth, the Luedeking–Piret equation for actinorhodin production and Luedeking–Piret-like equations for glucose and oxygen consumptions. The model appeared to provide a reasonable description for each parameter during the growth phase.

Molecular cloning and analysis of a pleiotropic regulatory gene locus from the nystatin producer Streptomyces noursei ATCC11455

A regulatory gene locus from Streptomyces noursei ATCC14455, the producer of the antifungal antibiotic nystatin, was cloned in Streptomyces lividans based on its ability to activate actinorhodin (Act) production in this host. Deletion and DNA sequencing analyses showed that a small gene, designated ssmA, located downstream of an afsR homologue (a known pleiotropic regulator) was responsible for the Act overproduction in S. lividans. Database searches for the ssmA gene product revealed its limited similarity to the AfsR2 regulatory protein from S. lividans and CREA catabolite repressor from Aspergillus nidulans. To study the effect of ssmA on nystatin production, this gene was either deleted from S. noursei genome, or placed under control of P ermE* promoter and introduced in S. noursei. The properties of the corresponding strains indicate that ssmA is involved in regulation of growth and antibiotic production only in the media with certain carbon sources.

An additional regulatory gene for actinorhodin production in Streptomyces lividans involves a LysR-type transcriptional regulator.

The sequence of a 4.8-kbp DNA fragment adjacent to the right-hand end of the actinorhodin biosynthetic (act) cluster downstream of actVB-orf6 from Streptomyces coelicolor A3(2) reveals six complete open reading frames, named orf7 to orf12. The deduced amino acid sequences from orf7, orf10, and orf11 show significant similarities with the following products in the databases: a putative protein from the S. coelicolor SCP3 plasmid, LysR-type transcriptional regulators, and proteins belonging to the family of short-chain dehydrogenases/reductases, respectively. The deduced product of orf8 reveals low similarities with several methyltransferases from different sources, while orf9 and orf12 products show no similarities with other known proteins. Disruptions of orf10 and orf11 genes in S. coelicolor appear to have no significant effect on the production of actinorhodin. Nevertheless, disruption or deletion of orf10 in Streptomyces lividans causes actinorhodin overproduction. The introduction of extra copies of orf10 and orf11 genes in an S. coelicolor actIII mutant restores the ability to produce actinorhodin. Transcriptional analysis and DNA footprinting indicate that Orf10 represses its own transcription and regulates orf11 transcription, expression of which might require the presence of an unknown inducer. No DNA target for Orf10 protein was found within the act cluster.

Alteration of the synthesis of the Clp ATP-dependent protease affects morphological and physiological differentiation in Streptomyces.

The genes of Streptomyces coelicolor A3(2) encoding catalytic subunits (ClpP) and regulatory subunits (ClpX and ClpC) of the ATP-dependent protease family Clp were cloned, mapped and characterized. S. coelicolor contains at least two clpP genes, clpP1 and clpP2, located in tandem upstream from the clpX gene, and at least two unlinked clpC genes. Disruption of the clpP1 gene in S. lividans and S. coelicolor blocks differentiation at the substrate mycelium step. Overexpression of clpP1 and clpP2 accelerates aerial mycelium formation in S. lividans, S. albus and S. coelicolor. Overproduction of ClpX accelerates actinorhodin production in S. coelicolor and activates its production in S. lividans.

A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2)

An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an M r of 29 422 showed the greatest similarity to the previously identified sporulation sigma factor (σ F) of Streptomyces coelicolor, and general stress response sigma factor (σ B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of −10 and −35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.

Actinorhodin production by Streptomyces coelicolor A3(2) in iron-restricted media.

Production of the polyketide antibiotic actinorhodin by Streptomyces coelicolor A3(2) was investigated using a defined medium with or without iron supplementation. Iron limitation was found to enhance the intracellular production and export of the pigmented antibiotic. The effect of iron deficiency was particularly pronounced when the bacterium was grown with nitrate instead of ammonium. Analysis of the excreted pigment led to the identification of the lactone form of actinorhodin, gamma-actinorhodin.

A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2).

An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an M(r) of 29,422 showed the greatest similarity to the previously identified sporulation sigma factor (sigma F) of Streptomyces coelicolor, and general stress response sigma factor (sigma B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of -10 and -35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.

The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster.

Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (orfA) codes for a protein that resembles several transport proteins. The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomyces antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the mtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of mtmR in a high-copy-number vector in S. argillaceus caused a 16-fold increase in mithramycin production. The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21. A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by S. argillaceus is proposed.

Actinorhodin and undecylprodigiosin production in wild-type and relA mutant strains of Streptomyces coelicolor A3(2) grown in continuous culture

The effects of growth rate and nutrient feed rate on the production of actinorhodin (Act) and undecylprodigiosin (Red) were determined in Streptomyces coelicolor A3(2) and in a congenic relA null-mutant known to be deficient in ppGpp synthesis and antibiotic production under conditions of nitrogen limitation. In the relA + strain, Act production was inversely related to specific growth rate in continuous cultures limited by glucose, ammonium, or phosphate, while Red biosynthesis was optimal at 0.05 h −1 regardless of the specific nutrient limitation. Production of Act and Red in the relA mutant was lower than that of the parental strain, particularly under conditions of glucose- and ammonium-limitation, indicating an important and general role for ppGpp in determining the onset of the antibiotic biosynthesis under conditions of nutrient limitation. At constant growth rate, but with varying nutrient feed rates, the specific rate of Act production was adversely influenced by increasing levels of glucose, ammonium, and phosphate, with phosphate having the greatest inhibitory effect. Under the same conditions, the specific rate of Red production was stimulated by increasing glucose levels, but markedly decreased by increased levels of phosphate.

Actinorhodin and undecylprodigiosin production in wild-type and relA mutant strains of Streptomyces coelicolor A3(2) grown in continuous culture.

The effects of growth rate and nutrient feed rate on the production of actinorhodin (Act) and undecylprodigiosin (Red) were determined in Streptomyces coelicolor A3(2) and in a congenic relA null-mutant known to be deficient in ppGpp synthesis and antibiotic production under conditions of nitrogen limitation. In the relA+ strain, Act production was inversely related to specific growth rate in continuous cultures limited by glucose, ammonium, or phosphate, while Red biosynthesis was optimal at 0.05 h-1 regardless of the specific nutrient limitation. Production of Act and Red in the relA mutant was lower than that of the parental strain, particularly under conditions of glucose- and ammonium-limitation, indicating an important and general role for ppGpp in determining the onset of the antibiotic biosynthesis under conditions of nutrient limitation. At constant growth rate, but with varying nutrient feed rates, the specific rate of Act production was adversely influenced by increasing levels of glucose, ammonium, and phosphate, with phosphate having the greatest inhibitory effect. Under the same conditions, the specific rate of Red production was stimulated by increasing glucose levels, but markedly decreased by increased levels of phosphate.

A silent ABC transporter isolated from Streptomyces rochei F20 induces multidrug resistance.

In the search for heterologous activators for actinorhodin production in Streptomyces lividans, 3.4 kb of DNA from Streptomyces rochei F20 (a streptothricin producer) were characterized. Subcloning experiments showed that the minimal DNA fragment required for activation was 0.4 kb in size. The activation is mediated by increasing the levels of transcription of the actII-ORF4 gene. Sequencing of the minimal activating fragment did not reveal any clues about its mechanism; nevertheless, it was shown to overlap the 3' end of two convergent genes, one of whose translated products (ORF2) strongly resembles that of other genes belonging to the ABC transporter superfamily. Computer-assisted analysis of the 3.4-kb DNA sequence showed the 3' terminus of an open reading frame (ORF), i.e., ORFA, and three complete ORFs (ORF1, ORF2, and ORFB). Searches in the databases with their respective gene products revealed similarities for ORF1 and ORF2 with ATP-binding proteins and transmembrane proteins, respectively, which are found in members of the ABC transporter superfamily. No similarities for ORFA and ORFB were found in the databases. Insertional inactivation of ORF1 and ORF2, their transcription analysis, and their cloning in heterologous hosts suggested that these genes were not expressed under our experimental conditions; however, cloning of ORF1 and ORF2 together (but not separately) under the control of an expressing promoter induced resistance to several chemically different drugs: oleandomycin, erythromycin, spiramycin, doxorubicin, and tetracycline. Thus, this genetic system, named msr, is a new bacterial multidrug ABC transporter.

Involvement of two A-factor receptor homologues in Streptomyces coelicolor A3(2) in the regulation of secondary metabolism and morphogenesis.

Nucleotide sequences homologous to arpA encoding the A-factor receptor protein (ArpA) of Streptomyces griseus are distributed in a wide variety of streptomycetes. Two genes, cprA and cprB, each encoding an ArpA-like protein were found and cloned from Streptomyces coelicolor A3(2). CprA and CprB shared 90.7% identity in amino acid sequence and both showed about 35% identity to ArpA. Disruption of cprA by use of an M13 phage-derived single-stranded vector resulted in severe reduction of actinorhodin and undecylprodigiosin production. In addition, the timing of sporulation in the cprA disruptants was delayed by 1 day. The cprA gene thus appeared to act as a positive regulator or an accelerator for secondary metabolite formation and sporulation. Consistent with this idea, introduction of cprA on a low-copy-number plasmid into the parental strain led to overproduction of these secondary metabolites and accelerated the timing of sporulation. On the other hand, cprB disruption resulted in precocious and overproduction of actinorhodin. However, almost no effect on undecylprodigiosin was detected in the cprB disruptants. Sporulation of the cprB disruptant began 1 day earlier than the parental strain. The cprB gene thus behaved as a negative regulator on actinorhodin production and sporulation. Consistent with this, extra copies of cprB in the parental strain caused reduced production of actinorhodin and delay in sporulation. It is thus concluded that both cprA and cprB play regulatory roles in both secondary metabolism and morphogenesis in S. coelicolor A3(2), just as the arpA/A-factor system in Streptomyces griseus.

Effect of sucrose on actinorhodin production by Streptomyces coelicolor A3(2)

The effect of sucrose on actinorhodin production by Streptomyces coelicolor was investigated using both defined and complex media. Both media contained glucose as the main carbon source and sucrose was included for osmotic balance. The production of antibiotic in the defined medium decreased with the addition of sucrose but, when sucrose was excluded from the complex medium, the culture did not produce actinorhodin. A sucrose concentration of 340 g/litre was optimal for actinorhodin production in the complex medium.

Molecular and functional analysis of the ribosomal L11 and S12 protein genes (rplK and rpsL) of Streptomyces coelicolor A3(2).

A RelC deletion mutant, KO-100, of Streptomyces coelicolor A3(2) has been isolated from a collection of spontaneous thiostrepton-resistant mutants. KO-100 grows as vigorously as the parent strain and possesses a 6-bp deletion within the rplK, previously termed relC. When the wild-type rplK gene was propagated on a low-copy-number vector in mutant KO-100, the ability to produce ppGpp, actinorhodin and undecylprodigiosin, which had been lost in the RelC mutant, was completely restored. Allele replacement by gene homogenotization demonstrated that the RelC mutation is responsible for the resistance to thiostrepton and the inactivation of ppGpp, actinorhodin and undecylprodigiosin production. Western blotting showed that ribosomes from the RelC mutant KO-100 contain only one-eighth the amount of L11 protein found in ribosomes of the parent strain. The impairment of antibiotic production in KO-100 could be rescued by the introduction of mutations that confer resistance to streptomycin (str), which result in alteration of Lys-88 in ribosomal protein S12 to Glu or Arg. No accompanying restoration of ppGpp synthesis was detected in these RelC str double mutants.

Production of actinorhodin byStreptomyces coelicolor A3(2) grown in chemostat culture

Streptomyces coelicolor was grown in variously limited chemostat cultures and the specific rate of extracellular actinorhodin production (q(actinorhodin)) was measured. The highest q(actinorhodin) values were observed in glucose- or ammonia-limited cultures, whereas almost no actinorhodin was produced in sulfate-, phosphate-, potassium-, or magnesium-limited cultures. The effect of the dilution rate on actinorhodin production was studied in glucose-limited cultures. It was found that q(actinorhodin) was highest at D = 0.06h(-1), which was well below the maximal D value tested (0.14 h(-1)). This explains why, in batch cultures, actinorhodin production starts at the onset of the stationary phase. It was also found that the use of nitrilotriacetate instead of citrate as a chelating agent had a negative effect on actinorhodin production. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 577-582, 1997.