Anabolic-catabolic glucose utilization and product formation of Streptomyces griseus

If in stationary growth phases appearing in the submerged cultivation of Streptomyces griseus several times the consumption rates of glucose and oxygen and the rates of streptomycin formation are put in relation, the following results are obtained: The yield coefficient Ysp of glucose was below 0.1. Since from the stoichiometric equation of turnover for the biosynthesis of the streptomycin follows that the substrate and the product are in the same weight relation, it was possible to check whether the quantity of glucose that does not appear in the streptomycin is used for the energy supplying synthesis or conservation reactions. As a characteristic value was built up the quotient qo3/qgluc that in total substrate oxidations takes the numerical value 6. This quotient varied between 2.0 and 6.0 so that anabolic side reactions during the production of secondary substances were concluded from. As possibilities are discussed syntheses of analytically disregarded primary metabolites or preliminary steps of the biosynthesis of streptomycin. Due to the decrease of the enthalpy production by anabolic reaction steps in stationary growth phases follows a physiological-energetical importance of microbial product syntheses with likely evolutive action.     

Intracellular levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) in cultures of Streptomyces griseus producing streptomycin.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were identified in the vegative mycelium of Streptomyces griseus. Adenosine 5'-diphosphate 3'-diphosphate (ppApp) and adenosine 5'-triphosphate 3'-diphosphate (pppApp) were not present but several other phosphorus-containing compounds which may have been inorganic polyphosphates were detected. During exponential growth of S. griseus the concentrations of ppGpp and pppGpp were several times higher than in the stationary stage. They fell sharply when exponential growth ended and then remained at an almost constant basal level. For the tetraphosphate the maximum concentration was about 50, and for the basal level about 10, pmol per millilitre of a culture with an optical density of 1.0. Production of streptomycin started several hours after exponential growth had ended and the concentrations of ppGpp and pppGpp had fallen. Streptomycin synthesis was delayed if the cells were resuspended just before production started in fresh medium lacking phosphate, but it was not delayed by glucose starvation. Both cultures, as well as cultures transferred to nitrogen-free medium, showed an immediate increase in ppGpp content to about four-fold the basal level. The results suggest that the guanosine polyphosphates do not directly control initiation of streptomycin production in S. griseus. Twelve additional species of Streptomyces examined all contained ppGpp and pppGpp.     

Intracellular cyclic adenosine 3',5'-monophosphate levels and streptomycin production in cultures of Streptomyces griseus.

Changes in the amount of cyclic 3',5'-adenosine monophosphate within the mycelium of Streptomyces griseus were measured as cultures progressed through trophophase and idiophase in a complex medium supporting growth and streptomycin synthesis. Concentrations were highest before the cultures entered stationary phase and had declined 90% by 5 h before the antibiotic was produced. This low conentration was maintained while the antibiotic accumulated during the idiophase. The results indicate that the onset of streptomycin synthesis is not directly mediated by an increase in intracellular cyclic 3',5'-adenosine monophosphate concentration, and thus that antibiotic production in S. griseus is not controlled by catabolite repression.     

Rsistance to streptomycin in a producing strain of Streptomyces griseus.

Streptomyces griseus S 104 was sensitive to streptomycin during exponential growth in a medium which, in the subsequent stationary phase, supported production of the antibiotic in yields above 200 mug/ml. When antibiotic production began cultures developed a tolerance toward their lethal metabolite. This was not due to an increase in pH associated with antibiotic production, since pH effects on streptomycin sensitivity in S. griseus were in the reverse direction. However, the degree of tolerance was directly related to the amount of cell material present. Streptomycin production caused no change in the proportion of resistant variants in the population, nor did it cause the severe inhibition of protein synthesis observed in non-producing cultures exposed to the antibiotic. The lack of an effect on protein synthesis is attributed to the absence of streptomycin with in the cytoplasm since soluble extracts from mycelium harvested in the production phase were inactive when bioassayed immediately after cell disruption. However, they developed antibacterial activity rapidly when heated, and more slowly when incubated at 25 degrees C. The addition of phosphatase inhibitors during incubation prevented the appearance of antibiotic activity, and it was concluded that a small amount of streptomycin phosphate is present in the mycelium during antibiotic production. Differences in (14C) streptomycin uptake suggested that the mycelium was appreciably less permeable to the antibiotic in the production phase than during exponential growth. However, a small amount was taken up and much of it was in the soluble fraction of disrupted cells. Bioassays showed that this 14C-labeled antibiotic within the cells had been partially inactivated, suggesting that conversion of streptomycin to an inactive derivative is involved in the mechanism which protects the organism from its metabolite.     

Cloning and characterization of the A-factor receptor gene from Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein control streptomycin production, streptomycin resistance, and aerial mycelium formation in Streptomyces griseus. The A-factor receptor protein (ArpA) was purified from a cell lysate of S. griseus IFO 13350. The NH2-terminal amino acid sequences of ArpA and lysyl endopeptidase-generated fragments were determined for the purpose of preparing oligonucleotide primers for cloning arpA by the PCR method. The arpA gene cloned in this way directed the synthesis of a protein having A-factor-specific binding activity when expressed in Escherichia coli under the control of the T7 promoter. The arpA gene was thus concluded to encode a 276-amino-acid protein with a calculated molecular mass of 29.1 kDa, as determined by nucleotide sequencing. The A-factor-binding activity was observed with a homodimer of ArpA. The NH2-terminal portion of ArpA contained an alpha-helix-turn-alpha-helix DNA-binding motif that showed great similarity to those of many DNA-binding proteins, which suggests that it exerts its regulatory function for the various phenotypes by directly binding to a certain key gene(s). Although a mutant strain deficient in both the ArpA protein and A-factor production overproduces streptomycin and forms aerial mycelium and spores earlier than the wild-type strain because of repressor-like behavior of ArpA, introduction of arpA into this mutant abolished simultaneously its streptomycin production and aerial mycelium formation. All of these data are consistent with the idea that ArpA acts as a repressor-type regulator for secondary metabolite formation and morphogenesis during the early growth phase and A-factor at a certain critical intracellular concentration releases the derepression, thus leading to the onset of secondary metabolism and aerial mycelium formation. The presence of ArpA-like proteins among Streptomyces spp., as revealed by PCR, together with the presence of A-factor-like compounds, suggests that a hormonal control similar to the A-factor system exists in many species of this genus.     

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture

Abstract Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h⁻¹ (15% of μ _max). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.     

Effect of pamamycin-607 on secondary metabolite production by Streptomyces spp

The effect of the aerial mycelium-inducing compound, pamamycin-607, on antibiotic production by several Streptomyces spp. was examined. Exposure to 6.6 μM pamamycin-607 stimulated by 2.7 fold the puromycin production by Streptomyces alboniger NBRC 12738, in which pamamycin-607 had first been isolated, and restored aerial mycelium formation. Pamamycin-607 also stimulated the respective production of streptomycin by S. griseus NBRC 12875 and that of cinerubins A and B by S. tauricus JCM 4837 by approximately 1.5, 1.7 and 1.9 fold. The antibiotic produced by Streptomyces sp. 91-a was identified as virginiamycin M(1), and its synthesis was enhanced 2.6 fold by pamamycin-607. These results demonstrate that pamamycin-607 not only restored or stimulated aerial mycelium formation, but also stimulated secondary metabolite production.     

Cloning and characterization of a gene involved in aerial mycelium formation in Streptomyces griseus.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is essentially required for aerial mycelium formation and streptomycin production in Streptomyces griseus. A DNA fragment which induced aerial mycelium formation and sporulation in an A-factor-deficient mutant strain, S. griseus HH1, was cloned from this strain on a high-copy-number plasmid. Subcloning and nucleotide sequencing revealed that one open reading frame with 218 amino acids, named AmfC, served as a multicopy suppressor of the aerial mycelium-defective phenotype of the A-factor-deficient strain. The amfC gene did not restore A-factor or streptomycin production, indicating that amfC is involved in aerial mycelium formation independently of secondary metabolic function. Disruption of the chromosomal amfC gene in the wild-type S. griseus strain caused a severe reduction in the abundance of spores but no effect on the shape or size of the spores. The infrequent sporulation of the amfC disruptant was reversed by introduction of amfC on a plasmid. The amfC-defective phenotype was also restored by the orf1590 gene but not by the amfR-amfA-amfB gene cluster. Nucleotide sequences homologous to the amfC gene were distributed in all of 12 Streptomyces species tested, including Streptomyces coelicolor A3(2). The amfC homolog of S. coelicolor A3(2) was cloned and its nucleotide sequence was determined. The AmfC products of S. griseus and S. coelicolor A3(2) showed a 60% identity in their amino acid sequences. Introduction of the amfC gene of S. coelicolor A3(2) into strain HH1 induced aerial mycelium formation and sporulation, which suggests that both play the same functional role in morphogenesis in the strains.     

Metabolic initiation of differentiation and secondary metabolism by Streptomyces griseus: significance of the stringent response (ppGpp) and GTP content in relation to A factor.

I investigated the significance of the intracellular accumulation of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and of the coordinated decrease in the GTP pool for initiating morphological and physiological differentiation of Streptomyces griseus, a streptomycin-producing strain. In solid cultures, aerial mycelium formation was severely suppressed by the presence of excess nutrients. However, decoyinine, a specific inhibitor of GMP synthetase, enabled the cells to develop aerial mycelia in the suppressed cultures at concentrations which only partially inhibited growth. A factor (2S-isocapryloyl-3S-hydroxymethyl-gamma-butyrolactone) added exogenously had no such effect. Decoyinine was also effective in initiating the formation of submerged spores in liquid culture. The ability to produce streptomycin did not increase but decreased drastically on the addition of decoyinine. This sharp decrease in streptomycin production was accompanied by a decrease in intracellular accumulation of ppGpp. A relaxed (rel) mutant was found among 25 thiopeptin-resistant isolates which developed spontaneously. The rel mutant had a severely reduced ability to accumulate ppGpp during a nutritional shift-down and also during postexponential growth and showed a less extensive decrease in the GTP pool than that in the rel+ parental strain. The rel mutant failed to induce the enzymes amidinotransferase and streptomycin kinase, which are essential for the biosynthesis of streptomycin. The abilities to form aerial mycelia and submerged spores were still retained, but the amounts were less, and for both the onset of development was markedly delayed. The decreased ability to produced submerged spores was largely restored by the addition of decoyinine. This was accompanied by an extensive GTP pool decrease. The rel mutant produced A factor normally, indicating that synthesis of A factor is controlled neither by ppGpp nor by GTP. Conversely, a mutant defective in A-factor synthesis accumulated as much ppGpp as did the parental strain. It was concluded that morphological differentiation of S. griseus results from a decrease in the pool of GTP, whereas physiological differentiation results from a more direct function of the rel gene product (ppGpp). It is also suggested that A factor may render the cell sensitive to receive and respond to the specified signal molecules, presumably ppGpp (for physiological differentiation) or GTP (for morphological differentiation).     

Genetic analysis of A-factor synthesis in Streptomyces coelicolor A3(2) and Streptomyces griseus

A-factor is a potent pleiotropic effector produced by Streptomyces griseus and is essential for streptomycin production and spore formation in this organism. Its production is widely distributed among various actinomycetes including Streptomyces coelicolor A3(2). Genetic analysis of A-factor production was carried out with S. coelicolor A3(2), and two closely linked loci for A-factor mutations (afsA and B) were identified between cysD and leuB on the chromosomal linkage map. In contrast, genetic crosses of A-factor-negative mutants of S. griseus, using a protoplast fusion technique, failed to give a fixed locus for A-factor gene(s) and suggested involvement of an extrachromosomal or transposable genetic element in A-factor synthesis in this organism.     

Induction of actinorhodin production by rpsL (encoding ribosomal protein S12) mutations that confer streptomycin resistance in Streptomyces lividans and Streptomyces coelicolor A3(2).

A strain of Streptomyces lividans, TK24, was found to produce a pigmented antibiotic, actinorhodin, although S. lividans normally does not produce this antibiotic. Genetic analyses revealed that a streptomycin-resistant mutation str-6 in strain TK24 is responsible for induction of antibiotic synthesis. DNA sequencing showed that str-6 is a point mutation in the rpsL gene encoding ribosomal protein S12, changing Lys-88 to Glu. Gene replacement experiments with the Lys88-->Glu str allele demonstrated unambiguously that the str mutation is alone responsible for the activation of actinorhodin production observed. In contrast, the strA1 mutation, a genetic marker frequently used for crosses, did not restore actinorhodin production and was found to result in an amino acid alteration of Lys-43 to Asn. Induction of actinorhodin production was also detected in strain TK21, which does not harbor the str-6 mutation, when cells were incubated with sufficient streptomycin or tetracycline to reduce the cell's growth rate, and 40 and 3% of streptomycin- or tetracycline-resistant mutants, respectively, derived from strain TK21 produced actinorhodin. Streptomycin-resistant mutations also blocked the inhibitory effects of relA and brgA mutations on antibiotic production, aerial mycelium formation or both. These str mutations changed Lys-88 to Glu or Arg and Arg-86 to His in ribosomal protein S12. The decrease in streptomycin production in relC mutants in Streptomyces griseus could also be abolished completely by introducing streptomycin-resistant mutations, although the impairment in antibiotic production due to bldA (in Streptomyces coelicolor) or afs mutations (in S. griseus) was not eliminated. These results indicate that the onset and extent of secondary metabolism in Streptomyces spp. is significantly controlled by the translational machinery.     

Identification of a cell wall channel of Streptomyces griseus: the channel contains a binding site for streptomycin

Cells of the Gram-positive actinomycete Streptomyces griseus were disrupted and the cell envelope was subjected to sucrose step-gradient centrifugation. The different fractions were analysed for NADH-oxidase activity and the formation of ion-permeable channels in lipid bilayers. Highest channel-forming activity and highest NADH-oxidase activity were found in different fractions. The cell wall fraction contained an ion-permeable channel with a single-channel conductance of 850 pS in 1 M KCl. The channel-forming protein, with an apparent molecular mass of 28 kDa, was purified to homogeneity using fast protein liquid chromatography after the extraction of whole cells with detergent. Single-channel experiments suggest that the cell wall channel is wide and water-filled. Titration experiments with streptomycin produced by S. griseus suggested that the cell wall channel binds this antibiotic with a half saturation constant of about 6 mM in 1 M KCl. The binding of streptomycin was found to be ionic strength dependent and the half saturation constant decreased to 60 microM at 0.1 M KCl. The results indicate that the 28 kDa protein represents the hydrophilic pathway through the cell wall of the Gram-positive bacterium S. griseus.     

The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation.

A-factor, 2-(6'-methylheptanoyl)-3R-hydroxymethyl-4-butanolide, is an autoregulator essential for streptomycin production and sporulation in Streptomyces griseus. S. griseus 2247 that requires no A-factor for streptomycin production or sporulation was found to have a defect in the A-factor-binding protein. This observation implied that the A-factor-binding protein in the absence of A-factor repressed the expression of both phenotypes in the wild-type strain. Screening among mutagenized S. griseus colonies for strains producing streptomycin and sporulating in the absence of A-factor yielded three mutants that were also deficient in the A-factor-binding protein. Reversal of the defect in the A-factor-binding protein of these mutants led to the simultaneous loss of streptomycin production and sporulation. These data suggested that the A-factor-binding protein played a role in repressing both streptomycin production and sporulation and that the binding of A-factor to the protein released its repression. Mutants deficient in the A-factor-binding protein began to produce streptomycin and sporulate at an earlier stage of growth than did the wild-type strain. These mutants produced approximately 10 times more streptomycin than did the parental strain. These findings are consistent with the idea that the intracellular concentration of A-factor determines the timing of derepression of the gene(s) whose expression is repressed by the A-factor-binding protein.     

Biosynthesis of fredericamycin A, a new antitumor antibiotic

Fredericamycin A (FM A), produced by a strain of Streptomyces griseus, represents a new structural class of antitumor antibiotics containing a spiro ring system. Studies on the producer organism showed that glucose in the fermentation medium is not utilized until late in the growth stage, just prior to synthesis of FM A. [14C]Glucose tracer experiments demonstrated that glucose is incorporated into FM A by catabolism to acetate. Biosynthetic enrichment of FM A with single- and double-labeled [13C]acetate showed that the entire carbon skeleton of the spiro ring system is derived from acetate. L-Methionine was shown to provide the only nonskeletal carbon in FM A, the methoxy carbon at position C-6. The direction of the polyketide chain and the position of the carbon lost during biosynthesis were established by using stable isotope experiments. A general model for FM A biosynthesis is proposed, and a possible scheme for the formation of the spiro carbon center is presented.     

Detection and properties of A-factor-binding protein from Streptomyces griseus.

The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The "inducing material" virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein.     

Molecular cloning and expression in Streptomyces lividans of a streptomycin 6-phosphotransferase gene from a streptomycin-producing microorganism

The gene encoding streptomycin 6-kinase involved in the self-resistance of the streptomycin-producing Streptomyces griseus HUT 6037 was cloned in the plasmid vector pIJ703. The resulting plasmid, pSP6, contained 2.5 kb inserts of S. griseus DNA. When streptomycin-susceptible S. lividans 1326 was retransformed with pSP6, all transformants produced streptomycin 6-kinase. Addition of streptomycin to the culture medium of S. lividans carrying pSP6 plasmid brought about a remarkable increase in streptomycin 6-kinase activity in the cell extracts. It is suggested from the results that the production of streptomycin 6-kinase in streptomycin producer was induced by streptomycin accumulated during cultivation.     

Effect of the A-factor on the adenylate level in Streptomyces griseus

The adenylate content of various strains of Streptomyces griseus was measured. With respect to the ATP and ADP content the strains capable of spore formation, streptomycin synthesis and A-factor (2-isocapryloyl-3-hydroxymethyl-4-hydroxybutyrolactone) synthesis differed from the A-factor deficient mutants. The addition of the A-factor to the recipient strains decreased the intracellular content of ATP and the ATP/ADP ratio. The strain which is not an A-factor recipient did not modify the ATP content when the A-factor was added to the medium.