Variations in levels of cAMP, DNA and RNA in Streptomyces alboniger under conditions of aerial mycelium formation and repression

Abstract The relationship between endogenous levels of cyclic adenosine 3',5'-monophosphate (cAMP) and the formation of aerial mycelia was investigated in Streptomyces alboniger under conditions of aerial mycelium formation and repression. The relationship between cellular levels of DNA and RNA and aerial mycelium formation was also investigated. In contrast to cellular differentiation in other Streptomyces , neither variations in cAMP, DNA or RNA levels were found to be associated with the development of aerial mycelia in S. alboniger . The regulation of adenylate cyclase in S. alboniger , however, was found to differ from that of Escherichia coli and related organisms in that glucose raised, rather than lowered, endogenous cAMP levels.     

Regulation of formation of aerial mycelia and spores of Streptomyces viridochromogenes.

Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more casein hydrolysate to the medium stimulated growth while completely repressing the formation of aerial mycelia and spores. This repression was temporary, as evidenced by the fact that transfer of the organisms to media not containing casein hydrolysate resulted in the appearance of aerial mycelia and spores. The effects of individual amino acids were tested. Glycine retarded growth and repressed formation of both aerial mycelia and spores. L-Aspartic acid, L-glutamic acid, and L-histidine stimulated or had little effect on growth and repressed formation of spores but not aerial mycelia. Repression by casein hydrolysate could not be attributed to the carbon/nitrogen ratio or the pH of the medium. Adding 1.25 to 2.5 mM adenine to the medium caused a reversal of the casein hydrolysate repression of aerial mycelium formation but did not reverse repression of sporulation. Dimethyladenine and 8-azaguanine had an effect similar to that of adenine, but a variety of other purine or pyrimidine derivatives had no effect on casein hydrolysate repression. The repression of aerial mycelium and spore formation by casein hydrolysate occurred only in media containing 15 mM or more phosphate. Aerial mycelia and spores were formed in media containing casein hydrolysate and 3 mM or less phosphate.     

Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts.

Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside antibiotics. The genes pac and dmpM, encoding a puromycin N-acetyl transferase and an O-demethyl puromycin O-methyltransferase, respectively, are tightly linked in the DNA of S. alboniger. The entire set of genes encoding the puromycin biosynthesis pathway was cloned by screening a gene library from S. alboniger, raised in the low copy number cosmid pKC505, with a DNA fragment containing pac and dmpM. Puromycin was identified by biochemical and physicochemical methods, including 1H NMR, in the producing transformants. This pathway was located in a single DNA fragment of 15 kb which included the resistance, structural and regulatory genes and was expressed when introduced into two heterologous hosts Streptomyces lividans and Streptomyces griseofuscus. In addition to pac and dmpM, two other genes have been identified in the pur cluster: pacHY, which determines an N-acetylpuromycin hydrolase and prg1, whose deduced amino acid sequence is significantly similar to that of degT, a Bacillus stearothermophilus pleiotropic regulatory gene.     

Simultaneous loss of multiple differentiated functions in aerial mycelium-negative isolates of streptomycetes.

Germination and outgrowth of spores of Streptomyces alboniger, Streptomyces scabies, and Streptomyces violaceus-ruber in the presence of intercalating dyes resulted in a high frequency (2 to 20%) of occurrence of aerial mycelium-negative (Amy-) isolates. Coincident with the appearance of the Amy- trait was the loss of several differentiated functions, including the characteristic pigments and earthy odor of the wild types. All S. alboniger, 27% of S. scabies, and 39% of the S. violaceus-ruber Amy- isolates were arginine auxotrophs. The missing enzyme step was identified as argininosuccinate synthetase by using a sensitive microassay for estimation of enzyme activity. The remainder of the S. scabies and S. violaceus-ruber isolates were prototrophs. In addition, S. alboniger Amy- isolates failed to produce or respond to the stimulator of aerial mycelium formation isolated from the wild type. The Amy- isolates did not revert to either Amy+ of Arg+. The lack of any detectable reversion, coupled with the high frequency of curing, supports the idea that a deletion of genetic material, possibly a plasmid, has occurred.     

New loci required for Streptomyces coelicolor morphological and physiological differentiation.

Streptomyces coelicolor colonies differentiate both morphologically, producing aerial spore chains, and physiologically, producing antibiotics as secondary metabolites. Single mutations, which block both aspects of differentiation, define bld (bald colony) genes. To identify new bld genes, mutagenized colonies were screened for blocks in the earliest stage of sporulation, the formation of aerial mycelia, and blocks in antibiotic synthesis. The mutations in 12 mutants were mapped; in each strain, the pleiotropic phenotype was due to a single mutation. Seven of the strains contained mutations in known bld loci, bldA and bldB. Three strains contained mutations in a new locus, bldG, and two contained mutations in another new locus, bldH. Like the previously defined bldA mutants, the bldG and bldH mutants were developmentally blocked on glucose. On a variety of carbon sources whose utilization was subject to glucose repression, the developmental blocks were partially relieved for bldG (and bldA) mutants and fully relieved for bldH mutants. These results are compatible with an hypothesis which suggests that there are two alternative controls on S. coelicolor differentiation, one of which is glucose repressible.     

The mechanism of resistance to puromycin and to the puromycin-precursor O-demethyl-puromycin in Streptomyces alboniger

Ribosomes from Streptomyces alboniger are sensitive in vitro to puromycin and, to a lesser extent, to the puromycin-precursor O-demethyl-puromycin. The puromycin-inactivating enzyme (puromycin N-acetyltransferase) from S. alboniger also N-acetylates O-demethyl-puromycin. This finding indicates that in certain antibiotic-producing organisms the antibiotic-inactivating enzymes may play a role in self-defence against toxic precursor molecules.     

Brom Cresol Green and Brom Phenol Blue as Indicators of Respiration Deficiency in Yeast

Strains of Saccharomyces which contained cells of respirationally normal (wild-type) and of respiration-deficient (RD) mutants were grown on untrient agar plates containing 20-23 mg/liter of either brom cresol green (BCG) or brom phenol blue (BPB). Glucose content of the media was varied experimentally in the range of 1.5-6%. After 2-4 days of incubation, normal colonies were very palely stained whereas RD colonies were drak green with BCG and dark blue with BPB. Optimum glucose content in the medium was 2-3% for S. cerevisiae, S. carlbergensis and S. chevalieri, but Fleischmann's (baker's) yeast developed the best color contrast with 4-5% glucose.     

Repression of biotin biosynthesis in Escherichia coli during growth on biotin vitamers.

A strain of Escherichia coli in which the lacZ gene was fused to the bioA promoter was constructed. Colonies of this strain formed Lac(+) colonies on low-biotin agar (1.6 to 4.1 nM) and Lac(-) colonies on high-biotin agar (41 nM). This lac-bio fusion strain was used to study the question of whether cells growing on the biotin vitamers d-biotin-d-sulfoxide (BDS) and dethiobiotin (DTB) generate enough biotin to give maximal repression of beta-galactosidase synthesis. Repression by high concentrations (400 nM) of BDS was almost maximal (about 96%), whereas DTB repression reached a saturation level of about 80% with increasing DTB concentrations. The levels of repression obtained with both vitamers were sufficient to cause the colonies to appear Lac(-). When the lac-bio fusion was transduced into lines carrying mutations (bis) that prevent reduction of BDS to biotin, the transductants were not repressed by added BDS. Repression by BDS is unlikely to result from accumulation of extracellular biotin-related substances because (i) washed bis(+) cells were not detectably derepressed when transferred into medium containing BDS and (ii) washed bis cells were not detectably repressed when transferred into medium in which bis(+) cells had grown. Lactose agar plates containing high concentrations of DTB or BDS comprise an efficient selective medium for bioB or bis mutants and were used to isolate spontaneous mutations of these genes. This method should be adaptable to the selection of mutations in any biosynthetic pathway subject to end-product repression.     

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.     

Biosynthesis of puromycin by Streptomyces alboniger: characterization of puromycin N-acetyltransferase

Puromycin N-acetyltransferase from Streptomyces alboniger inactivates puromycin by acetylating the amino position of its tyrosinyl moiety. This enzyme has been partially purified by column chromatography through DEAE-cellulose and Affigel Blue and characterized. It has an Mr of 23 000, as determined by gel filtration. In addition to puromycin, the enzyme N-acetylates O-demethylpuromycin, a toxic precursor of the antibiotic, and chryscandin, a puromycin analogue antibiotic. The Km values for puromycin and O-demethylpuromycin are 1.7 and 4.6 microM, respectively. The O-demethylpuromycin O-methyltransferase from S. alboniger, which apparently catalyzes the last step in the biosynthesis of puromycin [Rao, M. M., Rebello, P. F., & Pogell, B. M. (1969) J. Biol. Chem. 244, 112-118], also O-methylates N-acetyl-O-demethylpuromycin. The Km values of the methylating enzyme for O-demethylpuromycin and N-acetyl-O-demethylpuromycin are 260 and 2.3 microM, respectively. These findings suggest that O-demethylpuromycin, if present in S. alboniger, would be N-acetylated and then O-methylated to be converted into N-acetylpuromycin. It might even be possible that N-acetylation of the puromycin backbone takes place at an earlier precursor.     

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.     

Nutritional regulation of differentiation and synthesis of an exocytoplasmic deoxyriboendonuclease in Streptomyces antibioticus

Streptomyces antibioticus produces a cell-wall-located deoxyriboendonuclease (DNAase) the synthesis of which in submerged and surface cultures is related to the growth rate. DNAase synthesis always preceded aerial mycelium formation in surface cultures. Production of aerial mycelium began at the end of exponential growth or in the early stationary phase; it was absent in cultures grown on nutrient agar/glucose or in media with a high concentration of casein hydrolysate. These nutritional conditions also impaired production of the DNAase. External DNA substrates were not degraded by mycelium producing the DNAase. These observations lead us to suggest a role for the enzyme in the developmental cycle of S. antibioticus.     

Inhibition of Histoplasma capsulatum by Candida albicans and Other Yeasts on Sabouraud''s Agar Media

The inhibition of growth of Histoplasma capsulatum by Candida albicans and other yeasts on Sabouraud's agar was investigated. Histoplasma (yeast-phase inoculum) was grown alone and in mixtures with yeasts at 25 C for 4-week periods. As few as 10 colonies of C. albicans completely inhibited the growth of approximately 50,000 potential colonies of Histoplasma. The pH was determined in cultures of 36 colonies of Candida on media containing 1, 2, and 4% glucose by spotting the agar with pH indicators. A drop in the pH became noticeable in all three media about the 3rd day of incubation, and a pH of 3.5 was reached in about 7 days. Subsequently, the pH remained almost stationary in the 4% glucose-agar, rose slowly in the 2% glucose-agar, and rose sharply in the 1% glucose-agar. The growth of Histoplasma was inhibited completely at pH 4 and below. When the pH was controlled in mixed cultures, some growth of Histoplasma was obtained. Substitution of maltose for glucose delayed the development of acidity and allowed the appearance of numerous mycelial colonies in the presence of Candida. This growth was arrested as soon as the medium became acid. Four other species which also acidified the Sabouraud's medium effected similar inhibition. It was thus shown that severe and prolonged acidity produced by some yeasts in the sugar-rich Sabouraud's media is alone sufficient to completely inhibit Histoplasma during the standard 4-week incubation of specimens such as sputum.     

Ultraviolet radiation-induced neoplastic transformation of normal human cells,in vitro

Human foreskin cell cultures in scheduled DNA synthesis (S phase) of the cell cycle were exposed to UV irradiation at a dose of 10 J · m^?2 in the presence of insulin. These treated cell populations, when selectively passaged in a high amino acid supplemented complete growth medium (CM) after 20 Dulbecco's phosphate buffered saline (pH 6.8) (PDL), were able to be grown in soft agar. These treated cell populations were also grown in 1% serum supplemented growth medium and at 41°C in 10% serum supplemented growth medium. Cell populations 4–5 PDL after treatment exhibited altered colony morphology and altered lectin agglutination profiles but would not grow in soft agar. These events appeared to be associated with the early stages in the expression phase of the transformed phenotype. After 20 PDL, we observed that these cells would grow in soft agar at a frequency of 20 colonies/10⁵ cells seeded in soft agar. The cell populations derived from these colonies, when propagated and injected into the nude mice, formed myxofibromas at the injection sites rather than the type of tumor (fibrosarcoma) previously described for chemical carcinogen-induced neoplasms.     

Hapten-specific murine colony-forming B cells. III. Delineation of functional B cell subpopulations grown under different conditions

The influence of anti-immunoglobulin M (IgM) and anti-IgD on the ability of fluorescein (FL)-specific B cells to proliferate in a colony-forming assay, and of their progeny to further differentiate in response to different FL-antigens was studied. Splenic FL-specific B cells were purified on FL-gelatin plates and were then cultured in semisolid agar in the presence or absence of anti-mu, and anti-delta, or both. Experiments were performed under conditions of either sheep red blood cell (SRBC)-potentiated or SRBC + lipopolysaccharide (LPS)-potentiated colony growth. The resulting colonies were then tested in secondary filler cell-dependent microcultures for the ability to be triggered by different classes of FL-antigens to yield plaque-forming cells (PFC). Anti-delta inhibited 47% of colony growth under both agar culture conditions. Anti-mu inhibited 55% of colony growth in SRBC + LPS-potentiated agar cultures, and inhibited 72% if only SRBC was present. If anti-delta and anti-mu were added together, inhibition was nearly additive. When anti-Ig-treated colonies were tested for PFC responses against FL-polymerized flagellin (POL), both normal and anti-delta resistant colonies, grown under both agar culture conditions, responded well. Anti-mu resistant colonies were refractory to FL-POL challenge. Only normal or anti-delta resistant colonies grown in SRBC + LPS agar cultures were able to respond well to FL-Ficoll, whereas even normal SRBC-potentiated colonies responded poorly. All except SRBC-potentiated, anti-mu treated colonies were able to respond to nonspecific signals present in cultures containing FL-KLH and activated T cell help. These data suggest that addition of specific anti-Ig antibodies, and variation of agar culture conditions, can select for B cell subpopulations responsive only to certain types of antigens.     

Antibacterial activity of the metabolic by-products of a Veillonella species and Bacteroides fragilis

A Veillonella species and Bacteroides fragilis were isolated from the cecal contents of adult chickens. When growth on an agar medium supplemented with 0.4% glucose and adjusted to pH 6.5, mixed cultures containing Veillonella and B. fragilis inhibited the growth of Salmonella typhimurium; Salmonella enteritidis, Escherichia coli 0157:H7 and Pseudomonas aeruginosa. Decreasing the glucose concentration of the agar decreased the inhibitory activity of the mixed culture. Mixed cultures grown on agar media supplemented with 0.5% glucose and adjusted to pH 6.5, 7.0 or 7.5 also inhibited the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 and P. aeruginosa. However, increasing the pH of the agar decreased the inhibitory activity of the mixed culture. Pure cultures of Veillonella or B. fragilis did not inhibit the growth of S. typhimurium, S. enteritidis, E. coli 0157:H7 or P. aeruginosa on any of the agar supplemented with different concentrations of glucose or on any of the agar adjusted to different pH levels. The inhibitory activity of the mixed culture was correlated with the concentration of volatile fatty acids that were formed as B. fragilis metabolized glucose to produce succinate and acetate and as the succinate produced by B. fragilis was decarboxylated by Veillonella to produce propionate.     

Preparation of Mutants Resistant to Catabolite Repression of Trichoderma reesei

The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth’s cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (KDD-10 and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% Avicel alone. These results suggest that KDD-10 and DGD-16 are comparatively derepressed by glucose for cellulase production.     

Regulation and genetic enhancement of glucoamylase and pullulanase production in Clostridium thermohydrosulfuricum.

We studied the general mechanism for regulation of glucoamylase and pullulanase synthesis in Clostridium thermohydrosulfuricum. These amylases were expressed only when the organism was grown on maltose or other carbohydrates containing maltose units. Amylase synthesis was more severely repressed by glucose than by xylose. Catabolite repression-resistant mutants were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained glucose-starch agar plates. Amylases were produced in both wild-type and mutant strains when starch was added to cells growing on xylose but not when starch was added to cells growing on glucose. In both wild-type and mutant strains, glucoamylase and pullulanase were produced at high levels in starch-limited chemostats but not in glucose- or xylose-limited chemostats. Therefore, we concluded that amylase synthesis in C. thermohydrosulfuricum was inducible and subject to catabolite repression. The mutants produced about twofold more glucoamylase and pullulanase, and they were catabolite repression resistant for production of glucose isomerase, lactase, and isomaltase. The mutants displayed improved starch metabolism features in terms of enhanced rates of growth, ethanol production, and starch consumption.     

Regulation and genetic enhancement of beta-amylase production in Clostridium thermosulfurogenes.

We studied the general mechanism for regulation of beta-amylase synthesis in Clostridium thermosulfurogenes. beta-Amylase was expressed at high levels only when the organism was grown on maltose or other carbohydrates containing maltose units. Three kinds of mutants altered in beta-amylase production were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained starch-glucose agar plates. beta-Amylase was produced only when maltose was added to cells growing on sucrose in wild-type and catabolite repression-resistant mutant strains, but the differential rate of enzyme synthesis in constitutive mutants was constant regardless of the presence of maltose. In carbon-limited chemostats of wild-type and catabolite repression-resistant mutant stains, beta-amylase was expressed on maltose but not on glucose or sucrose. beta-Amylase synthesis was immediately repressed by the addition of glucose. Therefore, we concluded that beta-amylase synthesis in C. thermosulfurogenes was inducible and subject to catabolite repression. The addition of cAMP did not eliminate the repressive effect of glucose. The mutants were generally characterized in terms of beta-amylase production, growth properties, fermentation product formation, and alterations in glucose isomerase and glucoamylase activities. A hyperproductive mutant produced eightfold more beta-amylase on starch medium than the wild type and more rapidly fermented starch to ethanol.