Possible role of streptomycin released from spore cell wall of Streptomyces griseus

Vegetative mycelia and spores of the investigated high- and low-producer strains of Streptomyces griseus bound significant amounts (4%) of streptomycin, which could be removed by increasing ionic strength. The release of antibiotic from the spores was easier when the spores were germinating. This phenomenon is considered to play an ecological role. We suppose that the streptomycin released during the germination process may protect the young hyphae from the different bacteria growing in the microenvironment of the Streptomyces spores.     

Group-wise growth of Streptomyces in a medium containing streptomycin

Summary We have described the observation that Streptomyces griseus colonies grow group-wise on agar media containing streptomycin. We have found that this phenomenon is due to a substance (s) produced by germinating Str. griseus spores in media containing streptomycin, and this substance made the neighbouring spores more tolerant to increasing streptomycin concentrations. The substance is produced specifically by Str. griseus strains. The substance has probably a great molecular size, is thermolabile, not a nucleic acid and the applied enzymes did not inactivate it. Some investigated enzyme-poisons did not influence either its production or its effect on Str. griseus spores. We succeeded in carrying over the substance into liquid phase and separate it from the producing culture and this enables us to furterh purification and investigation of the substance.     

High molecular weight ribosomal ribonucleic acid from vegetative hyphae and spores of Streptomyces griseus.

High molecular weight ribosomal ribonucleic acids (rRNAs) were isolated from young vegetative cells and spores of a streptomycin non-producing Streptomyces griseus, and their electrophoretic mobility was compared to each other and to that of rRNAs of Escherichia coli K-12. The electrophoretic mobility of 23 and 16S rRNAs from vegetative cells and spores of S. griseus was identical, but the 23S rRNAs of streptomyces ribosomes migrated more slowly on polyacrylamide gel than those of E. coli ribosomes. Intact, electrophoretically homogenous rRNAs could be isolated from S. griseus (No. 45-H) only in the presence of diethyl 1 pyrocarbonate (DEP), and intact rRNAs could be obtained from spores only if DEP had been added before breaking the spores. Otherwise instead of two distinct bands, three were obtained on polyacrylamide gel.     

Characterization of an asporogenous mutant ofStreptomyces aureofaciens with increased resistance to several aminoglycoside antibiotics

An asporogenous spontaneous mutant of Streptomyces aureofaciens named ASR1 was selected on streptomycin gradient plates. The mutant is very stable and differs in ultrastructure and morphology, it is prototrophic but it lost the ability to grow well on soybean extract medium and produces one-tenth tetracyclines of the parent. The ASR1 mutant has a 3-4-fold increased resistance to streptomycin and is cross-resistant to other aminoglycosides. Comparison of the protein profiles from both strains on SDS gels revealed a very low expression of a 29.5 kDa protein in the ASR1 mutant which is overexpressed in both vegetative cells and spores of the parental strain.     

Sporulation of Streptomyces griseus in submerged culture.

A wild-type strain of Streptomyces griseus forms spores both on solid media (aerial spores) and in liquid culture (submerged spores). Both spore types are highly resistant to sonication, but only aerial spores are resistant to lysozyme digestion. Electron micrographs suggest that lysozyme sensitivity may result from the thinner walls of the submerged spores. Studies of the life cycle indicate that neither streptomycin excretion nor extracellular protease activity is required for sporulation: the analysis of mutants, however, suggests that antibiotic production may be correlated with the ability to sporulate. A method was devised to induce the rapid sporulation of S. griseus in a submerged culture. This method, which depends on nutrient deprivation, was used to determine that either ammonia or phosphate starvation can trigger sporulation and that the enzyme glutamine synthetase may be useful as a sporulation marker after phosphate deprivation.     

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.     

The effect of Streptomyces species on the survival of Salmonella in soil

Abstract A modified Chelex 100 ion-exchange extraction method was used to monitor streptomycete spores, streptomycete mycelia and Salmonella in soil. Salmonella dusseldorf in soil was inhibited by the bactericidal effect of streptomycin and by the growth of Streptomyces bikiniensis . The soil used in the experiments exerted an antimicrobial effect on S. dusseldorf .
Competition between S. dusseldorf, Streptomyces lividans TK24 and Stm. bikiniensis ATCC 11062 was monitored in soil. In sterile amended soil Stm. lividans increased the survival of S. dusseldorf , whereas survival was reduced in the presence of the known streptomycin producer, Stm. bikiniensis . In the presence of S. dusseldorf the production of spores and mycelia by Stm. bikiniensis was reduced, and Stm. lividans sporulation was reduced but mycelia production increased. Evidence was seen for a beneficial effect between S. dusseldorf and Stm. lividans mycelia.
In non-sterile unamended soil the survival of S. dusseldorf was increased in the presence of Stm. lividans , whereas Stm. bikiniensis had no effect. Stm. lividans and Stm. bikiniensis reduced the survival of S. dusseldorf in non-sterile amended soil, with the most dramatic reduction caused by Stm. bikiniensis . No such changes in the survival of S. dusseldorf were observed with non-sterile amended soil that had been treated with sludge. The presence of sludge in unamended soil increased the rate of Salmonella die-off. In unamended soil containing sludge the presence of Stm. lividans increased the survival of S. dusseldorf , whereas survival was reduced in the presence of Stm. bikiniensis . The data provided evidence of antibiosis in soil, relating to the possible production of streptomycin by Stm. bikiniensis .     

Inhibition of the respiration of dormant and swollenStreptomyces spores by chloramphenicol

Chloramphenicol produces a decrease in the respiratory quotient of dormant and swollenStreptomyces antibioticus spores of about 20–25% and 18–24%, respectively, in the absence of protein synthesis as measured by [³H]leucine incorporation and by chemical methods. Rifampin and streptomycin had no inhibitory effect on respiration, thus excluding the possibility of an effect of all protein synthesis inhibitors on respiration. The inhibition of respiration by chloramphenicol was not a consequence of an increase in mortality due to toxicity, nor was it influenced by the developmental stage of the spores. It seems that chloramphenicol affects the activity of some component(s) related to the electron transport chain ofS. antibioticus spores, situated before the cytochrome oxidase level.     

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.     

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.     

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).     

The nucleotide sequence of a streptomycin streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from a streptomycin producer

The nucleotide sequence of the DNA fragment containing the streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from the streptomycin-producer Streptomyces griseus strain HUT 6037 was determined. Analysis of the sequence revealed an open reading frame which could encode 325 amino acid residues. A biased codon usage pattern, reflecting the high G + C composition (approximately 74%) of Streptomyces DNA, was observed in the gene.     

A mutation at proline-115 in the A-factor receptor protein of Streptomyces griseus abolishes DNA-binding ability but not ligand-binding ability.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) and its specific receptor protein (ArpA) are required for streptomycin production and aerial mycelium formation in Streptomyces griseus. A mutant strain HO1 that produced streptomycin and formed aerial mycelium and spores was derived from an A-factor-deficient mutant, S. griseus HH1. The phenotypes of mutant HO1 were found to result from a single amino acid replacement of ArpA; the proline residue at position 115 in the wild-type ArpA was replaced by serine, yielding mutant ArpA (P115S). The mutant ArpA (P115S) was still able to form a homodimer and possessed A-factor-binding ability but lost the ability to bind DNA. The properties of P115S suggest that ArpA consists of two independently functional domains, one for A-factor binding and one for DNA binding, and that proline-115 plays an important role in DNA binding. This is in agreement with the idea that A-factor binding to the COOH-terminal domain of ArpA causes a subtle conformational change of the distal NH2-terminal DNA-binding domain, resulting in dissociation of ArpA from DNA.     

Molecular detection of streptomycin-producing streptomycetes in Brazilian soils.

Actinomycetes were isolated from soybean rhizosphere soil collected as two field sites in Brazil. All the isolates were identified as Streptomyces species and were screened for streptomycin production and the presence of two genes, strA and strB1, known to be involved in streptomycin biosynthesis in Streptomyces griseus. Antibiotic resistance profiles were determined for 53 isolates from cultivated and uncultivated sites, and approximately half the strains were streptomycin resistance. Clustering by the unweighted pair group method with averages indicated the presence of two major clusters, with the majority of resistant strains from cultivated sites being placed in cluster 1. Only representatives from this cluster contained strA. Streptomycetes containing strA and strB1 were phenotypically diverse, and only half could be assigned to known species. Sequence comparison of 16S rRNA and trpBA (tryptophan synthetase) genes revealed that streptomycin- producing streptomycetes were phylogenetically diverse. It appeared that a population of streptomycetes had colonized the rhizosphere and that a proportion of these were capable of streptomycin production.     

Cloning and characterization of a gene from Streptomyces griseus coding for a streptomycin-phosphorylating activity

Abstract Six different plasmids expressing streptomycin (SM) resistance and SM phosphotransferase were obtained by cloning genomic DNA from Streptomyces griseus into Streptomyces lividans . The phosphorylating enzymatic activity formed in S. lividans differed in several biochemical properties from the one in S. griseus , though the phosphorylated products were identical.     

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.     

Unstable genetic determinant of A-factor biosynthesis in streptomycin-producing organisms: cloning and characterization.

We cloned a DNA fragment directing synthesis of A-factor from the total cellular DNA of streptomycin-producing Streptomyces bikiniensis on the plasmid vector pIJ385 . Introduction of the recombinant plasmid ( pAFB1 ) into A-factor-deficient S. bikiniensis and Streptomyces griseus mutants led to A-factor production in the host cells, as a result of which streptomycin production, streptomycin resistance, and spore formation of these mutants were simultaneously restored. The plasmid pAFB1 also complemented both afsA and afsB mutations of Streptomyces coelicolor A3(2). These results indicated that the cloned DNA fragment contained the genetic determinant of A-factor biosynthesis. The cloned fragment, when carried on a multicopy vector plasmid, induced production of a large amount of A-factor in several Streptomyces hosts. In Southern blot DNA/DNA hybridization analyses with a trimmed 5-kilobase fragment containing the intact A-factor determinant as probe, total cellular DNA from A-factor-deficient mutants gave no positive hybridization. The DNA blot experiment also showed a wide distribution of sequences homologous to the S. bikiniensis A-factor determinant among most, but not all, A-factor-producing actinomycetes with a varying extent of homology and the absence of these sequences from most A-factor nonproducers .     

Sporulation of several species of Streptomyces in submerged cultures after nutritional downshift

Streptomyces griseus ATCC 10137, S. griseus IMRU 3570, S. griseus JI 2212, S. acrimycini JI 2236 and S. albus G sporulated abundantly in several liquid media after nutritional downshift. Spores formed in submerged cultures were viable and as thermoresistant as aerial spores. Scanning electron microscopy showed that submerged spores are morphologically similar to aerial spores. The sporulation of the Streptomyces strains tested in complex medium appeared to be triggered by phosphate nutritional downshift, induced by addition of Ca2+ to the medium. Spore-shaped bodies were formed by S. lividans JI 1326 and S. coelicolor JI 2280 when grown in complex medium supplemented with Ca2+ and proline. The thermoresistance of these spore-shaped bodies differed from that of aerial spores.     

The Production of Mannosidostreptomycinase

S ummary : A medium is described which supports the production of high levels of mannosidostreptomycinase (B'ase) activity by Streptomyces griseus. The enzyme can be utilized to convert streptomycin B to streptomycin A in normal fermentations.     

Enzymatic Phosphorylation of Streptomycin by Extracts of Streptomycin-producing Strains of Streptomyces

Extracts of post-exponential phase mycelia of Streptomyces bikiniensis ATCC 11062, and other streptomycin-producers, catalyze phosphorylation of streptomycin and dihydrostreptomycin with adenosine-5'-triphosphate. The phosphate is esterified with an -OH group of the streptidine moiety. It is suggested that O-phosphoryl-streptomycin might serve as an intracellular precursor of extracellular streptomycin or as a detoxification product of streptomycin or that it might serve an unknown physiological function in the producing organism.