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Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive 1 . The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species 1,2 . Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly 3,4 . Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria., (© 2024. The Author(s).)

Dihydropyriculol is a major secondary metabolite of Pyricularia oryzae. However, the biological activity of dihydropyriculol has not been reported. Here, we showed that dihydropyriculol has inhibitory activity against Streptomyces griseus. Localization analysis of dihydropyriculol revealed that dihydropyriculol could reach to S. griseus under confrontation culture. These results suggest that dihydropyriculol can be used as a chemical weapon against S. griseus., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Green tea is thought to be a primary source of folate in the Japanese diet, based on folate content analyzed by a microbiological assay. Green tea also contains high amount of catechins, in particular, epigallocatechin gallate (EGCg), which was demonstrated to be able to inhibit the digestive enzyme activities and microbial growth in the folate assay. In the present study, we examined whether tea catechins interfered with components of the folate assay for green tea. A marked inhibitory effect of EGCg on microbial growth was observed at an inhibitory concentration of higher than 10 μg/mL. Tea catechins without the galloyl moiety did not show an inhibitory effect. EGCg inhibited the activity of the three enzymes used for assay sample preparation at an inhibitory concentration of higher than 750 μg/mL for α-amylase, 1,000 μg/mL for protease, and 50 μg/mL for conjugase. However, with each step of the assay, the actual concentration of EGCg was decreased to below the inhibitory concentration of each analytical step. Lack of influence of EGCg on green tea folate assay was confirmed by an addition of folate standard in tea infusion. These results suggested that tea catechins have no practical impact on folate analysis in green tea, using the general microbiological assay.

Streptomyces griseus causes subcutaneous mycetomas in felines and dolphins; however, human mycetoma caused by S. griseus has not previously been reported. Hereby, we report a case of a 50-year-old female presenting with swelling in the left upper cervical region and the left cheek that lasted for 6 months. The fine needle aspiration (FNA) performed on the swelling yielded purulent material; on microscopy, actinomycosis was diagnosed. On culturing, the pus grew S. griseus. To the best of our knowledge, this is the first reported case of human mycetoma caused by S. griseus., (Copyright © 2013. Published by Elsevier B.V.)

Natural products remain the best sources of drugs and drug leads and serve as outstanding small-molecule probes to dissect fundamental biological processes. A great challenge for the natural product community is to discover novel natural products efficiently and cost effectively. Here we report the development of a practical method to survey biosynthetic potential in microorganisms, thereby identifying the most promising strains and prioritizing them for natural product discovery. Central to our approach is the innovative preparation, by a two-tiered PCR method, of a pool of pathway-specific probes, thereby allowing the survey of all variants of the biosynthetic machineries for the targeted class of natural products. The utility of the method was demonstrated by surveying 100 strains, randomly selected from our actinomycete collection, for their biosynthetic potential of four classes of natural products, aromatic polyketides, reduced polyketides, nonribosomal peptides, and diterpenoids, identifying 16 talented strains. One of the talented strains, Streptomyces griseus CB00830, was finally chosen to showcase the discovery of the targeted classes of natural products, resulting in the isolation of three diterpenoids, six nonribosomal peptides and related metabolites, and three polyketides. Variations of this method should be applicable to the discovery of other classes of natural products.

Addition of perfluorodecalin with gas-transporting function to the liquid medium during submerged cultivation of actinomycetes of the genus Streptomyces resulted in higher intensity and level of the biomass synthesis and increased production of streptomycin and daunorubicin. Addition of perfluorodecalin to the medium provided a 2.0-2.3-fold surpass of the maximum antibiotic production (achieved by the 120th-144th hours of the culture growth) vs. the antibiotic accumulation peaks in the control.

An oligopeptide permease family ATP-binding cassette (ABC) transporter encoded by SGR2418-SGR2414 was shown to be essential for aerial mycelium formation on glucose-containing media in Streptomyces griseus. In spite of only weak sequence similarity, the operon was equivalent to the bldK operon of Streptomyces coelicolor A3(2) in terms of chromosomal location and function. Transcription of the operon appeared not to be directly regulated by AdpA, a global regulator of morphological and physiological development in S. griseus, although it was affected by adpA inactivation. This study revealed that an ABC transporter was essential for aerial mycelium formation not only in S. coelicolor A3(2) but also in S. griseus, indicating that extracellular signaling by certain peptides should be conserved among different Streptomyces species., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Unlike other Streptomyces spp., the streptomycin producer Streptomyces griseus IFO13189 shows emergence of a small fraction of rsmG and rpsL mutants among spontaneous low- or high-level streptomycin-resistant mutants. rsmG, but not rpsL, mutants showed greater ability (two- to threefold) to produce streptomycin, accompanied by enhanced transcription of metK and strR, together with streptomycin biosynthetic genes, such as strB1, strD and strF, thus underlying the observed increase in streptomycin production in the rsmG mutants. Moreover, rsmG mutation was effective for activating the 'silent' or poorly expressed secondary metabolite-biosynthetic genes present in S. griseus.

Indolmycin, a potential antibacterial drug, competitively inhibits bacterial tryptophanyl-tRNA synthetases. An effort to identify indolmycin resistance genes led to the discovery of a gene encoding an indolmycin-resistant isoform of tryptophanyl-tRNA synthetase. Overexpression of this gene in an indolmycin-sensitive strain increased the indolmycin MIC 60-fold. Its transcription and distribution in various bacterial genera were assessed. The level of resistance conferred by this gene was compared to that of a known indolmycin resistance gene and to those of genes with resistance-conferring point mutations.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is a microbial hormone that triggers morphological differentiation and secondary metabolism in Streptomyces griseus. The effects of A-factor on global gene expression were determined by DNA microarray analysis of transcriptomes obtained with the A-factor-deficient mutant DeltaafsA. A-factor was added at a concentration of 25 ng ml(-1) to mutant DeltaafsA at the middle of the exponential growth phase, and RNA samples were prepared from the cells grown after A-factor addition for a further 5, 15 and 30 min, and 1, 2, 4, 8 and 12 h. The effects of A-factor on transcription of all protein-coding genes of S. griseus were evaluated by comparison of the transcriptomes with those obtained from cells grown in the absence of A-factor. Analysis of variance among the transcriptomes revealed that 477 genes, which were dispersed throughout the chromosome, were differentially expressed during the 12 h after addition of A-factor, when evaluated by specific criteria. Quality threshold clustering analysis with regard to putative polycistronic transcriptional units and levels of upregulation predicted that 152 genes belonging to 74 transcriptional units were probable A-factor-inducible genes. Competitive electrophoretic mobility shift assays using DNA fragments including putative promoter regions of these 74 transcriptional units suggested that AdpA bound 37 regions to activate 72 genes in total. Many of these A-factor-inducible genes encoded proteins of unknown function, suggesting that the A-factor regulatory cascade of S. griseus affects gene expression at a specific time point more profoundly than expected.

Ralstonia pickettii K50 (strain K50) is a denitrifying bacterium that produces low levels of N(2)O under aerobic conditions. In this study, we found that co-culturing of strain K50 with Streptomyces griseus significantly enhanced the denitrification activity of strain K50 in an artificial wastewater (AWW) system. Most factors that enhance denitrification activity were in the high molecular weight fraction of the cell-free broth of S. griseus, and were suggested to be extracellular proteases. Further investigation revealed that the cultivation of strain K50 in protease-treated AWW medium fully enhanced denitrification, and that a shortage of amino acids in the medium limited it. Among the 20 standard amino acids tested, only histidine had a significant effect in inducing denitrification by strain K50. Our results indicate that histidine is a novel inducer of bacterial denitrification.

We cloned and characterized sigH encoding a stress-response sigma factor, sigma(H), of Streptomyces griseus. Nucleotide sequencing of the sigH gene cluster revealed an identical gene organization as in the orthologous region of Streptomyces coelicolor A3(2). Transcriptional analysis by S1 nuclease mapping showed the presence of three tandem promoters (P1-P3) that direct transcription of sigH. The activity of P1 was markedly reduced in a sigH-depleted mutant, suggesting its dependence on sigma(H). P1 was induced by addition of 0.7 M NaCl, and P2 was induced by heat shock at 45 degrees C or addition of 4% ethanol. The sigH mutant of S. griseus showed conditional defect in aerial mycelium formation and streptomycin production depending on high concentration of glucose (>2%). Meanwhile, the wild-type strain of S. griseus introduced with rshA encoding a probable anti-sigma factor for sigma(H) on a high-copy-number plasmid was unable to perform development on media containing 1% glucose while it showed wild-type phenotype on media containing 1% maltose. RshA inhibited the sigma(H)-dependent in vitro run-off transcription at P1, which confirmed its role as a negative regulator for sigma(H). Analysis of RshA-sigma interaction by an Escherichia coli two-hybrid system showed specific interaction of RshA with sigma(H) and two related sigma factors, sigma(L) and sigma(F). We speculate that the high copy number of rshA represses morphological and physiological development of S. griseus through simultaneous inactivation of sigma(H) and related stress-response sigma factors, which play an essential role in the onset of cellular differentiation and antibiotic production on glucose media.

The 52-kDa protein, EshA, whose expression is controlled developmentally, is produced during the late growth phase of Streptomyces spp. We found that disruption of the eshA gene, which encodes the EshA protein, abolishes the aerial mycelium formation and streptomycin production in Streptomyces griseus when grown on an agar plate. The eshA disruptant KO-390 demonstrated a reduced amount of expression of the transcriptional activator strR, thus accounting for the failure to produce streptomycin. KO-390 was found to accumulate deoxynucleoside triphosphates at high levels, including dGTP, at late growth phase. The accumulation of dGTP was a cause for the impaired ability of KO-390 to produce aerial mycelium, because the ability to form aerial mycelium was completely repaired by addition of decoyinine, an inhibitor of GMP synthetase. The accumulation of dNTP in KO-390 coincided with a reduced rate of DNA synthesis. The developmental time frame of these phenomena in KO-390 matched a burst of EshA expression in the wild-type strain. In contrast to S. griseus, the eshA disruption did not affect the ability for Streptomyces coelicolor to form aerial mycelium and did not result in the aberrant accumulation of dNTP accompanied by arrest of DNA synthesis, implying qualitative differences in addition to quantitative differences between the two EshA proteins. We propose that the S. griseus EshA protein somehow positively affects (or regulates) the replication of DNA in wild-type cells at late growth phase but leads to aberrant phenotypes in mutant cells due to the disturbed DNA replication. The EshA protein was found to exist as a multimer ( approximately 20-mers) creating a cubic-like structure with a diameter of 27 nm and located predominantly in cytoplasm.

The effect of beta-lactam antibiotics that are known to inhibit cell wall biosynthesis and induce cell wall autolysis on the electrophysiological state of the plasma membrane in Streptomyces griseus was studied. Addition of various beta-lactam antibiotics induced a dose- and growth-stage-dependent depolarization of the membrane potential of Streptomyces griseus. The hydrolyzed biologically inactive derivative penicilloic acid had no depolarizing effect on the membrane potential. The ionophore gramicidin D, while depolarizing the membrane potential, also induced a dose-dependent increase in cell wall lysis. These observations suggest that alteration of the transmembrane potential could be an important signal in triggering cell wall autolysis of S. griseus., (©2002 Elsevier Science (USA).)

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.

In the presence of cefoxitin, which inhibits septum formation during sporulation, Streptomyces griseus is unable to sporulate, retaining the sonication sensitivity of nonsporulating hyphae. Cefoxitin- and sonication-resistant mutant SKK2600 was isolated and showed many morphological differences from its parental strain. A 3.6-kb DNA fragment that complemented the mutations of SKK2600 contained two open reading frames (ORFs), either of which could complement SKK2600. One ORF, designated ssfR, encoded a protein containing a potential DNA-binding helix-turn-helix motif close to its N terminus. SsfR is similar to members of a large family of transcriptional regulators, particularly IclR of Escherichia coli. The second ORF was identified as ssgA, a previously described sporulation gene from S. griseus (S. Kawamoto and J. C. Ensign, Actinomycetology 9:136-151, 1995). A point mutation of C to T seven nucleotides upstream of the UGA stop codon of ssfR was responsible for the phenotype of isolated mutant strain SKK2600. Surprisingly, this mutation should not change the primary structure of SsfR. The ssfR and ssgA disruption mutants were constructed and showed the "white" mutant phenotype, with some growth medium dependence. In addition, the ssfR null mutant sporulated ectopically in phosphate starvation medium.

Previous studies showed that some lactones have beta-lactamase inhibitory or antibacterial effects, others--like A-factor (a gamma-butyrolactone) and its derivatives--stimulate sporulation in Streptomyces griseus strains. Our experiments were aimed at exploring whether synthetic gamma-lactones had such effects. None of the seven gamma-lactones studied showed antibacterial activity, but two of them inhibited beta-lactamases isolated from various bacteria. These two gamma-lactones did not reduce colony formation of murine bone marrow cells in vitro, indicating that they were not toxic to proliferating mammalian cells. Four gamma-lactones, including the two inhibiting beta-lactamase, stimulated sporulation in the non-sporulating S. griseus bald 7 mutant. Further studies of gamma-lactones as potential inhibitors of beta-lactamase seem to be warranted.

Two Streptomyces griseus strains were isolated from different soil types. S. griseus CAG17 strain was isolated from an agricultural area with low organic matter but rich in phosphorus content and S. griseus 26K strain was isolated from a forest area rich in organic matter with a low phosphorus content. The survival and metabolic activity of these isolates were studied in dynamic sterile soil microcosm systems. The fitness of each isolate was studied by re-inoculation in a soil type different from its origin. Maximum percentage of germination and respiration rates occurred within the first 48 h after each soil turnover (removal and addition of certain soil volumes). Data suggested that S. griseus CAG17 survived better independently of the soil type in comparison with S. griseus 26K which sporulated within the first 12 h after inoculation. Incubation temperatures did affect the lifecycles in relation to soil type. For example, the lowest temperature tested, 22 degrees C, was more favourable for extended germination and adaptation in general but revealed lesser spore numbers in the 'foreign' soil environment. Monitoring metabolic activity by estimation of urease, phosphatases and dehydrogenase-specific activities, between 18 and 35 degrees C incubation temperatures, was a reliable method for studying the survival and growth of streptomycete populations in soil. Results also confirmed that respiration rate and enzyme-specific activity corresponded with spore counts in long-term experiments which were designed for the investigation of survival and growth of S. griseus CAG17. Under selective pressure by heavy metals, in soil microcosm systems, metabolic activity proved a useful tool for the investigation of streptomycete activity. These methods could also be applied in agricultural field studies for monitoring microbial populations under conditions where various 'pollutants' are present in soil samples.

A new type of beta-lactamase has been isolated and characterized in Streptomyces griseus NRRL B-2682. The enzyme has membrane-bound and extracellular forms. Biochemical characterization of some of the properties of the enzyme showed that it belongs to the class A group of penicillinases. Comparison of the membrane-bound and extracellular forms of the beta-lactamases suggests that they seem to be differently processed forms of the same enzyme. The N-terminal amino acid sequence of the extracellular form of the beta-lactamase showed a high degree of similarity to a D-aminopeptidase of another Streptomyces griseus strain. Secretion of the beta-lactamase was affected by the differentiation state of the strain since in spontaneous non-sporulating mutants only the membrane-bound form was present. In accordance with this when sporulation of the wild-type strain was inhibited it failed to secrete extracellular beta-lactamase. Addition of globomycin to the non-sporulating cells liberated the enzyme from the membrane, indicating that the protein is processed normally by signal peptidase II and a glyceride-thioether group, together with a fatty acid amide-linkage, is responsible for the attachment of the enzyme to the cellular membrane. Under sporulation-repressed conditions addition of peptidoglycan fragments and analogues or inhibition of cell wall biosynthesis by penicillin-G induced beta-lactamase secretion and also restored sporulation both in solid and submerged cultures. These results confirm that beta-lactamase secretion is tightly coupled to the sporulation process in S. griseus.

We used fluorescein-tagged beta-lactam antibiotics to visualize penicillin-binding proteins (PBPs) in sporulating cultures of Streptomyces griseus. Six PBPs were identified in membranes prepared from growing and sporulating cultures. The binding activity of an 85-kDa PBP increased fourfold by 10 to 12 h of sporulation, at which time the sporulation septa were formed. Cefoxitin inhibited the interaction of the fluorescein-tagged antibiotics with the 85-kDa PBP and also prevented septum formation during sporulation but not during vegetative growth. The 85-kDa PBP, which was the predominant PBP in membranes of cells that were undergoing septation, preferentially bound fluorescein-6-aminopenicillanic acid (Flu-APA). Fluorescence microscopy showed that the sporulation septa were specifically labeled by Flu-APA; this interaction was blocked by prior exposure of the cells to cefoxitin at a concentration that interfered with septation. We hypothesize that the 85-kDa PBP is involved in septum formation during sporulation of S. griseus.

m-Aminophenylboronic acid (APBA) inhibited the germination, growth and sporulation of Streptomyces griseus NRRL B-2682 in an age- and concentration-dependent manner in submerged and solid cultures. When added to cells or cell extracts it irreversibly inhibited NAD+-glycohydrolase and ADP-ribosyltransferase activity. ADP-ribosyltransferase was more sensitive, but inhibition was not complete, even in the presence of 10 mM APBA. The in vivo effects of the inhibitor correlated with its in vitro effect on ADP-ribosylation and on the profile of ADP-ribosylated endogenous proteins. The physiological importance of ADP-ribosyltransferase was supported by the observation that APBA strongly inhibited the growth of a non-sporulating and NAD+- glycohydrolase-negative mutant of the parental strain. The resistance of S. griseus NRRL B-2682 strains able to grow in the presence of APBA was due to permeability factors. A comparison of the ADP-ribosylated protein profiles of S. griseus NRRL B-2682 grown under various conditions showed similarities, but also specific differences. The results suggest that the ADP-ribosyltransferase of S. griseus NRRL B-2682 is an indispensable enzyme for growth and differentiation of the strain. It may regulate the activity of key enzymes or developmental proteins by responding to intra- and extracellular conditions.

Three genes (hrd) homologous to the rpoD gene of Escherichia coli, that encode sigma factor-like proteins, have been cloned from DNA of the candicidin-producing strain Streptomyces griseus IMRU 3570. They are located in different regions of the chromosome. Sequence analysis showed that the first one is analogous to the hrdB gene of S. coelicolor. The second showed high similarity to the hrdD gene of S. coelicolor and S. aureofaciens and is linked, as in S. coelicolor, to a N-acetyltransferase-encoding gene (nat) distantly related to the pat and bar genes that encode resistance to bialafos. The third showed no close homology with other known hrd genes from actinomycetes and has been named hrdT. Functional domains in the three S. griseus Hrd proteins are highly conserved in relation to those of the sigma 70 protein family. Northern analysis showed that hrdB is expressed as a 1.9-kb transcript during active growth in phosphate-rich medium, but it is less efficiently transcribed under sporulation conditions (phosphate-starved) or after a heat-shock treatment. Two other shorter transcripts of 1.2 and 0.7 kb were also detected with the same probe. The hrdD gene is transcribed as a single 1.1-kb transcript under sporulation conditions following nutritional shiftdown and, to a lower extent, during growth conditions in phosphate-rich medium. The hrdT gene is weakly transcribed (1.5-kb RNA) under all conditions tested. The hrd-encoded sigma factors probably recognize actinomycetes promoters (SEP type) with E. coli-like consensus sequences.

The antimicrobial activities of twenty-two substances structurally related to staurosporine, aglycone in the indolocarbazole and bis-indole series were examined against Streptomyces chartreusis and Streptomyces griseus, Bacillus cereus, Escherichia coli, Candida albicans and Botrytis cinerea. Inhibition of sporulation was examined also on the two species of Streptomyces. Unlike literature reports for efficient protein kinase inhibitors, staurosporine and K-252a, no evident correlation could be found either between protein kinase inhibitory potencies and inhibition of sporulation of the Streptomyces species, or protein kinase inhibitory potencies and growth of all microorganisms tested. A weak activity against C. albicans was observed for the chloro-indolocarbazole compounds as already reported for structurally related substances from the cyanobacterium Tolypothrix tjipanasensis.

A gene (nonR) conferring tetranactin resistance on the macrotetrolide-sensitive strain, Streptomyces lividans TK64, was isolated during a shotgun cloning experiment, in which chromosomal fragments from Streptomyces griseus were ligated into the vector pIJ699 and then introduced by transformation into S. lividans TK64. The sequence (3326 bp) of the cloned DNA revealed three complete open reading frames (ORFs) and one incomplete ORF encoded on one strand of the DNA. The nonR gene (designated here ORFA) encodes a polypeptide of 279 amino acids (Mr 30610) and contains a putative active site motif, GXSXG, characteristic of serine proteases and esterases. A functional role for the nonR gene product may involve the inactivation of the antibiotic through hydrolysis of one or more ester linkages in the macrotetrolide ring. The deduced product of the incomplete ORFX lying adjacent to ORFA showed 27.9% sequence identity with the C-terminal region of rat mitochondrial enoyl-CoA hydratase, and is possibly a macrotetrolide biosynthetic enzyme.

This study describes the bactericidal and sporicidal effects of four disinfectants on a geosmin-producing strain of Streptomyces griseus. The disinfectants investigated were chlorine, chloramine, chlorine dioxide and ozone. Chlorine and chlorine dioxide at concentrations around 1 mg/l were effective inactivators of both spore and mycelial propagules. Decimation times of less than 1 min were determined in each case. Both growth forms exhibited a high ozone demand, but decimation times resulting from an initial dose of around 2.5 mg/l were approximately 1.5 min. Monochloramine was comparatively less effective: at a concentration of approximately 1 mg/l the decimation times for spores and mycelia were 13.8 and 22.7 min, respectively.

We determined the nucleotide sequence of a 1794-bp BglII-BamHI fragment containing the kan gene (KmR) which encodes a novel aminoglycoside 3-N-acetyltransferase of Streptomyces griseus SS-1198PR. The sequence was found to contain four open reading frames (ORF) in the direction of transcription determined by low resolution S1 nuclease mapping. Except for the largest one, these ORF are unlikely to be the coding sequence of kan, because of their inability to confer kanamycin resistance. The largest ORF could encode a 30.8-kDa protein consisting of 284 amino acids (aa) with 93.3 mol% G + C in the third position of codons, and is preceded by potential ribosome-binding sites. Thus, the largest ORF was likely to represent the kan gene. The transcription start point of kan was located 84 bp upstream from the start codon. The -10 region showed similarity to the Escherichia coli consensus promoter motif, while no sequence similarity was found in the -35 region. The deduced aa sequence of kan showed 85% similarity with that of the aacC7 gene of Streptomyces rimosus forma paromomycinus [Lopez-Cabrera et al., J. Bacteriol. 171 (1989) 321-328] suggesting that these genes are derived from a common ancestral gene.

Two auxotrophic mutants of S. rimosus and S. griseus were obtained through nitrosoguanidine treatment. The equal volume of two protoplast containing 10(9)/ml were mixed and put in the fusion chamber of Shimadzu somatic hybridizer-SSH-C11. Fifteen seconds after protoplast pearl chains were formed through electrophoresis under high-frequency electric field (800 V/cm 1 MHz), it was turned into high DC pulse field (6 kV/cm) for 20 seconds immediately to make the protoplast to be fused. Colonies were raised on regeneration medium R3. 34971 colonies were selected and transferred to basic medium containing two antibiotics. Nine of them were prototrophic fusant with double resistance. They had good biological characteristics of parent strain, such as faster growth rate, higher productivity and stronger microbial activity. Bioautographic result showed that it can produce bioactive characteristics that their parents do not have.

S. griseus Kr. is a commercial strain producing grisin, an antibiotic of the streptothricin group used as a feed additive. It was shown earlier that genetic instability of the strain was very high which was evident from a high frequency of nonreverting Grn- Grns mutants. With densitographic analysis of chromosomal DNA electrophoregrams and DNA-DNA hybridization it was revealed that the molecular basis of the genetic instability of the S. griseus strain was deletion of a DNA fragment about 20 kb in size containing a grisin resistance gene. The resistance gene designated as gsr was cloned to S. lividans TK 64 within the plasmid vector pIJ699. The restriction map of a cloned DNA fragment with a gsr gene was constructed and its similarity to that of a nat gene resistant to norseothricin, another streptothricin was observed. Introduction of a gsr gene within the multicopy plasmid pIJ699 into S. griseus 212, a highly productive strain synthesiing the antibiotic, led to an increase in its resistance and productivity. Proceeding from the preliminary data on possible linkage of a gsr gene and grisin biosynthesis genes, it appeared possible to use the cloned gene as a molecular probe in cloning the biosynthesis genes.

The isolated cell wall of Streptomyces griseus 52-1 strain labelled with fluorescein isothiocyanate (FITC) and containing wall-bound autolytic enzyme was lysed as a function of different cations. The autolysis was accelerated by aminoglycoside antibiotics (streptomycin and the structurally closely related neomycin) which have a polycationic character. Since this strain is a streptomycin producer it is suggested that streptomycin may have a regulatory function on autolysis.

Two genes, aphE and orf, coding for putative Mr 29,000 and Mr 31,000, proteins respectively, were identified in the nucleotide sequence of a 2.8 kbp DNA segment cloned from Streptomyces griseus N2-3-11. The aphE gene expressed streptomycin (SM) resistance and a SM phosphorylating enzyme in S. lividans strains. The two genes were found to be in opposite direction and seemed to share a common region of transcription termination. The aphE gene shows significant homology to the aph gene, encoding aminoglycoside 3'-phosphotransferase, APH(3'), from the neomycin-producing S. fradiae. The enzymatic specificity of the aphE gene product was identified to be SM 3"-phosphotransferase, APH(3"). The primary structure of the APH(3") protein is closely related to the members of the APH(3') family of enzymes. However, the APH(3") enzyme did not detectably phosphorylate neomycin or kanamycin. There is only low similarity of the protein to the APH(6) group of SM phosphotransferases. An evolutionary relationship between antibiotic and protein kinases is proposed.

Mutants of Streptomyces griseus blocked in cytodifferentiation regained their capacity to form differentiated mycelia and/or anthracycline pigments in the presence of butyrolactone-type autoregulatory effectors such as trans-2-(6'-methylheptanol-1'-yl)-3-hydroxymethyl-4-butanolide+ ++. In the pertinent indicator strains, the effect has been correlated with the increase of lipid synthesis, with changes in the composition of lipid fraction and with the restoration of the production of neutral proteinases. The results suggest that autoregulatory butyrolactones from streptomycetes stimulate cytodifferentiation of their producers at an early stage of development.

Streptomyces cyaneofuscatus PRL 1642 producing valinomycin was shown to synthesize bioregulators of differentiation similar to A factor. The regulators stimulate spore formation and streptomycin synthesis in a Streptomyces griseus 1439 mutant deficient in A factor. Some S. cyaneofuscatus mutants respond to A factor addition into the medium by an increase in valinomycin synthesis and a change in morphological properties. The regulators from S. cyaneofuscatus are more effective toward mutants of this species than toward S. griseus mutants.

The metabolic function of NAD(P)-glycohydrolase in the streptomycin-producing Streptomyces griseus was investigated. Phospho-adenosinediphospho-ribose, the product of NAD(P)-glycohydrolase reaction was shown to interfere as a competitive inhibitor not only with the glucose-6-phosphate dehydrogenase (VORONINA et al. 1978) but also with the NADP-dependent isocitrate and 6-phospho-gluconate dehydrogenases. Inhibition kinetics were studied with isocitrate dehydrogenase from pig heart and 6-phosphogluconate dehydrogenase from yeast as well as with mycelial extracts of a mutant of S. griseus lacking NAD(P)-glycohydrolase.

The streptomycin sensitivity of ribosomes derived from a streptomycin-producing Streptomyces griseus was examined in a polyuridylic acid directed 14C-phenylalanine incorporating system. In order to get reproducible results it is essential to use cell-free extracts which do not inactivate streptomycin. This condition can be fulfilled by the combination of washed ribosomes of the streptomycin-producing strain and the 110 000 g supernatant of the streptomycin-nonproducing variant of S. griseus, because the streptomycin-phosphorylating activity can be washed out from ribosomes of younger streptomycin-producing cultures, and the streptomycin-nonproducing S. griseus does not have any streptomycin-inactivating capacity. In this amino acid polymerizing system the ribosomes of the streptomycin-producing strain were as sensitive to streptomycin as the ribosomes of the nonproducing variant or of Escherichia coli.

Addition of cyclic-AMP (c-AMP) to Streptomyces griseus fermentations inhibited candicidin formation. In a phosphate-free resting cell system, c-AMP inhibited net candicidin formation and incorporation of labeled propionate and p-aminobenzoic acid into the antibiotic but did not inhibit protein synthesis. All nucleotides tested, regardless of the position of the phosphate ester, were effective inhibitors; nucleosides and free bases were not. Inhibition occurred whether the nucleotide was added early or late. The results indicate that inhibition of antibiotic formation by exogenous nucleotides, including cyclic nucleotides, is similar to the effect produced by inorganic phosphate.

Streptomyces griseus 15 was subjected to the action of 1,4-bis-diazoacetyl butane (DAB) taken at concentrations of 10 to 50,000 micrograms/ml. Small doses (10-100 micrograms/ml) of DAB had no mutagenic action and activated the cultural growth (the viability and the survival rate of spores increased on solid media, while the biomass yield rised in liquid media). Experiments were conducted using the method of orthogonal planning of a bifactorial experiment, and the role of the exposure time rised with a decrease in the mutagen concentration.

The soil of potted ornamental plants as a reservoir for Aspergillus species pathogenic for man is of epidemiological and ecological interest. Isolation of A. niger as the sole hyphomycete from the soil of potted African violets (Saintpaulia ionantha, Gesneriaceae), prompted us to look for A. inger on the surface of the roots of this plant. Small pieces of the roots were inoculated in the nutrient-free agar-gel with and without antibiotics. On the antibiotic-free gel, a dense growth of Streptomyces griseus was consistently observed around the root pieces. But the gel fortified with antibiotics showed pure growth of only A. niger. One of the two strains of S. ariseus isolated from the African violet, showed inhibitory effect against a number of fungi including A. niger but the other strain had no effect on this fungus. The possible influence of streptomycetes on the occurrence of aspergilli in the soil of potted ornamental plants is discussed.

The genetic and biochemical basis of a 200-fold increase in kanamycin (KM)-resistance shown in Streptomyces griseus SS-1198PR generated by protoplast regeneration was investigated. A 15-kb Bcl I-DNA segment responsible for the KM-resistance was cloned into pIJ61 with Streptomyces lividans TK21 as host. The KM-resistance segment was then subcloned into pIJ702 as a 1.8-kb BamH I-Bgl II fragment with a BamH I site essential for the KM-resistance. Both S. lividans TK21 containing the cloned segments and S. griseus SS-1198PR showed multiple resistance to KM, dibekacin and gentamicin C complex. Cell free extracts from these strains inactivated the antibiotics in the presence of acetyl CoA in agreement with their resistance pattern. The structure of the inactivated KM-A was determined as 3-N-acetyl-KM-A indicating acetylation by an aminoglycoside acetyltransferase, AAC(3). The substrate range of the enzyme was unique and was designated AAC(3)-V. No genetic linkage was found between the cloned 15 kb Bcl I segment and the separately cloned streptomycin resistance gene (str) segment (3.8 kb Sph I fragment). The str genes cloned from both the parent (SS-1198) and the strain SS-1198PR were identical in their size, restriction site and function. In addition, these strains showed no significant difference in the total DNA digestion pattern. These results indicate that protoplast regeneration may cause a critical change in a specific region of DNA resulting in a high activity of an AAC(3) with a novel substrate range.

The effect of grisin on survival and variation of Actinomyces griseus producin grisin was studied. The efficiency of various concentrations of grisin on induction of variation according to the feature of the antibiotic production was compared. A possibility of increasing the productivity of strain. VNIIGenetics-115 by the use of the mutations of resistance to grisin is shown.

An str gene cluster containing at least four genes (strR, strA, strB, and strC) involved in streptomycin biosynthesis or streptomycin resistance or both was self-cloned in Streptomyces griseus by using plasmid pOA154. The strA gene was verified to encode streptomycin 6-phosphotransferase, a streptomycin resistance factor in S. griseus, by examining the gene product expressed in Escherichia coli. The other three genes were determined by complementation tests with streptomycin-nonproducing mutants whose biochemical lesions were clearly identified. strR complemented streptomycin-sensitive mutant SM196 which exhibited impaired activity of both streptomycin 6-phosphotransferase and amidinotransferase (one of the streptomycin biosynthetic enzymes) due to a regulatory mutation; strB complemented strain SD141, which was specifically deficient in amidinotransferase; and strC complemented strain SD245, which was deficient in linkage between streptidine 6-phosphate and dihydrostreptose. By deletion analysis of plasmids with appropriate restriction endonucleases, the order of the four genes was determined to be strR-strA-strB-strC. Transformation of S. griseus with plasmids carrying both strR and strB genes enhanced amidinotransferase activity in the transformed cells. Based on the gene dosage effect and the biological characteristics of the mutants complemented by strR and strB, it was concluded that strB encodes amidinotransferase and strR encodes a positive effector required for the full expression of strA and strB genes. Furthermore, it was found that amplification of a specific 0.7-kilobase region of the cloned DNA on a plasmid inhibited streptomycin biosynthesis of the transformants. This DNA region might contain a regulatory apparatus that participates in the control of streptomycin biosynthesis.

Recombinants between Streptomyces coelicolor A3(2) and Streptomyces griseus Kr-15 were obtained using methods of hybrid construction. Recombinant Rcg1, obtained from a cross between S. griseus and a S. coelicolor UF (SCPI-) strain, phenotypically resembled S. coelicolor UF strains and in crosses with a S. coelicolor NF donor strin produced recombinatn progeny at a frequency of 100%. Recominant Rcg3, like SCP1-carrying S. coelicolor strains, inhibited SCP1-strains of S. coelicolor and in crosses with a UF recipient strain of S. coelicolor generated recombinants at high frequency. In crosses between S. griseus and Rcgi the frequency of recombinant formation was increased about 100-fold relative to crosses between S. griseus and S. coelicolor. Effective transfer of S. grieseus and Rcg3 chromosomal markers into Rcg1 and S. coelicolor, respectively, indicated that S. griseus had donor properties. Studies of the ability of recombinants to support phage growth indicated that parental chromosomal fragments containing genes involved in control of phage-receptor formation and intracellular growth were present in the hybrids. Grisin-producing recombinants, capable of restricting phages attacking S. coelicolor and S. griseus, were obtained.

Cerulenin, an inhibitor of fatty acid synthesis, inhibits specifically the biosynthesis of the polyene macrolide candicidin by resting cells of Streptomyces. 50% inhibition was achieved with a cerulenin concentration of 1.5 mug/ml. Cells in which candicidin synthesis was inhibited for 10 h remained capable of candicidin synthesis after removal of the inhibitor. Cerulenin inhibits specifically the incorporation of [14C] propionate into candicidin but does not affect total protein or RNA synthesis. The uptake of [14C] propionate was not inhibited under conditions which totally prevented the incorporation of propionate into candicidin. Incorporation of p-amino[14C] benzoic acid NH2 [14C] BzO- into the aromatic moiety of candicidin was also inhibited by cerulenin. The inhibitory action of cerulenin was not reversed by exogenous fatty acids. Since cerulenin is known to block the condensation of malonyl-CoA subunits in the formation of fatty acids, it is concluded that the polyene macrolide candicidin is synthesized via the polyketide pathway by condensation steps similar to those occurring in fatty acid biosynthesis.

Ribosomes from an erythromycin-producing strain, Streptomyces erythreus, lacked affinity for erythromycin and were also resistant to other macrolide antibiotics (leucomycin, spiramycin, and tylosin) and to lincomycin, whereas Streptomyces griseus B(3) ribosomes were susceptible to all of these antibiotics.

The correlation between the biosynthesis of the cell wall and the formation of streptomycin (SM) in Streptomyces griseus, influenced by a specific inhibitor of cell wall synthesis, was investigated. Penicillin, in subinhibitory concentrations (1 to 5 mug/ml), was added to cultures of S. griseus in different stages of its life cycle. The inhibitor decreased SM production, when young cultures were treated; however, there was an increase in SM formation when penicillin was added to older cultures. The explanation for these findings is discussed in detail.