Acetylation of blasticidin S by its producing actinomycetes.

A blasticidin S-producing actinomycetes, Streptoverticillium sp. JCM 4673 possesses an enzyme activity which acetylates the drug in the presence of acetyl coenzyme A. The modified drug was biologically inactive when tested against protein synthesis in vivo and in vitro. Production of the enzyme which acetylates blasticidin S increases with formation of the antibiotic during cell growth.

Isolation and properties of a puromycin acetyltransferase from puromycin-producing Streptomyces alboniger.

Puromycin 2"-N-acetyltransferase was isolated from cell extracts of puromycin-producing Streptomyces alboniger KCC S-0309 by ammonium sulfate fractionation, heat treatment to eliminate contaminant proteins and chromatography on DEAE-Toyopearl 650S. After PAGE (polyacrylamide gel electrophoresis) of the final fraction, a single protein band corresponding to puromycin 2"-N-acetyltransferase was detected. The molecular weight of the enzyme determined by SDS-PAGE and Sephadex G-150 chromatography was about 21,000 and 85,000, respectively, suggesting that the enzyme consisted of four subunits. The isoelectric point and the optimum pH for reaction were 6.2 and 7.7, respectively. The Km values for puromycin and acetyl coenzyme A were 40 microM and 67 microM, respectively. The enzyme was thermostable up to 70 degrees C for 12 minutes. It was shown, by using an in vitro protein synthesizing system from a puromycin-susceptible organism S. flavotricini subsp. pseudochromogenes V-13-1, that the isolated puromycin 2"-N-acetyltransferase could protect polyphenylalanine synthesis from inhibition by puromycin.

Mechanism of self-protection in a puromycin-producing micro-organism.

Puromycin is a potent inhibitor of bacterial protein synthesis, but puromycin-producing Streptomyces alboniger KCC S-0309 is tolerant to the antibiotic in vivo. Puromycin bound to both 30S and 50S ribosomal subunits from S. alboniger and inhibited polyuridylate-directed polyphenylalanine synthesis by the ribosomes. However, the organism possessed a novel puromycin-inactivating enzyme which acetylated the antibiotic at the 2''-NH2 group of the O-methyltyrosine moiety.

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.

Retrostatin, a new specific enzyme inhibitor against avian myeloblastosis virus reverse transcriptase.

A novel enzyme inhibitor against RNA-directed DNA polymerase of avian myeloblastosis virus was produced by an isolate of a new streptomycete for which the name Streptomyces retrostaticus is proposed. This enzyme inhibitor, which was named retrostatin, did not inhibit DNA-directed DNA polymerase of Escherichia coli and DNA-directed RNA polymerase of Ehrlich ascites tumor cells. Retrostatin was produced by the microorganism together with streptonigrin. These two substances were extracted from the culture broth with ethyl acetate at acidic pH. Retrostatin is an acidic pH indicator and the free acid was recovered as a red powder. Retrostatin had weak antibiotic activities against Gram-positive bacteria and yeasts.

Purification and characterization of streptomycin 6-kinase, an enzyme implicated in self-protection of a streptomycin-producing micro-organism.

Streptomycin 6-kinase of the streptomycin-producing strain Streptomyces griseus HUT 6037 was purified by fractionation with (NH4)2SO4 and chromatography on DEAE-Sephadex A-25, hydroxyapatite and Sephadex G-100. After PAGE of the final fraction, a protein band corresponding to streptomycin 6-kinase was detected, together with a less intense band having no enzyme activity. Molecular weights determined by SDS-PAGE and by Sephadex G-100 chromatography were about 36000 and 38000, respectively, suggesting that the enzyme was a monomer. The isoelectric point of the enzyme was pH 6.6. Among the nucleoside 5'-triphosphates tested, ATP was the preferred phosphoryl donor. The Km values for streptomycin and ATP were 3.5 mM and 0.4 mM, respectively. The enzyme activity was strongly inhibited by EDTA and AgNO3. It was shown by using an in vitro protein-synthesizing system that purified streptomycin 6-kinase could protect polyphenylalanine synthesis of the streptomycin-susceptible S. griseus strain KSN from inhibition by streptomycin.

New antibiotics, carbazomycins A and B. III. Taxonomy and biosynthesis.

The carbazomycin-producing microorganism, strain H 1051-MY 10, was determined to a strain of Streptoverticillium ehimense. Biosynthesis of carbazomycin B was studied using 14C-labeled and 13C-enriched precursors in combination with 13C NMR spectroscopy. The C-2 carbon of [2-13C]trytophan was shown to be involved at the C-3 carbon in carbazomycin B and both carbons of [1,2-13C]acetate at the C-1 and C-10 moiety of the antibiotic. [CH3-13C]Methionine was involved at the methoxyl group but not at the methyl group on the C-2 carbon of the antibiotic. Neither of the labeled carbons, [1-14C]tryptophan nor [2,3-13C]propionic acid, was detected in the antibiotic, and a progenitor of the C-2 and C-11 moiety of the antibiotic has not been determined.

Mechanism of protection of protein synthesis against streptomycin inhibition in a producing strain.

The influence of streptomycin (SM) on protein synthesis in a SM-producing strain was investigated using polyuridylic acid-directed polyphenylalanine synthesis in cell-free extracts. Tolerance of protein synthesis to SM developed with increasing culture age of cells and could be attributed to a decrease in affinity of the ribosomes for SM and in increase in SM 6-kinase activity in cells. SM 6-phosphate produced from SM by SM 6-kinase did not bind to ribosomes and, furthermore, ribosome-bound SM was effectively released on phosphorylation with SM 6-kinase. Also a decrease in cell permeability to SM during the production phase may contribute in protecting protein synthesis from the antibiotic.

Susceptibility of protein synthesis to neomycin in neomycin-producing Streptomyces fradiae.

A cell-free protein-synthesizing system from Streptomyces fradiae was developed by preparing ribosomes and an S-150 fraction with precautions to prevent protease action. Using this system, the ribosomes of this organism were shown to be susceptible to its own product, neomycin.