We have isolated and studied the organization of Streptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity to S. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector plJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.

Azinomycins A and B, isolated from the culture broth of Streptomyces griseofuscus S 42227, were examined for antitumor activities against P388 leukemia, P815 mastocytoma, B-16 melanoma, Ehrlich carcinoma, Lewis lung carcinoma and Meth A fibrosarcoma. Azinomycin B was markedly effective against the intraperitoneally inoculated tumors such as P388 leukemia, B-16 melanoma and Ehrlich carcinoma. The intraperitoneal administration of azinomycin B showed 57% survivors for 45 days and 193 % ILS against P388 leukemia. For Ehrlich carcinoma, azinomycin B gave 161 % ILS and 63% survivors for 45 days, but solid tumors such as Lewis lung carcinoma and Meth A fibrosarcoma were not susceptible to repeated injection of this substance. AzinomycinA was somewhatless effective than azinomycin B for the tumor systems tested.

A strain of Streptomyces griseofuscus S42227 (FERM P-8443) was found to produce new antitumor antibiotics, called azinomycins A and B. The molecular formulas of azinomycins A and B were determined as C30H33N3O10 and C31H33N3O11, respectively. They were active against Gram-positive bacteria, Gram-negative bacteria and L5178Y cells in tissue culture.

No abstract

No abstract

The new piericidin group antibiotics, glucopiericidins A and B were isolated from the culture broth of Streptomyces pactum S48727 (FERMP-8117) as co-metabolite of piericidin Ai.The structures of glucopiericidins A and B were determined as piericidin Al9 10-O-fi-Dglucoside and piericidin Al9 3'-0-D-glucoside on the basis of their spectral and chemical properties , respectively.Glucopiericidins were more potent in inhibiting antibody formation than piericidin Ai in vitro. In addition, these substances showedbetter antimicrobial activities than piericidin AÂ cute toxicities of these substances in mice were lower than that of piericidin Ax. This indicates that D-glucose in glucopiericidin molecules is important in modulating their physiological activities.In the course of a screening for physiologically active substances, a strain of actinomycetes, S48727, was shown to produce new piericidin glucosides, glucopiericidins A and B in addition to the known antibiotic piericidin A^K They showed antimicrobial activity and in vitro inhibitory activity against antibody formation. This paper reports the taxomony of the producing organism and the fermentation, the isolation, structures and biological properties of glucopiericidins A and B.Taxonomy

Streptomyces hygroscopicus, which produces the glutamine synthetase inhibitor phosphinothricin, possesses at least two genes (glnA and glnB) encoding distinct glutamine synthetase isoforms (GSI and GSII) Glutamine synthetase (GS), a pivotal enzyme for nitrogen metabolism, is found in at least three distinct forms. In studies reported to date, one of these forms, GSI, has been primarily associated with procaryotes and another, GSII, has been associated with eucaryotes. A third type of GS has been recently found in the anaerobe Bacteriodes fragilis (22). These three types of enzymes are distinct in their primary as well as tertiary structures. GSI is composed of 12 subunits (443 to 474 amino acids each); GSII has 8 subunits (332 to 378 amino acids each) (see Table 2 for references); and the Bacteriodes fragilis enzyme has 6 subunits (729 amino acids) (22). Sequence alignments show that the GSI and GSII families are only 15% identical (40) and that GSI has an extended C terminus (see Fig. 6 for references), which includes an adenylylation site important for the posttranslational control of activity (43). Thermolability of GSII has been widely used to differentiate it from GSI (13,16,17).The strict association of one of these two enzyme families with procaryotes and the other with eucaryotes was first called into question by studies of nitrogen metabolism in nodulating bacteria such as Rhizobium (13,18,33), Agrobacterium (17), Bradyrhizobium (7), and Frankia (16)

A DNA sequence (brpA) which regulates the expression of the genes of the bialaphos biosynthesis pathway (bap) in Streptomyces hygroscopicus was identfied and characterized. A newly isolated nonproducing mutant (NP57) had a pleiotropic defect involving at least 6 of the 13 known bap genes; only the step 6 conversion could be detected. NP57 was more sensitive to bialaphos than its parent and had depressed levels of the demethylphosphinothricin acetyltransferase activity (step 10 in the pathway) which confers bialaphos resistance. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of extracts of this mutant showed that it lacked proteins corresponding to steps 5 and 10. NP57 lacked mRNAs for steps 5, 10, and 13. Bialaphos productivity of NP57 was restored by transformation with a plasmid containing a 5.9-kilobase DNA fragment which was adjacent to the structural gene cluster. Subconing experiments showed that a 1.3-kilobase fragment from this primary dlone restored all the defects of NP5. We conclude that brpA can activate the transcription of the bialaphos resistance gene as well as at least six other bap structural genes.Under certain nutritional conditions, usually associated with the stationary phase of growth, streptomycetes can activate alternative (secondary) metabolic pathways which divert the intermediates of primary metabolism into antibiotic production pathways. The hlature of the nutritional imbalance and the intracellular regulatory factor(s) which leads to this activation or derepression of antibiotic biosynthesis genes are not well understood. In the case of streptomycin biosynthesis, it has been shown that the pathway enzymes are coordinately regulated (32). The availability of genetic engineering techniques and cloning vectors for Streptomyces species makes it easier to study the genes which control antibiotic production.Molecular cloning experiments have shown that structural genes which code for enzymes in a given antibiotic biosynthesis pathway are clustered (2-5, 15, 20, 23, 30). Studies of polar insertion mutations have demonstrated that the methylenomycin (3) and actinorhodin (16) biosynthesis genes are expressed as polycistronic transcriptional units. In the case of methylenomycin (3), streptomycin (23), and actinorhodin (16), the activator gene(s) is located near the structural genes under its control. These genes, which regulate specific antibiotic pathways, are presumably regulated by systems which turn on secondary metabolism and morphological development.We studied the biosynthesis of bialaphos, a secondary metabolite of Streptomyces hygroscopicus and a commercially important herbicide. Bialaphos is a tripeptide which consists of two L-alanine residues and the L-glutarnic acid analog phosphinothricin (13). In S. hygroscopicus, bialaphos is degraded to phosphinothricin, which is a potent inhibitor of glutamine synthetase (1; unpublished data). The bar gene encodes demethylphosphinothricin acetyltransferase * Corresponding author.