Transcriptional regulation of bialaphos biosynthesis in Streptomyces hygroscopicus

Abstract

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.

The Aminonucleoside Antibiotic A201A is Inactivated by a Phosphotransferase Activity from Streptomyces Capreolus NRRL 3817, the Producing Organism

“…Alternatively, some intermediate may be the in vivo substrate for Ard2. This is the case in Streptomyces strains that produce streptomycin, which occurs via phosphorylated intermediates (Mansouri and Piepersberg, 1991), and in the synthesis of bialaphos and puromycin, where an intermediate is acetylated (Anzai et al, 1987;Vara et al, 1985). In these three cases the antibiotics are finally activated during or after cell exclusion by specific hydrolases which remove the inactivating group (Lacalle et a]., 1993;Mansouri and Piepersberg, 1991;Raibaud et al, 1991).…”

A relA/spoT Homologous Gene from Streptomyces coelicolor A3(2) Controls Antibiotic Biosynthetic Genes

“…For the first three compounds, it has been reported that their biosynthesis is controlled by the action of specific regulatory genes located within the particular biosynthetic clusters (4,(7)(8)(9). Such regulatory genes have also been identified in other antibiotic clusters, such as bialaphos (10), streptomycin (11,12), and daunorubicin (13).…”

Regulation of secondary metabolism in Streptomyces spp. and overproduction of daunorubicin in Streptomyces peucetius

“…Work with other species has established that the Streptomyces hygroscopicus brpA (1,43) and Streptomyces griseus strR (10,23,39,42) (9,53) genes also fall into one of these groups. Some of the features of these protein families resemble the well-studied two-component regulatory systems of other bacteria (13,48).…”