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A chromosomal determinant of Streptomyces hygroscopicus, functioning as a positive regulator of the bialaphos-resistance gene bar in the model strain S. lividans, was conjugatively transferred into a bialaphos (BAP) producer through pVGTB24 plasmid. The plasmid is unable to replicate autonomously in S. hygroscopicus, but integrates into its chromosome via homology with the fragment containing this determinant. Integration of pVGTB24 was accompanied by a decrease in resistance of S. hygroscopicus exconjugant clones to the self-produced antibiotic and also by a decrease in their antibiotic productivity.

A system for introduction of plasmids into industrial producers of antibiotics chlortetracycline and bialaphos using intergeneric conjugation of Escherichia coli and Streptomyces was developed. Low level stability of inheritance of autonomously replicating DNA in recipient strains was shown. Site-specific integration of the conjugative-integrative vector pTO1 provided stable plasmid maintenance within the chromosomes of Streptomyces aureofaciens and S. hygroscopicus. Phenomenon of disturbance in differentiation and antibiotic production, resulting from pTO1 integration into S. hygroscopicus chromosome, was discovered.

Streptomyces hygroscopicus ATCC21705 is an organism producing bialaphos, a promising ecologically safe antibiotic of peptide nature. An unknown determinant was cloned within the 1.4-kb SsH fragment of chromosomal DNA of S. hygroscopicus. Its presence along with the bar gene in the hybrid plasmid PVGB21 increased expression of the phenotype resistance to bialaphos in the recipient strain of S. lividans TK 64. Plasmid PVG B22 carrying the isolated SsH fragment did not provide resistance to the antibiotic. Transcription from the cloned fragment was revealed by RNA-DNA hybridization. It was shown that the cloned determinant had a positive action on bar gene transcription correlating with a significant increase in the activity of N-acetyltransferase in the mycelium of the transformant pVG B21.

The object of the study was a strain of Streptomyces hygroscopicus producing bialaphos, an antibiotic used as a herbicide, which is promising and ecologically safe. Molecular cloning of a bialaphos resistance gene (bar) was performed in the recipient strain, S. lividans TK64, within the 2.0-kb DNA fragment with the plasmid pIJ699. Introduction of a bar gene into another strain producing bialaphos, i.e. S. viridochromogenus Tu494, led to its higher constitutive resistance to bialaphos. The results confirmed the data on different regulation of bar (S. hygroscopicus) and pat (S. virido-chromogenus) resistance genes.

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Studies of regulation of antibiotic synthesis involved both basic and applied aspects of biology of antibiotic producers. This paper is a survey of published experimental data and key hypotheses for the regulation of antibiotic synthesis in bacteria of the genus Streptomyces. Summarized data on transcriptional regulatory systems of gene clusters responsible for biosynthesis of some antibiotics in these microorganisms are presented. Results of our previous research of the regulation of antibiotic bialaphos production in the S. hygroscopicus strain are considered as well. On the basis of these results, a hypothetical scheme of the organization of lower levels of the regulatory system of the bialaphos biosynthetic cluster was constructed. It is assumed that an integrase-like product of the brpB gene cloned by the authors mediates transcriptional initiation of this gene cluster.

Opportunities for application of integrative vectors carrying the attP site and the int gene of the temperate actinophage phi C31 for cloning genes in Streptomyces strains were demonstrated. The behavior of the integrative vectors pZAT22 and pTO1 in the model strain S. lividans TK64 and in the bialaphos-producing strain S. hygroscopicus, respectively, was characterized. Restriction maps of the S. lividans and S. hygroscopicus chromosomal regions containing attB sites were constructed. The bar gene of resistance to bialaphos was incorporated into the chromosome of the model strain S. lividans via the integrative pZAT22 vector, the level of expression of this gene within the chromosome of a heterologous host was determined. The possibility of amplification of the bar gene in the chromosome of the strain S. lividans was shown. The conjugative integrative vector pTO1, which carried the cloned bar gene, was introduced into the bialaphos-producing strain by intergeneric matings of Escherichia coli and S. hygroscopicus. The effect of an additional copy of the gene in the chromosome of S. hygroscopicus on strain resistance and productivity was studied.