Single-stranded DNA-binding proteins (SSBs) are required for repair, recombination and replication in all organisms. Eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues. To our knowledge, phosphorylation of SSBs in bacteria has not been reported. A systematic search for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (ScSSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation of SSBs is a conserved process of post-translational modification in taxonomically distant bacteria.

SUMMARY From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.

DNA-binding assays using mobility shift polyacrylamide gel electrophoresis revealed the presence of a protein that specifically bound to a restriction fragment -288 to -191 bp upstream from the transcriptional start point of strR, a regulatory gene for streptomycin biosynthesis in Streptomyces griseus. The binding site corresponded to an upstream activation sequence predicted from the results of in vivo promoter assays. The binding was greatly enhanced by 5 mM Mg2+. This binding was detected with the protein source only from the wild-type strain and not from an A-factor-deficient mutant strain. The exogenous supplementation of A-factor to the A-factor-deficient mutant strain caused the appearance of the protein in the DNA-binding assay. A synthetic nucleotide 52 bp in length (region from -293 to -242), which was synthesized on the basis of data obtained from both retardation assays with dissected DNA fragments and in vivo promoter assays, was retarded by the A-factor-dependent protein. In addition to this A-factor-dependent protein, at least three proteins with different recognition site affinities capable of binding to the upstream region of the strR promoter were detected. The binding of one of these proteins to both sides of the upstream activation sequence bound by the A-factor-dependent protein was completely abolished in the presence of ATP and Mg2+ in the incubation mixture. The region bound by these proteins showed anomalous electrophoretic mobility, like that of a bent DNA molecule, which is probably caused by the presence of many blocks consisting of A and T. The region bound by these proteins was found to be transcribed in the orientation opposite to that of strR.

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is a microbial hormone controlling streptomycin (Sm) production, Sm resistance and sporulation in Streptomyces griseus. In order to identify A-factor-dependent promoters in the Sm biosynthetic gene cluster, a new promoter-probe plasmid with a low copy number was constructed by using an extremely thermostable malate dehydrogenase gene as the reporter. Of the three promoters in the Sm production region that includes strR, aphD and strB, only the promoter of strR, which codes for a putative regulatory protein, was found to be directly controlled by A-factor. This was also confirmed by S1 nuclease mapping. The region essential for its A-factor-dependence was determined to be located 430-330 base pairs upstream of the transcriptional start point. Increase in the copy number of the strR promoter region did not lead to a corresponding increase in the total promoter activity, probably due to titration of a putative activator which binds to the enhancer-like region and controls the expression of the strR promoter. This putative activator is apparently distinct from the A-factor-receptor protein. The aphD gene, which encodes the major Sm resistance determinant, Sm-6-phosphotransferase, was transcribed mainly by read-through from the A-factor-dependent strR promoter; this accounts for the prompt induction of Sm resistance by A-factor.