Effect of phosphate on the expression of protein-Ser/Thr kinase pkg2 in Streptomyces granaticolor.

A time-correlated expression of eukaryotic-like protein Ser/Thr kinase Pkg2 of Streptomyces granaticolor was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by transcriptional fusion experiments. In a complex medium the activity of pkg2 promoter was constant during the life cycle. Direct RNA analysis proved the presence of corresponding pkg2 transcript. S1 nuclease protection analysis of the transcription initiation site showed that pkg2 gene is expressed as a leaderless mRNA. Under phosphate starvation the promoter activity was detectable merely in the early exponential phase. Under these conditions turning off of pkg2 promoter and cessation of pkg2 transcript level coincided with the start of granaticin production.

Evidence for phosphoprotein phosphatase in Streptomyces granaticolor.

The existence of phosphoprotein phosphatase (PPP) in aerial mycelium of S. granaticolor was demonstrated. Using inhibitors of serine and/or threonine PPP and specifically labeled substrate it was found that the PPP is of the serine and/or threonine type.

Pkg2, a novel transmembrane protein Ser/Thr kinase of Streptomyces granaticolor.

A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.

Characterisation of two putative protein Ser/Thr kinases from actinomycete Streptomyces granaticolor both endowed with different properties.

The structural genes, pkg4 and pkg3, encoding two putative protein serine/threonine kinases in Streptomyces granaticolor, have been cloned and sequenced. The genes were isolated after screening genomic sublibraries with specific probes obtained by PCR amplification of chromosomal DNA using degenerate primers which correspond to amino acid sequences highly conserved in eukaryotic protein Ser/Thr kinases. The sequences of these genes predict polypeptide chains of 761 and 780 amino acids for Pkg4 and Pkg3, respectively. The genes are separated by only 2 bp and therefore probably constitute an operon. pkg4, which is positioned upstream of pkg3, contains a UUALeu codon suggesting a developmental-dependent mode of expression. The amino-terminal half of both proteins clearly shares similarities with the family of protein Ser/Thr kinases. Both proteins studied also possess a region rich in Pro and Ala residues and a repeating motif of 11 amino acid residues, the function of which is unknown, in the carboxy-terminal domain. Expression of pkg4 in Escherichia coli gave rise to two different forms: a soluble protein autophosphorylated at threonine residues and an insoluble form phosphorylated at threonine and serine residues. In contrast, when pkg3 was expressed in E. coli, no autophosphorylation was detected either in vivo or in vitro.