afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptmyces coelicolor A3(2)

AfsR is a pleiotropic, global regulator that controls the production of actinorhodin, undecylprodigiosin and calcium-dependent antibiotic in Streptomyces coelicolor A3(2). AfsR, with 993 amino acids, is phosphorylated on serine and threonine residues by a protein serine/threonine kinase AfsK and contains an OmpR-like DNA-binding fold at its N-terminal portion and A- and B-type nucleotide-binding motifs in the middle of the protein. The DNA-binding domain, in-dependently of the nucleotide-binding domain, contributed the binding of AfsR to the upstream region of afsS that locates immediately 3′ to afsR and encodes a 63-amino-acid protein. No transcription of afsS in the ΔafsR background and restoration of afsS transcription by afsR on a plasmid in the same genetic background indicated that afsR served as a transcriptional activator for afsS. Interestingly, the AfsR binding site overlapped the promoter of afsS, as determined by DNase I protection assay and high-resolution S1 nuclease mapping. The nucleotide-binding domain contributed distinct ATPase and GTPase activity. The phosphorylation of AfsR by AfsK greatly enhanced the DNA-binding activity and modulated the ATPase activity. The DNA-binding ability of AfsR was independent of the ATPase activity. However, the ATPase activity was essential for transcriptional activation ofafsS, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. Thus, AfsR turns out to be a unique transcriptional factor, in that it is modular, in which DNA-binding and ATPase activities are physically separable, and the two functions are modulated by phosphorylation on serine and threonine residues

An AfsK/AfsR system involved in the response of aerial mycelium formation to glucose in Streptomyces griseus

In Streptomyces coelicolor A3(2), a protein serine/threonine kinase (AfsK) and its target protein (AfsR) control secondary metabolism. AfsK and AfsR homologues (AfsK-g and AfsR-g) from Streptomyces griseus showed high end-to-end similarity in amino acid sequence with the respective S. coelicolor A3(2) proteins, as determined by cloning and nucleotide sequencing. AfsK-g and a fusion protein between AfsK-g and thioredoxin (TRX–AfsK-g) produced in high yield as inclusion bodies in Escherichia coli were solubilized with urea, purified by column chromatography and then refolded to an active form by dialysis to gradually remove the urea. AfsR-g was also fused to glutathione S-transferase (GST–AfsR-g); the fusion product in the soluble fraction in E. coli was purified. Incubation of AfsK-g or TRX–AfsK-g in the presence of [γ-32P]ATP yielded autophosphorylated products containing phosphoserine and phosphothreonine residues. In addition, TRX–AfsK-g phosphorylated serine and threonine residues of GST–AfsR-g in the presence of [γ-32P]ATP. Disruption of chromosomal afsK-g had no effect on A-factor or streptomycin production, irrespective of the culture conditions. The afsK-g disruptants did not form aerial mycelium or spores on media containing glucose at concentrations higher than 1%, but did form spores on mannitol- and glycerol-containing media; this suggests that afsK-g is essential for morphogenesis in the presence of glucose. Introduction of afsK-g restored aerial mycelium formation in the disruptants. The phenotype of afsR-g disruptants was similar to that of afsK-g disruptants; introduction of afsR-g restored the defect in aerial mycelium formation on glucose-containing medium. Thus the AfsK/AfsR system in S. griseus is conditionally needed for morphological differentiation, whereas in S. coelicolor A3(2) it is conditionally involved in secondary metabolism

Protein serine/threonine kinases in signal transduction for secondary metabolism and morphogenesis in Streptomyces

A number of proteins in the Gram-positive bacterial genus Streptomyces are phosphorylated on their serine/threonine and tyrosine residues in response to developmental phases. AfsR is one of these proteins and acts as a transcriptional factor in both the regulation of secondary metabolism in Streptomyces coelicolor A3(2) and morphological differentiation in Streptomyces griseus. In S. coelicolor A3(2), AfsR is phosphorylated on its serine and threonine residues by more than three protein kinases whose kinase activity is enhanced by means of autophosphorylation on their serine and threonine residues. The degree of autophosphorylation of AfsK is regulated by KbpA which, by binding directly to the kinase domain of AfsK, inhibits its autophosphorylation. Phosphorylation of AfsR enhances its DNA-binding activity and causes it to bind the promoter elements, including –35, of afsS, thus resulting in activation of afsS transcription. ATPase activity of AfsR is essential for this transcriptional activation, probably because the energy available from ATP hydrolysis is required for the isomerization of the closed complex between AfsR and RNA polymerase to a transcriptionally competent open complex. afsS, encoding a 63-aminoacid protein, then activates transcription of actII-ORF4, a pathway-specific transcriptional activator in the actinorhodin biosynthetic gene cluster, in an as yet unknown way. Distribution of the afsK-afsR systems in a wide variety of Streptomyces species and the presence of many phosphorylated proteins in a given Streptomyces strain suggest that the signal transduction via not only two-component regulatory systems but also serine/ threonine kinases generally regulates secondary metabolism and morphogenesis in this genus

Site-directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6

AbstractKinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Alcaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between copper-containing proteins. Apparent values of kcat and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of D11K and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR