The polyphosphate kinase gene (ppk) from Streptomyces lividans, which encodes a 774-amino-acid protein (86.4 kDa) showing extensive homology to other bacterial polyphosphate kinases, was cloned by polymerase chain reaction (PCR) using oligonucleotides derived from the putative ppk gene from the closely related species, Streptomyces coelicolor. In vitro, the purified Ppk was shown to be able to synthesize the polyphosphate [poly(P)] from ATP (forward reaction) as well as to regenerate ATP from the poly(P) in the presence of an excess of ADP (reverse reaction). In conditions of poly(P) synthesis, a phosphoenzyme intermediate was detected, indicating an autophosphorylation of the enzyme in the presence of ATP. The ppk gene was shown to be transcribed as a monocistronic mRNA from a unique promoter. Its transcription was only detectable during the late stages of growth in liquid minimal medium. A mutant strain interrupted for ppk was characterized by increased production of the antibiotic actinorhodin on rich R2YE solid medium (0.37 mM KH2PO4 added). This production was enhanced on the same medium with no KH2PO4 added but was completely abolished by the addition of 1.48 mM KH2PO4. In the ppk mutant strain, this increased production correlated with enhanced transcription of actII-ORF4 encoding the specific activator of the actinorhodin pathway. In that strain, the transcription of redD and cdaR, encoding the specific activators of the undecylprodigiosin and calcium-dependent antibiotic biosynthetic pathways, respectively, was also increased but to a lesser extent. The enhanced expression of these regulators did not seem to be related to increased relA-dependent ppGpp synthesis, as no obvious increase in relA expression was observed in the ppk mutant strain. These results suggested that the negative regulatory effect exerted by Ppk on antibiotic biosynthesis was most probably caused by the repression exerted by the endogenous Pi, resulting from the hydrolysis of the poly(P) synthesized by Ppk, on the expression of the specific activators of the antibiotic biosynthetic pathways.
Anti-Bacterial Agents Base Sequence