The polyphosphate kinase plays a negative role in the control of antibiotic production in Streptomyces lividans

Abstract

SummaryThe 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 KH 2 PO 4 added). This production was enhanced on the same medium with no KH 2 PO 4 added but was completely abolished by the addition of 1.48 mM KH 2 PO 4 . 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.

Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms

“…Poly(P) appears to be essential for (p)ppGpp biosynthesis through the mprAB-sigE-relA signaling cascade during stationary phase in M. smegmatis (281,285), indicating that poly(P) acts upstream of RelA in these bacteria and not downstream, as in E. coli (286,287). Specifically, under growth-limiting conditions, poly(P) appears to act as a preferred phosphate donor for the two-component sensor histidine kinase MprB, which then phosphorylates the cognate response regulator MprA, facilitating transcription of the alternative RNA polymerase sigma factor sigE gene, thereby leading to increased transcription of relA and enhanced (p)ppGpp synthesis.…”

Polyphosphate and stress response in mycobacteria

“…This demonstrates that poly P acts upstream of (p)ppGpp in mycobacteria, and not downstream as in the other bacteria where the stringent response was previously studied. This includes the relatively close actinomycete Streptomyces lividans, in which the inactivation of ppk1 did not cause any alteration in rel expression or (p)ppGpp biosynthesis (Chouayekh and Virolle, 2002). Sureka et al also discovered that rel induction in stationary phase is under direct transcriptional control of the alternative sigma factor s E , whose structural gene is also induced under these conditions.…”

Regulation of ppk Expression and In Vivo Function of Ppk in Streptomyces lividans TK24

“…Recently the disruption of two loci (phoR/phoP and ppk), both related to phosphate metabolism, was shown to enhance greatly antibiotic production in the normally very weak producer Streptomyces lividans TK24 (7,10,28). The genes phoR and phoP encode a sensory kinase and a response regulator, respectively, constituting a two-component system for signaling and promoting the adaptation of the bacteria to P i limitation (10,28).…”

“…Since the supply of P i is reduced in a phoP mutant, the latter is prematurely starved of P i , and this starvation is thought to be responsible for the triggering of antibiotic biosynthesis (28). Strong antibiotic production was also observed upon disruption of the ppk gene of S. lividans (7). This gene encodes an enzyme catalyzing the reversible polymerization of the ␥ phosphate of ATP into polyphosphate (polyP).…”

“…This gene encodes an enzyme catalyzing the reversible polymerization of the ␥ phosphate of ATP into polyphosphate (polyP). This enzyme thus acts as a polyphosphate kinase when the ATP/ ADP ratio in the in vitro reaction mixture is high and as a nucleoside diphosphate kinase (NDPK), regenerating ATP from ADP and polyphosphate, when this ratio is low (7). Since the function of this enzyme, established in vitro, does not necessarily reflect its in vivo function, in this study, we attempted to determine the activity of Ppk in vivo, via analysis of the polyphosphate content of the cell.…”