Control of acetyl phosphate-dependent phosphorylation of the response regulator CiaR by acetate kinase in Streptococcus pneumoniae.

Sabrina Kaiser, Lisa Marie Hoppstädter, Kevser Bilici, Kevin Heieck, Reinhold Brückner

Microbiology (Reading, England) 2020 Apr

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The two-component regulatory system CiaRH of Streptococcus pneumoniae affects a large variety of physiological processes including ß-lactam resistance, competence development, maintenance of cell integrity, bacteriocin production, but also host colonization and virulence. The response regulator CiaR is active under a wide variety of conditions and the cognate CiaH kinase is not always needed to maintain CiaR activity. Using tetracycline-controlled expression of ciaR and variants, acetyl phosphate was identified in vivo as the alternative source of CiaR phosphorylation in the absence of CiaH. Concomitant inactivation of ciaH and the acetate kinase gene ackA led to very high levels of CiaR-mediated promoter activation. Strong transcriptional activation was accompanied by a high phosphorylation status of CiaR as determined by Phos-tag gel electrophoresis of S. pneumoniae cell extracts. Furthermore, AckA acted negatively upon acetyl phosphate-dependent phosphorylation of CiaR. Experiments using the Escherichia coli two-hybrid system based on adenylate cyclase reconstitution indicated binding of AckA to CiaR and therefore direct regulation. Subsequent in vitro CiaR phosphorylation experiments confirmed in vivo observations. Purified AckA was able to inhibit acetyl phosphate-dependent phosphorylation. Inhibition required the presence of ADP. AckA-mediated regulation of CiaR phosphorylation is the first example for a regulatory connection of acetate kinase to a response regulator besides controlling acetyl phosphate levels. It will be interesting to see if this novel regulation applies to other response regulators in S. pneumoniae or even in other organisms.

Citation

Sabrina Kaiser, Lisa Marie Hoppstädter, Kevser Bilici, Kevin Heieck, Reinhold Brückner. Control of acetyl phosphate-dependent phosphorylation of the response regulator CiaR by acetate kinase in Streptococcus pneumoniae. Microbiology (Reading, England). 2020 Apr;166(4):411-421