Activity of the response regulator CiaR in mutants of Streptococcus pneumoniae R6 altered in acetyl phosphate production

Abstract

The two-component regulatory system (TCS) CiaRH of Streptococcus pneumoniae is implicated in competence, ß-lactam resistance, maintenance of cell integrity, bacteriocin production, host colonization, and virulence. Depending on the growth conditions, CiaR can be highly active in the absence of its cognate kinase CiaH, although phosphorylation of CiaR is required for DNA binding and gene regulation. To test the possibility that acetyl phosphate (AcP) could be the alternative phosphodonor, genes involved in pyruvate metabolism were disrupted to alter cellular levels of acetyl phosphate. Inactivating the genes of pyruvate oxidase SpxB, phosphotransacetylase Pta, and acetate kinase AckA, resulted in very low AcP levels and in strongly reduced CiaR-mediated gene expression in CiaH-deficient strains. Therefore, alternative phosphorylation of CiaR appears to proceed via AcP. The AcP effect on CiaR is not detected in strains with CiaH. Attempts to obtain elevated AcP by preventing its degradation by acetate kinase AckA, were not successful in CiaH-deficient strains with a functional SpxB, the most important enzyme for AcP production in S. pneumoniae. The ciaH-spxB-ackA mutant producing intermediate amounts of AcP could be constructed and showed a promoter activation, which was much higher than expected. Since activation was dependent on AcP, it can apparently be used more efficiently for CiaR phosphorylation in the absence of AckA. Therefore, high AcP levels in the absence of CiaH and AckA may cause extreme overexpression of the CiaR regulon leading to synthetic lethality. AckA is also involved in a regulatory response, which is mediated by CiaH. Addition of acetate to the growth medium switch CiaH from kinase to phosphatase. This switch is lost in the absence of AckA indicating metabolism of acetate is required, which starts with the production of AcP by AckA. Therefore, AckA plays a special regulatory role in the control of the CiaRH TCS.

Streptococcus pneumoniae Senses a Human-like Sialic Acid Profile via the Response Regulator CiaR

“…The internal pool of AcPh, the high-energy intermediate of the acetate kinase phosphor transacetylase pathway, can also act as phosphor-donor. Intracellular AcPh levels are affected by the pyruvate oxidase SpxB (Marx et al, 2014). It is interesting to note that pyruvate, the source of AcPh in this pathway, is also a metabolite generated in the pneumococcal catabolism of Neu5Ac (Vimr and Troy, 1985), and probably also of Neu5Gc (Hopkins et al, 2013).…”

“…Marx et al . showed that a deletion of the spxB gene leads to a 2-fold reduction of ciaR transcription and its target genes (Marx et al, 2014). We found that not only ciaR was downregulated ( data not shown ), but also nanA transcription was reduced in a spxB -deletion mutant, supporting our hypothesis that CiaR (indirectly) controls transcription of the sialidase NanA.…”

“…The RR CiaR directly controls a number of promoters and genes, including the high-temperature requirement A gene htrA, enhancing resistance to oxidative stress, and has been shown to affect transcription of several small non-coding RNAs with different down-stream effects (Halfmann et al, 2007; Marx et al, 2010). Phosphorylation of CiaR is required for its regulatory activity, but internal acetyl phosphate (AcPh) may serve as phosphor-donor besides CiaH (Marx et al, 2014). …”

New Aspects of the Interplay between Penicillin Binding Proteins, murM , and the Two-Component System CiaRH of Penicillin-Resistant Streptococcus pneumoniae Serotype 19A Isolates from Hungary

“…However, cells containing ciaH232 reacted more strongly to the presence of acetate in the medium (79). In the present study, some new features of ciaH232 that are associated with cell morphology, β-lactam resistance, and cell wall composition were revealed.…”

Fine‐tuning of choline metabolism is important for pneumococcal colonization

“…CiaH is a membrane bound histidine kinase and CiaR is an intracellular response regulator which is activated by phosphorylation (Halfmann et al, 2011). This phosphorylation is mediated by CiaH (Halfmann et al, 2011) or by other intracellular phosphate donors such as acetyl-phosphate (Marx et al, 2014), depending on medium composition. We showed that P lic1P1 was inactive in the absence of CiaR (Fig.…”