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The juxtamembrane domain of StkP is phosphorylated and influences cell division in Streptococcus pneumoniae

ABSTRACT: Membrane eukaryotic-like Ser/Thr protein-kinases play a pivotal role in different aspects of bacterial physiology. In contrast to the diversity of their extracellular domains, their cytoplasmic catalytic domains are highly conserved. However, the function of a long juxtamembrane domain (JMD), which connects the catalytic domain to the transmembrane helix, remains elusive. In this study, we investigated the function of the JMD of the Ser/Thr protein-kinase StkP in cell division of Streptococcus pneumoniae. We observed that the deletion of the JMD did not affect StkP kinase activity. However, it impaired the ability of StkP to phosphorylate its endogenous substrates, which resulted in significant cell morphogenesis defects. Furthermore, multiple threonine residues were identified as being phosphorylated in the JMD. To investigate the functional significance of these phosphorylation sites, we conducted an integrative analysis, combining structural biology, proteomics and bacterial cell imaging. Our results revealed that the phosphorylation of the JMD did not affect the phosphorylation of StkP substrates. However, we observed that it influenced the timing of StkP localization at the division septum and the dynamics of cell constriction. We further demonstrated that phosphorylation of the JMD facilitated the recruitment of several cell division proteins, suggesting that it is required for the assembly of the division machinery at the division septum. In conclusion, this study demonstrates that the function of the JMD of StkP is modulated by phosphorylation and critical for the cell division of S. pneumoniae. These observations may serve as a model for understanding the regulatory function of other bacterial Ser/Thr protein-kinases.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Streptococcus Pneumoniae (ncbitaxon:1313)

SUBMITTER: Christophe Grangeasse

PROVIDER: MSV000096495 | MassIVE | Thu Nov 21 23:53:00 GMT 2024

REPOSITORIES: MassIVE

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Project description:Different transcriptome, metabolome, and phosphoproteome studies have already indicated a regulatory role of Ser/Thr phosphorylation in the central metabolism of S. aureus. However, it remains still unknown how this exactly tunes its metabolism. For that reason, Extreme Pathway Analysis was used to understand metabolic flux regulation by Ser/Thr phosphorylation. YANAsquare software and YANAvergence routine were chosen to calculate pathway activities of WT, ΔpknB, Δstp, and double mutant S. aureus NewmanHG strains. Gene expression data of S. aureus NewmanHG were collected taking the wild type and mutations of kinase (pknB), phosphatase (stp) or both (pknB/stp) phenotypes. The resulting flux changes were modelled with YANAvergence taking the different gene expression data and an established genome scale model of S. aureus metabolic modes as basis. The fluxes of the different strains were next calculated, taking the gene expression data into account. A concerted metabolic change of fluxes was observed. In particular the pathways for peptidoglycan, nucleotide, and aromatic amino acid synthesis as well as catabolism involving aspartate transaminase (GOT) changed significantly compared to the wild type. Single mutations of pknB and stp were compared to illustrate their different contribution to the phenotype, e.g. pyrimidine synthesis is dramatically impaired by pknB deletion but much less by loss of stp. In the double knock out strain, there is higher activity of peptidoglycan, purine, and aromatic amino acids synthesis from glucose, but lower activity of pyrimidine synthesis from glucose compared to WT. The results suggest a probable regulatory role of Ser/Thr phosphorylation in amino acid catabolism and the glycolysis/gluconeogenesis switch to provide the cell with sufficient cell wall components, nucleotides, and aromatic amino acids. Yvck is suggested to serve as regulatory protein linking Ser/Thr phosphorylation to the previous results of S. aureus metabolism. Moreover, Yvck seems in particular to be involved in the switch between glycolysis and gluconeogenesis. However, further studies based on experimental approaches are needed to demonstrate that Ser/Thr phosphorylation and Yvck are necessary for a correct cell growth under gluconeogenic conditions. The metabolic model allows not only a comprehensive representation of these changes for all involved pathways in the four strains, it further points out the function of the previously unannotated ycvk operon.. The relevance of these specific metabolic pathway adaptations regarding the pathogenicity of S.aureus and their contributions to the infection process will be analyzed and discussed.

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The juxtamembrane domain of StkP is phosphorylated and influences cell division in Streptococcus pneumoniae

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