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Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth.

ABSTRACT: The activity of the PrkC protein kinase is regulated in a sophisticated manner in Bacillus subtilis cells. In spores, in the presence of muropeptides, PrkC stimulates dormancy exit. The extracellular region containing PASTA domains binds peptidoglycan fragments to probably enhance the intracellular kinase activity. During exponential growth, the cell division protein GpsB interacts with the intracellular domain of PrkC to stimulate its activity. In this paper, we have reinvestigated the regulation of PrkC during exponential and stationary phases. We observed that, during exponential growth, neither its septal localization nor its activity are influenced by the addition of peptidoglycan fragments or by the deletion of one or all PASTA domains. However, Dynamic Light Scattering experiments suggest that peptidoglycan fragments bind specifically to PrkC and induce its oligomerization. In addition, during stationary phase, PrkC appeared evenly distributed in the cell wall and the deletion of one or all PASTA domains led to a non-activated kinase. We conclude that PrkC activation is not as straightforward as previously suggested and that regulation of its kinase activity via the PASTA domains and peptidoglycan fragments binding occurs when PrkC is not concentrated to the bacterial septum, but all over the cell wall in non-dividing bacillus cells.

SUBMITTER: Pompeo F

PROVIDER: S-EPMC5786024 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth.

Pompeo Frédérique F Byrne Deborah D Mengin-Lecreulx Dominique D Galinier Anne A

Scientific reports 20180126 1

The activity of the PrkC protein kinase is regulated in a sophisticated manner in Bacillus subtilis cells. In spores, in the presence of muropeptides, PrkC stimulates dormancy exit. The extracellular region containing PASTA domains binds peptidoglycan fragments to probably enhance the intracellular kinase activity. During exponential growth, the cell division protein GpsB interacts with the intracellular domain of PrkC to stimulate its activity. In this paper, we have reinvestigated the regulati ...[more]

PMID: 29374241

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Project description:Transmembrane Ser/Thr kinases containing extracellular PASTA (penicillin-binding protein [PBP] and Ser/Thr-associated) domains are ubiquitous among Actinobacteria and Firmicutes species. Such PASTA kinases regulate critical bacterial processes, including antibiotic resistance, cell division, cell envelope homeostasis, and virulence, and are sometimes essential for viability. Previous studies of purified PASTA kinase fragments revealed they are capable of autophosphorylation in vitro, typically at multiple sites on the kinase domain. Autophosphorylation of a specific structural element of the kinase known as the activation loop is thought to enhance kinase activity in response to stimuli. However, the role of kinase phosphorylation at other sites is largely unknown. Moreover, the mechanisms by which PASTA kinases are deactivated once their stimulus has diminished are poorly understood. Enterococcus faecalis is a Gram-positive intestinal bacterium and a major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, and such antimicrobials trigger enhanced phosphorylation of IreK in vivo Here we identify multiple sites of phosphorylation on IreK and evaluate their function in vivo and in vitro While phosphorylation of the IreK activation loop is required for kinase activity, we found that phosphorylation at a site distinct from the activation loop reciprocally modulates IreK activity in vivo, leading to diminished activity (and diminished antimicrobial resistance). Moreover, this site is important for deactivation of IreK in vivo upon removal of an activating stimulus. Our results are consistent with a model in which phosphorylation of IreK at distinct sites reciprocally regulates IreK activity in vivo to promote adaptation to cell wall stresses.IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes species and regulate critical processes, including antibiotic resistance, cell division, and cell envelope homeostasis. Previous studies of PASTA kinase fragments revealed autophosphorylation at multiple sites. However, the functional role of autophosphorylation and the relative impacts of phosphorylation at distinct sites are poorly understood. The PASTA kinase of Enterococcus faecalis, IreK, regulates intrinsic resistance to antimicrobials. Here we identify multiple sites of phosphorylation on IreK and show that modification of IreK at distinct sites reciprocally regulates IreK activity and antimicrobial resistance in vivo Thus, these results provide new insights into the mechanisms by which PASTA kinases can regulate critical physiological processes in a wide variety of bacterial species.

Project description:Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivoEnterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis, the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivoIMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The prevailing model for signaling by PASTA kinases proposes that the extracellular PASTA domains bind ligands to drive kinase dimerization, enhanced autophosphorylation, and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo We show that the PASTA kinase IreK of Enterococcus faecalis responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires the PASTA domains and phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling.

Dataset Information

Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth.

Publications

Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth.

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