Abstract
Protein phosphatase PstP is conserved throughout the Actinobacteria in a genetic locus related to cell wall synthesis and cell division. In many Actinobacteria it is the sole annotated serine threonine protein phosphatase to counter the activity of multiple serine threonine protein kinases. We used transcriptional knockdown, electron microscopy and comparative phosphoproteomics to investigate the putative dual functions of PstP as a specific regulator of cell division and as a global regulator of protein phosphorylation. Comparative phosphoproteomics in the early stages of PstP depletion showed hyperphosphorylation of protein kinases and their substrates, confirming PstP as a negative regulator of kinase activity and global serine and threonine phosphorylation. Analysis of the 838 phosphorylation sites that changed significantly, suggested that PstP may regulate diverse phosphoproteins, preferentially at phosphothreonine near acidic residues, near the protein termini, and within membrane associated proteins. Increased phosphorylation of the activation loop of protein kinase B (PknB) and of the essential PknB substrate CwlM offer possible explanations for the requirement for pstP for growth and for cell wall defects when PstP was depleted.
Highlights
Serine/threonine phosphorylation in bacteria is a ubiquitous reversible signal that controls cell cycle events, virulence, and responses to environmental cues[1]
The transmembrane Serine threonine (S/T) phosphatase PstP is conserved between M. smegmatis and M. tuberculosis (91% identity in the phosphatase domain), and this genetic locus is conserved throughout the Actinobacteria (Fig. 1) and is associated with cell division11. pstP is cotranscribed with genes involved in peptidoglycan synthesis rodA and pbpA12,13 and there is weak positive correlation of transcription with protein
PstP is required for normal cell wall synthesis and cell division, as demonstrated by the cell wall and morphological changes with loss of viability upon knockdown of the pstP operon
Summary
Serine/threonine phosphorylation in bacteria is a ubiquitous reversible signal that controls cell cycle events, virulence, and responses to environmental cues[1]. Studies of purified bacterial S/T phosphatases suggest that these are multi-specific[1,7], for example recombinant Mycobacterium tuberculosis PstP dephosphorylates itself, protein kinases PknB and PknA and phosphoproteins PbpA and GarA3,4,8. These in vitro studies necessarily lack any regulatory proteins and use non-physiological protein concentrations. PknB homologues, characterised by periplasmic PASTA domains (Penicillin-binding protein And Serine Threonine kinase Associated20), regulate many events in the cell cycle, as demonstrated by diverse phenotypes caused by gene disruption in Bacillus subtilis, Staphylococcus aureus, Corynebacterium glutamicum, Streptomyces coelicolor, M. tuberculosis and Streptococcus pneumoniae[21]. The mechanisms by which PknB regulates cell division are being elucidated and some may be organism-specific, for example PknB regulates peptidoglycan biosynthesis in M. tuberculosis by phosphorylation of several key enzymes and regulators including the regulatory protein CwlM22,23 and proposed lipid II flippase[18]
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