Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes

The physiological role of proteins phosphorylated on serine/threonine/tyrosine (Ser/Thr/Tyr) residues or the identity of the corresponding kinases and phosphatases is generally poorly understood in bacteria. As a first step in analysing the importance of such phosphorylation, we sought to establish the nature of the Ser/Thr/Tyr phosphoproteome in Bacillus subtilis, using in vivo labelling with [32P]‐orthophosphate, one‐unit pH 2‐DE, combined with MS. Highly reproducible 2‐D profiles of phosphoproteins were obtained with early stationary‐phase cells. The 2‐D profiles contained at least 80 clearly labelled spots in the pH range 4–7. Forty‐six spots were analysed by MS (confirmed in most cases by LC‐MS/MS), identifying a total of 29 different proteins, with 19 identified for the first time as bacterial phosphoproteins. These phosphoproteins are implicated in a wide variety of cellular processes, including carbon and energy metabolism, transport, stress and development. Significant changes to the profiles were obtained as a result of cold, heat or osmotic shock, demonstrating that, in stationary‐phase cells, the phosphoproteome is dynamic. An initial comparative study indicated that at least 25 [32P]‐labelled spots were also stained by Pro‐Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro‐Q.