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

Proteomics. 2006 Apr;6(7):2157-73. doi: 10.1002/pmic.200500352.


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 [(32)P]-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 [(32)P]-labelled spots were also stained by Pro-Q Diamond, with apparently six additional phosphoproteins uniquely detected by Pro-Q.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus subtilis / growth & development
  • Bacillus subtilis / metabolism*
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • Bacterial Proteins / physiology*
  • Cold Temperature
  • Culture Media
  • Electrophoresis, Gel, Two-Dimensional
  • Hot Temperature
  • Mass Spectrometry
  • Osmotic Pressure
  • Phosphates / metabolism
  • Phosphoproteins / chemistry*
  • Phosphoproteins / metabolism
  • Phosphoproteins / physiology*
  • Phosphorus Radioisotopes
  • Phosphorylation
  • Proteomics / methods*
  • Serine / chemistry
  • Serine / metabolism
  • Threonine / chemistry
  • Threonine / metabolism
  • Tyrosine / chemistry
  • Tyrosine / metabolism


  • Bacterial Proteins
  • Culture Media
  • Phosphates
  • Phosphoproteins
  • Phosphorus Radioisotopes
  • Threonine
  • Tyrosine
  • Serine