Dynamics of protein phosphorylation on Ser/Thr/Tyr in Bacillus subtilis.
The Ser/Thr/Tyr phosphoproteome of Bacillus subtilis was analyzed by a 2-D gel-based approach combining Pro-Q Diamond staining and [(33)P]-labeling. In exponentially growing B. subtilis cells 27 proteins could be identified after staining with Pro-Q Diamond and/or [(33)P]-labeling and one additional protein was labeled solely by [(33)P] resulting in a total of 28 potentially phosphorylated proteins. These proteins are mainly involved in enzymatic reactions of basic carbon metabolism and the regulation of the alternative sigma factor sigma(B). We also found significant changes of the phosphoproteome including increased phosphorylation and dephosphorylation rates of some proteins as well as the detection of four newly phosphorylated proteins in response to stress or starvation. For nine proteins, phosphorylation sites at serine or threonine residues were determined by MS. These include the known phosphorylation sites of Crh, PtsH, and RsbV. Additionally, we were able to identify novel phosphorylation sites of AroA, Pyk, and YbbT. Interestingly, the phosphorylation of RsbRA, B, C, and D, four proteins of a multicomponent protein complex involved in environmental stress signaling, was found during exponential growth. For RsbRA, B, and D, phosphorylation of one of the conserved threonine residues in their C-termini were verified by MS (T171, T186, T181, respectively).
Eymann
Christine
C
Institute for Microbiology, Ernst-Moritz-Arndt-University Greifswald, Greifswald, Germany.
Becher
Dörte
D
Bernhardt
Jörg
J
Gronau
Katrin
K
Klutzny
Anja
A
Hecker
Michael
M
D001412:Bacillus subtilis / Q000737:chemistry* / Q000502:physiology; D001426:Bacterial Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D007525:Isoelectric Focusing; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry*; D012694:Serine / Q000737:chemistry*; D013194:Staining and Labeling; D013912:Threonine / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D001426:Bacterial Proteins; D010761:Phosphorus Radioisotopes; D011506:Proteins; D013912:Threonine; D014443:Tyrosine; D012694:Serine