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 [(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.
Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay Cedex, France.
Alain
A
Lévine
Françoise
F
Vannier
Cédric
C
Absalon
Lauriane
L
Kuhn
Peter
P
Jackson
Elaine
E
Scrivener
Valérie
V
Labas
Joëlle
J
Vinh
Patrick
P
Courtney
Jérôme
J
Garin
Simone J
SJ
Séror
D001412:Bacillus subtilis / Q000254:growth & development / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D003080:Cold Temperature; D003470:Culture Media; D015180:Electrophoresis, Gel, Two-Dimensional; D006358:Hot Temperature; D013058:Mass Spectrometry; D009997:Osmotic Pressure; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D003470:Culture Media; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D013912:Threonine; D014443:Tyrosine; D012694:Serine