Dynamics of protein phosphorylation on Ser/Thr/Tyr in Bacillus subtilis

Abstract

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).

The Phosphoproteome of the Minimal Bacterium Mycoplasma pneumoniae

“…In B. subtilis, there are different results concerning the pyruvate dehydrogenase. One study reports phosphorylation of PdhB (12), whereas PdhC and PdhD were found to be phosphoproteins in another work (9). In contrast, none of the subunits is phosphorylated in E. coli, Campylobacter jejuni, and L. lactis (11,13,52).…”

“…This exceeds the percentage of phosphorylated proteins in other bacteria by far. For example, only 2.5% of all proteins of B. subtilis are known to be phosphorylated on a serine, threonine, or tyrosine residue (9,10,12). This raises the question whether protein phosphorylation is more common in M. pneumoniae than in other bacteria.…”

“…The gels were subsequently stained with Pro-Q Diamond (Invitrogen) and Flamingo TM fluorescent dye (Bio-Rad) to visualize the phosphoproteins and all proteins, respectively. Image analysis, spot quantification, and determination of putative phosphorylated protein spots were performed as described previously (9). The determination of phosphorylated proteins was based on the Pro-Q/Flamingo log ratio.…”

Analytical strategies for phosphoproteomics

“…Phosphoproteins separated by gel electrophoresis can also be visualized by the use of a phosphospecific stain such as Pro-Q Diamond [16][17][18][19]. Alternatively, phosphoproteins can be radioactively labeled using 32 P or 33 P [16,17] and detected by autoradiography, which is the most sensitive method available, but may not be compatible with downstream analytical procedures such as MS. Radioactive labeling is possible both in vivo as well as in vitro. In in vivo studies, cells are incubated with 32 P, however, the presence of endogenous ATP pools within cells can interfere with the incorporation of the label and result in inefficient radioactive labeling [20].…”

Evolutionary Constraints of Phosphorylation in Eukaryotes, Prokaryotes, and Mitochondria

“…Recent phosphoproteomics studies of Bacillus subtilis, Escherichia coli, and Lactococcus lactis described around 100 phosphorylation sites on serine, threonine, and tyrosine in each of these species (13)(14)(15). Bacterial phosphorylation sites can change in response to environmental conditions (16).…”