The Serine/Threonine/Tyrosine Phosphoproteome of the Model Bacterium Bacillus subtilis

Abstract

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification in eukaryotes, but little is known about its extent and function in prokaryotes. Although protein kinases and phosphatases have been predicted and identified in a variety of bacterial species, classical biochemical approaches have so far revealed only a few substrate proteins and even fewer phosphorylation sites. Bacillus subtilis is a model Gram-positive bacterium in which two-dimensional electrophoresis-based studies suggest that the Ser/Thr/Tyr phosphorylation should be present on more than a hundred proteins. However, so far only 16 phosphorylation sites on eight of its proteins have been determined, mostly in in vitro studies. Here we performed a global, gel-free, and site-specific analysis of the B. subtilis phosphoproteome using high accuracy mass spectrometry in combination with biochemical enrichment of phosphopeptides from digested cell lysates. We identified 103 unique phosphopeptides from 78 B. subtilis proteins and determined 78 phosphorylation sites: 54 on serine, 16 on threonine, and eight on tyrosine. Detected phosphoproteins are involved in a wide variety of metabolic processes but are enriched in carbohydrate metabolism. We report phosphorylation sites on almost all glycolytic and tricarboxylic acid cycle enzymes, several kinases, and members of the phosphoenolpyruvate-dependent phosphotransferase system. This significantly enlarged number of bacterial proteins known to be phosphorylated on Ser/Thr/Tyr residues strongly supports the emerging view that protein phosphorylation is a general and fundamental regulatory process, not restricted only to eukaryotes, and opens the way for its detailed functional analysis in bacteria. Molecular & Cellular Proteomics 6:697-707, 2007.

Phosphoproteome Analysis of E. coli Reveals Evolutionary Conservation of Bacterial Ser/Thr/Tyr Phosphorylation

“…However, recent large scale genomic sequencing combined with the Global Ocean Sampling project (2) revealed that microbial eukaryotic-like protein kinases outnumber histidine kinases, traditionally considered the canonical microbial kinases (3). Likewise, our recent global and site-specific proteomics study of Ser/Thr/Tyr phosphorylation in Bacillus subtilis, a model Gram-positive bacterium, detected more than 100 phosphorylation events on 78 proteins, many of them classified as essential (4). Combined with previous two-dimensional gel studies using 32 P labeling (5-7), this strongly suggests that Ser/Thr/Tyr protein phosphorylation is common in bacterial cell, although at much lower levels than in its eukaryotic counterpart, and that it may be important in the regulation of bacterial cellular processes.…”

Global dynamics of Escherichia coli phosphoproteome in central carbon metabolism under changing culture conditions

“…Evidence has accumulated confirming that S/T/Y phosphorylation is highly present in bacteria and has important regulatory roles [26-39, 41, 61]. Pioneering work by Macek and co-workers opened new avenues to identify hundreds of phosphorylation events in bacteria [14][15][16]. These studies led to important breakthroughs in the field of bacterial signalling, such as cross-talk between a S/T/Y kinase and a two-component H/D kinase found to be activated by YbdM-dependent phosphorylation [62].…”

“…Strong Cation Exchange (SCX) was performed as described elsewhere [14]. according to the manufacturer's protocol.…”

“…Mass spectrometry based phosphoproteomic studies in Escherichia coli (E. coli), Bacillus subtilis (B. subtilis) and Lactococcus lactis (L. lactis) [14][15][16] confirmed earlier evidence from gel based proteomics [17,18] that S/T/Y phosphorylation is far more widespread in bacteria than suggested by the few well-known examples, e.g., HPr kinase/phosphorylases [19][20][21][22] and 4 isocitrate dehydrogenase (Icd) [23][24][25]. Since then, investigations have flourished; reports have emerged of S/T/Y phosphorylation in Klebsiella pneumoniae [26], Pseudomonas spp.…”

Two-dimensional gel electrophoresis in bacterial proteomics

“…Using colorimetric Pro-Q Diamond® (Invitrogen) staining, 32 P/ 33 P-radiolabeling or with the aid of phosphoprotein specific antibodies, phosphoproteins resolved on the 2-D gel can be revealed. 2-DE methods have been used to analyze the phosphoproteomes of several bacterial species, such as E. coli, Corynebacterium glutamicum, Neisseria meningitidis and B. subtilis (Cortay et al, 1986;Bendt et al, 2003;Macek et al, 2007;Bernardini et al, 2011). The phosphorylated proteins identified were involved in a variety of different cellular processes, such as central metabolism, with many having housekeeping functions.…”