A comprehensive two-dimensional map of cytosolic proteins ofBacillus subtilis

Abstract

Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is an excellent experimental tool for physiological studies opening a new perspective for understanding overall cell physiology. This is the intriguing outcome of a method introduced by Klose and O'Farrell independently 25 years ago. Physiological proteomics requires a 2-D reference map on which most of the main proteins were identified. In this paper, we present such a reference map with more than 300 entries for Bacillus subtilis proteins with an isoelectric point (pI) between 4 and 7. The most abundant proteins of exponentially growing cells were compiled and shown to perform mainly housekeeping functions in glycolysis, tricarboxylic acid cycle (TCC), amino acid biosynthesis and translation as well as protein quality control. Furthermore, putative post-translational modifications were shown at a large scale, with 47 proteins in total forming more than one spot. In a few selected cases evidence for phosphorylation of these proteins is presented. The proteome analysis in the standard pI range was complemented by either stretching the most crowded regions in a narrow pH gradient 4.5-5.5, or by adding other fractions of the total B. subtilis proteome such as alkaline proteins as well as extracellular proteins. A big challenge for future studies is to provide an experimental protocol covering the fraction of intrinsic membrane proteins that almost totally escaped detection by the experimental procedure used in this study.

Bacillus subtilisDuring Feast and Famine: Visualization of the Overall Regulation of Protein Synthesis During Glucose Starvation by Proteome Analysis

“…The DNA-array data represent a more complete description of the changes in gene expression, but the proteomic data allow a more convenient (visual) inspection of the gene expression patterns, which is quite sufficient for getting an overview of cell physiology. Furthermore, the proteomic approach, which more closely reflects the final level of gene expression, depicts reactions that can never be visualized by using the DNA-array techniques, such as the large number of proteins still present and probably active in nongrowing cells but no longer being synthesized (the green proteins), or posttranslational reactions such as protein modification, stability, or protein targeting not considered in this study (see Antelmann et al 2001;Büttner et al 2001). The dual channel imaging technique ) alone, visualizing synthesis and accumulation of proteins, is an excellent tool for describing not only the fate of single regulons but also the fate of each single protein during the growth and development process, including proteins that are no longer being synthesized in nongrowing cells.…”

“…At present our B. subtilis master gel contains ∼ 600 entries organized in a B. subtilis 2D protein database named Sub2D (Büttner et al 2001;Werner and Bernhardt 1998; accessible via http://microbio2.biologie.uni-greifswald.de:8880/ sub2d.htm). However, this master gel only provides the experimental tool for physiological proteomics visualizing the physiological state of a cell at the level of proteins.…”

“…2, Gel 1) illustrates that during this growth phase mainly vegetative proteins are synthesized (autoradiogram, red) and accumulated (densitogram, green; both together, yellow), among them the main catabolic enzymes for glucose utilization, enzymes synthesizing amino acids, proteins, and nucleotides, as well as proteins of the translational machine, and so on (Fig. 2, Gel 1; Büttner et al 2001). As long as sufficient glucose is available to support exponential growth, the synthesis and amount of protein show a reasonable correlation (Fig 3A).…”

Proteome-wide analysis of the functional roles of bacilysin biosynthesis in Bacillus subtilis

“…A first comprehensive map of the vegetative B. subtilis proteome was published by Büttner et al (2001), who were able to identify more than 300 cytosolic proteins by standard 2D-PAGE located in an analytical window of pI 4-7. Three years later Eymann et al (2004) expanded the cytosolic proteome map of growing cells to a total number of 693 proteins in the region pI 4-7.…”

Towards the proteome of the marine bacteriumRhodopirellula baltica: Mapping the soluble proteins

“…1). As known from proteomic studies with other bacteria, enzymes of these central pathways belong to the most abundant protein spots on 2-D gels [16,17]. No evidence was obtained for the presence of the Entner-Doudoroff pathway, which agrees with the bioinformatical prediction [4].…”