Proteomes of Growing and Nongrowing Cells

What is valid for B. subtilis is also true for S. aureus: only a part of the genome is active under definite life circumstances, and the proteomes of growing cells will be different from the proteomes of nongrowing cells. First of all a vegetative pro-teome map has to be established for S. aureus as the basis for physiological studies. A "theoretical proteome map" simply derived from the genome sequence shows two major peaks, a neutral-acid peak and a more alkaline one. Whereas in the alkaline region most of the ribosomal proteins and membrane proteins should be expected from their pi values, the majority of vegetative proteins such as metabolic enzymes are located in the main proteomic window pi 4-7. This main window provides a good starting point for physiological studies. On the way towards the entire proteome, however, we must not ignore that fact that not only alkaline proteins, but also cell-surface-bound or even extracellular proteins have to be considered in addition to the main fractions of cytosolic proteins. Analyzing the majority of intrinsic membrane proteins requires the establishment of gel-free proteomes (see chapter 3.1).

Neutral/weakly acid and alkaline proteins of S. aureus strains COL and 8325 from midexponential phase cells growing in tryptone soy broth at 37 °C were analyzed by Cordwell et al. [26]. From the 347 protein spots, 266 proteins were identified, corresponding to approximately 12% of the proteome. Recently we provided a proteome map of growing cells of S. aureus COL with 460 entries [27] (Kohler et al., in press) (Fig. 3.8). Our data on the gel-based proteomics of S. aureus are being integrated into a comprehensive proteome database, Staph-2D (http://microbio2. biologie.uni-greifswald.de:8880/sub2d.htm). In a recent study a combined proteo-mic and transcriptomic approach was used to analyze the gene expression pattern in postexponential cells of S. aureus N315. Five hundred ninety-one proteins, corresponding to 23% of the proteome, were identified by gel-based and gel-free pro-teomics [28].

Despite the fact that the main metabolic pathways can be derived from the genome sequence, only very limited information about its regulation is available. Many metabolic enzymes have been identified on the proteome map of growing cells; most of the metabolic pathways have been covered by the proteomic approach [27] (Kohler et al., in press), offering the chance to analyze the regulation of entire metabolic routes in the postgenome era (see Fig. 3.9). As in B. subti-lis, the regulation of core carbon catabolism is a good model for such studies, because almost all glycolytic or TCA cycle enzymes belonging to the most abundant vegetative proteins have been visualized by proteomics. Glycolysis is activated by glucose excess or by a shift from aerobic to anaerobic conditions that also triggers repression of the TCA cycle and strong induction of fermentation/overflow metabolisms (Fig. 3.10). The pyruvate formate lyase (Pfl) is one of the most abundant enzymes induced by the anaerobic shift. The arginine deiminase pathway providing additional ATP molecules with energy limitation is also activated under anaerobic conditions, but only in the absence of glucose [27]. In addition to meta-

Fig. 3.8 Reference 2-D map of cytosolic proteins of S. aureus COL in a pI range of 4-7. Protein extracts of cells grown in TSB medium at the exponential and stationary growth phases and under anaerobic conditions were mixed and separated on 2-D gels. Protein spots are labeled with protein names according to the S. aureus N315 database. Protein extracts were stained with colloidal Coomassie brilliant blue.

Fig. 3.8 Reference 2-D map of cytosolic proteins of S. aureus COL in a pI range of 4-7. Protein extracts of cells grown in TSB medium at the exponential and stationary growth phases and under anaerobic conditions were mixed and separated on 2-D gels. Protein spots are labeled with protein names according to the S. aureus N315 database. Protein extracts were stained with colloidal Coomassie brilliant blue.

bolic enzymes, a global regulator, SrrA, known to be involved in anaerobic gene regulation was also induced by the anaerobic shift. These data represent an example of the opportunities to gain information about the regulation of cell physiology by systematic application of physiological proteomics.

From a physiological point of view, proteins produced in response to growth-restricting stimuli in their environment are of crucial significance for survival in nature, because stress and starvation are the rule and not the exception in natural ecosystems. The proteomes of nongrowing cells are probably more heterogeneous than proteomes of growing cells, because most of the stress/starvation stimuli induce a great number of stress/starvation proteins organized in an adaptational gene expression network of nongrowing cells. Following a similar approach as for B. subtilis, proteins newly induced or repressed by environmental stimuli can be allocated to stress or starvation stimulons that can be dissected into single regulons analyzing mutants in global regulators by a combined proteomic and tran-scriptomic approach. Color coding and related techniques can be used to define overlapping areas within the adaptational network.

3 Fig. 3.9 Assignment of proteins identified in S. aureus COL to biochemical pathways and other essential cellular components. Proteins that have not been identified in the 2-D gel images thus far are colored green. A Purine and pyrimidine metabolism, B glycolysis, pentose phosphate shunt, and citric acid cycle, C oxidative stress resistance, D ATPase components, E proteolysis, F components of the translational machinery, G amino acid metabolism, H fatty acid synthesis and metabolism of cell wall components, and I biotin metabolism. (This figure also appears with the color plates.)

Q tricarboxylic acid cycle a fermentation

• regulation a SrrA

Aerofeic gfowtfi

30 min anaefOtHC growth

Fig. 3.10 Protein pattern of cells of S. aureus COL grown under aerobic (green) and anaerobic (red) conditions in synthetic medium. Cells were pulse-labeled (5 min) with 35S-l-methionine under aerobic conditions and 30 min after imposition of anaerobic growth conditions. Radioactively labeled proteins were visualized by the phosphoimaging technique. Proteins whose synthesis was increased after shifting to anaerobic growth conditions are shown in red (e.g., enzymes involved in glycolysis and fermentation) and those whose synthesis was decreased are shown in green (e.g., enzymes involved in the TCA cycle). (This figure also appears with the color plates.)

Only a few data are available that demonstrate the power of proteomics for analyzing the stress or starvation responses. Preliminary proteome data have been published on the heat stress response. A temperature shift from 37 °C to 48 °C induced the production of at least eight proteins, among them GroEL and GroES [29]. Our unpublished data showed mainly the heat induction of the HrcA and CtsR regulon, represented by induction of the GroEL/S- and DnaK machinery as well as of the Clp proteins. In contrast to B. subtilis, the groES/L and dnaK operon are regulated by the two heat shock regulators, HrcA and CtsR. They act together synergistically to maintain low base levels of expression of these operons in the absence of stress [30]. Surprisingly, the members of the rB-dependent response, strongly heat-inducible in B. subtilis, are not induced in heat-stressed cells of S. aureus grown in synthetic medium.

Hydrogen peroxide treatment induced only a few proteins in S. aureus. Some protein spots that seemed to be newly induced were found as the result of a protein shift to a more acidic position, caused by oxidation of cysteine residues to sulfonic acid (Fig. 3.11) [31]. The glyceraldehyde-3-phosphate dehydrogenase behaved in this way, being inactivated by this irreversible oxidative damage. In parallel with the inactivation of the enzyme there was a drop in the ATP level followed by a cessation of growth. After 40min adaptation the enzyme "shifted back," and in parallel the ATP level increased again, followed by resumption of growth. This "enzyme reactivation" was due to a newly synthesized Gap protein. Repair of fully oxidized sulfhydryl groups is probably impossible from an energetic point of view.

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