Highly Precise Quantification of Protein Molecules per Cell During Stress and Starvation Responses in Bacillus subtilis
2014; Elsevier BV; Volume: 13; Issue: 9 Linguagem: Inglês
10.1074/mcp.m113.035741
ISSN1535-9484
AutoresSandra Maaβ, Gerhild Wachlin, Jörg Bernhardt, Christine Eymann, Vincent Fromion, Katharina Riedel, Dörte Becher, Michael Hecker,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoSystems biology based on high quality absolute quantification data, which are mandatory for the simulation of biological processes, successively becomes important for life sciences. We provide protein concentrations on the level of molecules per cell for more than 700 cytosolic proteins of the Gram-positive model bacterium Bacillus subtilis during adaptation to changing growth conditions. As glucose starvation and heat stress are typical challenges in B. subtilis' natural environment and induce both, specific and general stress and starvation proteins, these conditions were selected as models for starvation and stress responses. Analyzing samples from numerous time points along the bacterial growth curve yielded reliable and physiologically relevant data suitable for modeling of cellular regulation under altered growth conditions. The analysis of the adaptational processes based on protein molecules per cell revealed stress-specific modulation of general adaptive responses in terms of protein amount and proteome composition.Furthermore, analysis of protein repartition during glucose starvation showed that biomass seems to be redistributed from proteins involved in amino acid biosynthesis to enzymes of the central carbon metabolism. In contrast, during heat stress most resources of the cell, namely those from amino acid synthetic pathways, are used to increase the amount of chaperones and proteases. Analysis of dynamical aspects of protein synthesis during heat stress adaptation revealed, that these proteins make up almost 30% of the protein mass accumulated during early phases of this stress. Systems biology based on high quality absolute quantification data, which are mandatory for the simulation of biological processes, successively becomes important for life sciences. We provide protein concentrations on the level of molecules per cell for more than 700 cytosolic proteins of the Gram-positive model bacterium Bacillus subtilis during adaptation to changing growth conditions. As glucose starvation and heat stress are typical challenges in B. subtilis' natural environment and induce both, specific and general stress and starvation proteins, these conditions were selected as models for starvation and stress responses. Analyzing samples from numerous time points along the bacterial growth curve yielded reliable and physiologically relevant data suitable for modeling of cellular regulation under altered growth conditions. The analysis of the adaptational processes based on protein molecules per cell revealed stress-specific modulation of general adaptive responses in terms of protein amount and proteome composition. Furthermore, analysis of protein repartition during glucose starvation showed that biomass seems to be redistributed from proteins involved in amino acid biosynthesis to enzymes of the central carbon metabolism. In contrast, during heat stress most resources of the cell, namely those from amino acid synthetic pathways, are used to increase the amount of chaperones and proteases. Analysis of dynamical aspects of protein synthesis during heat stress adaptation revealed, that these proteins make up almost 30% of the protein mass accumulated during early phases of this stress. Recently technical approaches in systems biology have become more and more important for the life science community. Successful modeling of biological pathways as part of these approaches strongly depends on quantitative, high-quality, and validated data sets (1.Aebersold R. Molecular Systems Biology: a new journal for a new biology?.Mol. Syst. Biol. 2005; 1: 2005.0005Crossref PubMed Scopus (13) Google Scholar). Proteins are an important part of these attempts to uncover the systemic properties of biological systems as they represent the central players in the complex cellular metabolic and adaptational network (2.Souchelnytskyi S. Bridging proteomics and systems biology: What are the roads to be traveled?.Proteomics. 2005; 5: 4123-4137Crossref PubMed Scopus (51) Google Scholar). Although relative protein quantification methods allow for comparison of protein abundances in samples and to characterize the proteome dynamics in cellular systems, these data are not sufficient for mathematical modeling in systems biology. Furthermore, the availability of protein concentrations at the proteome level can provide new insights in what is going on in the cell upon stress, and thus enable us to better understand how cells adapt to changing conditions. Knowing intracellular protein concentrations is essential in order to obtain a real mass balance leading to evaluation of the costs of running an active metabolic pathway or expressing enzymes for stress responses. In order to provide suitable proteomic data for systems biology, techniques for global absolute quantification of proteins recently emerged. These approaches make use of quantitative Western blotting (3.Ghaemmaghami S. Huh W.K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Global analysis of protein expression in yeast.Annu. Rev. Plant Physiol. Plant Mol. Biol. 2003; 41: 55-75Google Scholar), mass spectrometry (4.Ishihama Y. Schmidt T. Rappsilber J. Mann M. Hartl F.U. Kerner M.J. Frishman D. Protein abundance profiling of the Escherichia coli cytosol.BMC genomics. 2008; 9: 102Crossref PubMed Scopus (358) Google Scholar, 5.Malmström J. Beck M. Schmidt A. Lange V. Deutsch E.W. Aebersold R. Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans.Nature. 2009; 460: 762-765Crossref PubMed Scopus (342) Google Scholar), or merge traditional two-dimensional polyacrylamide gel electrophoresis (two-dimensional PAGE) 1The abbreviations used are: two-dimensional PAGE, two-dimensional polyacrylamide gel electrophoresis; PTS, phosphotransferase system; SRM, selected reaction monitoring; S/N, signal to noise; TCC, tricarboxylic acid cycle. 1The abbreviations used are: two-dimensional PAGE, two-dimensional polyacrylamide gel electrophoresis; PTS, phosphotransferase system; SRM, selected reaction monitoring; S/N, signal to noise; TCC, tricarboxylic acid cycle. and mass spectrometry (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.Anal. Chem. 2011; 83: 2677-2684Crossref PubMed Scopus (77) Google Scholar) to determine cellular protein concentrations on a global scale. Entirely mass spectrometry-based strategies have recently convincingly demonstrated the capacity to quantify about half of the predicted proteome of Leptospira interrogans (5.Malmström J. Beck M. Schmidt A. Lange V. Deutsch E.W. Aebersold R. Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans.Nature. 2009; 460: 762-765Crossref PubMed Scopus (342) Google Scholar) and therefore provide more comprehensive data for systems biology than two-dimensional PAGE based methods (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.Anal. Chem. 2011; 83: 2677-2684Crossref PubMed Scopus (77) Google Scholar, 7.Baudouin-Cornu P. Lagniel G. Chédin S. Labarre J. Development of a new method for absolute protein quantification on two-dimensional gels.Proteomics. 2009; 9: 4606-4615Crossref PubMed Scopus (7) Google Scholar). Although these gel-based methods are biased toward high-abundant proteins, usage of MS-calibrated two-dimensional gels allows distinguishing even between different protein isoforms caused by post-translational modifications without missing values along a time course experiment. Moreover, two-dimensional PAGE is a well-established method for easy and convenient visualization of main metabolic pathways and the most obvious adaptational responses. Until now, approaches for large-scale absolute protein quantification have had a strong technological focus. Only a few predominantly physiological applications have been reported (5.Malmström J. Beck M. Schmidt A. Lange V. Deutsch E.W. Aebersold R. Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans.Nature. 2009; 460: 762-765Crossref PubMed Scopus (342) Google Scholar, 8.Lu P. Vogel C. Wang R. Yao X. Marcotte E.M. Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation.Nat. Biotechnol. 2007; 25: 117-124Crossref PubMed Scopus (911) Google Scholar, 9.Schmidt A. Beck M. Malmström J. Lam H. Claassen M. Campbell D. Aebersold R. Absolute quantification of microbial proteomes at different states by directed mass spectrometry.Mol. Syst. Biol. 2011; 7: 510Crossref PubMed Scopus (79) Google Scholar). In this study, we provide absolute protein concentrations of the bacterial model organism Bacillus subtilis during stress and starvation adaptation. As model for different stress conditions, we selected the best-studied responses during heat stress and glucose starvation. A comparative analysis of different stress conditions allows to differentiate between general, nonspecific adaptive responses ensuring survival during a wide spectrum of conditions and specific stress adaptation by differential expression of particular regulons facilitating a direct interaction with the stimulus (10.Bernhardt J. Weibezahn J. Scharf C. Hecker M. Bacillus subtilis during feast and famine: visualization of the overall regulation of protein synthesis during glucose starvation by proteome analysis.Genome Res. 2003; 13: 224-237Crossref PubMed Scopus (110) Google Scholar). The general adaptive response of B. subtilis is governed by global regulators such as the alternative RNA polymerase sigma factor Sigma B or the main stringent factor RelA. Induction of SigB-dependent genes provides cells with a multiple, nonspecific and preventive stress resistance (11.Hecker M. Völker U. General stress response of Bacillus subtilis and other bacteria.Adv. Microb. Physiol. 2001; 44: 35-91Crossref PubMed Scopus (256) Google Scholar, 12.Price C.W. Fawcett P. Cérémonie H. Su N. Murphy C.K. Youngman P. Genome-wide analysis of the general stress response in Bacillus subtilis.Mol. Microbiol. 2001; 41: 757-774Crossref PubMed Scopus (224) Google Scholar). The SigB-dependent general stress response in B. subtilis is induced by a different set of stress and starvation stimuli. Thereby, environmental stresses, like heat shock, activate the phosphatase RsbU via a signal transduction pathway that involves additional regulatory proteins to dephosphorylate the anti-anti-sigma-factor RsbV that subsequently releases SigB (13.Yang X. Kang C.M. Brody M.S. Price C.W. 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In order to investigate this adaptational response we have chosen the model heat stress. Already described specific heat-shock induced genes of B. subtilis belong to the HrcA regulon (20.Schulz A. Schumann W. hrcA, the first gene of the Bacillus subtilis dnaK operon encodes a negative regulator of class I heat shock genes.J. Bacteriol. 1996; 178: 1088-1093Crossref PubMed Google Scholar), the CtsR regulon (21.Derré I. Rapoport G. Msadek T. CtsR, a novel regulator of stress and heat shock response, controls clp and molecular chaperone gene expression in gram-positive bacteria.Mol. Microbiol. 1999; 31: 117-131Crossref PubMed Scopus (316) Google Scholar, 22.Derré I. Rapoport G. Devine K. Rose M. Msadek T. ClpE, a novel type of HSP100 ATPase, is part of the CtsR heat shock regulon of Bacillus subtilis.Mol. Microbiol. 1999; 32: 581-593Crossref PubMed Scopus (90) Google Scholar, 23.Krüger E. Hecker M. 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Radioactive pulse-labeling experiments with 35S-methionine were carried out to investigate protein synthesis of B. subtilis during glucose starvation. Therefore three independent biological replicates of B. subtilis 168 trp+ (32.Jules M. Le Chat L. Aymerich S. Le Coq D. The Bacillus subtilis ywjI (glpX) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme.J. Bacteriol. 2009; 191: 3168-3171Crossref PubMed Scopus (18) Google Scholar) were grown in M9 minimal medium at 37 °C under vigorous agitation. The composition of M9 was: 0.1% (w/v) glucose, 0.1% (w/v) L-malate, CaCl2 * 2H2O (0.015 g L−1), MgSO4 * 7H2O (0.246 g L−1), Na2HPO4 * 2H2O (8.5 g L−1), and KH2PO4 (3 g L−1), NaCl (0.5 g L−1), NH4Cl (1 g L−1) and trace elements (10 ml stock solution for 1 L medium). The trace element stock solution consisted of (per liter): ZnCl2 (0.170 g), CuCl2 * 2H2O (0.043 g), MnCl2 * 4H2O (0.100 g), CoCl2 * 6H2O (0.060 g), and Na2MoO4 * 2H2O (0.060 g). Cultures were pulse-labeled for 5 min each with 10 μCi of L-[35S]-methionine per ml at an OD600 nm of 0.4 - 0.5 (for control), at maximal OD and at several time points (15, 30, 60, and 120 min) after the transition to glucose starvation as described previously (10.Bernhardt J. Weibezahn J. Scharf C. Hecker M. Bacillus subtilis during feast and famine: visualization of the overall regulation of protein synthesis during glucose starvation by proteome analysis.Genome Res. 2003; 13: 224-237Crossref PubMed Scopus (110) Google Scholar). In combination with protein amounts derived from fluorescence stained two-dimensional gels (see below) it can be investigated which proteins are actually synthesized at a specific time point and which protein amounts have already been accumulated. For starvation experiments B. subtilis 168 trp+ (32.Jules M. Le Chat L. Aymerich S. Le Coq D. The Bacillus subtilis ywjI (glpX) Gene Encodes a Class II Fructose-1,6-Bisphosphatase, Functionally Equivalent to the Class III Fbp Enzyme.J. Bacteriol. 2009; 191: 3168-3171Crossref PubMed Scopus (18) Google Scholar) was cultivated as mentioned above. Samples were harvested in exponential phase (OD600 nm 0.5), in transient phase, at maximal OD and 60, 120, 180, and 240 min after entering the stationary phase triggered by glucose exhaustion (Fig. 1). For heat stress experiments, cells were cultivated in minimal medium (33.Stülke J. Hanschke R. Hecker M. Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool.J. Gen. Microbiol. 1993; 139: 2041-2045Crossref PubMed Scopus (201) Google Scholar) and stressed by a sudden temperature shift to 52 °C. Cells were harvested shortly before (control) and at 10, 30, and 60 min after continuous heat shock (Fig. 1). For every experiment, three independent biological replicates were analyzed. After harvesting and centrifuging cells were resuspended in TE-Buffer (10 mm Tris, 1 mm EDTA, pH 7.5) and mechanically disrupted using the Precellys 24 homogenizator (PeqLab, Germany; 3 × 30 s at 6.5 m s−1). Protein concentration of extracts was determined using a ninhydrin-based assay (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.Anal. Chem. 2011; 83: 2677-2684Crossref PubMed Scopus (77) Google Scholar, 34.Starcher B. A ninhydrin-based assay to quantitate the total protein content of tissue samples.Anal. Biochem. 2001; 292: 125-129Crossref PubMed Scopus (298) Google Scholar). Two-dimensional PAGE was performed as previously described (35.Büttner K. Bernhardt J. Scharf C. Schmid R. Mäder U. Eymann C. Antelmann H. Völker A. Völker U. Hecker M. A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis.Electrophoresis. 2001; 22: 2908-2935Crossref PubMed Scopus (186) Google Scholar) in five technical replicates. 100 μg protein was loaded onto 18 cm IPG strips (pH 4–7, GE-Healthcare). After two-dimensional PAGE gels were fixed with 40% (v/v) ethanol and 10% (v/v) acetic acid for 1 to 2 h and subsequently stained with FlamingoTM. Stained gels were scanned (Typhoon 9400, GE-Healthcare) and their images analyzed employing Delta2D 4.2 software (Decodon GmbH, Germany). For all spots detected on the gel the spot volume was assigned to proteins, exported from the software and subsequently used for calibration of two-dimensional gels as described earlier (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.Anal. Chem. 2011; 83: 2677-2684Crossref PubMed Scopus (77) Google Scholar). Protein samples were reduced, alkylated and digested as previously described (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.Anal. Chem. 2011; 83: 2677-2684Crossref PubMed Scopus (77) Google Scholar). Samples were spiked with heavy peptides of anchor proteins used for calibration of two-dimensional gels to a final concentration of 5–25 fmol μl−1. A detailed list of used peptides and their optimized transition parameters has been published elsewhere (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.Anal. Chem. 2011; 83: 2677-2684Crossref PubMed Scopus (77) Google Scholar). LC-MS analyses was performed as described previously (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.Anal. Chem. 2011; 83: 2677-2684Crossref PubMed Scopus (77) Google Scholar). All raw files were processed using MultiQuantTM 1.1 software (Applied Biosystems, Foster City, CA). A peptide ratio of native and heavy species was based on three transitions that were weighted according to their signal to noise (S/N) ratios before being averaged. Accordingly, S/N weighted peptide ratios were combined to the final protein ratio. Based on the added amount of heavy peptides, the absolute quantity of target anchor proteins could be calculated. Absolute amounts of targeted anchor proteins obtained by SRM were used to calibrate two-dimensional gels of the same sample in order to obtain absolute abundance of all proteins visible on this gel. Amounts of multiple spots of the same protein were added up. Standard deviations for proteins represented by multiple spots were calculated using error propagation. Final standard deviation was calculated using a random effect model (36.Sahai H. Ojeda M.M. Analysis of Variance for Random Models, Volume 2: Unbalanced Data: Theory, Methods, Applications, and Data Analysis. Springer, 2004: 331-342Google Scholar). Bacterial cell size and cell disruption efficiency were determined as previously published (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.Anal. Chem. 2011; 83: 2677-2684Crossref PubMed Scopus (77) Google Scholar). Volumes of the rod-shaped cells were calculated assuming a cylinder and two hemispheres without subtracting any values for the cell envelope. At least 100 cells were dimensioned for every sample. Therefore, a standard deviation could be calculated for each analyzed population (supplemental Table S6). Determination of absolute protein abundances by combining accurate targeted mass spectrometry with the resolving power of two-dimensional PAGE provides a global view on the concentrations of a large number of proteins. Although the derived quantification data across biological replicates can be at most a highly accurate determination of molecular counts we will use in the following the common term "absolute quantification." In this study we present a comprehensive proteomic data set applicable for bioinformatic modeling of B. subtilis' stress and starvation responses. As models of starvation and stress responses we chose glucose starvation and heat stress. Therefore we determined protein concentration at seven different time points along the bacterial growth curve for cells under glucose starvation and at four time points during heat stress (Fig. 1). Whereas glucose starvation causes a complete stop of cell growth, 52 °C heat stress leads to a drop of growth rate from 1.2/h during exponential growth to 0.5/h during heat stress. After preparation of a cell count calibrated protein sample by determination of cell titer, cell disruption efficiency and protein content according to previously published protocols (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.Anal. Chem. 2011; 83: 2677-2684Crossref PubMed Scopus (77) Google Scholar, 34.Starcher B. A ninhydrin-based assay to quantitate the total protein content of tissue samples.Anal. Biochem. 2001; 292: 125-129Crossref PubMed Scopus (298) Google Scholar), isotopically labeled peptides of anchor proteins were spiked in the sample in known concentrations. Digestion and targeted SRM acquisition led to determination of protein concentration for all anchor proteins in the total protein sample. Hence, the absolute amount of anchor protein on fluorescently stained two-dimensional gels prepared from the same sample could be calculated and was used for calibration of these two-dimensional images by relating spot intensities to the ones of anchor proteins (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D. Efficient, global-scale quantification of absolute protein amounts by integration of targeted mass spectrometry and two-dimensional gel-based proteomics.Anal. Chem. 2011; 83: 2677-2684Crossref PubMed Scopus (77) Google Scholar). This enabled determination of protein concentration for all proteins detectable on the two-dimensional gels. Detectable soluble cytosolic proteins have an isoelectric point between 4 and 7 and a molecular weight of 10–150 kDa. In order to provide reliable data, only anchor proteins with less than 15% CV among four technical replicates of MS analysis and less than 35% CV between five technical two-dimensional gel replicates were used for calibration of two-dimensional gels. Noncovalently binding fluorescent dyes (e.g. Flamingo, Krypton) bind proteins in an amount proportional to the protein size (6.Maass S. Sievers S. Zühlke D. Kuzinski J. Sappa P.K. Muntel J. Hessling B. Bernhardt J. Sietmann R. Völker U. Hecker M. Becher D
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