Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MSE)

Muntel, Jan; Fromion, Vincent; Goelzer, Anne; Maaß, Sandra; Mäder, Ulrike; Büttner, Knut; Hecker, Michael; Becher, Dörte

doi:10.1074/mcp.m113.032631

Cited by 88 publications

(84 citation statements)

References 53 publications

(48 reference statements)

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“…Additionally the integration of nonprotein parameters, such as known polysaccharides or lipids would be of great interest in order to gain new insight on, for example, the membrane composition during different stress conditions. Unfortunately, until now the absolute quantification of membrane proteins is still a challenge because of the need for a complex sample preparation (51). Furthermore, absolute quantification of nonproteinogenic components would be inevitable in this context.…”

Section: Discussionmentioning

confidence: 99%

Highly Precise Quantification of Protein Molecules per Cell During Stress and Starvation Responses in Bacillus subtilis

Maaß

Wachlin

Bernhardt

et al. 2014

Molecular & Cellular Proteomics

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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, highquality, and validated data sets (1). 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).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), mass spectrometry (4, 5), or merge traditional two-dimensional polyacrylamide gel elec...

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Section: Discussionmentioning

confidence: 99%

Highly Precise Quantification of Protein Molecules per Cell During Stress and Starvation Responses in Bacillus subtilis

Maaß

Wachlin

Bernhardt

et al. 2014

Molecular & Cellular Proteomics

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“…However, this model of Rho activity cannot be directly extrapolated to Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus, given the low cellular abundance of Rho and low Rho/RNAP ratio in these bacteria Maass et al, 2011;Nicolas et al, 2012;Muntel et al, 2014). In Bacillus subtilis, the cellular level of Rho estimated using immunoblotting analysis does not exceed 50 hexamers per cell .…”

Section: Regulation Of Gene Expressionmentioning

confidence: 99%

“…In Bacillus subtilis, the cellular level of Rho estimated using immunoblotting analysis does not exceed 50 hexamers per cell . Similarly, the copy number of Rho determined by the absolute quantification of the Bacillus subtilis cytosolic proteome does not exceed 80 Rho hexamers per cell, which corresponds to *0.8 % of RNAP (Muntel et al, 2014). Measurement of Bacillus subtilis Rho cellular abundance using a GFP-Rho fusion estimated the Rho hexamers at *5 % of the level of RNAP (Nicolas et al, 2012).…”

Section: Regulation Of Gene Expressionmentioning

confidence: 99%

Transcription termination factor Rho: a hub linking diverse physiological processes in bacteria

et al. 2016

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Factor-dependent termination of transcription in bacteria relies on the activity of a specific RNA helicase, the termination factor Rho. Rho is nearly ubiquitous in bacteria, but the extent to which its physiological functions are conserved throughout the different phyla remains unknown. Most of our current knowledge concerning the mechanism of Rho's activity and its physiological roles comes from the model micro-organism Escherichia coli, where Rho is essential and involved in the control of several important biological processes. However, the rather comprehensive knowledge about the general mechanisms of action and activities of Rho based on the E. coli paradigm cannot be directly extrapolated to other bacteria. Recent studies performed in different species favour the view that Rho-dependent termination plays a significant role even in bacteria where Rho is not essential. Here, we summarize the current state of the ever-increasing knowledge about the various aspects of the physiological functions of Rho, such as limitation of deleterious foreign DNA expression, control of gene expression, suppression of pervasive transcription, prevention of R-loops and maintenance of chromosome integrity, focusing on similarities and differences of the activities of Rho in various bacterial species. Introduction Transcription termination is a critical step in gene regulation in all living organisms. In bacteria, transcription termination is well known to be essential for the generation of different types of functional RNAs, the definition of the boundaries of the transcriptional units, the release of RNA polymerase (RNAP) and the regulation of gene expression via the mechanism known as transcription attenuation (Peters et al., 2011;Santangelo & Artsimovitch, 2011).However, recent studies have revealed new roles of transcriptional termination, e.g. those linked to the maintenance of genome integrity or degradation of untranslated mRNAs. Particular attention is now paid to transcription termination due to its crucial role in the control of pervasive transcription. This type of genome-wide transcription, not associated with annotated genome features such as protein-coding genes, is a universal phenomenon for all the three domains of life and viruses (Georg & Hess, 2011;Wade & Grainger, 2014). In eukaryotes, pervasive transcription arises mainly from bidirectional promoters that synthesize both mRNA and diverse non-coding RNAs, but this phenomenon is also controlled by selective transcriptional termination (Kapranov et al., 2007;Schulz et al., 2013). Recently, an essential role of transcription termination in the control of pervasive transcription was demonstrated for both Gram-positive and Gram-negative model micro-organisms Bacillus subtilis and Escherichia coli (Nicolas et al., 2012;Peters et al., 2012).In bacteria, transcription termination is achieved by two mechanisms: factor-independent (intrinsic) and factordependent termination. Intrinsic termination is strongly associated with sequence-specific signals characterized by ...

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“…Meanwhile the majority of proteins synthesized in bacteria are identified for model microorganisms such as Bacillus subtilis 23, Escherichia coli 45, Leptospira interrogans 67, Mycoplasma pneumoniae 89, Mycobacterium tuberculosis 1011, Saccharomyces cerevisiae 1213, and Staphylococcus aureus 14. Becher and co-workers identified the greater part of proteins synthesized in exponentially growing and glucose-starved cells of S. aureus 14.…”

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confidence: 99%

Costs of life - Dynamics of the protein inventory of Staphylococcus aureus during anaerobiosis

Zühlke

Dörries

Bernhardt

et al. 2016

Sci Rep

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Absolute protein quantification was applied to follow the dynamics of the cytoplasmic proteome of Staphylococcus aureus in response to long-term oxygen starvation. For 1,168 proteins, the majority of all expressed proteins, molecule numbers per cell have been determined to monitor the cellular investments in single branches of bacterial life for the first time. In the presence of glucose the anaerobic protein pattern is characterized by increased amounts of glycolytic and fermentative enzymes such as Eno, GapA1, Ldh1, and PflB. Interestingly, the ferritin-like protein FtnA belongs to the most abundant proteins during anaerobic growth. Depletion of glucose finally leads to an accumulation of different enzymes such as ArcB1, ArcB2, and ArcC2 involved in arginine deiminase pathway. Concentrations of 29 exo- and 78 endometabolites were comparatively assessed and have been integrated to the metabolic networks. Here we provide an almost complete picture on the response to oxygen starvation, from signal transduction pathways to gene expression pattern, from metabolic reorganization after oxygen depletion to beginning cell death and lysis after glucose exhaustion. This experimental approach can be considered as a proof of principle how to combine cell physiology with quantitative proteomics for a new dimension in understanding simple life processes as an entity.

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Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MSE)

Cited by 88 publications

References 53 publications

Highly Precise Quantification of Protein Molecules per Cell During Stress and Starvation Responses in Bacillus subtilis

Highly Precise Quantification of Protein Molecules per Cell During Stress and Starvation Responses in Bacillus subtilis

Transcription termination factor Rho: a hub linking diverse physiological processes in bacteria

Costs of life - Dynamics of the protein inventory of Staphylococcus aureus during anaerobiosis

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