Jörg Bernhardt | Universität Greifswald (original) (raw)

Papers by Jörg Bernhardt

Research paper thumbnail of A comprehensive proteome map of growingBacillus subtilis cells

PROTEOMICS, 2004

The proteome of growing cells of Bacillus subtilis was analyzed in order to provide the basis for... more The proteome of growing cells of Bacillus subtilis was analyzed in order to provide the basis for its application in microbial physiology. DNA arrays were used to calculate the number of genes transcribed in growing cells. From the 4100 B. subtilis genes, 2515 were actively transcribed in cells grown under standard conditions. From these genes 1544 proteins should be covered by our standard gel system pI 4-7. Using this standard gel system and supplementary zoom gels (pI 5.5-6.7, 5-6, 4.5-5.5, and 4-5) 693 proteins which are expressed in growing cells were detected that cover more than 40% of the vegetative proteome predicted for this region. Particularly broad coverage and thus comprehensive monitoring will be possible for central carbohydrate metabolism (glycolysis, pentose phosphate shunt, and citric acid cycle), amino acid synthesis pathways, purine and pyrimidine metabolism, fatty acid metabolism, and main cellular functions like replication, transcription, translation, and cell wall synthesis. Comparing the theoretical pI and Mr values with those experimentally determined a reasonable correlation was found for the majority of protein spots. By a color code outliers with dramatic deviations in charge or mass were visualized that may indicate post-translational modifications. In addition to the cytosolic neutral and alkaline proteins, 130 membrane proteins were found relying on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation in combination with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) techniques. The vegetative proteome containing 876 proteins in total is now ready for physiological applications. Two main proteome fractions (pI 4-7 and zoom gel pI 4.5-5.5) should be sufficient for such high-throughput physiological proteomics.

Research paper thumbnail of Dual channel imaging of two-dimensional electropherograms inBacillus subtilis

Electrophoresis, 1999

The allocation of proteins to stimulons and regulons is an essential step towards the understandi... more The allocation of proteins to stimulons and regulons is an essential step towards the understanding of the global regulation of the expression of entire genomes. The computer-aided evaluation and matching of two-dimensional protein gels loaded with radioactively labeled proteins from exponentially growing or stressed cells is a useful but time-consuming procedure for the description of stimulons and regulons. This paper describes the dual-channel image analysis that offers the opportunity to visualize the content and synthesis rate of a whole set of bacterial proteins on a single electropherogram. By pulse-labeling with L-[35S]methionine, the protein synthesis pattern (red color) can be directly compared with the protein level pattern (green color). Because matching of other gels can be avoided, this new technique is useful for the rapid search for proteins that belong to different stimulons or regulons. This approach was tested for the identification of proteins of heat stress or oxidative stress stimulons. Proteins that were induced by heat or oxidative stress colored red while proteins whose synthesis was switched off by the stress factor colored green. Proteins that were continuously synthesized before and after the imposition of stress retained their yellow color. The advantages and possible pitfalls of the technique are discussed.

Research paper thumbnail of A comprehensive two-dimensional map of cytosolic proteins ofBacillus subtilis

ELECTROPHORESIS, 2001

Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followed by spot iden... more 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.*

Research paper thumbnail of Gel-based proteomics of Gram-positive bacteria: A powerful tool to address physiological questions

PROTEOMICS, 2008

In this review, we demonstrate the power of gel-based proteomics to address physiological questio... more In this review, we demonstrate the power of gel-based proteomics to address physiological questions of bacteria. Although gel-based proteomics covers a subpopulation of proteins only, fundamental issues of a bacterial cell such as almost all metabolic pathways or the main signatures of stress and starvation responses can be analyzed. The analysis of the synthesis pattern of single proteins, e.g., in response to environmental changes, requires gel-based proteomics because only this technique can compare protein synthesis and amount in the same 2-D gel. Moreover, highly sophisticated software packages facilitate the analysis of the regulation of the main metabolic enzymes or the stress/starvation responses, PTMs, protein damage/repair, and degradation and finally protein secretion mechanisms at a proteome-wide scale. The challenge of proteomics whose panorama view shows events never seen before is to select the most interesting issues for detailed follow up studies. This "road map of proteomics" from proteome data via new hypothesis and finally novel molecular mechanisms should lead to exciting information on bacterial physiology. However, many proteins escape detection by gel-based procedures, such as membrane or low abundance proteins. The smart combination of gel-free and gel-based approaches is the "state of the art" for physiological proteomics of bacteria.

Research paper thumbnail of Towards the entire proteome of the model bacterium Bacillus subtilis by gel-based and gel-free approaches

With the emergence of mass spectrometry in protein science and the availability of complete genom... more With the emergence of mass spectrometry in protein science and the availability of complete genome sequences, proteomics has gone through a rapid development. The soil bacterium Bacillus subtilis, as one of the first DNA sequenced species, represents a model for Gram-positive bacteria and its proteome was extensively studied throughout the years. Having the final goal to elucidate how life really functions, one basic requirement is to know the entirety of cellular proteins. This review presents how far we have got in unraveling the proteome of B. subtilis. The application of gel-based and gel-free technologies, the analyses of different subcellular proteome fractions, and the pursuance of various physiological strategies resulted in a coverage of more than one-third of B. subtilis theoretical proteome.

Research paper thumbnail of Common versus noble Bacillus subtilis differentially responds to air and argon gas plasma

PROTEOMICS, 2013

The applications of low-temperature plasma are not only confined to decontamination and steriliza... more The applications of low-temperature plasma are not only confined to decontamination and sterilization but are also found in the medical field in terms of wound and skin treatment. For the improvement of already established and also for new plasma techniques, in-depth knowledge on the interactions between plasma and microorganism is essential. In an initial study, the interaction between growing Bacillus subtilis and argon plasma was investigated by using a growth chamber system suitable for low-temperature gas plasma treatment of bacteria in liquid medium. In this follow-up investigation, a second kind of plasma treatment-namely air plasma-was applied. With combined proteomic and transcriptomic analyses, we were able to investigate the plasma-specific stress response of B. subtilis toward not only argon but also air plasma. Besides an overlap of cellular responses due to both argon and air plasma treatment (DNA damage and oxidative stress), a variety of gas-dependent cellular responses such as growth retardation and morphological changes were observed. Only argon plasma treatments lead to a phosphate starvation response whereas air plasma induced the tryptophan operon implying damage by photooxidation. Biological findings were supported by the detection of reactive plasma species by optical emission spectroscopy and Fourier transformed infrared spectroscopy measurements.

Research paper thumbnail of Visual account of protein investment in cellular functions

Proceedings of the National Academy of Sciences, 2014

Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held ... more Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held desire to measure absolute protein abundances on a genome-wide scale. However, can this knowledge be translated into a clearer picture of how cells invest their protein resources? This article aims to give a broad perspective on the composition of proteomes as gleaned from recent quantitative proteomics studies. We describe proteomaps, an approach for visualizing the composition of proteomes with a focus on protein abundances and functions. In proteomaps, each protein is shown as a polygon-shaped tile, with an area representing protein abundance. Functionally related proteins appear in adjacent regions. General trends in proteomes, such as the dominance of metabolism and protein production, become easily visible. We make interactive visualizations of published proteome datasets accessible at www.proteomaps.net. We suggest that evaluating the way protein resources are allocated by various organisms and cell types in different conditions will sharpen our understanding of how and why cells regulate the composition of their proteomes.

Research paper thumbnail of Altering gene expression by aminocoumarins: the role of DNA supercoiling in Staphylococcus aureus

BMC genomics, 2014

Background: It has been shown previously that aminocoumarin antibiotics such as novobiocin lead t... more Background: It has been shown previously that aminocoumarin antibiotics such as novobiocin lead to immediate downregulation of recA expression and thereby inhibit the SOS response, mutation frequency and recombination capacity in Staphylococcus aureus. Aminocoumarins function by inhibiting the ATPase activity of DNA gyrase subunit B with a severe impact on DNA supercoiling. Results: Here, we have analysed the global impact of the DNA relaxing agent novobiocin on gene expression in S. aureus. Using a novobiocin-resistant mutant, it became evident that the change in recA expression is due to gyrase inhibition. Microarray analysis and northern blot hybridisation revealed that the expression levels of a distinct set of genes were increased (e.g., recF-gyrB-gyrA, the rib operon and the ure operon) or decreased (e.g., arlRS, recA, lukA, hlgC and fnbA) by novobiocin. The two-component ArlRS system was previously found to decrease the level of supercoiling in S. aureus. Thus, downregulation of arlRS might partially compensate for the relaxing effect of novobiocin. Global analysis and gene mapping of supercoiling-sensitive genes did not provide any indication that they are clustered in the genome. Promoter fusion assays confirmed that the responsiveness of a given gene is intrinsic to the promoter region but independent of the chromosomal location.

Research paper thumbnail of Visual account of protein investment in cellular functions

Proceedings of the National Academy of Sciences of the United States of America, 2014

Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held ... more Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held desire to measure absolute protein abundances on a genome-wide scale. However, can this knowledge be translated into a clearer picture of how cells invest their protein resources? This article aims to give a broad perspective on the composition of proteomes as gleaned from recent quantitative proteomics studies. We describe proteomaps, an approach for visualizing the composition of proteomes with a focus on protein abundances and functions. In proteomaps, each protein is shown as a polygon-shaped tile, with an area representing protein abundance. Functionally related proteins appear in adjacent regions. General trends in proteomes, such as the dominance of metabolism and protein production, become easily visible. We make interactive visualizations of published proteome datasets accessible at www.proteomaps.net. We suggest that evaluating the way protein resources are allocated by various organisms and cell types in different conditions will sharpen our understanding of how and why cells regulate the composition of their proteomes.

Research paper thumbnail of Highly precise quantification of protein molecules per cell during stress and starvation responses in Bacillus subtilis

Molecular & cellular proteomics : MCP, 2014

Systems biology based on high quality absolute quantification data, which are mandatory for the s... more 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.

Research paper thumbnail of Global proteome analysis of vancomycin stress in Staphylococcus aureus

International journal of medical microbiology : IJMM, 2013

Vancomycin is one of the few remaining treatment options for multi resistant Staphylococcus aureu... more Vancomycin is one of the few remaining treatment options for multi resistant Staphylococcus aureus infections. Several transcriptomics and proteomics studies have investigated the bacterium's cellular response to vancomycin, but quantitative proteomic studies have been limited in the number of proteins and restricted to certain sub-cellular compartments so far. Here, we combined the enrichment of different proteomic sub-fractions with in vivo metabolic labeling and shotgun proteomics to analyze the vancomycin induced stress response. Quantitative data for approximately 1400 proteins could be obtained, covering the majority of cytosolic as well as membrane localized proteins, cell surface associated and extracellular proteins. Besides major adaptive processes induced by limited growth of the cells due to the sublethal vancomycin exposure, specific cellular responses are seen on proteome level, e.g. the specific increase of proteins synthesizing amino acids which are essential for the peptidoglycan synthesis or the decrease of most proteins with a virulence related function. Most important, the influence on regulatory targets of the two-component system VraSR as the main regulatory system known for cell wall stress as well as for global regulons like SigB and SaeR was detected.

Research paper thumbnail of Common versus noble Bacillus subtilis differentially responds to air and argon gas plasma

Proteomics, 2013

The applications of low-temperature plasma are not only confined to decontamination and steriliza... more The applications of low-temperature plasma are not only confined to decontamination and sterilization but are also found in the medical field in terms of wound and skin treatment. For the improvement of already established and also for new plasma techniques, in-depth knowledge on the interactions between plasma and microorganism is essential. In an initial study, the interaction between growing Bacillus subtilis and argon plasma was investigated by using a growth chamber system suitable for low-temperature gas plasma treatment of bacteria in liquid medium. In this follow-up investigation, a second kind of plasma treatment–namely air plasma–was applied. With combined proteomic and transcriptomic analyses, we were able to investigate the plasma-specific stress response of B. subtilis toward not only argon but also air plasma. Besides an overlap of cellular responses due to both argon and air plasma treatment (DNA damage and oxidative stress), a variety of gas-dependent cellular responses such as growth retardation and morphological changes were observed. Only argon plasma treatments lead to a phosphate starvation response whereas air plasma induced the tryptophan operon implying damage by photooxidation. Biological findings were supported by the detection of reactive plasma species by optical emission spectroscopy and Fourier transformed infrared spectroscopy measurements.

Research paper thumbnail of Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis

Proceedings of the National Academy of Sciences of the United States of America, 2012

Reversible protein phosphorylation is an important and ubiquitous protein modification in all liv... more Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. Here we report that protein phosphorylation on arginine residues plays a physiologically significant role. We detected 121 arginine phosphorylation sites in 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidence that protein arginine phosphorylation has a functional role and is involved in the regulation of many critical cellular processes, such as protein degradation, motility, competence, and stringent and stress responses. Our results suggest that in B. subtilis the combined activity of a protein arginine kinase and phosphatase allows a rapid and reversible regulation of protein activity and that protein arginine phosphorylation can play a physiologically important and regulatory role in bacteria.

Research paper thumbnail of Global relative and absolute quantitation in microbial proteomics

Current opinion in microbiology, 2012

Proteomic studies are designed to yield either qualitative information on proteins (identificatio... more Proteomic studies are designed to yield either qualitative information on proteins (identification, distribution, posttranslational modifications, interactions, structure and function) or quantitative information (abundance, distribution within different localizations, temporal changes in abundance due to synthesis and degradation or both). To this end these studies can draw upon a wide range of qualitative and quantitative gel-based and gel-free techniques. This review summarizes current proteomic workflows for global relative or absolute protein quantitation and their application in microbial physiology.

Research paper thumbnail of Life and death of proteins: a case study of glucose-starved Staphylococcus aureus

Molecular & cellular proteomics : MCP, 2012

The cellular amount of proteins not only depends on synthesis but also on degradation. Here, we e... more The cellular amount of proteins not only depends on synthesis but also on degradation. Here, we expand the understanding of differential protein levels by complementing synthesis data with a proteome-wide, mass spectrometry-based stable isotope labeling with amino acids in cell culture analysis of protein degradation in the human pathogen Staphylococcus aureus during glucose starvation. Monitoring protein stability profiles in a wild type and an isogenic clpP protease mutant revealed that 1) proteolysis mainly affected proteins with vegetative functions, anabolic and selected catabolic enzymes, whereas the expression of TCA cycle and gluconeogenesis enzymes increased; 2) most proteins were prone to aggregation in the clpP mutant; 3) the absence of ClpP correlated with protein denaturation and oxidative stress responses, deregulation of virulence factors and a CodY repression. We suggest that degradation of redundant, inactive proteins disintegrated from functional complexes and thereby amenable to proteolytic attack is a fundamental cellular process in all organisms to regain nutrients and guarantee protein homeostasis.

Research paper thumbnail of Proteomics approaches for the analysis of enriched microbial subpopulations and visualization of complex functional information

Current opinion in biotechnology, 2013

Advances in the separation of microbial subpopulations and in proteomics technologies have paved ... more Advances in the separation of microbial subpopulations and in proteomics technologies have paved the way for the global molecular characterization of microbial cells that share common functional characteristics. Quantitative characterization of the dynamics of microbial proteomes enables an unprecedented view of the adaptive responses of microbes to environmental stimuli or during interaction with other species or host cells. However, the intrinsic complexity of such data requires sophisticated visualization methods for the display, mining, interpretation and efficient exploitation of these data resources. In this review, we discuss how new approaches in data visualization such as streamgraphs, network graphs or Voronoi treemaps are being used in the field to provide new insights into the functional complexity of microbial cells, populations and multispecies consortia.

Research paper thumbnail of A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria

Methods (San Diego, Calif.), 2013

The development of a mass spectrometric workflow for the sensitive identification and quantitatio... more The development of a mass spectrometric workflow for the sensitive identification and quantitation of the kinetics of changes in metaproteomes, or in particular bacterial pathogens after internalization by host cells, is described. This procedure employs three essential stages: (i) SILAC pulse-chase labeling and infection assay; (ii) isolation of bacteria by GFP-assisted cell sorting; (iii) mass spectrometry-based proteome analysis. This approach displays greater sensitivity than techniques relying on conventional cell sorting and protein separation, due to an efficient combination of a filtration-based purification and an on-membrane digestion. We exemplary describe the use of the workflow for the identification and quantitation of the proteome of 10⁶ cells of Staphylococcus aureus after internalization by S9 human bronchial epithelial cells. With minor modifications, the workflow described can be applied for the characterization of other host-pathogen pairs, permitting identification and quantitation of hundreds of bacterial proteins over a time range of several hours post infection.

Research paper thumbnail of Data visualization in environmental proteomics

Proteomics, 2013

From raw data to gene expression profiles, from single cultures to complex microbial communities,... more From raw data to gene expression profiles, from single cultures to complex microbial communities, environmental proteomics works with data of different complexity levels that need to be interpreted in detail or in its entirety. Although data visualization is closely connected with data analysis approaches, this work will solely focus on data visualization. Complementing traditional tools such as bar charts or line graphs, scientists and visualization professionals have been provided sophisticated visualization tools. Many rules and concerns regarding the display of single but also complex data will be reviewed and discussed. Visual approaches such as microcharts, heat maps, stream graphs, and tree maps will be brought to the reader's attention and demonstrated by utilizing real data sets.

Research paper thumbnail of Data visualization in environmental proteomics

Proteomics, 2013

Soil- and litter-borne microorganisms vitally contribute to biogeochemical cycles. However, chang... more Soil- and litter-borne microorganisms vitally contribute to biogeochemical cycles. However, changes in environmental parameters but also human interferences may alter species composition and elicit alterations in microbial activities. Soil and litter metaproteomics, implying the assignment of soil and litter proteins to specific phylogenetic and functional groups, has a great potential to provide essential new insights into the impact of microbial diversity on soil ecosystem functioning. This article will illuminate challenges and perspectives of current soil and litter metaproteomics research, starting with an introduction to an appropriate experimental design and state-of-the-art proteomics methodologies. This will be followed by a summary of important studies aimed at (i) the discovery of the major biotic drivers of leaf litter decomposition, (ii) metaproteomics analyses of rhizosphere-inhabiting microbes, and (iii) global approaches to study bioremediation processes. The review will be closed by a brief outlook on future developments and some concluding remarks, which should assist the reader to develop successful concepts for soil and litter metaproteomics studies.

Research paper thumbnail of A Gene at 3338on theBacillus subtilisChromosome Encodes the Newly Identified sB-Dependent General Stress Protein GspA

Research paper thumbnail of A comprehensive proteome map of growingBacillus subtilis cells

PROTEOMICS, 2004

The proteome of growing cells of Bacillus subtilis was analyzed in order to provide the basis for... more The proteome of growing cells of Bacillus subtilis was analyzed in order to provide the basis for its application in microbial physiology. DNA arrays were used to calculate the number of genes transcribed in growing cells. From the 4100 B. subtilis genes, 2515 were actively transcribed in cells grown under standard conditions. From these genes 1544 proteins should be covered by our standard gel system pI 4-7. Using this standard gel system and supplementary zoom gels (pI 5.5-6.7, 5-6, 4.5-5.5, and 4-5) 693 proteins which are expressed in growing cells were detected that cover more than 40% of the vegetative proteome predicted for this region. Particularly broad coverage and thus comprehensive monitoring will be possible for central carbohydrate metabolism (glycolysis, pentose phosphate shunt, and citric acid cycle), amino acid synthesis pathways, purine and pyrimidine metabolism, fatty acid metabolism, and main cellular functions like replication, transcription, translation, and cell wall synthesis. Comparing the theoretical pI and Mr values with those experimentally determined a reasonable correlation was found for the majority of protein spots. By a color code outliers with dramatic deviations in charge or mass were visualized that may indicate post-translational modifications. In addition to the cytosolic neutral and alkaline proteins, 130 membrane proteins were found relying on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation in combination with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) techniques. The vegetative proteome containing 876 proteins in total is now ready for physiological applications. Two main proteome fractions (pI 4-7 and zoom gel pI 4.5-5.5) should be sufficient for such high-throughput physiological proteomics.

Research paper thumbnail of Dual channel imaging of two-dimensional electropherograms inBacillus subtilis

Electrophoresis, 1999

The allocation of proteins to stimulons and regulons is an essential step towards the understandi... more The allocation of proteins to stimulons and regulons is an essential step towards the understanding of the global regulation of the expression of entire genomes. The computer-aided evaluation and matching of two-dimensional protein gels loaded with radioactively labeled proteins from exponentially growing or stressed cells is a useful but time-consuming procedure for the description of stimulons and regulons. This paper describes the dual-channel image analysis that offers the opportunity to visualize the content and synthesis rate of a whole set of bacterial proteins on a single electropherogram. By pulse-labeling with L-[35S]methionine, the protein synthesis pattern (red color) can be directly compared with the protein level pattern (green color). Because matching of other gels can be avoided, this new technique is useful for the rapid search for proteins that belong to different stimulons or regulons. This approach was tested for the identification of proteins of heat stress or oxidative stress stimulons. Proteins that were induced by heat or oxidative stress colored red while proteins whose synthesis was switched off by the stress factor colored green. Proteins that were continuously synthesized before and after the imposition of stress retained their yellow color. The advantages and possible pitfalls of the technique are discussed.

Research paper thumbnail of A comprehensive two-dimensional map of cytosolic proteins ofBacillus subtilis

ELECTROPHORESIS, 2001

Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followed by spot iden... more 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.*

Research paper thumbnail of Gel-based proteomics of Gram-positive bacteria: A powerful tool to address physiological questions

PROTEOMICS, 2008

In this review, we demonstrate the power of gel-based proteomics to address physiological questio... more In this review, we demonstrate the power of gel-based proteomics to address physiological questions of bacteria. Although gel-based proteomics covers a subpopulation of proteins only, fundamental issues of a bacterial cell such as almost all metabolic pathways or the main signatures of stress and starvation responses can be analyzed. The analysis of the synthesis pattern of single proteins, e.g., in response to environmental changes, requires gel-based proteomics because only this technique can compare protein synthesis and amount in the same 2-D gel. Moreover, highly sophisticated software packages facilitate the analysis of the regulation of the main metabolic enzymes or the stress/starvation responses, PTMs, protein damage/repair, and degradation and finally protein secretion mechanisms at a proteome-wide scale. The challenge of proteomics whose panorama view shows events never seen before is to select the most interesting issues for detailed follow up studies. This "road map of proteomics" from proteome data via new hypothesis and finally novel molecular mechanisms should lead to exciting information on bacterial physiology. However, many proteins escape detection by gel-based procedures, such as membrane or low abundance proteins. The smart combination of gel-free and gel-based approaches is the "state of the art" for physiological proteomics of bacteria.

Research paper thumbnail of Towards the entire proteome of the model bacterium Bacillus subtilis by gel-based and gel-free approaches

With the emergence of mass spectrometry in protein science and the availability of complete genom... more With the emergence of mass spectrometry in protein science and the availability of complete genome sequences, proteomics has gone through a rapid development. The soil bacterium Bacillus subtilis, as one of the first DNA sequenced species, represents a model for Gram-positive bacteria and its proteome was extensively studied throughout the years. Having the final goal to elucidate how life really functions, one basic requirement is to know the entirety of cellular proteins. This review presents how far we have got in unraveling the proteome of B. subtilis. The application of gel-based and gel-free technologies, the analyses of different subcellular proteome fractions, and the pursuance of various physiological strategies resulted in a coverage of more than one-third of B. subtilis theoretical proteome.

Research paper thumbnail of Common versus noble Bacillus subtilis differentially responds to air and argon gas plasma

PROTEOMICS, 2013

The applications of low-temperature plasma are not only confined to decontamination and steriliza... more The applications of low-temperature plasma are not only confined to decontamination and sterilization but are also found in the medical field in terms of wound and skin treatment. For the improvement of already established and also for new plasma techniques, in-depth knowledge on the interactions between plasma and microorganism is essential. In an initial study, the interaction between growing Bacillus subtilis and argon plasma was investigated by using a growth chamber system suitable for low-temperature gas plasma treatment of bacteria in liquid medium. In this follow-up investigation, a second kind of plasma treatment-namely air plasma-was applied. With combined proteomic and transcriptomic analyses, we were able to investigate the plasma-specific stress response of B. subtilis toward not only argon but also air plasma. Besides an overlap of cellular responses due to both argon and air plasma treatment (DNA damage and oxidative stress), a variety of gas-dependent cellular responses such as growth retardation and morphological changes were observed. Only argon plasma treatments lead to a phosphate starvation response whereas air plasma induced the tryptophan operon implying damage by photooxidation. Biological findings were supported by the detection of reactive plasma species by optical emission spectroscopy and Fourier transformed infrared spectroscopy measurements.

Research paper thumbnail of Visual account of protein investment in cellular functions

Proceedings of the National Academy of Sciences, 2014

Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held ... more Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held desire to measure absolute protein abundances on a genome-wide scale. However, can this knowledge be translated into a clearer picture of how cells invest their protein resources? This article aims to give a broad perspective on the composition of proteomes as gleaned from recent quantitative proteomics studies. We describe proteomaps, an approach for visualizing the composition of proteomes with a focus on protein abundances and functions. In proteomaps, each protein is shown as a polygon-shaped tile, with an area representing protein abundance. Functionally related proteins appear in adjacent regions. General trends in proteomes, such as the dominance of metabolism and protein production, become easily visible. We make interactive visualizations of published proteome datasets accessible at www.proteomaps.net. We suggest that evaluating the way protein resources are allocated by various organisms and cell types in different conditions will sharpen our understanding of how and why cells regulate the composition of their proteomes.

Research paper thumbnail of Altering gene expression by aminocoumarins: the role of DNA supercoiling in Staphylococcus aureus

BMC genomics, 2014

Background: It has been shown previously that aminocoumarin antibiotics such as novobiocin lead t... more Background: It has been shown previously that aminocoumarin antibiotics such as novobiocin lead to immediate downregulation of recA expression and thereby inhibit the SOS response, mutation frequency and recombination capacity in Staphylococcus aureus. Aminocoumarins function by inhibiting the ATPase activity of DNA gyrase subunit B with a severe impact on DNA supercoiling. Results: Here, we have analysed the global impact of the DNA relaxing agent novobiocin on gene expression in S. aureus. Using a novobiocin-resistant mutant, it became evident that the change in recA expression is due to gyrase inhibition. Microarray analysis and northern blot hybridisation revealed that the expression levels of a distinct set of genes were increased (e.g., recF-gyrB-gyrA, the rib operon and the ure operon) or decreased (e.g., arlRS, recA, lukA, hlgC and fnbA) by novobiocin. The two-component ArlRS system was previously found to decrease the level of supercoiling in S. aureus. Thus, downregulation of arlRS might partially compensate for the relaxing effect of novobiocin. Global analysis and gene mapping of supercoiling-sensitive genes did not provide any indication that they are clustered in the genome. Promoter fusion assays confirmed that the responsiveness of a given gene is intrinsic to the promoter region but independent of the chromosomal location.

Research paper thumbnail of Visual account of protein investment in cellular functions

Proceedings of the National Academy of Sciences of the United States of America, 2014

Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held ... more Proteomics techniques generate an avalanche of data and promise to satisfy biologists' long-held desire to measure absolute protein abundances on a genome-wide scale. However, can this knowledge be translated into a clearer picture of how cells invest their protein resources? This article aims to give a broad perspective on the composition of proteomes as gleaned from recent quantitative proteomics studies. We describe proteomaps, an approach for visualizing the composition of proteomes with a focus on protein abundances and functions. In proteomaps, each protein is shown as a polygon-shaped tile, with an area representing protein abundance. Functionally related proteins appear in adjacent regions. General trends in proteomes, such as the dominance of metabolism and protein production, become easily visible. We make interactive visualizations of published proteome datasets accessible at www.proteomaps.net. We suggest that evaluating the way protein resources are allocated by various organisms and cell types in different conditions will sharpen our understanding of how and why cells regulate the composition of their proteomes.

Research paper thumbnail of Highly precise quantification of protein molecules per cell during stress and starvation responses in Bacillus subtilis

Molecular & cellular proteomics : MCP, 2014

Systems biology based on high quality absolute quantification data, which are mandatory for the s... more 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.

Research paper thumbnail of Global proteome analysis of vancomycin stress in Staphylococcus aureus

International journal of medical microbiology : IJMM, 2013

Vancomycin is one of the few remaining treatment options for multi resistant Staphylococcus aureu... more Vancomycin is one of the few remaining treatment options for multi resistant Staphylococcus aureus infections. Several transcriptomics and proteomics studies have investigated the bacterium's cellular response to vancomycin, but quantitative proteomic studies have been limited in the number of proteins and restricted to certain sub-cellular compartments so far. Here, we combined the enrichment of different proteomic sub-fractions with in vivo metabolic labeling and shotgun proteomics to analyze the vancomycin induced stress response. Quantitative data for approximately 1400 proteins could be obtained, covering the majority of cytosolic as well as membrane localized proteins, cell surface associated and extracellular proteins. Besides major adaptive processes induced by limited growth of the cells due to the sublethal vancomycin exposure, specific cellular responses are seen on proteome level, e.g. the specific increase of proteins synthesizing amino acids which are essential for the peptidoglycan synthesis or the decrease of most proteins with a virulence related function. Most important, the influence on regulatory targets of the two-component system VraSR as the main regulatory system known for cell wall stress as well as for global regulons like SigB and SaeR was detected.

Research paper thumbnail of Common versus noble Bacillus subtilis differentially responds to air and argon gas plasma

Proteomics, 2013

The applications of low-temperature plasma are not only confined to decontamination and steriliza... more The applications of low-temperature plasma are not only confined to decontamination and sterilization but are also found in the medical field in terms of wound and skin treatment. For the improvement of already established and also for new plasma techniques, in-depth knowledge on the interactions between plasma and microorganism is essential. In an initial study, the interaction between growing Bacillus subtilis and argon plasma was investigated by using a growth chamber system suitable for low-temperature gas plasma treatment of bacteria in liquid medium. In this follow-up investigation, a second kind of plasma treatment–namely air plasma–was applied. With combined proteomic and transcriptomic analyses, we were able to investigate the plasma-specific stress response of B. subtilis toward not only argon but also air plasma. Besides an overlap of cellular responses due to both argon and air plasma treatment (DNA damage and oxidative stress), a variety of gas-dependent cellular responses such as growth retardation and morphological changes were observed. Only argon plasma treatments lead to a phosphate starvation response whereas air plasma induced the tryptophan operon implying damage by photooxidation. Biological findings were supported by the detection of reactive plasma species by optical emission spectroscopy and Fourier transformed infrared spectroscopy measurements.

Research paper thumbnail of Global impact of protein arginine phosphorylation on the physiology of Bacillus subtilis

Proceedings of the National Academy of Sciences of the United States of America, 2012

Reversible protein phosphorylation is an important and ubiquitous protein modification in all liv... more Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. Here we report that protein phosphorylation on arginine residues plays a physiologically significant role. We detected 121 arginine phosphorylation sites in 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidence that protein arginine phosphorylation has a functional role and is involved in the regulation of many critical cellular processes, such as protein degradation, motility, competence, and stringent and stress responses. Our results suggest that in B. subtilis the combined activity of a protein arginine kinase and phosphatase allows a rapid and reversible regulation of protein activity and that protein arginine phosphorylation can play a physiologically important and regulatory role in bacteria.

Research paper thumbnail of Global relative and absolute quantitation in microbial proteomics

Current opinion in microbiology, 2012

Proteomic studies are designed to yield either qualitative information on proteins (identificatio... more Proteomic studies are designed to yield either qualitative information on proteins (identification, distribution, posttranslational modifications, interactions, structure and function) or quantitative information (abundance, distribution within different localizations, temporal changes in abundance due to synthesis and degradation or both). To this end these studies can draw upon a wide range of qualitative and quantitative gel-based and gel-free techniques. This review summarizes current proteomic workflows for global relative or absolute protein quantitation and their application in microbial physiology.

Research paper thumbnail of Life and death of proteins: a case study of glucose-starved Staphylococcus aureus

Molecular & cellular proteomics : MCP, 2012

The cellular amount of proteins not only depends on synthesis but also on degradation. Here, we e... more The cellular amount of proteins not only depends on synthesis but also on degradation. Here, we expand the understanding of differential protein levels by complementing synthesis data with a proteome-wide, mass spectrometry-based stable isotope labeling with amino acids in cell culture analysis of protein degradation in the human pathogen Staphylococcus aureus during glucose starvation. Monitoring protein stability profiles in a wild type and an isogenic clpP protease mutant revealed that 1) proteolysis mainly affected proteins with vegetative functions, anabolic and selected catabolic enzymes, whereas the expression of TCA cycle and gluconeogenesis enzymes increased; 2) most proteins were prone to aggregation in the clpP mutant; 3) the absence of ClpP correlated with protein denaturation and oxidative stress responses, deregulation of virulence factors and a CodY repression. We suggest that degradation of redundant, inactive proteins disintegrated from functional complexes and thereby amenable to proteolytic attack is a fundamental cellular process in all organisms to regain nutrients and guarantee protein homeostasis.

Research paper thumbnail of Proteomics approaches for the analysis of enriched microbial subpopulations and visualization of complex functional information

Current opinion in biotechnology, 2013

Advances in the separation of microbial subpopulations and in proteomics technologies have paved ... more Advances in the separation of microbial subpopulations and in proteomics technologies have paved the way for the global molecular characterization of microbial cells that share common functional characteristics. Quantitative characterization of the dynamics of microbial proteomes enables an unprecedented view of the adaptive responses of microbes to environmental stimuli or during interaction with other species or host cells. However, the intrinsic complexity of such data requires sophisticated visualization methods for the display, mining, interpretation and efficient exploitation of these data resources. In this review, we discuss how new approaches in data visualization such as streamgraphs, network graphs or Voronoi treemaps are being used in the field to provide new insights into the functional complexity of microbial cells, populations and multispecies consortia.

Research paper thumbnail of A proteomics workflow for quantitative and time-resolved analysis of adaptation reactions of internalized bacteria

Methods (San Diego, Calif.), 2013

The development of a mass spectrometric workflow for the sensitive identification and quantitatio... more The development of a mass spectrometric workflow for the sensitive identification and quantitation of the kinetics of changes in metaproteomes, or in particular bacterial pathogens after internalization by host cells, is described. This procedure employs three essential stages: (i) SILAC pulse-chase labeling and infection assay; (ii) isolation of bacteria by GFP-assisted cell sorting; (iii) mass spectrometry-based proteome analysis. This approach displays greater sensitivity than techniques relying on conventional cell sorting and protein separation, due to an efficient combination of a filtration-based purification and an on-membrane digestion. We exemplary describe the use of the workflow for the identification and quantitation of the proteome of 10⁶ cells of Staphylococcus aureus after internalization by S9 human bronchial epithelial cells. With minor modifications, the workflow described can be applied for the characterization of other host-pathogen pairs, permitting identification and quantitation of hundreds of bacterial proteins over a time range of several hours post infection.

Research paper thumbnail of Data visualization in environmental proteomics

Proteomics, 2013

From raw data to gene expression profiles, from single cultures to complex microbial communities,... more From raw data to gene expression profiles, from single cultures to complex microbial communities, environmental proteomics works with data of different complexity levels that need to be interpreted in detail or in its entirety. Although data visualization is closely connected with data analysis approaches, this work will solely focus on data visualization. Complementing traditional tools such as bar charts or line graphs, scientists and visualization professionals have been provided sophisticated visualization tools. Many rules and concerns regarding the display of single but also complex data will be reviewed and discussed. Visual approaches such as microcharts, heat maps, stream graphs, and tree maps will be brought to the reader's attention and demonstrated by utilizing real data sets.

Research paper thumbnail of Data visualization in environmental proteomics

Proteomics, 2013

Soil- and litter-borne microorganisms vitally contribute to biogeochemical cycles. However, chang... more Soil- and litter-borne microorganisms vitally contribute to biogeochemical cycles. However, changes in environmental parameters but also human interferences may alter species composition and elicit alterations in microbial activities. Soil and litter metaproteomics, implying the assignment of soil and litter proteins to specific phylogenetic and functional groups, has a great potential to provide essential new insights into the impact of microbial diversity on soil ecosystem functioning. This article will illuminate challenges and perspectives of current soil and litter metaproteomics research, starting with an introduction to an appropriate experimental design and state-of-the-art proteomics methodologies. This will be followed by a summary of important studies aimed at (i) the discovery of the major biotic drivers of leaf litter decomposition, (ii) metaproteomics analyses of rhizosphere-inhabiting microbes, and (iii) global approaches to study bioremediation processes. The review will be closed by a brief outlook on future developments and some concluding remarks, which should assist the reader to develop successful concepts for soil and litter metaproteomics studies.

Research paper thumbnail of A Gene at 3338on theBacillus subtilisChromosome Encodes the Newly Identified sB-Dependent General Stress Protein GspA