Dörte Becher

Substrate-controlled succession of marine bacterioplankton populations induced by a phytoplankton bloom.

Blooming Succession Algal blooms in the ocean will trigger a succession of microbial predators and scavengers. Teeling et al. (p. 608) used a combination of microscopy, metagenomics, and metaproteomics to analyze samples from a North Sea diatom bloom over time. Distinct steps of polysaccharide degradation and carbohydrate uptake could be assigned to clades of Flavobacteria and Gammaproteobacteria, which differ profoundly in their transporter profiles and their uptake systems for phosphorus. The phytoplankton/bacterioplankton coupling in coastal marine systems is of crucial importance for global carbon cycling. Bacterioplankton clade succession following phytoplankton blooms may be predictable enough that it can be included in models of global carbon cycling. Seasonal diatom growth in the North Sea results in a temporal succession of metabolically specialized bacteria. Phytoplankton blooms characterize temperate ocean margin zones in spring. We investigated the bacterioplankton response to a diatom bloom in the North Sea and observed a dynamic succession of populations at genus-level resolution. Taxonomically distinct expressions of carbohydrate-active enzymes (transporters; in particular, TonB-dependent transporters) and phosphate acquisition strategies were found, indicating that distinct populations of Bacteroidetes, Gammaproteobacteria, and Alphaproteobacteria are specialized for successive decomposition of algal-derived organic matter. Our results suggest that algal substrate availability provided a series of ecological niches in which specialized populations could bloom. This reveals how planktonic species, despite their seemingly homogeneous habitat, can evade extinction by direct competition.

Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis

Outside In Acquisition and analysis of large data sets promises to move us toward a greater understanding of the mechanisms by which biological systems are dynamically regulated to respond to external cues. Now, two papers explore the responses of a bacterium to changing nutritional conditions (see the Perspective by Chalancon et al.). Nicolas et al. (p. 1103) measured transcriptional regulation for more than 100 different conditions. Greater amounts of antisense RNA were generated than expected and appeared to be produced by alternative RNA polymerase targeting subunits called sigma factors. One transition, from malate to glucose as the primary nutrient, was studied in more detail by Buescher et al. (p. 1099) who monitored RNA abundance, promoter activity in live cells, protein abundance, and absolute concentrations of intracellular and extracellular metabolites. In this case, the bacteria responded rapidly and largely without transcriptional changes to life on malate, but only slowly adapted to use glucose, a shift that required changes in nearly half the transcription network. These data offer an initial understanding of why certain regulatory strategies may be favored during evolution of dynamic control systems. A horizontal analysis reveals the breadth of genes turned on and off as nutrients change. Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Global Network Reorganization During Dynamic Adaptations of Bacillus subtilis Metabolism

Outside In Acquisition and analysis of large data sets promises to move us toward a greater understanding of the mechanisms by which biological systems are dynamically regulated to respond to external cues. Now, two papers explore the responses of a bacterium to changing nutritional conditions (see the Perspective by Chalancon et al.). Nicolas et al. (p. 1103) measured transcriptional regulation for more than 100 different conditions. Greater amounts of antisense RNA were generated than expected and appeared to be produced by alternative RNA polymerase targeting subunits called sigma factors. One transition, from malate to glucose as the primary nutrient, was studied in more detail by Buescher et al. (p. 1099) who monitored RNA abundance, promoter activity in live cells, protein abundance, and absolute concentrations of intracellular and extracellular metabolites. In this case, the bacteria responded rapidly and largely without transcriptional changes to life on malate, but only slowly adapted to use glucose, a shift that required changes in nearly half the transcription network. These data offer an initial understanding of why certain regulatory strategies may be favored during evolution of dynamic control systems. A vertical analysis reveals that a simple switch of one food for another evokes changes at many levels. Adaptation of cells to environmental changes requires dynamic interactions between metabolic and regulatory networks, but studies typically address only one or a few layers of regulation. For nutritional shifts between two preferred carbon sources of Bacillus subtilis, we combined statistical and model-based data analyses of dynamic transcript, protein, and metabolite abundances and promoter activities. Adaptation to malate was rapid and primarily controlled posttranscriptionally compared with the slow, mainly transcriptionally controlled adaptation to glucose that entailed nearly half of the known transcription regulation network. Interactions across multiple levels of regulation were involved in adaptive changes that could also be achieved by controlling single genes. Our analysis suggests that global trade-offs and evolutionary constraints provide incentives to favor complex control programs.

A Comprehensive Proteomics and Transcriptomics Analysis of Bacillus subtilis Salt Stress Adaptation

In its natural habitats, Bacillus subtilis is exposed to changing osmolarity, necessitating adaptive stress responses. Transcriptomic and proteomic approaches can provide a picture of the dynamic changes occurring in salt-stressed B. subtilis cultures because these studies provide an unbiased view of cells coping with high salinity. We applied whole-genome microarray technology and metabolic labeling, combined with state-of-the-art proteomic techniques, to provide a global and time-resolved picture of the physiological response of B. subtilis cells exposed to a severe and sudden osmotic upshift. This combined experimental approach provided quantitative data for 3,961 mRNA transcription profiles, 590 expression profiles of proteins detected in the cytosol, and 383 expression profiles of proteins detected in the membrane fraction. Our study uncovered a well-coordinated induction of gene expression subsequent to an osmotic upshift that involves large parts of the SigB, SigW, SigM, and SigX regulons. Additionally osmotic upregulation of a large number of genes that do not belong to these regulons was observed. In total, osmotic upregulation of about 500 B. subtilis genes was detected. Our data provide an unprecedented rich basis for further in-depth investigation of the physiological and genetic responses of B. subtilis to hyperosmotic stress.

A Proteomic View of an Important Human Pathogen – Towards the Quantification of the Entire Staphylococcus aureus Proteome

The genome sequence is the “blue-print of life,” but proteomics provides the link to the actual physiology of living cells. Because of their low complexity bacteria are excellent model systems to identify the entire protein assembly of a living organism. Here we show that the majority of proteins expressed in growing and non-growing cells of the human pathogen Staphylococcus aureus can be identified and even quantified by a metabolic labeling proteomic approach. S. aureus has been selected as model for this proteomic study, because it poses a major risk to our health care system by combining high pathogenicity with an increasing frequency of multiple antibiotic resistance, thus requiring the development of new anti-staphylococcal therapy strategies. Since such strategies will likely have to target extracellular and surface-exposed virulence factors as well as staphylococcal survival and adaptation capabilities, we decided to combine four subproteomic fractions: cytosolic proteins, membrane-bound proteins, cell surface-associated and extracellular proteins, to comprehensively cover the entire proteome of S. aureus. This quantitative proteomics approach integrating data ranging from gene expression to subcellular localization in growing and non-growing cells is a proof of principle for whole-cell physiological proteomics that can now be extended to address physiological questions in infection-relevant settings. Importantly, with more than 1700 identified proteins (and 1450 quantified proteins) corresponding to a coverage of about three-quarters of the expressed proteins, our model study represents the most comprehensive quantification of a bacterial proteome reported to date. It thus paves the way towards a new level in understanding of cell physiology and pathophysiology of S. aureus and related pathogenic bacteria, opening new avenues for infection-related research on this crucial pathogen.

Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis

Functional genomics of the Gram-positive model organism Bacillus subtilis reveals valuable insights into basic concepts of cell physiology. In this study, we monitor temporal changes in the proteome, transcriptome and extracellular metabolome of B. subtilis caused by glucose starvation. For proteomic profiling, a combination of in vivo metabolic labelling and shotgun mass spectrometric analysis was carried out for five different proteomic subfractions (cytosolic, integral membrane, membrane, surface and extracellular proteome fraction), leading to the identification of ∼52% of the predicted proteome of B. subtilis. Quantitative proteomic and corresponding transcriptomic data were analysed with Voronoi treemaps linking functional classification and relative expression changes of gene products according to their fate in the stationary phase. The obtained data comprise the first comprehensive profiling of changes in the membrane subfraction and allow in-depth analysis of major physiological processes, including monitoring of protein degradation.

S-Bacillithiolation Protects Against Hypochlorite Stress in Bacillus subtilis as Revealed by Transcriptomics and Redox Proteomics

Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In Bacillus subtilis the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate, BSH), termed as S-bacillithiolation. Here we have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid in B. subtilis. The expression profile of NaOCl stress is indicative of disulfide stress as shown by the induction of the thiol- and oxidative stress-specific Spx, CtsR, and PerR regulons. Thiol redox proteomics identified only few cytoplasmic proteins with reversible thiol-oxidations in response to NaOCl stress that include GapA and MetE. Shotgun-liquid chromatography-tandem MS analyses revealed that GapA, Spx, and PerR are oxidized to intramolecular disulfides by NaOCl stress. Furthermore, we identified six S-bacillithiolated proteins in NaOCl-treated cells, including the OhrR repressor, two methionine synthases MetE and YxjG, the inorganic pyrophosphatase PpaC, the 3-d-phosphoglycerate dehydrogenase SerA, and the putative bacilliredoxin YphP. S-bacillithiolation of the OhrR repressor leads to up-regulation of the OhrA peroxiredoxin that confers together with BSH specific protection against NaOCl. S-bacillithiolation of MetE, YxjG, PpaC and SerA causes hypochlorite-induced methionine starvation as supported by the induction of the S-box regulon. The mechanism of S-glutathionylation of MetE has been described in Escherichia coli also leading to enzyme inactivation and methionine auxotrophy. In summary, our studies discover an important role of the bacillithiol redox buffer in protection against hypochloric acid by S-bacillithiolation of the redox-sensing regulator OhrR and of four enzymes of the methionine biosynthesis pathway.

Gel-free and Gel-based Proteomics in Bacillus subtilis A Comparative Study

The proteome of exponentially growing Bacillus subtilis cells was dissected by the implementation of shotgun proteomics and a semigel-based approach for a particular exploration of membrane proteins. The current number of 745 protein identifications that was gained by the use of two-dimensional gel electrophoresis could be increased by 473 additional proteins. Therefore, almost 50% of the 2500 genes expressed in growing B. subtilis cells have been demonstrated at the protein level. In terms of exploring cellular physiology and adaptation to environmental changes or stress, proteins showing an alteration in expression level are of primary interest. The large number of vegetative proteins identified by gel-based and gel-free approaches is a good starting point for comparative physiological investigations. For this reason a gel-free quantitation with the recently introduced iTRAQ™ (isobaric tagging for relative and absolute quantitation) reagent technique was performed to investigate the heat shock response in B. subtilis. A comparison with gel-based data showed that both techniques revealed a similar level of up-regulation for proteins belonging to well studied heat hock regulons (SigB, HrcA, and CtsR). However, additional datasets have been obtained by the gel-free approach indicating a strong heat sensitivity of specific enzymes involved in amino acid synthesis.

The Phosphoproteome of the Minimal Bacterium Mycoplasma pneumoniae ANALYSIS OF THE COMPLETE KNOWN SER/THR KINOME SUGGESTS THE EXISTENCE OF NOVEL KINASES

Mycoplasma pneumoniae belongs to the Mollicutes, the group of organisms with the smallest genomes that are capable of host-independent life. These bacteria show little regulation in gene expression, suggesting an important role for the control of protein activities. We have studied protein phosphorylation in M. pneumoniae to identify phosphorylated proteins. Two-dimensional gel electrophoresis and mass spectrometry allowed the detection of 63 phosphorylated proteins, many of them enzymes of central carbon metabolism and proteins related to host cell adhesion. We identified 16 phosphorylation sites, among them 8 serine and 8 threonine residues, respectively. A phosphoproteome analysis with mutants affected in the two annotated protein kinase genes or in the single known protein phosphatase gene suggested that only one protein (HPr) is phosphorylated by the HPr kinase, HPrK, whereas four adhesion-related or surface proteins were targets of the protein kinase C, PrkC. A comparison with the phosphoproteomes of other bacteria revealed that protein phosphorylation is evolutionarily only poorly conserved. Only one single protein with an identified phosphorylation site, a phosphosugar mutase (ManB in M. pneumoniae), is phosphorylated on a conserved serine residue in all studied organisms from archaea and bacteria to man. We demonstrate that this protein undergoes autophosphorylation. This explains the strong conservation of this phosphorylation event. For most other proteins, even if they are phosphorylated in different species, the actual phosphorylation sites are different. This suggests that protein phosphorylation is a form of adaptation of the bacteria to the specific needs of their particular ecological niche.

S-Cysteinylation Is a General Mechanism for Thiol Protection of Bacillus subtilis Proteins after Oxidative Stress

S-Thiolation is crucial for protection and regulation of thiol-containing proteins during oxidative stress and is frequently achieved by the formation of mixed disulfides with glutathione. However, many Gram-positive bacteria including Bacillus subtilis lack the low molecular weight (LMW) thiol glutathione. Here we provide evidence that S-thiolation by the LMW thiol cysteine represents a general mechanism in B. subtilis. In vivo labeling of proteins with [35S]cysteine and nonreducing two-dimensional PAGE analyses revealed that a large subset of proteins previously identified as having redox-sensitive thiols are modified by cysteine in response to treatment with the thiol-specific oxidant diamide. By means of multidimensional shotgun proteomics, the sites of S-cysteinylation for six proteins could be identified, three of which are known to be S-glutathionylated in other organisms.