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.
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.
The Ser/Thr/Tyr phosphoproteome of Bacillus subtilis was analyzed by a 2-D gel-based approach combining Pro-Q Diamond staining and ³³P-labeling. In exponentially growing B. subtilis cells 27 proteins ...could be identified after staining with Pro-Q Diamond and/or ³³P-labeling and one additional protein was labeled solely by ³³P resulting in a total of 28 potentially phosphorylated proteins. These proteins are mainly involved in enzymatic reactions of basic carbon metabolism and the regulation of the alternative sigma factor σB. We also found significant changes of the phosphoproteome including increased phosphorylation and dephosphorylation rates of some proteins as well as the detection of four newly phosphorylated proteins in response to stress or starvation. For nine proteins, phosphorylation sites at serine or threonine residues were determined by MS. These include the known phosphorylation sites of Crh, PtsH, and RsbV. Additionally, we were able to identify novel phosphorylation sites of AroA, Pyk, and YbbT. Interestingly, the phosphorylation of RsbRA, B, C, and D, four proteins of a multicomponent protein complex involved in environmental stress signaling, was found during exponential growth. For RsbRA, B, and D, phosphorylation of one of the conserved threonine residues in their C-termini were verified by MS (T171, T186, T181, respectively).
Summary
Aromatic organic compounds that are present in the environment can have toxic effects or provide carbon sources for bacteria. We report here the global response of Bacillus subtilis 168 to ...phenol and catechol using proteome and transcriptome analyses. Phenol induced the HrcA, σB and CtsR heat‐shock regulons as well as the Spx disulfide stress regulon. Catechol caused the activation of the HrcA and CtsR heat‐shock regulons and a thiol‐specific oxidative stress response involving the Spx, PerR and FurR regulons but no induction of the σB regulon. The most surprising result was that several catabolite‐controlled genes are derepressed by catechol, even if glucose is taken up under these conditions. This derepression of the carbon catabolite control was dependent on the glucose concentration in the medium, as glucose excess increased the derepression of the CcpA‐dependent lichenin utilization licBCAH operon and the ribose metabolism rbsRKDACB operon by catechol. Growth and viability experiments with catechol as sole carbon source suggested that B. subtilis is not able to utilize catechol as a carbon‐energy source. In addition, the microarray results revealed the very strong induction of the yfiDE operon by catechol of which the yfiE gene shares similarities to glyoxalases/bleomycin resistance proteins/extradiol dioxygenases. Using recombinant His6‐YfiEBs we demonstrate that YfiE shows catechol‐2,3‐dioxygenase activity in the presence of catechol as the metabolite 2‐hydroxymuconic semialdehyde was measured. Furthermore, both genes of the yfiDE operon are essential for the growth and viability of B. subtilis in the presence of catechol. Thus, our studies revealed that the catechol‐2,3‐dioxygenase YfiE is the key enzyme of a meta cleavage pathway in B. subtilis involved in the catabolism of catechol.
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.
Lichens are recognized by macroscopic structures formed by a heterotrophic fungus, the mycobiont, which hosts internal autotrophic photosynthetic algal and/or cyanobacterial partners, referred to as ...the photobiont. We analyzed the structure and functionality of the entire lung lichen Lobaria pulmonaria L. Hoffm. collected from two different sites by state-of-the-art metaproteomics. In addition to the green algae and the ascomycetous fungus, a lichenicolous fungus as well as a complex prokaryotic community (different from the cyanobacteria) was found, the latter dominated by methanotrophic Rhizobiales. Various partner-specific proteins could be assigned to the different lichen symbionts, for example, fungal proteins involved in vesicle transport, algal proteins functioning in photosynthesis, cyanobacterial nitrogenase and GOGAT involved in nitrogen fixation, and bacterial enzymes responsible for methanol/C1-compound metabolism as well as CO-detoxification. Structural and functional information on proteins expressed by the lichen community complemented and extended our recent symbiosis model depicting the functional multiplayer network of single holobiont partners. Our new metaproteome analysis strongly supports the hypothesis (i) that interactions within the self-supporting association are multifaceted and (ii) that the strategy of functional diversification within the single lichen partners may support the longevity of L. pulmonaria under certain ecological conditions.
Purpose: Increasing incidence of onychomycosis and tinea pedis in humans of industrialized countries together with deep tissue infections are a therapeutic challenge in clinical mycology. For a ...better understanding of the pathology and immunology of infection, the authors analyze the exoproteomes of three reference strains of the most common clinical dermatophyte species (Trichophyton rubrum, Trichophyton interdigitale, Arthroderma benhamiae) and of Trichophyton strains isolated from affected patients.
Experimental design: Extracellular proteins of those in vitro grown strains are separated via 2D High Performance Electrophoresis and identified by mass spectrometry to find proteins with provoked host immune reactivity.
Results: More than 80 secreted proteins including virulence factors such as peptidases and other hydrolases are identified. By Western blotting with respective patient sera, up to 31 proteins with significant antigen–antibody reactions are detected in comparison with control sera, for example, peptidases as well as several oxidoreductases. One protein, beta‐glucosidase F2SZI9 seems to be a commonly processed antigen in all Trichophyton infections.
Conclusions and clinical relevance: These first global exoproteome data of three dermatophyte species can be a stepping stone on the way to further study the molecular mechanisms of Trichophyton pathogenicity‐associated traits. Possible candidates for potential new diagnostic methods or vaccination have to be validated in further investigations.
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