Michael Hecker

..subtilis...

..abstract:..

..subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins...

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..The smart combination of gel-free and gel-based approaches is the "state of the art" for physiological proteomics of bacteria...

..aureus can be identified and even quantified. In addition, posttranslational processes such as protein secretion, modification, processing, damages, and degradation can be adequately studied...

..A comprehensive understanding of this crucial stress response is essential not only for bacterial physiology but also for applied microbiology, including pathogenicity and pathogen control...

..Together our data show that the thiol-(S)-alkylation reaction with protein and non-protein thiols is the in vivo mechanism for thiol depletion and quinone toxicity in B. subtilis and most likely also in other bacteria...

..These analyses confirmed our DNA microarray data and furthermore revealed different regulatory groups of sigmaB-dependent genes...

..Moreover, we show that in those low GC, Gram+ bacteria, which lack the McsA/McsB complex, the Zn finger protein ClpE is able to sense and respond to oxidative stress, also resulting in CtsR inactivation...

..Data of this study indicate that, unlike in B. subtilis, phosphate starvation in B. licheniformis does not induce the SigmaB-dependent general stress response...

..subtilis. These novel thiol-stress-responsive mechanisms are highly conserved among Gram-positive bacteria and are thought to have evolved to detoxify quinone-like electrophiles...

..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...

..For sigB mutants in different strains, different effects on the T cell-mitogenic potential were observed depending on the SAg gene repertoire of the isolates...

..We speculate that AdhR is redox-regulated via thiol-(S)-alkylation by aldehydes and that AdhA and YraA are specifically involved in reduction of aldehydes and degradation or repair of damaged thiol-containing proteins respectively...

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..Redox regulation of CatR occurs independently of YodB, and no protein interaction was detected between CatR and YodB in vivo using protein cross-linking and mass spectrometry...

..g., SACOL0201, SACOL2360, and SACOL2658) were found to be upregulated during anaerobic growth, indicating a role in the regulation of anaerobic gene expression...

..Metabolomic analyses support the induction of the glyoxylate cycle during oxidative stress in B. licheniformis...

..Moreover, the DeltaykvE mutant displayed a 2-MHQ and catechol resistant phenotype. YkvE was renamed as MhqR controlling a 2-MHQ and catechol-resistance regulon of B. subtilis...

..g., PflB, Ald1, IlvA1) or in the menD mutant (arc operon). Only genes involved in nitrate respiration and the ald1 operon seem to be additionally regulated by a depletion of oxygen in the hemB and/or menD mutant...

..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...

..This study reveals a thiol-disulfide switch model of redox-regulation for the YodB repressor to sense electrophilic compounds in vivo...

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..licheniformis have been analyzed under different growth conditions allowing comparisons of the extracellular proteomes and conclusions regarding similarities and differences of the protein secretion mechanisms between the two species...

..Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type...

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..Nevertheless, a common signature of 11 conserved S. aureus proteins, recognized in at least half of the bacteremic patients, was identified. All patients mounted a dynamic antibody response to a subset of these proteins...

..In contrast, enzymes of the TCC and gluconeogenesis are increased at the stationary phase. With this comprehensive proteome map we have an essential tool for a better understanding of cell physiology of the human pathogen, S. aureus...

..subtilis theoretical proteome...

..Due to the strict sigma(B)-dependent expression of YqgZ it was renamed to MgsR (modulator of the general stress response)...

..Obviously, NO itself does not cause general protein thiol oxidations. In contrast, exposure of cells to NO prior to peroxide stress diminished the irreversible thiol oxidation caused by hydrogen peroxide...

..This proteomic approach provides an overview of cell adaptation to an osmotic upshift in B. subtilis visualizing the most dramatic changes in the protein synthesis pattern...

..Finally, evidence is presented that the antibiotic nitrofurantoin mediates the formation of oxidized thiols in B. subtilis...

..e., arlRS, saeRS, sarA, sarR, sarS, and sigB), as well as genes directly involved in the virulence of S. aureus (i.e., fnbA, epiE, epiG, and seb)...

..We conclude that the distinct biological behavior of egc and non-egc superantigens is not due to their intrinsic properties, which are very similar, but caused by their differential release by S. aureus...

..The expression profiles determined with the electrical chip and the optimized protocol were, in most cases, comparable with the profiles determined by real-time RT-PCR measurements...

..Both, SILAC as well as (14)N/(15)N-labeling are compatible with 2-DE, 2-D LC-MS/MS, and GeLC-MS/MS and thus will allow comprehensive simultaneous interrogation of cytoplasmic and enriched membrane proteomes...

..These alterations in levels of metabolic enzymes were paralleled by more pronounced changes in the concentrations of associated metabolites, in particular, intermediates of the glycolysis and several amino acids...

..Among them were yvyD, citA and the putative methylcitrate shunt genes mmgD, mmgE and yqiQ. However, both starvation conditions did not induce a general SigmaB-dependent stress response...

..Limitation of phosphate, carbon and nitrogen sources results in the secretion of specific proteins that may be involved in counteracting the starvation...

..Interestingly, especially genes with a high codon adaptation index highly overlap with anaerobically induced genes...

..Proteolysis of putative non-substrate-bound or "unemployed" proteins appears to be a characteristic feature of S. aureus in order to access nutrients as an important survival strategy under starvation conditions...

..It is demonstrated that the acoABCL operon is mainly responsible for acetoin and 2,3-butanediol degradation in B. licheniformis...

..aureus RsbU. Collectively, the data suggest that the activity of the S. aureus RsbU and hence SigB may be subjected to different regulation in comparison to that in B. subtilis...

..baltica, were detected in the surface proteome. Additionally, 8 proteins with a novel protein structure combining a conserved type IV pilin/N-methylation domain and a planctomycete-typical DUF1559 domain were identified...

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..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...

..Many genes encoding proteins of so far unknown functions could be assigned to specifically or commonly induced genes...

..Our approach revealed 28 surface-associated proteins not previously reported as SigB-dependent or -influenced, expanding the group of surface-associated proteins and virulence factors modulated by SigB...

..Moreover, the data imply that a secondary oxidative stress seems to be a common component of the severe stresses tested...

..The protein pattern of the RsbU+ strain (RN6390 RsbU+) was very similar to that of the Deltaagr/DeltarsbU mutant strain (RN6911) indicating an opposing effect of agr and rsbU on the expression of the same genes...

..We demonstrate that the mRNA analysis by means of the electric DNA-chip gives similar results compared to the micro array analysis and the real time RT-PCR technique...

..Several genes encoding proteins of unknown function could be assigned to one of these groups...

..During glucose limitation, the utilization of the alternative energy source acetoin keeps the protein synthesis machinery of B. subtilis cells active and thus allows for a long lasting acoA-controlled expression of recombinant genes...

..aureus. Since the activity of lactate dehydrogenase enables S. aureus to resist NO stress and thus the innate immune response, our data suggest that deactivation of Rex is a prerequisite for this phenomenon...

..Thus, the high antibody prevalence in humans is likely not induced by short-term nasal colonization, and presumably minor infections are required to trigger anti-S. aureus antibody responses...

..subtilis cells, thus representing approximately 69% of all transcribed IMPs...

..Importantly, the extensive variability of secreted virulence factors in S. aureus also suggests that development of protective vaccines against this pathogen requires a carefully selected combination of invariably produced antigens...

..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...

..These observations indicate the involvement of ClpE in global protein disaggregation. As a characteristic structural element of ClpE, the N-terminal zinc finger domain was proven to be essential for basal in vitro ATPase activity...

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..Our data indicate that the damaged Gap protein was not repaired...

..The genes encoding enzymes of the pentose phosphate pathway are expressed both in the presence and the absence of glucose, as suggested by the central role of this pathway in generating anabolic precursors...

..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)...

..This chapter gives an overview of current RNA analysis techniques. The bacterial stress and starvation responses, which are of potential importance for industrial microbial bioprocesses are summarised...

..subtilis fermentation processes. The data suggest for example acoA as a marker gene for glucose limitation or glnA as an indicator for nitrogen limitation...

..Thus, its function in sulfate assimilation renders TrxA an essential protein in growing B. subtilis cells...

..Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated...

..Proteins that are obviously nonfunctional, unprotected, or even "unemployed" seem to be recognized and proteolyzed by Clp proteases when the resources for growth become limited...

..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...

..Integrating this information into complex pathway models a comprehensive survey of the symbiotic physiology was established...

..These data indicate very critical interactions of membrane-sensory proteins with phospholipids and they demonstrate the power of membrane proteomics for the characterization of bacterial physiology and pathogenicity...

..Thus, the concentration of sigma(B) after stress is not high enough to compensate for its relatively low affinity for E, and it seems that additional mechanisms must be invoked to account for the binding of sigma(B) to E after stress...

..A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp...

..The ycnDE operon was most strongly induced by chromanon. Mutational analyses revealed that the putative hydroquinone-specific dioxygenases MhqO and MhqA confer resistance to 2-MHQ in B. subtilis...

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..The data indicate that proteomics can be a valuable tool to describe the physiological state of B. licheniformis cell populations, e.g., of cells growing in a bioreactor...

..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...

..We conclude that the general stress response of B. subtilis is induced by rifampin depending on RsbP activity and that loss of SigB function causes increased sensitivity to the antibiotic...

..Its function as a regulatory metabolic checkpoint resembles that of homoserine trans-succinylase (MetA) in E. coli which is similarly ATP-dependently degraded...

..Currently, it is possible to identify and quantify more than 50% of the predicted proteome in different cellular subfractions. In this review, we summarize the development of B. subtilis proteomics during the past 25 years...

..Analysis of the half-life of stress-inducible clp mRNAs during exponential growth and heat shock revealed precise regulation of the synthesis of each Clp protein at the posttranscriptional level as well to meet the needs of B. subtilis...

..Instead, the overproduction of degradative enzymes in both of these mutants was reflected in the extracellular proteome...

..Loss of RsbX function increases the fraction of phosphorylated RsbS and doubly phosphorylated RsbRA in unstressed cells. We propose that RsbX both maintains the ready state of the stressosome prior to stress and restores it post-stress...

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..7%). A comparison of the various methods disclosed the one-dimensional gel-LC and the shaving approach to be highly complementary techniques. A combination of them will reveal a most comprehensive view on membrane proteomes...

..Induction of proteins involved in defense against oxidative and heat stress indicates a change in redox balance and reactive oxygen species concentrations...

..We conclude that WapA processing products are substrates for the extracellular proteases which are stabilized in the absence of sigD due to an impaired cell wall turnover...

..Determination of sporulation frequencies confirmed the physiological relevance of the expression data...

..In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages...

..McsB represents therefore a new kind of tyrosine kinase, driven by a guanidino phosphotransferase domain...

..Such a method should prove useful in the process of antibiotic drug discovery...

..Stressing its importance for the C. albicans OSR, all 12 proteins were also induced after oxidative challenge by hydrogen peroxide or diamide...

..Finally, new approaches to determine the metaproteome of uncultured microbial consortia from marine habitats are discussed...

..In addition, the previously predicted as cytoplasmic protein oxalate decarboxylase OxdC was identified as the most abundant cell wall protein which was induced at the transcriptional level due to the acidic conditions caused by phytate...