Biomedical subjects

Michael Hecker

Publications and source records attributed to Michael Hecker.

At least 19 recordsLinked to original sources

PubMed2007

Physiological proteomics of the uncultured endosymbiont of Riftia pachyptila.

The bacterial endosymbiont of the deep-sea tube worm Riftia pachyptila has never been successfully cultivated outside its host. In the absence of cultivation data, we have taken a proteomic approach based on the metagenome sequence to study the metabolism of this peculiar microorganism in detail. As one result, we found that three major sulfide oxidation proteins constitute approximately 12% of the total cytosolic proteome, which highlights the essential role of these enzymes for the symbiont's energy metabolism. Unexpectedly, the symbiont uses the reductive tricarboxylic acid cycle in addition to the previously identified Calvin cycle for CO2 fixation.

Animals↗

PubMed2007

Proteomic profiling of cellular stresses in Bacillus subtilis reveals cellular networks and assists in elucidating antibiotic mechanisms of action.

Proteomic profiling provides a global view of the protein composition of the cell. In contrast to the static nature of the genome sequence, which provides the blueprint for all protein-based cellular building blocks, the proteome is highly dynamic. The protein composition is constantly adjusting to facilitate survival, growth, and reproduction in an ever-changing environment. In a quest to understand the regulation of cellular networks in bacteria and the role of individual proteins in the adaptation process, the proteomic response to stress and starvation was analyzed in wild-type and mutant strains. The knowledge derived from these proteomic studies was applied to investigating the bacterial response to antibiotics. It was found that proteomics presents a powerful tool for hypothesis generation regarding antibiotic mechanism of action.

Anti-Bacterial Agents↗

PubMed2007

Global gene expression profiling of Bacillus subtilis in response to ammonium and tryptophan starvation as revealed by transcriptome and proteome analysis.

The global gene expression profile of Bacillus subtilis in response to ammonium and tryptophan starvation was analyzed using transcriptomics and proteomics which gained novel insights into these starvation responses. The results demonstrate that both starvation conditions induce specific, overlapping and general starvation responses. The TnrA regulon, the glutamine synthetase (glnA) as well as the sigma(L)-dependent bkd and roc operons were most strongly and specifically induced after ammonium starvation. These are involved in the uptake and utilization of ammonium and alternative nitrogen sources such as amino acids, gamma-aminobutyrate, nitrate/nitrite, uric acid/urea and oligopeptides. In addition, several carbon catabolite-controlled genes (e.g. acsA, citB), the alpha-acetolactate synthase/-decarboxylase alsSD operon and several aminotransferase genes were specifically induced after ammonium starvation. The induction of sigma(F)- and sigma(E)-dependent sporulation proteins at later time points in ammonium-starved cells was accompanied by an increased sporulation frequency. The specific response to tryptophan starvation includes the TRAP-regulated tryptophan biosynthesis genes, some RelA-dependent genes (e.g. adeC, ald) as well as spo0E. Furthermore, we recognized overlapping responses between ammonium and tryptophan starvation (e.g. dat, maeN) as well as the common induction of the CodY and sigma(H) general starvation regulons and the RelA-dependent stringent response. Many genes encoding proteins of so far unknown functions could be assigned to specifically or commonly induced genes.

Ammonium Sulfate↗

PubMed2006

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 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.

Bacillus subtilis↗

PubMed2006

An acetoin-regulated expression system of Bacillus subtilis.

An expression system, which is based on the promoter of the acoABCL operon of Bacillus subtilis was developed and characterized. The acoABCL operon codes for the acetoin dehydrogenase complex, which is the major enzyme system responsible for the catabolism of acetoin in B. subtilis. Besides weak organic acids, the neutral overflow metabolite acetoin is metabolized by the cells in the early stationary phase. Transcription of reporter gene fusions with the acoA promoter of this operon is strongly repressed by glucose but induced by acetoin as soon as the preferred carbon source glucose is exhausted. The co-expression of an additional copy of the regulator gene acoR led to more than twofold higher activity of the acoA promoter. It is demonstrated that the induction of this promoter in growing cells with acetoin is possible with non-phosphotransferase system sugars as carbon and energy source and in a ccpA mutant background. Moreover, it could be shown that the activity of the acoA-directed expression system correlates with the level of acetoin in the medium. 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.

Acetoin↗

PubMed2006

The sigmaB regulon in Staphylococcus aureus and its regulation.

The Staphylococcus aureus genome codes for a sigma factor that shows close sequence similarity to the alternative sigma factor sigmaB of Bacillus subtilis. However, of the proteins controlling the activity of sigmaB in B. subtilis only RsbU, RsbV, and RsbW are encoded in the staphylococcal genome. Therefore, the regulation of the sigmaB activity must differ between these two bacterial species. The present study was designed (i) to describe the sigmaB regulon and (ii) to identify stimuli leading to an activation of sigmaB-dependent transcription. All conditions under which sigmaB was activated in S. aureus (heat shock, addition of MnCl2 or NaCl, alkaline shock) required the presence of RsbU, a positive regulator of sigmaB. In contrast to B. subtilis, a drop in the cellular ATP level caused by the addition of carbonyl cyanide m-chlorophenylhydrazone did not lead to an activation of sigmaB in S. aureus. Moreover, ethanol, a strong inductor of sigmaB activity in B. subtilis, also failed to induce sigmaB in S. aureus. Expression of sigB and sigmaB-dependent genes was enhanced following entry into stationary phase of cells grown in complex medium (LB medium). Our DNA microarray data indicated that 122 genes are positively regulated by sigmaB under alkaline stress conditions. Interestingly, only 12% of these genes have an orthologue in the B. subtilis sigmaB regulon, suggesting that the function of the sigmaB regulon in S. aureus is different from that in B. subtilis. We could show that sigmaB of S. aureus, in contrast to B. subtilis, may have a function in more basic cellular processes such as cell envelope composition, membrane transport processes and intermediary metabolism. sigmaB-dependent genes identified by the DNA microarray approach were subjected to detailed transcriptional analyses using primer extension and Northern blot techniques. These analyses confirmed our DNA microarray data and furthermore revealed different regulatory groups of sigmaB-dependent genes.

Adaptation, Physiological↗

PubMed2006

Staphylococcus aureus carriers neutralize superantigens by antibodies specific for their colonizing strain: a potential explanation for their improved prognosis in severe sepsis.

Staphylococcus aureus is one of the most common causes of hospital-acquired infections. At the same time, 25% of healthy persons are symptom-free S. aureus carriers, and they have an increased risk of developing nosocomial S. aureus septicemia. Paradoxically, their prognosis is much better than that of noncarriers. We compared the antibody profiles for carriers and noncarriers toward S. aureus superantigens. In carriers, we found high titers of neutralizing antibodies specific for those superantigens that are expressed by their colonizing strain. The results show that carriage status confers strain-specific humoral immunity, which may contribute to protection during S. aureus septicemia.

Antibodies, Bacterial↗

PubMed2006

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.

Bacillus subtilis↗

PubMed2006

The extracellular proteome of Bacillus licheniformis grown in different media and under different nutrient starvation conditions.

The now finished genome sequence of Bacillus licheniformis DSM 13 allows the prediction of the genes involved in protein secretion into the extracellular environment as well as the prediction of the proteins which are translocated. From the sequence 296 proteins were predicted to contain an N-terminal signal peptide directing most of them to the Sec system, the main transport system in Gram-positive bacteria. Using 2-DE the extracellular proteome of B. licheniformis grown in different media was studied. From the approximately 200 spots visible on the gels, 89 were identified that either contain an N-terminal signal sequence or are known to be secreted by other mechanisms than the Sec pathway. The extracellular proteome of B. licheniformis includes proteins from different functional classes, like enzymes for the degradation of various macromolecules, proteins involved in cell wall turnover, flagellum- and phage-related proteins and some proteins of yet unknown function. Protein secretion is highest during stationary growth phase. Furthermore, cells grown in complex medium secrete considerably higher protein amounts than cells grown in minimal medium. Limitation of phosphate, carbon and nitrogen sources results in the secretion of specific proteins that may be involved in counteracting the starvation.

Amino Acid Sequence↗

PubMed2006

Salt stress adaptation of Bacillus subtilis: a physiological proteomics approach.

The adaptation to osmotic stress is crucial for growth and survival of Bacillus subtilis in its natural ecosystem. Dual channel imaging and warping of 2-D protein gels were used to visualize global changes in the protein synthesis pattern of cells in response to osmotic stress (6% NaCl). Many vegetative enzymes were repressed in response to salt stress and derepressed after resumption of growth. The enzymes catalyzing the metabolic steps from glucose to 2-oxoglutarate, however, were almost constantly synthesized during salt stress despite the growth arrest. This indicates an enhanced need for the proline precursor glutamate. The synthesis of enzymes involved in sulfate assimilation and in the formation of Fe-S clusters was also induced, suggesting an enhanced need for the formation or repair of Fe-S clusters in response to salt stress. One of the most obvious changes in the protein synthesis profile can be followed by the very strong induction of the SigB regulon. Furthermore, members of the SigW regulon and of the PerR regulon, indicating oxidative stress after salt challenge, were also induced. 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.

Adaptation, Physiological↗

PubMed2006

Proteomic dissection of potential signal recognition particle dependence in protein secretion by Bacillus subtilis.

The bacterial signal recognition particle (SRP)-dependent pathway is believed to be a major targeting route for membrane proteins, as well as for subsets of secretory proteins. The present studies were aimed at an assessment of the role of two key components of SRP, namely Ffh and FtsY, in protein secretion by the Gram-positive bacterium Bacillus subtilis. Our results show that both components are important for the extracellular accumulation of proteins containing known signal peptides. Remarkably, extracellular accumulation of individual proteins was affected to different extents by depletion of Ffh or FtsY, at least under the conditions tested. Moreover, the observed Ffh or FtsY dependence of certain secretory proteins did not seem to correlate with signal peptide length or hydrophobicity. Although it is presently difficult to distinguish between direct and indirect effects, these findings suggest that other, yet unidentified, determinants in secretory proteins are also important for their SRP dependence. High-level production of homologous and heterologous secretory proteins was shown to result in elevated cellular Ffh and FtsY levels. This phenomenon is, most likely, due to post-transcriptional regulation. In conclusion, the present proteomic dissection of SRP-dependent extracellular protein accumulation provides exciting leads to identify novel determinants for interactions between secretory proteins and SRP.

Bacillus subtilis↗

PubMed2006

Impact of the accessory gene regulatory system (Agr) on extracellular proteins, codY expression and amino acid metabolism in Staphylococcus epidermidis.

The quorum-sensing system Agr is part of a complex regulatory network of gene expression in staphylococci. This study presents the effect of an agr mutation on a biofilm-forming Staphylococcus epidermidis isolate by employing proteome and transcriptome analysis. The agr mutant exhibited a significantly lowered amount of extracellular proteins: amongst others SspA, AtlE, GehD and the phenol soluble modulins PSM1/2. Cytoplasmic proteome analysis and expression profiling indicated that the agr inactivation led to a strongly altered regulation of metabolism and virulence. Most strikingly, expression of CodY, a global regulator of virulence and stationary phase gene expression, was decreased in the agr mutant. In this respect, homologous genes known to be controlled by CodY in Bacillus subtilis and Lactococcus lactis were found to be up-regulated in the S. epidermidis agr mutant. The combined data show that wild-type and agr mutant differ with respect to amino acid biosynthesis and oligopeptide transport, carbohydrate utilization, as well as GMP and IMP interconversion. Due to the varying physiological properties S. epidermidis agr mutants, which often occur spontaneously, might be capable of colonizing alternative ecological niches in the human host and could, therefore, have an advantage in adapting to changing environmental conditions.

Amino Acids↗

PubMed2006

The phosphate-starvation response of Bacillus licheniformis.

The phosphate-starvation stimulon of Bacillus licheniformis was analyzed at the transcriptional and translational level. The comparison of the transcriptome and the proteome demonstrated that this specific starvation response of B. licheniformis is partially similar to that of B. subtilis. However, it is also shown that B. licheniformis has evolved its own strategies to cope with this nutrient limitation. By means of the secretome analysis the phytase was identified as the most abundant protein under phosphate-starvation conditions. Data of this study indicate that, unlike in B. subtilis, phosphate starvation in B. licheniformis does not induce the SigmaB-dependent general stress response.

6-Phytase↗

PubMed2006

Guidelines for the next 10 years of proteomics.

In the last ten years, the field of proteomics has expanded at a rapid rate. A range of exciting new technology has been developed and enthusiastically applied to an enormous variety of biological questions. However, the degree of stringency required in proteomic data generation and analysis appears to have been underestimated. As a result, there are likely to be numerous published findings that are of questionable quality, requiring further confirmation and/or validation. This manuscript outlines a number of key issues in proteomic research, including those associated with experimental design, differential display and biomarker discovery, protein identification and analytical incompleteness. In an effort to set a standard that reflects current thinking on the necessary and desirable characteristics of publishable manuscripts in the field, a minimal set of guidelines for proteomics research is then described. These guidelines will serve as a set of criteria which editors of PROTEOMICS will use for assessment of future submissions to the Journal.

Biomarkers↗

PubMed2006

Proteome signatures for stress and starvation in Bacillus subtilis as revealed by a 2-D gel image color coding approach.

In this paper we have defined proteome signatures of Bacillus subtilis in response to heat, salt, peroxide, and superoxide stress as well as after starvation for ammonium, tryptophan, glucose, and phosphate using the 2-D gel-based approach. In total, 79 stress-induced and 155 starvation-induced marker proteins were identified including 50% that are not expressed in the vegetative proteome. Fused proteome maps and a color coding approach have been used to define stress-specific regulons that are involved in specific adaptative functions (HrcA for heat, PerR and Fur for oxidative stress, RecA for peroxide, CymR and S-box for superoxide stress). In addition, starvation-specific regulons are defined that are involved in the uptake or utilization of alternative nutrient sources (TnrA, sigmaL/BkdR for ammonium; tryptophan-activated RNA-binding attenuation protein for tryptophan; CcpA, CcpN, sigmaL/AcoR for glucose; PhoPR for phosphate starvation). The general stress or starvation proteome signatures include the CtsR, Spx, sigmaL/RocR, sigmaB, sigmaH, CodY, sigmaF, and sigmaE regulons. Among these, the Spx-dependent oxidase NfrA was induced by all stress conditions indicating stress-induced protein damages. Finally, a subset of sigmaH-dependent proteins (sporulation response regulator, YvyD, YtxH, YisK, YuxI, YpiB) and the CodY-dependent aspartyl phosphatase RapA were defined as general starvation proteins that indicate the transition to stationary phase caused by starvation.

Bacillus subtilis↗

PubMed2006

Direct mass spectrometric identification of ABCB1 (P-glycoprotein/MDR1) from the apical membrane fraction of human placenta using fourier transform ion cyclotron mass spectrometry.

Identification and quantification of proteins from human tissue by electrophoresis and subsequent mass spectrometry (MS) has been mainly restricted to high abundance, non-membrane-bound molecules. Pharmacologically relevant structures such as cytochrome P450 enzyme, drug transporters and receptors were not accessible by this technology. We developed a method to identify an integral membrane-bound protein (ABCB1) from human placenta using Fourier transform ion cyclotron (FTICR)-MS. Apical and basal membrane fractions were enriched from term human placenta using differential centrifugation. Following SDS-page these fractions were cleaved with trypsin, separated by nano-HPLC and subjected to FTICR-MS. We identified a total of 70 and 89 proteins in the apical and basal membranes, respectively. Among these proteins, and restricted to the apical membrane, was the transport protein ABCB1, with 10 peptides identified by MS, covering a total of 10% of the entire protein. The study describes a method suitable for direct monitoring of membrane-bound proteins from human tissue using FTICR-MS.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2006

pSM19035-encoded zeta toxin induces stasis followed by death in a subpopulation of cells.

The toxin-antitoxin operon of pSM19035 encodes three proteins: the omega global regulator, the epsilon labile antitoxin and the stable zeta toxin. Accumulation of zeta toxin free of epsilon antitoxin induced loss of cell proliferation in both Bacillus subtilis and Escherichia coli cells. Induction of a zeta variant (zetaY83C) triggered stasis, in which B. subtilis cells were viable but unable to proliferate, without selectively affecting protein translation. In E. coli cells, accumulation of free zeta toxin induced stasis, but this was fully reversed by expression of the epsilon antitoxin within a defined time window. The time window for reversion of zeta toxicity by expression of epsilon antitoxin was dependent on the initial cellular level of zeta. After 240 min of constitutive expression, or inducible expression of high levels of zeta toxin for 30 min, expression of epsilon failed to reverse the toxic effect exerted by zeta in cells growing in minimal medium. Under the latter conditions, zeta inhibited replication, transcription and translation and finally induced death in a fraction (approximately 50 %) of the cell population. These results support the view that zeta interacts with its specific target and reversibly inhibits cell proliferation, but accumulation of zeta might lead to cell death due to pleiotropic effects.

Antitoxins↗

PubMed2006

Differential gene expression in response to phenol and catechol reveals different metabolic activities for the degradation of aromatic compounds in Bacillus subtilis.

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, sigmaB 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 sigmaB 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-YfiE(Bs) 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.

Bacillus subtilis↗