Alkaline proteins of Bacillus subtilis: First steps towards a two-dimensional alkaline master gel

Ohlmeier, Steffen; Scheffold, Christian; Hecker, Michael

doi:10.1002/1522-2683(200011)21:17<3701::aid-elps3701>3.3.co;2-x

Cited by 18 publications

(24 citation statements)

References 21 publications

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“…7. However, optimization of the separation conditions for alkaline proteins required much more efforts than standard pI range separations in the neutral or acidic pI range [23][24][25]. The analysis of the theoretical proteome of B. subtilis also revealed that most of the proteins with more than two membrane-spanning domains are located in the alkaline region with a relatively high M r (Fig.…”

Section: Towards the Total Proteome: Analysis Of The Alkaline Pi Regisupporting

confidence: 62%

“…However, among the 42 alkaline proteins identified so far (Fig. 7), no intrinsic membrane protein was found [25] confirming the data of many laboratories that membrane proteins can only occasionally be visualized with the current 2-D electrophoresis technology; especially proteins with multiple membrane-spanning domains escape detection [26]. This observation also gains support when the grand average hydropathy (GRAVY) values [27] of all proteins potentially encoded by the B. subtilis genome sequence are compared with those of the proteins identified on 2-D gels thus far.…”

Section: Towards the Total Proteome: Analysis Of The Alkaline Pi Regimentioning

confidence: 99%

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A comprehensive two-dimensional map of cytosolic proteins ofBacillus subtilis

Büttner¹,

Bernhardt²,

Scheffold³

et al. 2001

Electrophoresis

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217

152

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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.

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Section: Towards the Total Proteome: Analysis Of The Alkaline Pi Regisupporting

confidence: 62%

Section: Towards the Total Proteome: Analysis Of The Alkaline Pi Regimentioning

confidence: 99%

A comprehensive two-dimensional map of cytosolic proteins ofBacillus subtilis

Büttner¹,

Bernhardt²,

Scheffold³

et al. 2001

Electrophoresis

Self Cite

217

152

View full text Add to dashboard Cite

show abstract

“…Most of these new genes, whose expression seems to be controlled by (p)ppGpp, were found only by transcriptome analysis. Failure to detect certain gene products by 2D gel analysis may be due to the fact that certain subproteomic fractions, e.g., those with membrane-spanning domains, extremely alkaline proteins (66), or secreted proteins (3) were not considered in our analysis. Negative stringent control.…”

Section: Discussionmentioning

confidence: 99%

“…Transcriptional units (predicted and validated), with data obtained from the Subtilist database at http://genolist.pasteur.fr/SubtiList/ (59, 60), are also indicated. Asterisks: ‫,ء‬ identified by MALDI-TOF MS (this study); ‫,ءء‬ identified by MALDI-TOF MS (66). Other proteins were reallocated from the Sub2D 2D database (http://microbio2.biologie.uni-greifswald.de:8880/sub2d.htm [8]).…”

Section: Protein Synthesis Rplo Ribosomal Protein L15 Rpsj-rplc-d-w-bmentioning

confidence: 99%

Bacillus subtilisfunctional genomics: global characterization of the stringent response by proteome and transcriptome analysis

et al. 2002

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The stringent response in Bacillus subtilis was characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type and relA mutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response to DL-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in the relA mutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknown y-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

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“…Several excellent proteomic studies of B. subtilis have been carried out. Notable among these are a recent alkaline 2D map and a comprehensive 2D map (12,61). These studies focused on the analysis of soluble proteins from vegetative cells.…”

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confidence: 99%

Proteomic Analysis of the Spore Coats of Bacillus subtilis and Bacillus anthracis

et al. 2003

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The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat.

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Alkaline proteins of Bacillus subtilis: First steps towards a two-dimensional alkaline master gel

Cited by 18 publications

References 21 publications

A comprehensive two-dimensional map of cytosolic proteins ofBacillus subtilis

A comprehensive two-dimensional map of cytosolic proteins ofBacillus subtilis

Bacillus subtilisfunctional genomics: global characterization of the stringent response by proteome and transcriptome analysis

Proteomic Analysis of the Spore Coats of Bacillus subtilis and Bacillus anthracis

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