Publications

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21 Publications visible to you, out of a total of 21

Abstract (Expand)

In this review, we demonstrate the power of gel-based proteomics to address physiological questions of bacteria. Although gel-based proteomics covers a subpopulation of proteins only, fundamental issues of a bacterial cell such as almost all metabolic pathways or the main signatures of stress and starvation responses can be analyzed. The analysis of the synthesis pattern of single proteins, e.g., in response to environmental changes, requires gel-based proteomics because only this technique can compare protein synthesis and amount in the same 2-D gel. Moreover, highly sophisticated software packages facilitate the analysis of the regulation of the main metabolic enzymes or the stress/starvation responses, PTMs, protein damage/repair, and degradation and finally protein secretion mechanisms at a proteome-wide scale. The challenge of proteomics whose panorama view shows events never seen before is to select the most interesting issues for detailed follow up studies. This "road map of proteomics" from proteome data via new hypothesis and finally novel molecular mechanisms should lead to exciting information on bacterial physiology. However, many proteins escape detection by gel-based procedures, such as membrane or low abundance proteins. The smart combination of gel-free and gel-based approaches is the "state of the art" for physiological proteomics of bacteria.

Authors: , Haike Antelmann, Knut Büttner, Jörg Bernhardt

Date Published: 13th Nov 2008

Publication Type: Not specified

Abstract (Expand)

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease-deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin-3 show that chemical protease inhibition can result in improved production of protease-sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.

Authors: Lidia Westers, Helga Westers, Geeske Zanen, Haike Antelmann, , David Noone, Kevin M Devine, , Wim J Quax

Date Published: 12th Jun 2008

Publication Type: Not specified

Abstract (Expand)

Quinones and alpha,beta-unsaturated carbonyls are naturally occurring electrophiles that target cysteine residues via thiol-(S)-alkylation. We analysed the global expression profile of Bacillus subtilis to the toxic carbonyls methylglyoxal (MG) and formaldehyde (FA). Both carbonyl compounds cause a stress response characteristic for thiol-reactive electrophiles as revealed by the induction of the Spx, CtsR, CymR, PerR, ArsR, CzrA, CsoR and SigmaD regulons. MG and FA triggered also a SOS response which indicates DNA damage. Protection against FA is mediated by both the hxlAB operon, encoding the ribulose monophosphate pathway for FA fixation, and a thiol-dependent formaldehyde dehydrogenase (AdhA) and DJ-1/PfpI-family cysteine proteinase (YraA). The adhA-yraA operon and the yraC gene, encoding a gamma-carboxymuconolactone decarboxylase, are positively regulated by the MerR-family regulator, YraB(AdhR). AdhR binds specifically to its target promoters which contain a 7-4-7 inverted repeat (CTTAAAG-N4-CTTTAAG) between the -35 and -10 elements. Activation of adhA-yraA transcription by AdhR requires the conserved Cys52 residue in vivo. 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.

Authors: Thi Thu Huyen Nguyen, Warawan Eiamphungporn, Ulrike Mäder, Manuel Liebeke, , , John D Helmann, Haike Antelmann

Date Published: 23rd Dec 2008

Publication Type: Not specified

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