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Deconvoluting the complexity of Bacillus subtilis disulfide stress responses by redox-state and absolute abundance quantification of extracellular, membrane and cytosolic proteins

ABSTRACT: To understand cellular mechanisms of stress management, omics data constitute a powerful resource. However, the availability of absolute quantitative data on protein abundances is still a serious limiting factor. This knowledge gap increments especially when protein abundances need to be compared across different cell compartments. In the present study, we aimed to provide deeper insights in the proteomic responses of the model Gram-positive bacterium Bacillus subtilis to disulfide stress. For this purpose, proteome-wide absolute abundances were determined with focus on different subcellular locations (cytosol and membrane) and the extracellular medium, and these were combined with redox state determination. To achieve the quantification of secreted proteins in the culture medium, a simple and straightforward protocol for the absolute quantification of extracellular proteins in bacteria was developed. Extracellular proteins, which are highly diluted in the medium, were concentrated using StrataClean beads together with a set of standard proteins that were used to determine the extent of the concentration step. The resulting dataset offers new insights into the protein abundances in different subcellular compartments and the extracellular medium, as well as a proteome-wide redox-state determination. Altogether, our study provides a quantitative understanding of disulfide stress management, protein production and secretion in B. subtilis.

INSTRUMENT(S): LTQ Orbitrap Velos, TSQ Vantage

ORGANISM(S): Bacillus Subtilis Subsp. Subtilis Str. 168

SUBMITTER: Sandra Maass

LAB HEAD: Dörte Becher

PROVIDER: PXD042394 | Pride | 2024-02-05

REPOSITORIES: Pride

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Project description:The obligate anaerobe, spore forming bacterium Clostridioides difficile (formerly Clostridium difficile) causes nosocomial and community acquired diarrhea often associated with antibiotic therapy. Major virulence factors of the bacterium are the two large clostridial toxins TcdA and TcdB. The production of both toxins was found strongly connected to the metabolism as well as the nutritional status in the environment. Here, we systematically investigated the changes of the gene regulatory, proteomic and metabolic networks of C. difficile 630Δerm underlying the adaptation to the non-growing state in the stationary phase. Integrated data from time-resolved transcriptome, proteome and metabolome investigations performed under defined growth conditions uncovered multiple adaptation strategies. Overall changes in the cellular processes included the downregulation of ribosome production, lipid metabolism, cold shock proteins, spermine biosynthesis, and glycolysis and in the later stages of riboflavin and coenzyme A biosynthesis. In contrast, several fermentation pathways, different chaperones and the cysteine, serine and pantothenate biosynthesis were upregulated. Focused on the Stickland amino acid fermentation and the central carbon metabolism, we demonstrated the ability of C. difficile to replenish its favored amino acid cysteine by a pathway from the glycolysis substrate 3-phosphoglycerate via L-serine. Following the growth course, the reductive equivalent pathways used were sequentially shifted from proline via leucine/phenylalanine to the central carbon metabolism first using butanoate fermentation and then to lactate. The toxin production was found correlated mainly to fluxes of the central carbon metabolism. Toxin formation in the supernatant was detected when the flux changed from butanoate to lactate synthesis in the late stationary phase. The holistic view derived from the combination of transcriptome, proteome and metabolome data allowed us to uncover the major metabolic strategies that are used by the clostridial cells to maintain its cellular integrity and ensure survival under starvation conditions.

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Deconvoluting the complexity of Bacillus subtilis disulfide stress responses by redox-state and absolute abundance quantification of extracellular, membrane and cytosolic proteins

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