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Integrative metabolomics and proteomics allow the global intracellular characterization of Bacillus subtilis cells and spores.

ABSTRACT: Reliable and comprehensive multi-omics analysis is essential for researchers to understand and explore complex biological systems more completely. Bacillus subtilis is a model organism for Gram-positive spore-forming bacteria, and in-depth insight into the physiology and molecular basis of spore formation and germination in this organism requires advanced multilayer molecular datasets generated from the same sample. In this study, we evaluated two monophasic methods for polar and nonpolar compound extraction (acetonitrile/methanol/water; isopropanol/water, and 60% ethanol), and two biphasic methods (chloroform/methanol/water, and methyl tert-butyl ether/methanol/water) on coefficients of variation of analytes, identified metabolite composition and the quality of proteomics profiles. The analytical workflow comprises (1) parallel metabolite and protein extraction, (2) monophasic or biphasic sample extraction, (3) proteomics comparison and (4) multi-omics-based data visualization. The 60% EtOH protocol proved to be the easiest in sample processing and more amenable to automation. Collectively, we annotated 505 and 484 metabolites and identified 1665 and 1562 proteins in B. subtilis vegetative cells and spores, respectively. We also show differences between vegetative cells and spores from a multi-omics perspective and demonstrated that an integrative multi-omics analysis can be implemented from one sample using the 60% EtOH protocol. Correlation analysis further demonstrated that the metabolome and proteome datasets were highly correlated in content for components annotated to be part of the pyrimidine metabolism pathway. The results obtained by the 60% EtOH protocol provide a comprehensive insight into differences in the metabolic and protein makeup of B. subtilis vegetative cells and spores.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Bacillus Subtilis (ncbitaxon:1423)

SUBMITTER: Gertjan Kramer

PROVIDER: MSV000092140 | MassIVE | Sat Jun 10 03:20:00 BST 2023

SECONDARY ACCESSION(S): PXD042868

REPOSITORIES: MassIVE

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Project description:Description Bacillus subtilis forms highly resistant, metabolically inactive spores upon nutrient limitation. These endospores pose challenges to the food and medical sectors. Spores reactivate their metabolism upon contact with germinants through germination and outgrowth, and then develop into vegetative cells. However, the mechanism of the activation of the molecular machinery that triggers spore germination and outgrowth is unclear. To gain further insight into spore germination and outgrowth, the transcriptome and proteome changes during Bacillus subtilis spore conversion to vegetative cells were analyzed. The transcriptome analysis also allowed us to trace the different functional groups of genes expressed during this conversion. For each time-point sampled, the change in the spore proteome was quantitatively monitored relative to the reference proteome of 15N metabolically labelled vegetative cells. Of the quantified proteins, 60 percent are common to vegetative cells and spores, indicating that spores have a minimal set of proteins sufficient for the resumption of metabolism upon completion of germination. The shared proteins thus represent the most basic survival kit for spore-based life. Until the phase transition, defined as the completion of germination, we observed no significant change in the proteome or the transcriptome. Our analysis identified 34 abundant mRNA transcripts in the dormant spores, at least 31 of which were rapidly degraded after the phase transition. We observed 3152 differentially expressed genes, and demonstrated with our mass spectrometry analyses the differential expression of 323 proteins. Our data show that 173 proteins from dormant spores, both proteins unique to spores and proteins shared with vegetative cells, are lost after completion of germination. Further analysis is required to functionally interpret the observed protein loss. The observed diverse timing of the synthesis of different protein sets reveals a putative core-strategy of the revival of life starting from the B. subtilis spore.

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Integrative metabolomics and proteomics allow the global intracellular characterization of Bacillus subtilis cells and spores.

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