The conserved WhiA protein influences the fatty acid composition of the Bacillus subtilis cell membrane
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ABSTRACT:
PROVIDER: PRJNA497441 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8075141.fastq.gz | Fastqsanger.gz | |||
| SRR8075142.fastq.gz | Fastqsanger.gz | |||
| SRR8075143.fastq.gz | Fastqsanger.gz | |||
| SRR8075144.fastq.gz | Fastqsanger.gz | |||
| SRR8075145.fastq.gz | Fastqsanger.gz |
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Project description:The conserved WhiA protein family is present in most Gram-positive bacteria and plays a role in cell division. WhiA contains a DNA-binding motive and has been identified as a transcription factor in the actinomycetes. In Bacillus subtilis, a deletion of whiA influences cell division and chromosome segregation. However, WhiA does not seem to function as a transcription factor in this organism, and it is still unclear how WhiA influences these processes. The B. subtilis whiA gene is part of an operon and flanked by two metabolic genes, including yvcK required for growth under gluconeogenic conditions, and the gene encoding the catabolite repressor Crh. Therefore, B. subtilis WhiA might play a role in carbon metabolism which could indirectly affect cell division and chromosome maintenance. To study this, we followed a metabolomics approach and measured exo-metabolome flux during growth on different carbon sources, using nuclear magnetic resonance spectroscopy. The carbon utilization of wild type and whiA mutant cells was similar, however, the whiA mutant exhibited depleted pools of branched-chain fatty acid precursors and higher levels of acetate and 2-oxoglutarate. Transcriptome data could not link these effects to gene regulatory differences, but the reduction in branched-chain fatty acid precursors influenced the fatty acid composition of the cell membrane. In conclusion, WhiA does not participate in carbon catabolite regulation, but affects fatty acid composition of the membrane by an as yet unknown mechanism. We speculate that the effect on the membrane might be related to the pleiotropic phenotype of the B. subtilis whiA mutant.
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Project description:Engineering bacterial cells to contain defined fatty acid compositions within their cell membranes establishes an in vivo experimental platform for membrane biophysics. Yet before fully realizing the potential of this system, it is prudent to understand the systemic changes induced by the labeling procedure itself. In this work, the systemic induced changes in cell are studied via analysis of cellular membrane compositions and shotgun proteomic analysis to compare expression levels of selected cellular proteins; in response to the labeling of Bacillus subtilis using cerulenin to inhibit fatty acid synthesis and subsequently providing selected fatty acids exogenously to be incorporated by the cell. Changes in the expression of enzymes involved in fatty acid biosynthesis and degradation are observed and consistent with expectations. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phophatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Beyond this, changes to cell wall enzymes and isoprenoid lipid production are also seen, which may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down regulated in the labeled cells, as was the actin-like protein mreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored between an enforced cell membrane fatty acid composition and lipid headgroup and raft cytoskeletal associated proteins.
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Project description:Engineering bacterial cells to contain defined fatty acid compositions within their cell membranes establishes an in vivo experimental platform for membrane biophysics. Yet before fully realizing the potential of this system, it is prudent to understand the systemic changes induced by the labeling procedure itself. In this work, the systemic induced changes in cell are studied via analysis of cellular membrane compositions and shotgun proteomic analysis to compare expression levels of selected cellular proteins; in response to the labeling of Bacillus subtilis using cerulenin to inhibit fatty acid synthesis and subsequently providing selected fatty acids exogenously to be incorporated by the cell. Changes in the expression of enzymes involved in fatty acid biosynthesis and degradation are observed and consistent with expectations. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phophatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Beyond this, changes to cell wall enzymes and isoprenoid lipid production are also seen, which may influence membrane organization, and indeed the well-known lipid raft-associated protein flotillin was found to be substantially down regulated in the labeled cells, as was the actin-like protein mreB. Taken as a whole, this study provides a greater depth of understanding for this important cell membrane experimental platform and presents a number of new connections to be explored between an enforced cell membrane fatty acid composition and lipid headgroup and raft cytoskeletal associated proteins.
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