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Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF)
Assessing the structure of living microbial cell membranes is a challenging analytical goal. The cell membrane is defined by its transverse structure, an approximately 5 nm-thick selectively permeable bilayer that serves many important cellular functions. Compositionally complex, dynamic, and organi...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9634059/ https://www.ncbi.nlm.nih.gov/pubmed/36339713 http://dx.doi.org/10.3389/fmolb.2022.1011981 |
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author | Nickels, Jonathan D. Bonifer, Kyle S. Tindall, Rachel R. Yahya, Ahmad Tan, Luoxi Do, Changwoo Davison, Brian H. Elkins, James G. |
author_facet | Nickels, Jonathan D. Bonifer, Kyle S. Tindall, Rachel R. Yahya, Ahmad Tan, Luoxi Do, Changwoo Davison, Brian H. Elkins, James G. |
author_sort | Nickels, Jonathan D. |
collection | PubMed |
description | Assessing the structure of living microbial cell membranes is a challenging analytical goal. The cell membrane is defined by its transverse structure, an approximately 5 nm-thick selectively permeable bilayer that serves many important cellular functions. Compositionally complex, dynamic, and organized in both the transverse and lateral dimensions, understanding the cell membrane structure—and the role that structure plays in cellular function, communication, and environmental sensing is an active scientific effort. Previously, we have devised a novel isotopic labeling approach for membrane lipids to enable direct in vivo structural studies of the cell membrane in the Gram-positive bacterium, Bacillus subtilis, using small-angle neutron scattering. This was accomplished through a genetic inhibition of fatty acid (FA) degradation (ΔfadN) and a chemical inhibition of FA biosynthesis using cerulenin, an irreversible inhibitor of type II fatty acid synthases. Here, we improve upon the previous system by introducing a dCas9/sgRNA-fabF complex that blocks transcription of the essential fabF gene when under xylose induction. This leads to greater sensitivity to cerulenin in the mutant strain (JEBS102) and more robust cell growth when supplementary FAs are introduced to the culture medium. A subtle change in FA uptake is noted when compared to the prior labeling strategy. This is seen in the gas chromatography/mass spectrometry (GC/MS) data as a higher ratio of n16:0 to a15:0, and manifests in an apparent increase in the membrane thickness determined via neutron scattering. This represents an improved method of isotopic labeling for the cell membrane of Bacillus subtilis; enabling improved investigations of cellular uptake and utilization of FAs, cell membrane structure and organization as a phenotypic response to metabolic and environmental changes. |
format | Online Article Text |
id | pubmed-9634059 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-96340592022-11-05 Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF) Nickels, Jonathan D. Bonifer, Kyle S. Tindall, Rachel R. Yahya, Ahmad Tan, Luoxi Do, Changwoo Davison, Brian H. Elkins, James G. Front Mol Biosci Molecular Biosciences Assessing the structure of living microbial cell membranes is a challenging analytical goal. The cell membrane is defined by its transverse structure, an approximately 5 nm-thick selectively permeable bilayer that serves many important cellular functions. Compositionally complex, dynamic, and organized in both the transverse and lateral dimensions, understanding the cell membrane structure—and the role that structure plays in cellular function, communication, and environmental sensing is an active scientific effort. Previously, we have devised a novel isotopic labeling approach for membrane lipids to enable direct in vivo structural studies of the cell membrane in the Gram-positive bacterium, Bacillus subtilis, using small-angle neutron scattering. This was accomplished through a genetic inhibition of fatty acid (FA) degradation (ΔfadN) and a chemical inhibition of FA biosynthesis using cerulenin, an irreversible inhibitor of type II fatty acid synthases. Here, we improve upon the previous system by introducing a dCas9/sgRNA-fabF complex that blocks transcription of the essential fabF gene when under xylose induction. This leads to greater sensitivity to cerulenin in the mutant strain (JEBS102) and more robust cell growth when supplementary FAs are introduced to the culture medium. A subtle change in FA uptake is noted when compared to the prior labeling strategy. This is seen in the gas chromatography/mass spectrometry (GC/MS) data as a higher ratio of n16:0 to a15:0, and manifests in an apparent increase in the membrane thickness determined via neutron scattering. This represents an improved method of isotopic labeling for the cell membrane of Bacillus subtilis; enabling improved investigations of cellular uptake and utilization of FAs, cell membrane structure and organization as a phenotypic response to metabolic and environmental changes. Frontiers Media S.A. 2022-10-21 /pmc/articles/PMC9634059/ /pubmed/36339713 http://dx.doi.org/10.3389/fmolb.2022.1011981 Text en Copyright © 2022 Nickels, Bonifer, Tindall, Yahya, Tan, Do, Davison and Elkins. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Molecular Biosciences Nickels, Jonathan D. Bonifer, Kyle S. Tindall, Rachel R. Yahya, Ahmad Tan, Luoxi Do, Changwoo Davison, Brian H. Elkins, James G. Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF) |
title | Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF) |
title_full | Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF) |
title_fullStr | Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF) |
title_full_unstemmed | Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF) |
title_short | Improved chemical and isotopic labeling of biomembranes in Bacillus subtilis by leveraging CRISPRi inhibition of beta-ketoacyl-ACP synthase (fabF) |
title_sort | improved chemical and isotopic labeling of biomembranes in bacillus subtilis by leveraging crispri inhibition of beta-ketoacyl-acp synthase (fabf) |
topic | Molecular Biosciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9634059/ https://www.ncbi.nlm.nih.gov/pubmed/36339713 http://dx.doi.org/10.3389/fmolb.2022.1011981 |
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