Dataset Information

Impact of Fatty-Acid Labeling of Bacillus subtilis Membranes on the Cellular Lipidome and Proteome.

ABSTRACT: Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an in vivo experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was paired with proteomics to assess how the proteome changes in response to the directed incorporation of exogenous FAs into the membrane of Bacillus subtilis. Key findings from this analysis include an alteration in lipid headgroup distribution, with an increase in phosphatidylglycerol lipids and decrease in phosphatidylethanolamine lipids, possibly providing a fluidizing effect on the cell membrane in response to the induced change in membrane composition. Changes in the abundance of enzymes involved in FA biosynthesis and degradation are observed; along with changes in abundance of cell wall enzymes and isoprenoid lipid production. The observed changes 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 in regard to modulating cell membrane FA composition and its effects on lipid headgroup and raft/cytoskeletal associated proteins.

SUBMITTER: Nickels JD

PROVIDER: S-EPMC7243436 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Impact of Fatty-Acid Labeling of <i>Bacillus subtilis</i> Membranes on the Cellular Lipidome and Proteome.

Nickels Jonathan D JD Poudel Suresh S Chatterjee Sneha S Farmer Abigail A Cordner Destini D Campagna Shawn R SR Giannone Richard J RJ Hettich Robert L RL Myles Dean A A DAA Standaert Robert F RF Katsaras John J Elkins James G JG

Frontiers in microbiology 20200515

Developing cultivation methods that yield chemically and isotopically defined fatty acid (FA) compositions within bacterial cytoplasmic membranes establishes an <i>in vivo</i> experimental platform to study membrane biophysics and cell membrane regulation using novel approaches. Yet before fully realizing the potential of this method, it is prudent to understand the systemic changes in cells induced by the labeling procedure itself. In this work, analysis of cellular membrane compositions was pa ...[more]

PMID: 32499768

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Project description:Many bacterial species typically live in complex three-dimensional biofilms, yet much remains unknown about differences in essential processes between nonbiofilm and biofilm lifestyles. Here, we created a CRISPR interference (CRISPRi) library of knockdown strains covering all known essential genes in the biofilm-forming Bacillus subtilis strain NCIB 3610 and investigated growth, biofilm colony wrinkling, and sporulation phenotypes of the knockdown library. First, we showed that gene essentiality is largely conserved between liquid and surface growth and between two media. Second, we quantified biofilm colony wrinkling using a custom image analysis algorithm and found that fatty acid synthesis and DNA gyrase knockdown strains exhibited increased wrinkling independent of biofilm matrix gene expression. Third, we designed a high-throughput screen to quantify sporulation efficiency after essential gene knockdown; we found that partial knockdowns of essential genes remained competent for sporulation in a sporulation-inducing medium, but knockdown of essential genes involved in fatty acid synthesis exhibited reduced sporulation efficiency in LB, a medium with generally lower levels of sporulation. We conclude that a subset of essential genes are particularly important for biofilm structure and sporulation/germination and suggest a previously unappreciated and multifaceted role for fatty acid synthesis in bacterial lifestyles and developmental processes. IMPORTANCE For many bacteria, life typically involves growth in dense, three-dimensional communities called biofilms that contain cells with differentiated roles held together by extracellular matrix. To examine how essential gene function varies between vegetative growth and the developmental states of biofilm formation and sporulation, we created and screened a comprehensive library of strains using CRISPRi to knockdown expression of each essential gene in the biofilm-capable Bacillus subtilis strain 3610. High-throughput assays and computational algorithms identified a subset of essential genes involved in biofilm wrinkling and sporulation and indicated that fatty acid synthesis plays important and multifaceted roles in bacterial development.

Project description:Bacillus subtilis is a soil-dwelling bacterium that can form biofilms, or communities of cells surrounded by a self-produced extracellular matrix. In biofilms, genetically identical cells often exhibit heterogeneous transcriptional phenotypes, so that subpopulations of cells carry out essential yet costly cellular processes that allow the entire population to thrive. Surprisingly, the extent of phenotypic heterogeneity and the relationships between subpopulations of cells within biofilms of even in well-studied bacterial systems like B. subtilis remains largely unknown. To determine relationships between these subpopulations of cells, we created 182 strains containing pairwise combinations of fluorescent transcriptional reporters for the expression state of 14 different genes associated with potential cellular subpopulations. We determined the spatial organization of the expression of these genes within biofilms using confocal microscopy, which revealed that many reporters localized to distinct areas of the biofilm, some of which were co-localized. We used flow cytometry to quantify reporter co-expression, which revealed that many cells "multi-task," simultaneously expressing two reporters. These data indicate that prior models describing B. subtilis cells as differentiating into specific cell types, each with a specific task or function, were oversimplified. Only a few subpopulations of cells, including surfactin and plipastatin producers, as well as sporulating and competent cells, appear to have distinct roles based on the set of genes examined here. These data will provide us with a framework with which to further study and make predictions about the roles of diverse cellular phenotypes in B. subtilis biofilms. IMPORTANCE Many microbes differentiate, expressing diverse phenotypes to ensure their survival in various environments. However, studies on phenotypic differentiation have typically examined only a few phenotypes at one time, thus limiting our knowledge about the extent of differentiation and phenotypic overlap in the population. We investigated the spatial organization and gene expression relationships for genes important in B. subtilis biofilms. In doing so, we mapped spatial gene expression patterns and expanded the number of cell populations described in the B. subtilis literature. It is likely that other bacteria also display complex differentiation patterns within their biofilms. Studying the extent of cellular differentiation in other microbes may be important when designing therapies for disease-causing bacteria, where studying only a single phenotype may be masking underlying phenotypic differentiation relevant to infection outcomes.

Dataset Information

Impact of Fatty-Acid Labeling of Bacillus subtilis Membranes on the Cellular Lipidome and Proteome.

Publications

Impact of Fatty-Acid Labeling of <i>Bacillus subtilis</i> Membranes on the Cellular Lipidome and Proteome.

Similar Datasets

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