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.
Highlights
Biological membranes fulfill many critical roles in the cell; the details of which are rooted in its structure, composition, biochemistry, and biophysical properties
The control of fatty acid (FA) incorporation in B. subtilis using the cerulenin method was tested by gas chromatography/mass spectrometry (GC/MS) analysis of FA methyl esters (FAMEs) extracted from two aliquots of B. subtilis cells containing a deletion of the fadN gene which were cultured in a minimal M9 glucose medium prepared with H2O (Figure 1)
This regulation is in expected to be associated with an accumulation of FA precursors, yet it is unclear how subsequent rescue of cell growth with exogenous FAs will impact FabF expression and other enzymes within the FA biosynthesis pathway in response to accumulation of acyl-ACP and free FAs due to exogenous addition
Summary
Biological membranes fulfill many critical roles in the cell; the details of which are rooted in its structure, composition, biochemistry, and biophysical properties. Perhaps foremost among current questions of the cell membrane center around the existence, composition, size and roles of lipid rafts. The lipid raft hypothesis (Simons and Ikonen, 1997) invokes lateral organization of membrane lipids and proteins into distinct domains in the plane of the membrane to facilitate the assembly and regulation of multimolecular complexes. This hypothesis provides a compelling rationale for numerous observations relating to membrane trafficking, endocytosis, signal transduction, and other processes (Simons and Toomre, 2000; Simons and Ehehalt, 2002; Shaw, 2006; Allen et al, 2007). Experimental evidence describing lipid rafts has been largely inferential due to the limitations of traditional techniques, and it is widely believed that these features are nanoscopic, as well as transient (Mukherjee and Maxfield, 2004; Lingwood and Simons, 2010), making them difficult to detect in vivo
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