Bacterial Lipid Composition Analysis

The lipid compositions of bacterial strains were determined following labelling with [1-¹⁴C]acetate or [³⁵S]sulfate (Amersham Biosciences). Cultures (1 ml) of wildtype and mutant strains were inoculated from pre-cultures grown in the same medium. After addition of 0.5 μCi of [¹⁴C]acetate (60 mCi mmol⁻¹) or [³⁵S]sulfate to each culture, the cultures were incubated for 4 h. The cells were harvested by centrifugation, washed with 500 μl of water once, resuspended in 100 μl of water and then lipids were extracted according to Bligh and Dyer (1959) (link). Aliquots of the lipid extracts were spotted on high-performance TLC silica gel 60 plates (Merck, Poole, UK) and were separated in two dimensions using chloroform/methanol/ammonium hydroxide (140:60:10, vol./vol./vol.) as a mobile phase for the first dimension and chloroform/methanol/glacial acetic acid/acetone/water (130:10:10:20:3, vol./vol./vol./vol./vol.) for the second dimension. To visualize the membrane lipids, developed two-dimensional TLC plates were exposed to autoradiography film (Kodak) or to a PhosphorImager screen (Amersham Biosciences). The individual lipids were quantified using ImageQuant software (Amersham Biosciences) (Vences-Guzman et al., 2011 (link)).