Bacterial Lipid Composition Analysis

The lipid compositions of bacterial strains were determined following labeling with [1-¹⁴C]acetate (Amersham Biosciences). Cultures (1 ml) of Burkholderia s.l. strains were inoculated from precultures grown in the same medium. After addition of 1 μCi of [¹⁴C]acetate (60 mCi mmol-1) to each culture, the cultures were incubated overnight. Cells were harvested by centrifugation, washed with 500 μl water once, resuspended in 100 μl water, and then lipids were extracted according to Bligh and Dyer (Bligh and Dyer, 1959 (link)). Aliquots of the lipid extracts were spotted on high performance TLC silica gel 60 plates (Merck, Poole, UK) and separated in two dimensions using chloroform/methanol/water (16:4:1, v/v/v) as a mobile phase for the first dimension and chloroform/methanol/acetic acid (15:3:2, v/v/v) as a mobile phase for the second dimension (Tahara and Fujiyoshi, 1994 (link)). To visualize 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-Guzmán et al., 2011 (link)).