Figure 2. A genetic screen identified ceramide synthesis enzymes.

(a-d) Negative ion ESI/MS shows the [M + Cl]⁻ ions of the lipids emerging at 2 to 3 min. Ceramide species are labelled with a red dot and the modified lipid moiety is designated with a red-dashed oval. Note that MS/MS analysis of ceramide from C. crescentus shows that the desaturation occurs on the acyl chain (see Extended Data Fig. 1e); however, we have not determined the precise position of the double bond. In this, and all subsequent figures, the structural cartoons only indicate which acyl chain is desaturated, but not the exact position of the double bond. Relative quantification of all the major lipid species for each mass spectrum is available in Supplementary Table 3. (a) Lipids were extracted from wild-type, Δccna_01222, and ccna_01222-complemented cells. (b) Lipids were extracted from Δccna_01212 and ccna_01212-complemented cells. (c) Recombinant CCNA_01212 was incubated with 40 μM 3-KDS and 50 μM C16:0-CoA for 1 hr and the reaction product was analyzed by normal phase LC/ESI-MS in negative ion mode. (d) Lipids were extracted from Δccna_00202 and ccna_00202-complemented cells. (e) Based on the MS data above, we propose the following model for bacterial ceramide synthesis. The genes comprising this synthetic pathway are in close proximity in the genome (see Extended Data Fig. 2b). (f) Cells expressing the indicated fluorescently-tagged proteins were grown overnight in the presence of inducer. GspG-mCherry and TAT-mCherry are control inner-membrane and periplasmic proteins, respectively. Control and permeabilized cells were imaged by fluorescence microscopy to monitor the loss of fluorescence upon permeabilization. The results are the overlay of phase and fluorescent images. Scale bar = 5 μm.