Figure 2 shows the TLC profiles of wild-type PAO1 and the olsA∷lux mutant. In P. aeruginosa PAO1, the major lipid produced under phosphate-rich conditions is likely phosphatidylethanolamine based on a similar mobility of control phosphatidylethanolamine. However, a novel lipid species was produced under phosphate-limiting conditions, along with a significant reduction in phosphatidylethanolamine

(Fig. 2). This novel lipid band was detected with iodine staining of total lipids (data not shown) and by ninhydrin staining for amino group-containing lipids (Fig. 2a). In the olsA∷lux mutant grown under phosphate-limiting conditions, there was no production of this novel phosphate-regulated lipid species and a corresponding increase in phosphatidylethanolamine Navitoclax production (Fig. 2a). The TLC profiles of both strains under phosphate-rich conditions were similar, where phosphatidylethanolamine was the predominant lipid in the membrane (Fig. 2a). The olsA gene was cloned into a medium copy plasmid and introduced into the olsA∷lux mutant, which restored the production of OLs under phosphate-limiting

growth conditions (Fig. 2b). To determine the identity of the novel phosphate-regulated lipid, this band was purified from the TLC plate and analyzed by MS. A positive-ion mode electrospray analysis of the purified lipid revealed major CAL101 signals at 625, 651 and 665 m/z (Fig. 3a). Using the 115 m/z ion characteristic of ornithine (Geiger et al., 1999; Aygun-Sunar et al., 2006), it was possible to determine which of the observed signals corresponded Glycogen branching enzyme to OLs according to the general structure shown in the inset in Fig. 3b. A cluster

of signals from 598 to 706 m/z all contained the 115 m/z ion, strongly implicating the three major signals and several minor less intense signals as molecular ions of OLs. Further confirmation for the presence of a cluster of OLs with varying acyl chains was achieved by MS/MS analysis of each of the major molecular ions. From the molecular anion signal, it is possible to determine the total number of carbon atoms in the two acyl chains and the number of unsaturated bonds (or cyclopropane rings); in the case of the 623.4 molecular anion signal, this corresponded to 32 : 0 (Fig. 4a). A major signal occurs upon cleavage of the terminal fatty acid (see inset) that is characteristic of the OL structure. Cleavage of the terminal fatty acid in Fig. 4a produced a 255 m/z fatty acyl anion of 16 : 0, and the expected signal of 367 was a dominant cleavage product. From these data, it is evident that the amido chain must also be 16 : 0. Further, a 131 m/z cleavage product occurs as expected for OLs (Aygun-Sunar et al., 2006). Similar MS/MS analysis of the 649.6 m/z signal produced two major fragment ions of 367 and 393 m/z, indicating the occurrence of two OLs of the same mass (34 : 1).