The TLC solvent system is indicated. Ori, origin; SF, solvent front. The presence of GPLs was probed for in lipid samples from Ms WT + pCP0, Ms ΔgplH + pCP0, and Ms ΔgplH + pCP0-gplH (complemented strain) by GC-MS analysis as well. The pCP0-bearing strains, Ms WT + pCP0 and Ms ΔgplH + pCP0, rather than their check details respective plasmid-free parental strains, were used in these experiments so that the WT, the mutant, and the complemented strain could all be cultured under identical conditions (i.e., kanamycin-containing

growth medium) for comparative analysis by GC-MS. Representative results from the GC-MS analysis are selleckchem shown in Figure 6. This analysis probed for the presence of the alditol acetate derivatives of the characteristic glycosyl residues of Ms GPLs as a fingerprint indicator of the presence of GPLs in the lipid samples analyzed [47]. The GC-MS analysis of samples from Ms WT + pCP0 revealed the expected m/z peak array consistent with the characteristic presence of alditol acetate derivatives of the 2,3,4-trimethyl-rhamnose, 3,4-dimethyl-rhamnose and 6-deoxy-talose components of GPLs [7, 8, 47]. Conversely, these alditol acetate derivatives were not detected

by GC-MS analysis of samples from Ms ΔgplH + pCP0. The samples from the complemented strain, Ms ΔgplH + pCP0-gplH, displayed an m/z peak array comparable to that of Ms WT + pCP0 and consistent with the presence of the alditol acetate derivatives

originating from OSI-906 manufacturer GPLs (not shown). Overall, the results of the GC-MS analysis and the results of the TLC analysis are in agreement with each other and, coupled with our genetic complementation-controlled analysis, conclusively demonstrate that gplH is essential for production of GPLs. Figure 6 GC-MS analysis of alditol acetate derivatives of the glycosyl residues of GPLs. (A) Total ion count chromatographs displaying the presence or absence of alditol acetates in extracted lipid samples from the strains indicated. (B) Mass spectra showing fragmentation pattern fingerprints RVX-208 demonstrating alditol acetate identity in peaks labeled 2,3,4-trimethyl- rhamnose (1), 3,4-dimethyl-rhamnose (2), and 6-deoxy-talose (3) from Ms WT + pCP0. Equivalent spectra were observed for the samples of Ms ΔgplH + pCP0-gplH (not shown). The selective ion monitoring MS analysis of the mutant strain Ms ΔgplH revealed that the strain lacks the alditol acetate derivatives. For illustration clarity, only the m/z values of selected diagnostic molecular ions are indicated in the spectra. These molecular ions arise from the fragmentation patterns of the corresponding alditol acetates as displayed next to each spectrum.