A comprehensive insight into the lipid composition of Myxococcus xanthus by UPLC-ESI-MS

Abstract

This article is available online at http://www.jlr.org that allows researchers to investigate the lipid repertoire of these bacteria as a whole, in order to unveil alterations in the lipid composition on the molecular level as a function of different metabolic conditions or growth stages.In recent years, progress has been made in elucidating the biosynthesis and putative biological importance of myxobacterial FAs including branched-chain ( 6 ), straightchain ( 7 ), unsaturated ( 8 ), and hydroxylated FAs, as well as sphingoid species ( 9 ), particularly with regard to the best studied myxobacterial model organism Myxococcus xanthus . In these studies, methanolysis-derived FA methyl esters (FAMEs) or selected lipid classes were analyzed by means of GC-MS; there is thus only limited information about the complete lipidome of M. xanthus . High temperature GC-MS was applied to unhydrolyzed lipid extracts in order to elucidate the structures and relative abundance of neutral lipids ( 10 ) and unusual iso-branched alkyldiacylglycerols ( 4 ), molecules that exhibit signaling functions in this organism ( 5 ). HPLC ESI-MS/MS experiments were used to determine changes in the glycerophosphoethanolamine (PE) composition of mutant strains with inactivated acyltransferases ( 11 ) on the one hand, and to determine the ratio of certain ether PEs to acyl PEs during the process of starvation-induced fruiting body formation ( 4 ) on the other hand. In a recent publication on the myxobacterial ether lipid biosynthesis and the importance of these lipids for the myxobacterial life cycle ( 12 ), all these methods had to be combined in order to examine the effects of gene inactivations on the lipid composition of the respective mutants, as the occurrence and abundance of Myxobacteria are a globally occurring order of soildwelling Gram-negative ␦ -proteobacteria that attracts interest due to a wide range of remarkable features including cooperative motility, predation, simple multicellularity [including fungi-like fruiting body formation ( 1 )], and as producers of a wide range of secondary metabolites ( 2 ). Additionally, they exhibit a complex pattern of primary metabolites, with fatty acyl species and associated lipids the most well-studied. Some of these lipids are involved in processes like chemotaxis or starvation-induced fruiting body formation, either as biomarkers or chemotactic signals ( 3-5 ). However, to date, no methodology has been described This work was supported in part by the Emmy Noether program of the Deutsche Forschungsgemeinschaft (DFG).

Neutral and Phospholipids of the Myxococcus xanthus Lipodome during Fruiting Body Formation and Germination

“…After identification of the lipid species by retention time and tandem mass spectrometry, the acquired data were separated into the different lipid classes, resulting in 43 triacylglycerols (TAGs), 7 diacylglycerols (DAGs), 32 phosphatidylethanolamines (PEs), 12 phosphatidylglycerols (PGs), 3 ceramides (Cers), and 4 ceramide phosphoinositol (Cer-PI) species (see Tables S1 and S2 in the supplemental material). In contrast to previous analyses, we could not identify cardiolipins in the lipid extracts, probably because of the minute amount of cardiolipins, which made up less than 1% of total lipids (5,20) and which were thus present at levels below the detection limit. Internal standards for the analysis in both ionization modes allowed correction for possible deviations caused by were chosen as internal standards for the negative and positive ionization modes, respectively, as these lipids are absent in the lipid profile of M. xanthus and PEs and TAGs represent the two most abundant lipid classes in the profile.…”

“…Sample extractions and high-performance liquid chromatography (HPLC) measurements were performed in triplicate as described previously (5). Immediately before the extraction procedure, PE(12:0/12:0) and TAG(18:0/18:0/ 18:0) were added to the dried cells as internal standards to a final concentration of 50 M.…”

“…In lipidomics, several applications describing the analysis of single lipid classes have been published in the past (2-4). Lorenzen et al developed an ultraperformance liquid chromatography (UPLC)-electrospray ionization (ESI)-MS method applicable for the analysis of whole-lipid extracts for their neutral and phospholipid contents, as exemplarily demonstrated for the Gram-negative deltaproteobacterium Myxococcus xanthus (5). Like most myxobacteria, M. xanthus forms spore-containing fruiting bodies upon amino acid depletion (6,7).…”

“…To generate a complete overview of the lipidome profile of M. xanthus during development and myxospore germination, we applied the previously described lipidome analysis (5) to M. xanthus at different stages of its complex life cycle (6, 7) using the lipid compound list already generated by tandem mass spectrometry (5). In addition, different mutant strains with developmental defects were analyzed.…”

Myxococcus CsgA, Drosophila Sniffer, and human HSD10 are cardiolipin phospholipases

“…These data point to the glycerol 2 ′ -OH being the CsgA target. CL and PG both contain this moiety and were examined as possible substrates, as CL is abundant in Myxococcus where it is found almost exclusively in the inner membrane (Orndorff and Dworkin 1980;Lorenzen et al 2014). Both CL and PG are excellent substrates for CsgA (Fig.…”