Profiling and tandem mass spectrometry analysis of aminoacylated phospholipids in Bacillus subtilis .

Cationic modulation of the dominantly negative electrostatic structure of phospholipids plays an important role in bacterial response to changes in the environment. In addition to zwitterionic phosphatidylethanolamine, Gram-positive bacteria are also abundant in positively charged lysyl-phosphatidylglycerol. Increased amounts of both types of lipids render Gram-positive bacterial cells more resistant to cationic antibiotic peptides such as defensins.  Lysyl and alanyl-phosphatidylglycerol as well as alanyl-cardiolipin have also been studied by mass spectroscopy. Phospholipids modified by other amino acids have been discovered by chemical analysis of the lipid lysate but have yet to be studied by mass spectroscopy. We exploited the high sensitivity of modern mass spectroscopy in searching for substructures in complex mixtures to establish a sensitive and thorough screen for aminoacylated phospholipids. The search for deprotonated aminoacyl anions in lipid extracted from Bacillus subtilis strain 168 yielded strong evidence as well as relative abundance of aminoacyl-phosphatidylglycerols, which serves as a crude measure of the specificity of aminoacyl-phosphatidylglycerol synthase MprF. No aminoacyl-cardiolipin was found. More importantly, the second most abundant species in this category is D-alanyl-phosphatidylglycerol, suggesting a possible role in the D-alanylation pathway of wall- and lipo-teichoic acids.

Characterization of N-Succinylation of L-Lysylphosphatidylglycerol in Bacillus subtilis Using Tandem Mass Spectrometry.

Phospholipids generally dominate in bacterial lipids. The negatively charged nature of phospholipids renders bacteria susceptible to cationic antibiotic peptides. In comparison with Gram-negative bacteria, Gram-positive bacteria in general have much less zwitterionic phosphatidylethanolamine. However, they are known for producing aminoacylated phosphatidylglycerol (PG), especially positively charged L-lysyl-PG, which is catalyzed by lysyl-PG synthase MprF, which appears to have a broad range of specificity for L-aminoacyl transfer RNAs. In addition, many Gram-positive bacteria also have a dlt-gene-coded D-alanylation pathway for lipoteichoic acids and wall teichoic acids covalently attached to a glycolipid or peptidoglycan. D-Alanylation also masks the dominant negative charge of the phosphate-rich polymers of teichoic acids. Using mass spectrometry, we have recently observed that precursor scans in negative mode for deprotonated amino acid fragments were most sensitive for ester-linked amino acids. Such a scan for precursors generating an m/z 145 lysyl anion revealed lysyl-PG as well as an additional species 100 m/z units greater than lysyl-PG. This unexpected species corresponded precisely to the expected mass of N-succinylated lysyl-PG. Tandem mass spectrometry revealed a precise match to the fragmentation pattern of this putative new species. PG, lysyl-PG, and N-succinyl-lysyl-PG may form a complete loop of charge reversal from -1 to +1 and then back to -1. Analogous charge reversal by N-succinylation of lysine residues in the bacterial as well as eukaryotic proteomes has been recently discovered as a major posttranslational modification. Such modification in bacterial lipids is possibly catalyzed by an enzyme homologous to the enzymes that modify lysine residues in proteins. Graphical Abstract ᅟ.