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

Abstract

Discuss this article AbstractCationic 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 strain 168 yielded strong evidence as well Bacillus subtilis 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

“…On the other hand, with either the phosphate group or the hydroxyl group in the PG backbone as the nucleophile, the noncyclic m/z 245 ion can be produced (Figure 3, reaction 4). This m/z 245 anion is equivalent to prominent fatty acyl fragments observed for phosphoglyceride anions [18] or aminoacyl anion dissociated from aminoacylated PG [14].…”

“…The fragmentation pattern of the putative Nsuccinylated lysyl-PG resembles that of PG [18] and aminoacylated PG [14] and can be fully explained by dissociations at multiple ester bonds and the amide bond between lysine and succinic acid residues (see the electronic supplementary material). For instance, phosphoglyceride anions all undergo collision-induced dissociation to produce prominent fragments of fatty acyl anions, aminoacyl anions, and cyclized and intact head group anions.…”

“…We have recently developed a sensitive MS profiling method by searching for precursors of deprotonated amino acid ions in bacterial lipids [14]. The search for the m/z 145 lysyl anion revealed a cluster of lysyl-PG as expected, with the dominant species being 32:0 lysyl-PG at m/z 849.…”

“…Unlike lipids extracted from the wild-type strain, which had two major fatty acyl compositions of 30:0 and 32:0 for PG, alanyl-PG, and lysyl-PG, lipids from the mutant strain had only one major composition of 32:0. The most sensitive methods for profiling aminoacyl-PGs were the precursor scans for deprotonated amino acid fragments [14]. Lipids from B. subtilis were extremely abundant in lysyl-PG and somewhat rich in alanyl-PG [14].…”

“…The most sensitive methods for profiling aminoacyl-PGs were the precursor scans for deprotonated amino acid fragments [14]. Lipids from B. subtilis were extremely abundant in lysyl-PG and somewhat rich in alanyl-PG [14]. .…”