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

Abstract

Abstract. 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 Nsuccinylated 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.

Integrated Succinylome and Metabolome Profiling Reveals Crucial Role of S-Ribosylhomocysteine Lyase in Quorum Sensing and Metabolism of Aeromonas hydrophila*

“…Proteomics is a recently developed powerful approach for investigating protein PTM sites (22)(23)(24). Here, we combined high-affinity purification with high sensitivity mass spectrometry technologies to identify the entire succinylome of A. hydrophila ATCC7966.…”

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances

“…32 For example, the LysPGS from Agrobacterium tumefaciens and the AlaPGS from C. perfringens specifically use Lys-tRNA and Ala-tRNA respectively, while the aaPGS from Bacillus subtilis utilizes both Lys- and Ala-tRNA as amino acid donors in vitro . 32 Interestingly both Lys-PG and Ala-PG were initially detected in the membrane of B. subtilis , 33 but a recent report suggests that D-Ala-PG and not L -Ala-PG is formed in B. subtilis by an aaPGS independent pathway. 34 One paralog in Enterococcus faecium (referred to as RakPGS or MprF2) displays a more relaxed specificity for its aa-tRNA and can utilize Ala-, Lys-, and Arg-tRNAs.…”

“…A recent report showed that Lys-PG in B. subtilis is used to produce N-succinylated Lys-PG. 33 The biologic significance of and enzyme responsible for this modification are not known. However, it was proposed that a homolog of the enzyme responsible for protein succinylation, recently discovered in bacteria, may be accountable for Lys-PG modification.…”

Comparative study on nutrient depletion-induced lipidome adaptations in Staphylococcus haemolyticus and Staphylococcus epidermidis

“…Small amounts of lysyl-DAG were detected in S. epidermidis lipids by a neutral loss scan for 146 amu lysine in positive mode (supplementary data). S. haemolyticus , however, produced N-succinyl-lysyl-PG as described previously 22 . In both lipidomes, PG was the predominant species followed by lysyl-PG, DGDG, and DAG (Fig.…”

“…subtilis 21 . An ampicillin-resistant bacterium that produced N-succinyl-lysyl-phosphatidylglycerol 22 was identified as S . haemolyticus , a species that drew immediate attention to other Staphylococci pathogens.…”