Peptidoglycan Compositional Analysis of Mycobacterium Smegmatis Using High-resolution LC-MS

Peptidoglycan (PG) is the exoskeleton of bacterial cells and is required for their viability, growth, and cell division. Unlike most bacteria, mycobacteria possess an atypical PG characterized by a high degree of unique linkages and chemical modifications which most likely serve as important determinants of virulence and pathogenesis in mycobacterial diseases. Despite this important role, the chemical composition and molecular architecture of mycobacterial PG have yet to be fully determined. Here we determined the chemical composition of PG from Mycobacterium smegmatis using high-resolution liquid chromatography-mass spectrometry. Purified cell walls from the stationary phase were digested with mutanolysin and compositional analysis was performed on 130 muropeptide ions that were identified using an in silico PG library. The relative abundance for each muropeptide ion was measured by integrating the extracted-ion chromatogram. The percentage of crosslink per PG subunit was measured at 45%. While both 3→3 and 4→3 transpeptide cross-linkages were found in PG dimers, a high abundance of 3→3 linkages was found associated with the trimers. Approximately 43% of disaccharides in the PG of M. smegmatis showed modifications by acetylation or deacetylation. A significant number of PG trimers are found with a loss of 41.00 amu that is consistent with N-deacetylation, whereas the dimers show a gain of 42.01 amu corresponding to O-acetylation of the PG disaccharides. This suggests a possible role of PG acetylation in the regulation of cell wall homeostasis in M. smegmatis. Collectively, these data report important novel insights into the ultrastructure of mycobacterial PG.