Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating β-1,4-linked disaccharide of N-acetylglucosamine and N-acetylmuramic acid appended with a highly conserved stem peptide. In Gram-negative bacteria, PG is assembled in the cytoplasm and exported into the periplasm where it undergoes considerable maturation, modification, or degradation depending on the growth phase or presence of environmental stressors. These modifications serve important functions in diverse processes, including PG turnover, cell elongation/division, and antibiotic resistance. Conventional methods for analyzing PG composition are complex and time-consuming. We present here a streamlined MS-based method that combines differential analysis with statistical 1D annotation approaches to quantitatively compare PGs produced in planktonic- and biofilm-cultured Pseudomonas aeruginosa We identified a core assembly of PG that is present in high abundance and that does not significantly differ between the two growth states. We also identified an adaptive PG assembly that is present in smaller amounts and fluctuates considerably between growth states in response to physiological changes. Biofilm-derived adaptive PG exhibited significant changes compared with planktonic-derived PG, including amino acid substitutions of the stem peptide and modifications that indicate changes in the activity of amidases, deacetylases, and lytic transglycosylases. The results of this work also provide first evidence of de-N-acetylated muropeptides from P. aeruginosa The method developed here offers a robust and reproducible workflow for accurately determining PG composition in samples that can be used to assess global PG fluctuations in response to changing growth conditions or external stimuli.
Keywords: Pseudomonas aeruginosa (P. aeruginosa); bacterial cell wall; biofilm; mass spectrometry (MS); microbiology; murine; peptidoglycan; peptidoglycomics; planktonic; sacculi.
© 2020 Anderson et al.
Conflict of interest statement
The authors declare that they have no conflicts of interest with the contents of this article
Peptidoglycan Compositional Analysis of Enterococcus Faecalis Biofilm by Stable Isotope Labeling by Amino Acids in a Bacterial CultureJD Chang et al. Biochemistry 57 (7), 1274-1283. PMID 29368511.Peptidoglycan (PG) is a major component of the cell wall in Enterococcus faecalis. Accurate analysis of PG composition provides crucial insights into the bacterium's cell …
Helicobacter Pylori Peptidoglycan Modifications Confer Lysozyme Resistance and Contribute to Survival in the HostG Wang et al. mBio 3 (6), e00409-12. PMID 23221800.Pathogenic bacteria evade host antibacterial enzymes by a variety of mechanisms, which include resisting lytic enzymes abundant in the host. Enzymatic modifications to pe …
High-Resolution Analysis of the Peptidoglycan Composition in Streptomyces CoelicolorLT van der Aart et al. J Bacteriol 200 (20). PMID 30061355.The bacterial cell wall maintains cell shape and protects against bursting by turgor. A major constituent of the cell wall is peptidoglycan (PG), which is continuously mo …
Cell Wall Hydrolases in Bacteria: Insight on the Diversity of Cell Wall Amidases, Glycosidases and Peptidases Toward PeptidoglycanA Vermassen et al. Front Microbiol 10, 331. PMID 30873139. - ReviewThe cell wall (CW) of bacteria is an intricate arrangement of macromolecules, at least constituted of peptidoglycan (PG) but also of (lipo)teichoic acids, various polysac …
Cell-wall Recycling and Synthesis in Escherichia Coli and Pseudomonas Aeruginosa - Their Role in the Development of ResistanceS Dhar et al. J Med Microbiol 67 (1), 1-21. PMID 29185941. - ReviewThe bacterial cell-wall that forms a protective layer over the inner membrane is called the murein sacculus - a tightly cross-linked peptidoglycan mesh unique to bacteria …