Abstract
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.
Highlights
Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating -1,4 –linked disaccharide of N-acetylglucosamine and N-acetylmuramic acid appended with a highly conserved stem peptide
Planktonic and biofilm cultures were initially inoculated with 5 ϫ 108 cfu of P. aeruginosa PAO1, and after 96 h of growth, four times more cellular biomass was obtained, which is consistent with observed reductions in growth rate in biofilm cultures [25,26,27,28]
The quantity of purified sacculi obtained from the planktonic cultures (58.6 Ϯ 5.7 mg of sacculi) when compared with biofilm cultures (13.7 Ϯ 1.6 mg of sacculi) remained constant per weight of cellular biomass, indicating that the reduction in purified sacculi in biofilm cultures was due to reduced growth rate
Summary
Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating -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. Numerous lytic enzymes have been identified that cleave at various locations in the PG structure [7, 8] These enzymes include peptidases and amidases that cleave the peptide side chain, as well as glucosaminidases, muramidases, and lytic transglycosylases that cleave the glycan chain. The replacement of the terminal D-alanine with D-serine or D-lactate has been shown to impart resistance to some antimicrobials in Gram-positive organisms [13,14,15]
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