Genome-wide peptidoglycan profiling of Vibrio cholerae

Abstract

Most bacteria cells are protected by a peptidoglycan cell wall. Defining the chemical structure of the peptidoglycan has been instrumental to characterize cell wall associated proteins and to illuminate the mode of action of cell wall-acting antibiotics. However, a major roadblock for a comprehensive understanding of peptidoglycan homeostasis has been the lack of methods to conduct large-scale, systematic studies. Here we have developed and applied an innovative high throughput peptidoglycan analytical pipeline to analyze the entire non-essential, arrayed mutant library of Vibrio cholerae. The unprecedented breadth of these analyses revealed that peptidoglycan homeostasis is preserved by a large percentage of the genome organized in complex networks that functionally link peptidoglycan features with genetic determinants. As an example, we discovered a novel bifunctional penicillin-binding protein in V. cholerae. Collectively, genome-wide peptidoglycan profiling provides a fast, easy, and unbiased method for systematic identification of the genetic determinants of peptidoglycan synthesis and remodeling.

Genetic interaction mapping reveals functional relationships between peptidoglycan endopeptidases and carboxypeptidases

“…To further test this, we purified sacculi from the ΔdacA1 mutant and ΔdacA1 murA :: murA L35F and analyzed them by LC/MS. The mutation in murA resulted in a significant decrease in the relative PG amount, which usually correlates with decreased PG synthesis [ 24 ] ( S8 Fig ). These results further corroborate the hypothesis that the rescue of the ΔdacA1 mutant by the hypomorphic mutations in the mur genes is due to a reduction in PG synthesis.…”

Peptidoglycan remodeling in response to cell wall acting antibiotics inBacillus subtilis

“…High-throughput PG isolation, muropeptide separation and quantification were realized essentially as described in (Hernandez et al 2022). Briefly, sacculi were isolated by resuspension of 500μl cell pellet in LB with 1.6% SDS and autoclaving 15 min at 120°C and 1 atm.…”

“…Here, we have performed the first comprehensive analysis of the effect of PG-targeting antibiotics in the composition and structure of the bacterial CW. Using a high-throughput approach for the preparation and analysis of PG samples (Hernandez et al 2022), we studied the changes in the sacculus of B. subtilis in response to exposure to lysozyme and to six antibiotics targeting different steps of the PG biosynthetic pathway at different sub-lethal concentrations and growing conditions. Notably, our study reveals that cells adapt to the presence of PG synthesis inhibitors: after long exposure times, cultures reach similar optical densities as untreated cultures and restore their pool of intracellular soluble PG precursors.…”