Investigation of Functional-Dependencies Between PG Hydrolases and The Bifunctional Class A PBPs Across Stress Conditions in E. coli

Abstract

Gram-negative bacteria possess a slender peptidoglycan layer, known as sacculus, positioned between the cytoplasmic and outer membrane. This layer serves as protection against osmotic challenges. Peptidoglycan hydrolases play a crucial role in breaking bonds within the sacculus to facilitate the insertion of newly synthesized peptidoglycan strands. The precise regulation of peptidoglycan synthases and hydrolases is essential to prevent cell lysis, but to date we do not know the detailed molecular mechanism how PG synthases and hydrolases coordinate their activities. To study this, we investigated the genetic interactions of the two PG synthases mrcA (encodes PBP1A) and mrcB (encodes PBP1B) with genes encoding for peptidoglycan amidases and endopeptidases under various envelope stress conditions. Our comprehensive analysis of the genetic interaction network revealed only a limited number of hydrolase gene deletions that led to reduced fitness in the absence of PBP1A or PBP1B. This suggests that none of the amidases or endopeptidases is strictly indispensable for the functioning of class A PBPs, highlighting the robustness of the peptidoglycan growth mechanism. Notably, we observed a significant decrease in the fitness of ∆mrcB cells under high salt stress. This reduction was attributed to the diminished peptidoglycan synthesis activity of PBP1A at elevated salt concentrations, underscoring the impact of environmental conditions on bacterial cell fitness. We also present an approach that utilizes peptidoglycan sacculi labelled with fluorescein-5 isothiocyanate (FITC) to analyze PG hydrolases under controlled laboratory conditions. Within this procedure, we isolate soluble hydrolytic byproducts released by PG hydrolases from the non-soluble FITC-PG sacculi. These byproducts are then measured and set in relation to the total available substrate. This method allows to assay PG hydrolase activity through end-point or time-course assays. Here we assess the amount of fluorescence released from covalently-linked FITC-PG as a result of amidases hydrolytic activity in the presence of two distinct proteins (NlpI and DolP) using optimized FITC release assay to accommodate a 96-well format and compare their activity to a control Lysozyme. However, our analysis suggests that NlpI indirectly impacts AmiA/AmiC activation via amidases regulators EnvC and NlpD, meanwhile DolP is likely not to be NlpD upstream regulator but might function indirectly through yet undiscovered mechanism.