Investigation of Functional-Dependencies Between PG Hydrolases and The Bifunctional Class A PBPs Across Stress Conditions in E. coli
Date
2024-01-04
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University of Birmingham
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.
Description
Keywords
peptidoglycan Hydrolases, and Bifunctional Class A PBPs