Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth.

Emily C A Goodall,Christopher Icke,Zihao Yang,Wee Boon Tan,Isabel Warner,Gabriela Boelter,Adam F Cunningham,Waldemar Vollmer,Karthik Pullela,Antoine Delhaye,Ian R Henderson,Alun Jones,Bing Zhang,Matthias Winkle,Robert G Parton,Jack A Bryant,Rochelle Da Costa,Georgia L Isom,James Rae,Eva Heinz,Manuel Banzhaf,Jeff Cole ,Shu‐Sin Chng ,Mark A T Blaskovich ,Jessica L Rooke ,Jean‐Francois Collet

doi:10.1371/journal.pgen.1009586

Abstract

The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

Highlights

The bacterial cell envelope plays a fundamental role in protection, host interaction, energy generation, expulsion of toxic substances and coordination of growth and cell division
Using a unique combination of high throughput chemical genetic screens we identified yhcB, a conserved gene of unknown function, required for the maintenance of cell envelope integrity in Escherichia coli
We posited that if the transposon disrupts a gene required for the maintenance of cell envelope integrity, the resulting mutant will become more susceptible to polymyxin B, and the gene would be identified through our Transposon insertion sequencing (TIS) screen as these mutants will be outcompeted during growth; identifiable as a depletion in transposon-insertions

Summary

Introduction

The bacterial cell envelope plays a fundamental role in protection, host interaction, energy generation, expulsion of toxic substances and coordination of growth and cell division. The OM is an asymmetrical bilayer of phospholipids and lipopolysaccharide (LPS), which forms a strong permeability barrier conferring resistance to many toxic antimicrobials. Both membranes are studded with integral membrane proteins and peripheral lipoproteins that facilitate cellular functions [2,3,4]. Each component of the cell envelope must be synthesised and assembled in a coordinated fashion to maintain cell envelope homeostasis and viability Understanding how this complex envelope is synthesised and maintained is instrumental to understanding how to disrupt its function, to kill the bacterium, or render the organism susceptible to otherwise ineffective treatments

Methods

Results

Discussion

Conclusion