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Published on: January 27, 2021
A pmrA constitutive mutant sensitizes Escherichia coli to deoxycholic acid
Jamie M Froelich1, Khoa Tran, Daniel Wall
1Department of Microbiology, Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, San Diego, CA 92121, USA.
Abstract:
An Escherichia coli mutant was isolated and shown to be polymyxin B resistant. Mapping and sequence analysis revealed a missense mutation at codon 53 within the pmrA (basR) gene that results in a G-to-V substitution. Fusions of promoters from the pmrC, yibD, and pmrH genes with the lacZ reporter showed that they were constitutively expressed in pmrA53 cells. In pmrA+ strains, these promoters were induced by iron and zinc, while a DeltapmrA mutation blocked induction. The PmrA regulon regulates genes whose products remodel the composition and charge of lipid A and hence the barrier properties of the outer membrane. Along these lines, the pmrA53 mutant was also found to be hypersensitive to the anionic bile detergent deoxycholic acid.
Insights
A novel Escherichia coli mutation in the pmrA gene confers resistance to polymyxin B. This genetic alteration constitutively expresses key genes involved in outer membrane remodeling, impacting bacterial cell surface properties.
Area of Science:
- Microbiology
- Bacterial genetics
- Molecular biology
Background:
- Polymyxin B is a critical antibiotic targeting the outer membrane of Gram-negative bacteria.
- The pmrA (basR) gene plays a role in regulating bacterial outer membrane properties.
- Understanding resistance mechanisms is crucial for combating antibiotic challenges.
Purpose of the Study:
- To characterize a polymyxin B resistant Escherichia coli mutant.
- To elucidate the molecular basis of resistance conferred by a specific pmrA mutation.
- To investigate the impact of this mutation on gene expression and outer membrane characteristics.
Main Methods:
- Isolation and genetic mapping of a polymyxin B resistant mutant.
- DNA sequencing to identify mutations within the pmrA gene.
- Construction of promoter-lacZ reporter fusions to assess gene expression.
- Analysis of gene regulation by PmrA under different conditions (metal ions, mutations).
Main Results:
- A missense mutation (G53V) was identified in the pmrA gene of the resistant mutant.
- The pmrA53 mutation led to constitutive expression of pmrC, yibD, and pmrH genes.
- These genes, part of the PmrA regulon, are normally induced by iron and zinc.
- The mutant exhibited hypersensitivity to deoxycholic acid, indicating altered outer membrane properties.
Conclusions:
- The G53V substitution in PmrA disrupts its regulatory function, leading to constitutive expression of genes involved in lipid A modification.
- Altered lipid A composition affects outer membrane charge and barrier function, contributing to polymyxin B resistance and deoxycholic acid sensitivity.
- This study highlights the complex role of the PmrA regulon in bacterial envelope homeostasis and antibiotic resistance.
