Molecular approaches to understand the effect of acetic acid on Escherichia coli

Alatar, Fatemah (2022). Molecular approaches to understand the effect of acetic acid on Escherichia coli. University of Birmingham. Ph.D.

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Abstract

Acetic acid has long been known for its antibacterial activity which can be used to treat infected burn wounds. However, there is a lack of a detailed mechanism on acetic acid's effect on E. coli at a molecular level. To learn more about this, we have used Transposon Directed Insertion Sequencing (TraDIS) to investigate the molecular mechanisms by which acetic acid acts as an antibacterial agent, by identifying non-essential genes whose loss alters the fitness of different strains of E. coli. We grew transposon libraries in three different strains of E. coli (uropathogenic E. coli EO499 (serotype 131), uropathogenic UTI89, and the lab strain MG1655) in M9 media + 0.2% casamino acids and 0.2% glucose at neutral pH 7 and mildly acidic pH 5.5, with or without acetic acid. Libraries were sequenced pre- and post-growth, using a transposon-specific primer to generate positions and frequencies for each transposon. RPKMS and insertion indices were generated by a TraDIS pipeline. To determine the impact of acetic acid on gene fitness value, the numbers of reads before and after the stress were compared for each gene in each strain. This enables us to identify genes where transposon inserts lead to a decrease or increase of fitness under acetic acid stress. Comparing the results between the strains will enable the identification of both strain-specific genes and genes shared between strains that have a role in fitness under acetic acid stress.
This project consists of two parts. In the first part, we evaluated the roles of candidate genes identified in a previously generated EO499 TraDIS library under acetic acid. Eight of these were depleted under acetic acid stress and they were chosen for further study: nuoM, nuoG, sucA, sthA, pitA, apaH, rssB and ytfP. Because of the difficulties of constructing gene deletions in the uropathogenic strain for validating the TraDIS results, we tested the relative fitness of the corresponding gene deletion mutants from the Keio library (in lab strain BW25113), with the growth conditions used for EO499. Interestingly, only a few knockouts showed a reduction in relative fitness in competitions at pH 5.5 with acetic acid. This may occur due to the differences between strains used in TraDIS and competition. To overcome this issue, we have also isolated transposon mutants from E. coli EO499 transposon library to determine relative fitness.
In the second part, we have optimized and constructed a UTI89 transposon library. The three E. coli (EO499, MG1655 and UTI89) transposon libraries were subjected to acetic acid stress as described, for a passaged for a time course of five days. Only day one and day five were sequenced. For the analysis, several bioinformatic pipelines were used for E. coli genome annotation and sequencing analysis. TraDIS allowed us to identify essential genes in three E. coli strains. The results presented here show which genes tend to be enriched or depleted under acetic acid.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):