Use of high-density transposon libraries to analyze oxidative protein folding and protein translocation in Escherichia coli

Jiang, Chen (2025). Use of high-density transposon libraries to analyze oxidative protein folding and protein translocation in Escherichia coli. University of Birmingham. Ph.D.

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Abstract

Transposon mutagenesis is a widely used method to introduce random mutations into bacterial genomes. Transposon directed insertion-site sequencing (TraDIS) combines transposon mutagenesis with next-generation sequencing to identify genes required for viability under experimental conditions, which provides linkage of phenotype to genotype. In this study, the Tn5-based transposon mutant libraries were used to investigate the oxidative protein folding in Escherichia coli (E. coli) cytoplasm and the azide resistance in E. coli K-12 strain MG1655. SHuffle strains are engineered to produce disulfide bonded proteins to high yields within the cytoplasm. TraDIS was used to analyze the physiology differences between SHuffle strains and their wild type strains by comparing the essential genes in each strain. SHuffle B was identified to have nearly two-fold unique essential genes that are mainly involved in cell response to oxidative stress, suggesting that the SHuffle mutations might cause more oxidative stress in E. coli B. The transposon libraries were further used to probe the genomic requirement to suppress the temperature sensitivity of SHuffle B. Insertion disrupting genes involved in aerobic respiration decreased susceptibility of SHuffle B to higher temperatures. TraDIS was also used to investigate the possible function of trigger factor (TF) and DsbC by determine the essential genes of E. coli strains containing ∆tig and ∆dsbC mutations, which increase susceptibility to sodium azide. Since the mechanism of azide was yet unknown, six transposon mutations conferred azide-resistance were isolated from the transposon library. All of affected genes were involved in ribosomal biogenesis. SecA migrated as two isoforms in SDS-PAGE, the large isoform was fulllength SecA and the small isoform was determined to be C-terminal tail truncated SecA. Expression of both isoforms increased in the isolated azide-resistant mutants in the presence of azide. However, only the small isoform accumulated in the azide-resistant secA mutants in the presence of azide, suggesting that the expression of the small isoform could affect the susceptibility to azide and two isoforms might have distinct physiological roles in vivo. The results presented highlight the potential applications of transposon mutagenesis in dealing with complex biological questions.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):