A genetic interaction study of the β-barrel assembly pathway

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Staunton, Kara A. (2021). A genetic interaction study of the β-barrel assembly pathway. University of Birmingham. Ph.D.

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Abstract

In Escherichia coli, the BAM complex is the β-barrel assembly machinery that is essential for the incorporation of proteins (OMPs) into the outer membrane. It consists of two essential components, BamA and BamD and three non-essential components, BamB, BamC and BamE. SurA, Skp and DegP are believed to be chaperones that transport OMPs to the BAM complex. The first aim of this project was to determine whether the non-essential BAM components have distinct physiological roles. A second aim was to determine whether the three putative chaperones are functionally redundant. A third aim was to reveal how OMP biogenesis coordinates with other cell envelope pathways. To address these unknowns, transposon-directed insertion-site sequencing (TraDIS) was utilised to identify genes that are essential in mutants defective in bamB, bamC, bamE, surA, skp and degP but not in the parent E. coli K-12 TraDIS library. In the ΔbamB, ΔbamC and ΔbamE TraDIS libraries, 29, 39 and 17 conditionally essential genes were identified, respectively. There were clear differences between these three datasets. Thus, the functions of BamB, BamC and BamE do not overlap. In the ΔsurA, Δskp and ΔdegP TraDIS libraries, 54, 9 and 44 conditionally essential genes were identified, respectively. Seventeen genes were conditionally essential in both the ΔsurA and ΔdegP TraDIS libraries, which suggests that the function of these two proteins partially overlap. In contrast, there were differences between the conditionally essential genes in the ΔsurA and Δskp datasets. Thus, the functions of SurA and Skp are not redundant. The TraDIS data also demonstrated that loss of genes involved in the synthesis and incorporation of heptose into lipopolysaccharide, combined with loss of bamB, surA or degP, is lethal to the cell. These LPS defects increased membrane fluidity, which decreased BAM activity. Cell death occurs due to a lack of OMP insertion into the OM. This study also identified synthetic lethality between surA and genes involved in the synthesis of enterobacterial common antigen and between members of the BAM pathway and the gene dapF, which is involved in peptidoglycan synthesis. Thus, OMP biogenesis requires a network of components and impairments in OMP biogenesis has negative consequences on other pathways.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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