All ETDs from UAB

Advisory Committee Chair

Patrick N Higgins

Advisory Committee Members

David A Schneider

Sunnie R Thompson

Tim M Townes

Janet L Yother

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Bacterial chromosomes are maintained under constant negative supercoiled stress that influences almost all cellular processes including DNA replication, transcription, segregation, site-specific and homologous recombination, and other DNA transposition. The average supercoiling density of the chromosome is generated by gyrase, and supercoil equilibrium is maintained by two other topoisomerases topo I and topo IV. The supercoils generated by gyrase can diffuse over 10 kb domains. The other half of the chromosome's supercoil structure is connected to abundant nucleoid-associated proteins (NAPs). My goal was to characterize factors other than gyrase that participate in generating or regulating supercoil dynamics in Escherichia coli and Salmonella Typhimurium. We discovered that transcription and gyrase catalytic speeds are matched and the two processes are mechanistically linked. Mutation in gyrase with a slow catalytic cycle decreased both diffusible supercoiling and transcription elongation rates. Mutation in RNAP ß′-subunit slowed transcription rates, which resulted in increased levels of diffusible supercoiling. To study the effect of NAPs on diffusible supercoiling, γδ resolution efficiency in WT, hupA, hupB, and hupAhupB double mutants were measured. The resolution efficiency (diffusible supercoiling density) was maintained better in cells that make HupA homodimers than HupB homodimers. Resolution efficiency dropped 7-fold in strains carrying deletions of both hupA and hupB. Surprisingly, inhibition of transcription did not increase resolution efficiency as it does in WT and single mutants. I propose a new model in which histone-like protein (HU) enhances dynamic chromosome slithering and branching. When HU is absent, the chromosome collapses into a liquid-crystalline form that is good for long term DNA stability but is inhibitory for site-specific recombination and DNA transcription, resulting in decreased growth rate. Finally, I have made E. coli strains with the supercoil sensor inserted at 9 positions in its genome. Resolution data suggest E. coli generates a chromosome with 15% higher diffusible supercoil levels than Salmonella. Perhaps because of this supercoil difference, deletions in FIS are much more severe in Salmonella than in E. coli, which indicates that the supercoil difference causes a different essential gene landscape in these two closely related bacterial species.

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