Characterizing Suppressor Mutations of bamD7ΔbamE

Author/​Artist
Morss, Alexandra [Browse]
Format
Senior thesis
Language
English
Description
70 pages

Availability

Available Online

Copies in the Library

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    Details

    Advisor(s)
    Silhavy, Tom [Browse]
    Department
    Princeton University. Department of Molecular Biology [Browse]
    Class year
    2013
    Restrictions note
    Walk-in Access. This thesis can only be viewed on computer terminals at the Mudd Manuscript Library.
    Summary note
    Like all Gram-negative bacteria, the cell envelope of Escherichia coli contains an inner and outer membrane. The outer membrane (OM) is extremely important in protecting the cell from the harsh environment in which it lives. In order for nutrients to be brought into the cell, outer membrane proteins (OMPs) in the form of β-barrels provide channels through which these molecules can move. OMPs are assembled by the Bam complex, which is made of a β-barrel protein (BamA) and 4 lipoproteins (BamBCDE). BamA and BamD are both essential for growth and have been found to physically interact with each other. The BamA-D interaction is stabilized by BamE. BamA cycles between different conformations, and both BamD and BamE are involved in controlling this process. Mutations in bamA, bamD, or bamE can disrupt the BamA-BamD interaction and alter the conformation of BamA, thereby compromising OMP assembly. In particular, the temperature-sensitive bamA5 mutation can be suppressed by an intragenic mutation within bamD, called bamD7. On its own, the bamD7 mutant does not display OM defects. However, a bamD7 ΔbamE double mutant is sensitive to antibiotics and growth at high temperatures, and is defective in OMP assembly. To understand the functions of BamD and BamE, we selected for spontaneous antibiotic and temperature resistant mutants of bamD7 ΔbamE. We then categorized them and chose the strongest suppressors based on resistance to antibiotics and restoration of OMP levels. Determining the location of the mutations in the best suppressors will allow us to better understand how the Bam complex functions to create the protective OM.
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