Date of Award

1992

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Randall C. Gayda

Abstract

FtsA is an essential cell division protein which is synthesized in minute amounts in Escherichia coli. To study the effects of overexpressing ftsA on the phenotype of E. coli cells, DNA fragments encoding the ftsA gene were subcloned downstream of a lac or a tac promoter in two plasmids. High-level expression of the ftsA gene from these promoters inhibited normal cell septation and caused the cells to become long, nonseptate filaments. Continued overexpression of ftsA resulted in the filaments developing spherical bulges up to 4 um in diameter. It is suggested that these bulges may emanate from septation sites because they were evenly spaced in relation to one another and to the cell poles. Observations of thin sections by electron microscopy demonstrated that these bulges contained small electron dense regions and large electron-lucent plate-like inclusions. A finding that the bulging filamentous cells contain more hexosamine per mass than control cells suggests that abnormal peptidoglycan synthesis might be occurring. FtsA protein was isolated from ftsA-overexpressing cells for the purpose of raising monoclonal antibodies. Mice were immunized with an FtsA fraction and their spleen cells were fused to Sp2/0-AG14 mouse myeloma cells. Hybrid cells were screened and two clones were positively identified as FtsA monoclonal antibody producers by enzyme-linked immunosorbant assays (ELISA) and Western blotting. The isolation of FtsA monoclonal antibodies provided a way to determine the average number of FtsA molecules per cell, that was between 50 and 200. In contrast, the concentration of FtsA normalized to total cell protein was constant over a wide range of growth rates. This finding supports the hypothesis of FtsA protein, being a stoichiometric component in septation.

Pages

101

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