Title

Evidence for a Direct Link between the Tol-Pal Protein Complex and Gram Negative Bacteria Cell Division via an Interaction between TolQ and the Divisome Protein FtsN

Date of Award

2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Ray Larsen, Dr.

Second Advisor

Adam Miller, Dr. (Committee Member)

Third Advisor

Roudabeh Jamasbi, Dr. (Committee Member)

Fourth Advisor

Rex Lowe, Dr. (Committee Member)

Fifth Advisor

Vipaporn Phuntumart, Dr. (Committee Member)

Abstract

The TolQ protein functions to couple cytoplasmic membrane-derived energy to support outer membrane processes in Gram negative bacteria. With other products of the widely-conserved tol-pal gene cluster, TolQ has been linked to the process of bacterial cell division. When present in excess, TolQ disrupts cell division, leading to filamentous growth of Escherichia coli. The potential role of TolQ in Gram negative cell division was investigated by a number of methods, including growth assays and immunoblot, two-hybrid, and mutational analyses. This filamentation phenotype is specific for TolQ over-expression independent of TolA and TolR levels, with the degree of filamentation directly proportional to TolQ levels. Over-expression of E. coli TolQ in closely related species indicates that this property of TolQ is not E. coli specific, as excess TolQ leads to a comparable phenotype in other Gram negatives. Bacterial two-hybrid analysis indicates a potential in vivo interaction between TolQ and the divisome protein FtsN, ostensibly one that competitively diverts FtsN from functioning efficiently during late-stage cell division. Filamentation resulting from TolQ over-expression can be suppressed in cells when FtsN is concurrently expressed in excess. Mutational analysis of the 19 amino acid TolQ N-terminus suggests that specific residues within and/or conformation of the extreme N-terminal region of TolQ are essential for filamentation. Results of this study indicate that the link between the Tol-Pal proteins and cell division is mediated in part through a direct interaction between the periplasmic regions of TolQ and FtsN, with a possible role for the Tol system in stabilization of the divisome during Gram negative cell division. These findings are presented in this dissertation along with a broader consideration of a role for the Tol-Pal complex in cell division and a dynamic nature for the bacterial cell division apparatus itself.