THE L-IDONIC ACID PATHWAY OF ESCHERICHIA COLI

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

By

Christoph Lawrence Bausch, B.S.

*****

The Ohio State University

2003

Dissertation Committee: Approved by Professor John Reeve, Advisor

Professor Tyrrell Conway Adviser Professor Tina Henkin Dept. Microbiology Professor Robert Tabita

ABSTRACT

Early studies of D-gluconate catabolism in Escherichia coli revealed two similar systems thought to be involved with gluconate catabolism. The main system,

GntI, is well characterized and known to be the primary pathway for gluconate catabolism. The subsidiary system, GntII, shares genetic similarities to the GntI system and was predicted to encode a secondary gluconate pathway. The experiments presented in this dissertation reveal that the GntII system is a novel pathway for the catabolism of

L-idonate in E. coli and describe the biochemistry, physiology, gene organization, and mechanisms of transcription regulation. The overall process of L-idonate metabolism is as follows: L-idonate is transported into the cell by IdnT and is oxidized by IdnD to the intermediate 5-ketogluconate (5KG), which is subsequently reduced by IdnO to gluconate, then phosphorylated by IdnK. An intermediate of the pathway, D-gluconate, serves to induce the edd-eda operon encoding the Entner-Doudoroff pathway, which further catabolizes the 6-phosphogluconate. The enzymes of the L-idonate pathway are encoded by the IdnR regulon, comprised of two operons: idnDOTR and divergently transcribed idnK. This regulon is induced by L-idonate or 5KG, with both operons being expressed at similar levels. The regulatory region located between idnD and idnK contains the transcriptional start sites, promoters, and single binding sites for IdnR and cAMP-CRP, as well as an UP element sequence. Transcription occurs through a CRP-

ii dependent Class III mechanism. The regulators for the L-idonate and D-gluconate pathways, IdnR and GntR bind to the same palindromic sequence, providing the foundation for cross talk. Discrimination between the IdnR and GntR regulons is achieved through preferential binding of GntR to operators of the gluconate regulon and

IdnR to the operator of the idonate regulon through interactions influenced by their respective inducers. Thus, a GntI system mutation is complemented by the GntII pathway enzymes through the intracellular accumulation of the inducer for L-idonate catabolism.

iii

Dedicated to my friends and family

iv ACKNOWLEDGMENTS

I wish to thank my advisor, Tyrrell Conway, for his encouragement, intellectual

support and advice. I would also like to express my sincere appreciation for his patience and understanding.

I thank John Reeve for accepting advisory duties for me the last 2 years and for his time correcting this document, and to those on my committee, Tina Henkin and

Robert Tabita, for their insightful criticisms and suggestions.

I thank David Eeles for his assistance in organizing meetings and paperwork that were necessary for me to complete this dissertation.

I thank April Anderson of the Department of Botany and Microbiology at the

University of Oklahoma, who painstakingly helped edit this dissertation.

I would like to also express my most sincere appreciation to:

Kenneth Nickerson of the Department of Biology at the University of Nebraska-

Lincoln, who encouraged me to pursue a doctorate in Microbiology.

Alan J. Wolfe of the Department of Microbiology and Immunology at Loyola

University- Chicago for his advice on my project and for providing CRP mutant variant plasmids.

v Christine M. Beatty of the Department of Microbiology and Immunology at

Loyola University-Chicago for her advice regarding EMSA experiments