Date of Award

5-14-2010

Document Type

Thesis

Degree Name

Bachelor of Arts

First Advisor

Slonczewski, Joan

Abstract

Escherichia coli is a highly acid resistant enteric bacteria that can colonize the intestine by surviving the extremely acidic conditions of the stomach. It is hypothesized that this acid resistance contributes to the low infectious dose of pathogenic E. coli such as O157:H7 or Shigella. The assays used to investigate acid resistance systems are conducted under aerobic exposure to extreme acid. However, in natural environments such as the human stomach, E. coli can experience anaerobic conditions. Under anaerobic growth conditions, E. coli express four hydrogenase (Hyd) isoenzymes that catalyze the reversible oxidation of molecular hydrogen. Previously, semi-aerobic exposure to extreme acid revealed that a deletion of all four hydrogenases eliminated acid resistance. In order to determine whether production or consumption hydrogenases were contributing to the loss of acid resistance, a hydrogen-specific Clark-type electrode was used to characterize the pH-dependence of hydrogen production and consumption. Hyd-3-dependent hydrogen production is induced 70-fold from pH 6.5 to 5.5, and hycE (Hyd-3) expression increased 3-fold from pH 7.0 to 5.5. Neither an external or internal pH shift immediately affected Hyd-3 enzymatic activity. Hyd-2-dependent hydrogen consumption increased less than 2-fold from pH 5.5 to 8.0, suggesting it is not as strongly pH-dependent as hydrogen production. Anaerobic cultures exposed to extreme acid revealed that a mutant deficient for Hyd-3 lost 20-fold acid resistance at pH 2.0. Aerobic cultures exposed to extreme acid showed no loss of acid resistance. These findings suggest the importance of hydrogen production by Hyd-3 in the anaerobic acid resistance system in E. coli.

Comments

Includes bibliographical references: 33-42

Share

COinS