Szi Fei Feng

Date of Award

8-2000

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Dr. Silvia Rossbach

Second Advisor

Dr. Susan Stapleton

Third Advisor

Dr. Bruce Bejcek

Access Setting

Masters Thesis-Open Access

Abstract

This thesis describes the isolation and characterization of a region containing several genes involved in metal tolerance in the common soil bacterium, Pseudomonas fluorescens ATCC 13525. The DNA region was identified in a cadmium and zinc sensitive mutant, 45-B10, which contains a lacZ-based reporter gene transposon Tn5-B20 inserted in the chromosome. The DNA surrounding the transposon was cloned from this mutant strain and DNA sequencing of this region revealed seven genes that share similarity with genes whose products are involved in the cation-proton antiporter efflux system of other Gram-negative bacteria. Complete DNA sequence was obtained from four of these genes, czyO, czyR, czyS and czyA. Defined mutations in each of these genes were generated in the chromosome of P. fluorescens via gene replacements. The gene replacement mutants were found to be more sensitive to cadmium or zinc compared to wild type. The comparison of DNA similarity and the phenotypic analysis of the mutants suggest that these genes play a role in metal homeostasis in P. fluorescens.

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