MIAMI UNIVERSITY The Graduate School

Certificate for Approving the Dissertation

We hereby approve the Dissertation of J. Allen Easton

Candidate for the Degree: Doctor of Philosophy

______

Director

(Dr. Michael W. Crowder) ______Reader (Dr. John W. Hawes) ______Reader (Dr. Chris A. Makaroff) ______Reader (Dr. Gary A. Lorigan) ______Graduate School Representative (Dr. Gary Janssen) ABSTRACT

IDENTIFICATION AND CHARACTERIZATION OF ZN(II)- RESPONSIVE GENES AND PROTEINS IN E. COLI

By J. Allen Easton

Transition metal ion homeostasis is absolutely crucial for the survival of all organisms. Zinc (Zn(II)) is perhaps one of the most important, yet least studied transition metals. Previous studies indicate that intracellular Zn(II) levels in E. coli are in the low millimolar range, yet there is less than one “free” Zn(II) ion per cell. All of the intracellular Zn(II) must then be bound and Zn(II) must be delivered from transporters and inserted into Zn(II)-. The cytoplasmic transport of transition metals, such as , , nickel, manganese, and arsenic, is accomplished by a group of proteins called metallochaperones. No such metallochaperone has been identified for Zn(II). In an effort to identify the Zn(II) metallochaperones in E. coli, proteomic and genomic studies were conducted. Proteomic studies were used to probe for the time-dependent response of E. coli to stress by Zn(II) excess. Genomic studies were used to probe for the transcriptional response of E. coli to stress by Zn(II) excess and deficiency. Several Zn(II)-metallochaperone candidates were identified, and these proteins were cloned, over-expressed, purified, and characterized. Trigger factor was found to be down-regulated at the proteomic level in response to excess Zn(II). Over-expression and characterization of trigger factor show that it tightly binds 0.5 Zn(II)/monomer; however, spectroscopic studies showed that Zn(II) binding is most likely adventitious. GatY/GatZ Zn(II)-responsive proteins that are part of the galactitol catabolic pathway. GatY was over-expressed and shown to bind 2 Zn(II) equivalents per . GatZ, reported to be necessary for GatY function, was tested for Zn(II)- binding and shown to not bind Zn(II). A transcript found to be highly up-regulated was ykgM. We cloned and over-expressed YkgM to elucidate why it is highly responsive to Zn(II). We determined that YkgM does not bind Zn(II), and may substitute for Zn(II)-containing ribosomal protein L31 in Zn(II)-limiting conditions. ZnuA was cloned, over-expressed, purified, and characterized. We found that ZnuA tightly binds 2 equivalents of Zn(II) per monomer. Our proteomic and genomic data suggest that there are no soluble, cytoplasmic Zn(II) metallochaperones in E. coli. Based on this conclusion, a novel model is hypothesized that explains Zn(II) transport in E. coli cytoplasm.

Identification and Characterization of Zn(II)-responsive Genes and

Proteins in E. coli

A DISSERTATION

Submitted to the Faculty of

Miami University in partial

Fulfillment of the requirements

For the degree of

Doctor of Philosophy

Department of and Biochemistry

By

J. Allen Easton

Miami University

Oxford, OH

2007

Dissertation Director: Dr. Michael W. Crowder

TABLE OF CONTENTS

Chapter 1: Introduction 1.1 The importance of Metal Ions 1 1.2 The importance of Zinc 3 1.3 Zinc homeostasis in eukaryotes 3 1.4 Zinc homeostasis in prokaryotes 6 1.5 Discovery of metallochaperones 9 1.6 Proteomic and genomic techniques 11 1.6.1 Proteomics 11 1.6.2 Genomics 18 1.7 Chapters of the dissertation 19 1.8 References 22

Chapter 2: Time-dependent Translational Response of E. coli to Excess Zn(II) 28 2.1 Summary 29 2.2 Introd