Year of Award

2010

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Other Degree Name/Area of Focus

Integrative Microbiology and Biochemistry

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

Michael F. Minnick

Commitee Members

J. Stephen Lodmell, D. Scott Samuels, Willard Granath, Jr., Keith K. Parker

Keywords

Bartonella, hemin, iron, regulation, Trench fever

Abstract

Bartonella quintana, a Gram-negative bacterial pathogen, causes Trench fever, bacillary angiomatosis and endocarditis. Transmitted by the human body louse (Pediculus humanus corporis), the agent has a tropism for erythrocytes in humans. In vitro growth requires an extraordinary concentration of hemin, and genomic analyses indicate several potential uptake systems and iron-responsive regulators. Transcription of the hbp genes (hemin binding protein genes) is responsive to alterations in available hemin and an HbpA homolog in B. henselae reportedly functions as a hemin receptor in E. coli hemA strain EB53. B. quintana hbpA was not able to complement EB53, indicating that it is not a hemin receptor. A functional hemin receptor and coordinate uptake system is encoded by the hemin utilization (hut) locus. B. quintana hutA was able to complement a hemA mutation in E. coli EB53 and was shown to be TonB-dependent using an isogenic E. coli hemA tonB strain.

Fur (ferric uptake regulator) has been described as a global iron-responsive regulator in &gamma-proteobacteria. If expression is forced, B. quintana fur is able to complement an E. coli fur mutant, but an endogenous promoter for the gene could not be located and native expression in B. quintana was not detected. Overexpression of the iron response regulator (Irr), a Fur family member, in B. quintana repressed hut locus transcription. Previous studies showed that Irr interacted with a consensus motif, the H-box, in the promoter of the hbp genes. A region with homology to the H-box consensus is present in the divergent promoter between hutA and tonB and in the promoter region of hemS.

The fate of hemin in the bacterial cytoplasm is not well understood. HemS is a potential hemin storage/degradation enzyme. Initial characterization indicates that HemS is able to bind hemin in a 1:1 fashion with an estimated dissociation constant (Kd) of 5.9 + 1.7 &muM. Complementation analyses using Corynebacterium ulcerans CU712hmuO&delta strain have not been successful but future experiments plan to use an E. coli chuS strain. These studies have characterized the principal hemin uptake system of B. quintana, identified its transcriptional regulator, and initiated investigation of a potential heme oxygenase.

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© Copyright 2010 Nermi Lee Parrow