Date on Master's Thesis/Doctoral Dissertation

12-2001

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Austin, Faye E.

Committee Co-Chair (if applicable)

Geoghegan, Thomas

Committee Member

Geoghegan, Thomas

Committee Member

Stout, Robert

Committee Member

Doyle, Ronald

Committee Member

Streips, Uldis

Author's Keywords

Lyme disease; Borrelia burgdorferi; Transcriptional control in bacteria; Bacterial responses to stress and environmental signals; SecA-dependent protein secretion

Abstract

Borrelia burgdorferi, the causative agent of Lyme disease, has been characterized as a microaerophilic spirochete. O2 consumption and utilization potentially yield reactive oxygen intermediates, such as superoxide, hydroxyl radicals, and hydrogen peroxide. This study investigated the expression of the sod gene, which encodes the only, identified oxidative defense mechanism in B. burgdorferi. Using primer extension analysis and RT-PCR, it was found that sod and secA are organized as a single transcriptional unit under the control of σ70-like promoter upstream of the secA open reading frame. Generally, gene expression decreases with increased distance from the promoter; however, secA expression was observed to be relatively lower amounts than sod. Both genes were expressed in all phases of growth, with greatest expression observed in stationary phase. Because transcription of most sod genes is tightly regulated by iron concentration, the secA-sod gene expression was analyzed in borrelial cells grown in varying amounts of metal. Iron-restriction did not significantly affect growth nor did it affect transcription of secA or sod. Likewise, no major differences in transcription were observed with restricting or supplementing media with manganese. To test the possibility that the borrelial SOD may function with either iron or manganese as a cofactor as in cambialistic SODs, SOD activity was assayed and the highest activity was observed in increased manganese concentrations. Protein expression profiles of Sh-2-82 cultured in metal-defined media were investigated by one- and two-dimensional gel electrophoresis. Approximately 15 proteins were differentially expressed in response to metal concentration.

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