Semester

Spring

Date of Graduation

2005

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Nyles W. Charon.

Abstract

Spirochetes are unique bacteria that cause a variety of diseases. Motility, provided by periplasmic flagella (PFs), is a virulence factor for spirochetes. This work focuses on PFs from Borrelia burgdorferi and Brachyspira hyodysenteriae. PFs consist of three main parts: basal body, hook, and filament. Monoclonal antibodies were developed to attempt identification of individual FlaB filament proteins in B. hyodysenteriae .;The PF filament of B. burgdorferi consists of a major flagellin, FlaB, and a minor flagellin, FlaA. FlaB constitutes 10-14%, whereas FlaA contributes less than 0.5%, of total cellular protein. Although these flagellins co-precipitate, the location of FlaA on a PF is unknown. FlaA accumulates at only 13% of wild type levels in the flaB null mutant, MC-1. Because mRNA levels are not negatively altered, FlaA regulation occurs at a posttranscriptional level. As FlaA is degraded over time, protein turnover may be a mechanism responsible for reduced FlaA accumulation in MC-1.;The flagellar hook has structural and regulatory roles in many bacteria. The PF hook gene, flgE, is required for motility, wave-like cell morphology, and PF synthesis in B. burgdorferi. The flgE null mutant, SC-1, has markedly reduced levels of filament proteins FlaB and FlaA. Because flaB and flaA transcripts are not negatively altered, filament protein synthesis is regulated at a posttranscriptional level. As seen in MC-1, FlaA is degraded over time in SC-1. Conversely, FlaB is stable indicating that protein turnover is not a significant mechanism for maintaining reduced FlaB accumulation in SC-1.;In addition to a loss of FlgE in SC-1, a series of high molecular weight bands reactive to hook antisera was lost. This "ladder" was present in wild type strains B31 and N40, and also in MC-1 cells, all of which presumably assemble the PF hook. The ladder is absent in SC-1 and fliF null basal body mutants in which the PF hook cannot assemble. Furthermore, the ladder is present in purified PFs and in isolated hook-basal body complexes from B31 PFs. The ladder suggests that these bands are FlgE aggregates that are strongly associated, perhaps by cross-linking. The nature of FlgE protein association is currently unknown.

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