Doctoral Dissertations

Date of Award

12-1991

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

T. C. Montie

Committee Members

B. Mullin, D. Bemis, S. Riggsby

Abstract

This dissertation was built around a central theme of developing a better understanding of the flagellin filament protein of P. aeruginosa by cloning the flagellin gene, and characterizing a posttranslational modification in the flagellin protein. To detect the flagellin gene within a recombinant clone of the pLAFR1 - PAO1 library, the recombinant clones were triparentally mated into fla- mutants and scored for their ability to complement the mutant. Motility spreading assays were performed determine if complementation had occurred, and confirmed by colony blot assays. Electron microscopy and amino acid analyses showed the structure and make-up of the flagella from the cells containing clones to be nearly identical with wild type flagella. Following isolation of positive recombinant clones, two different approaches were employed to determine which region of the 25 kb PAO1 fragment was necessary for expression of the flagella. First, deletion mapping was utilized. The positive clones were further digested with restriction enzymes and religated and triparentally mated back into P. aeruginosa fla- cells, and complementation was scored. The fla gene is located within a 5.0 kb Xho I fragment. The second approach involved transposon mutagenesis with Tn501. The sites of the transposon insertions was mapped by analyses of restriction digests. A gene replacement technique was utilized, and the loss of flagellar structure was characterized by motility spreading assays and colony blotting. The other major portion of this research was identification and characterization of the posttranslational modification occurring in the flagellin filament of P. aeruginosa. Both a- and b-type purified flagellin from P. aeruginosa grown in radiolabeled phosphate were shown to be phosphorylated. Initial characterization of labeled flagellin was by autoradiograms of SDS-PAGE gels and immunoblots with anti-flagella antibody. Thin layer electrophoresis (TLE) of partial acid hydrolyzed flagella filaments revealed that the radioactivity was in phosphotyrosine. This finding was unusual because a phosphorylated tyrosine is not prevalent in prokaryotes. Intact flagella filaments have been shown to react specifically with antiphosphotyrosine monoclonal antibody. Also, the labeled filaments were susceptible to phosphatase, indicating a monophosphoester linkage. The biological role of these modifications remains unknown.

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