Masters Theses

Date of Award

8-1997

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Thomas C. Montie

Committee Members

Neil Quigley, Liz Howell, David Bemis

Abstract

The major structural protein of the Pseudomonas aeruginosa flagellum is the protein, flagellin. Pseudomonas aeruginosa strains express two types of flagellin proteins: b-type that have a molecular weight (Mr) of 53kDa and a more heterologous a-type ranging in Mr from 45 to 52kDa. The purpose of this research is two fold. It was of interest firstly, to make a comparative study of b-type genes and secondly, to test whether a PCR technique could be used to extend this comparison to Fla- strains. To facilitate these objectives, a PCR technique was modified and employed to amplify most of the N- and C-terminus and all of the central variable region of the Pseudomonas aeruginosa flagellin genes. A PCR gene fragment size of 1.02kb corresponds to the flagellin a-type gene whereas an amplified gene fragment of 1.25kb corresponds to the flagellin b-type gene (Winstanley et al., 1996). To confirm and extend these correlations, we performed monoclonal antibody colony immunoblots on a number of motile Fla+ and non-motile Fla- strains and isolated and sequenced amplified gene fragments of 4 Fla- isolates and the wild type M2. The Fla- phenotype exhibited absence of reaction with type specific monoclonal antibodies, These results suggest that little or no flagellin is expressed in these non-motile mutants or that a mutation in the fliC gene is located in the Mab reactive epitope in the flagellin protein. The amino acid sequence results of strain M2 (Fla+, b-type) showed almost complete identity (99.7%) with the Fla+ b-type PA01, emphasizing the homologous nature of this group of flagellins. Variation in the b-type flagellins range from a single amino acid difference to a maximum of 5 amino acids in a Fla- CF isolate. In analyzing the M2 protein sequence, we have found 3 potential sites for linear Mab epitopes in the b-type flagellin that are absent in the a-type flagellins. The conserved nature of the b-type genes enabled one to rapidly detect mutations that might occur in the Fla- gene sequences. Only one of the fliC gene sequences provided evidence which linked mutations in the structural gene to the Fla- phenotype. A stop codon was found in the N-terminal region of CF isolate 448bb. Mutations at the regulatory level could explain the other flagellin deficiencies. The a-type flagellin protein sequences exhibited less identity amongst P. aeruginosa strains. Results show evidence for protein sequence variation in the a-type flagellins. In strain PA103 (Fla-, a-type), there were structural differences which involved two distinct deletion regions and three additional substitution regions, in comparison to the wild type a-type strain PAK. These sequence variations corresponded precisely to those of another GenBank a-type P. aeruginosa CF isolate K701 (Fla+) to which the PA103 fliC gene shares a high degree of homology. Location of mutations in a-type flagellins of Fla- strains is more difficult because of their decreased sequence homology compared to the b-types. The PCR protocol described here, cannot only be used to identify and study the fliC gene and differentiate a- and b-type flagellins, but also it can be used to rapidly detect and type the fliC gene in Fla- strains that do not react with monoclonal antibodies.

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