Molecular Approaches in Attempts to Identify the Flagellin b-type Gene in Clone pKW52

Author

Yolanda F. Kirkpatrick, University of Tennessee, Knoxville

Date of Award

5-1996

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Thomas Montie

Committee Members

Beth C. Mullin, David A. Bemis

Abstract

Pseudomonas aeruginosa is an important pathogen for susceptible individuals particularly those immunocompromised due to the prolonged use of drugs, noscomial infections, burns, or cancer. In addition, P. aeruginosa infections are high among cystic fibrosis patients. P. aeruginosa presents a unique health concern and challenge for researchers due to its multiple virulence factors and the increasing number of patients developing P. aeruginosa infections. P. aeruginosa produces a variety of toxins and enzymes. The flagellum of P. aeruginosa allows the bacterium to be motile. Motility has enabled the P. aeruginosa bacterium to rapidly colonize the host's body.

The major structural component of the P. aeruginosa flagellum is the protein, flagellin. P. aeruginosa flagellin was categorized two major groups, designated a and b via slide and tube agglutination assays and immunofluorescence technique. The a -type flagellin is composed of a major a₀antigenic component and one to three subantigen types, a₁, a₂, a₃, and a₄. The a-type flagellins have molecular weights of 45,000 to 52,000. The b-type flagellin appear to be antigenically homogenous and have a molecular weight of 53,000. It is the centermost region of the flagellin that is antigenic and highly variable even within one species.

Oligonucleotides 1N and 1C, derived from the N-terminal and C-terminal nucleotide sequence of the cloned P. aeruginosa (strain PAK) flagellin a-type gene, annealed to clone pKW52 (flagellin b-type) and an approximately 600bp fragment was amplified by PCR. The 600bp fragment was cloned in vector pCRII, and a partial nucleotide and amino acid sequence of the resulting clone pKWII was determined. Southern hybridization with radiolabeled probes was used in attempts to locate and identify the flagellin b-type gene in pKW52 and pKWII, respectively. The 1.7kb flagellin (a-type) gene probe did not hybridize to pKW52 and pKWII under low and high stringency hybridization conditions. In immunological studies of pKW52 flagellin antigen expression was detected in E. coli cells harboring clone pKW52. Flagellin expression was not detected in E. coli cells harboring clone pKWII.

The results of this thesis research suggest: 1) based on Southern hybridization experiments the sequence identity between the PAK 1.7kb flagellin a-type gene and clone pKW52 is estimated to be less than 90%, 2) the expression of flagellin b-type protein in E. coli cells harboring pKW52 suggested that clone pKW52 may contain the flagellin b-type gene, 3) colony immunoblotting experiments with antiflagellin antibodies correlated with Southern hybridization results demonstrating that pKWII did not contain the flagellin b-type gene, and 4) PCR and Southern hybridization experiments are molecular biology techniques that may be used to locate the flagellin b-type in clone pKW52.

Recommended Citation

Kirkpatrick, Yolanda F., "Molecular Approaches in Attempts to Identify the Flagellin b-type Gene in Clone pKW52. " Master's Thesis, University of Tennessee, 1996.
https://trace.tennessee.edu/utk_gradthes/4330