Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Medical Sciences - Pathobiological Sciences

First Advisor

Ronald L. Thune

Abstract

The pyruvate formate-lyase enzyme (Pfl), a key enzyme in alcoholic fermentation, catalyzes the anaerobic conversion of pyruvate into acetyl-CoA and formate in prokaryotes. The pyruvate formate-lyase gene (pfl) of the facultative anaerobe Aeromonas hydrophila was cloned and sequenced. Subsequently, the Pfl was characterized based on the deduced amino acid sequence and evaluated in vivo following insertion mutagenesis. Finally, the pathogenesis of a defined mutant was compared to wild type A. hydrophila in channel catfish. The pfl of A. hydrophila was identified from two recombinant pBluescript SK(-) clones excised from a $\lambda$ ZAP II phage library. Both clones carried the same 9.15 Kbp insert, oriented in opposite directions, and expressed two polypeptide of 85 & 82 KDa sizes regardless of isopropyl-B-D-thiogalactoside induction. Dideoxy sequencing of a 4.8 Kbp subclone expressing the two immunoreactive polypeptides identified a 2280 base open reading frame (ORF), with 70% sequence homology to the E. coli pfl. The ORF encodes an 85.5 KDa protein containing 760 amino acid residues with 85.5% homology to the E. coli Pfl peptide sequence. The Aeromonas Pfl secondary structure consists of 46% $\alpha$-helices and 39% $\beta$-sheets. Catalytically important adjacent cysteine residues in E. coli Pfl at positions 419-420 were also located at the same positions in the A. hydrophila Pfl. A free radical identified at glycine-735 residue in the cloned enzyme was also in the same position as in E. coli Pfl. A candidate trypsin cleavage site, located between arginine residues at 624-625 positions in A. hydrophila Pfl, was confirmed by the presence of a 68 KDa trypsin resistant fragment. Insertion mutagenesis of the pfl with a kanamycin resistant marker (kan) resulted in isolating an obligate aerobic A. hydrophila pfl mutant strain, which was characterized phenotypically by anaerobic growth on pyruvate and antibiotic resistance profile. Polymerase chain reaction experiments confirmed the insertion of the kan marker into the mutant chromosome and the occurrence of a double-crossover homologous recombination event. No significant difference was noticed in the LD$\sb{50}$ in channel catfish injected with either the parent or the mutant strain, indicating that the Pfl may not play an important role in the pathogenesis of acute A. hydrophila infections.

Pages

127

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