Isolation and characterization of extracellular protein export genes of Aeromonas hydrophila

Jiang, Bo (1991) Isolation and characterization of extracellular protein export genes of Aeromonas hydrophila. Masters thesis, Memorial University of Newfoundland.

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Abstract

Two transposon insertion mutants of Aeromonas hydrophila C5.84 and L1.97, which were defective in extracellular protein production, were characterized. Each of the mutations caused pleiotropic effects: the mutants were unable to secrete any of the major proteins normally extracellularly exported by the wild-type strain Ah65 but instead accumulated these proteins in the periplasm. This suggests that the inactivated genes in these two mutants were essential for the general extracellular protein export process. Besides the effects on protein secretion, the mutation in LI.97 also changed the outer membrane structure of the mutant and rendered the cell unusually fragile. -- By using the transposon antibiotic resistance genes as selective makers, two wild-type chromosomal fragments (11 kb and 15 kb respectively) corresponding to the transposon insertion regions in LI.97 and C5.84 were isolated. When transferred back into the mutants, the cloned DNA complemented the defects in the corresponding mutants and restored the normal secretory phenotype to the transconjugants. The mutation in C5.84 was also partially complemented by the heterologous 11 kb fragment corresponding to the L1.97 mutation. -- By further subcloning, deletion analysis, DNA sequencing and functional complementation studies, an extracellular export gene (exeE) located on the 11 kb fragment was identified and characterized. The exeE gene potentially encoded a 55 KD polypeptide. The predicted protein product contained a hydrophobic transmembrane segment and was thus classified as an integral membrane protein. -- Interestingly, exeE, the gene essential for general extracellular protein export in Aeromonas hydrophila, was found to be highly homologous to pulE, a gene involved in the pullulanase secretion in Klebsiella pneumoniae. Furthermore, the genes located both upstream and downstream of exeE, i.e. exeD, exeF and exeG also shared extensive similarities with pulD, pulF and pulG, suggesting that this cluster of exe genes has similar functions as the pul genes and are all required for extracellular protein export. These results provided strong evidence that the protein secretion machineries are conserved between two completely different gram-negative bacteria: Aeromonas hydrophila and Klebsiella pneumoniae.

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