Development of a Genetic System for DNA Transfer into Aeromonas Hydrophila

Aeromonas hydrophila is a gram-negative bacterium and opportunistic pathogen that usually inhabits aquatic ecosystems throughout the world. A. hydrophila strains can cause diseases in humans, fish, and other animals. Several virulence factors, such as aerolysin, have been studied extensively and their role in pathogenesis has been identified. However, several factors predicted to be involved in pathogenesis, such as chitinase, have not been yet investigated. To determine the role of these additional virulence factors, a genetic system for the isolation of mutants needs to be developed. I have used a plasmid that contains the mini-transposon himarRB1 to generate transposon-induced mutations in A. hydrophila. Unfortunately, the efficiency of transposition was extremently low. One possibility for this low efficiency is the digestion of DNA by the A. hydrophila restriction system. The A. hydrophila genome, however, does not appear to encode a restriction/modification system, suggesting that DNA modification and restriction may be performed by a novel system in this bacterium. To begin to analyze this system, total chromosomal DNA was isolated from A. hydrophila cells and digested with different restriction enzymes. The results obtained will allow us to identify enzyme restriction sites that are protected in A. hyrophila, and thus will help identify the type of DNA restriction system this organism possesses. We expect that identification of the A. hydrophila restriction/modification system will allow us to develop better tools for the isolation and analysis of A. hydrophila mutants