Internalization of oligodeoxyribonucleotides by Vibrio parahaemolyticus.

Abstract

The bacterium Vibrio parahaemolyticus was tested for its ability to internalize unmodified as well as modified DNA oligomers without attempting to permeabilize the cells. These experiments were conducted to establish whether it may be feasible to employ antisense oligomers for control of gene expression in Vibrio species without heat-shocking or electroporating the cells. The bacterium was found to bind radiolabeled synthetic oligodeoxyribonucleotides that were added to culture media. Incorporation of a phosphorothioate oligomer into subcellular regions was determined following cellular fractionation. The phosphorothioate was recovered primarily from the periplasm and peptidoglycan layer of the bacterium; however, a significant fraction was recovered from the bacterial cytosol. The extent of uptake depended on both the concentration of oligomer as well as culture medium selected. A maximum of 2.1 x 10(6) oligomers/cell was achieved when a 12-mer phosphorothioate oligomer (10 microM) was added to bacterial cultures in an artificial seawater (Instant Ocean) medium. Several terminally modified oligomers were found to become associated with bacterial cells, albeit less efficiently than the phosphorothioate. None of the oligomers tested was toxic to the bacteria at 0.1 microM, and the phosphorothioate was only marginally toxic at 10 microM. Stability of the oligomers in extracellular and cell-associated fractions was evaluated by PAGE; even after 8 hr of incubation intact phosphorothioate oligomer could be found in both components.