Use of Reporter Gene Technology to Probe the Periplasmic Environment of Gram Negative Bacteria using Bdellovibrio Bacteriovorus.

Abstract

Bdellovibrio is a small Gram negative bacterium that preys upon a broad range of other Gram negative organisms. This unique prokaryote has a life cycle that alternates between two distinct stages. The first is a motile attack phase, in which the flagellated bacterium is free to move around its environment. Approximately three out of every hundred collisions with a prey cell results in the attachment of the attacking Bdellovibrio . Once connected, the invading bacterium enters the periplasm of the prey cell. Inside the periplasm, Bdellovibrio begins the growth phase of its life cycle by utilizing host macromolecules to elongate, split into a number a predatory cells, and lyse the host cell. The progeny are released into the surrounding environment to repeat the cycle. The plasmid pProU-gfp is a fusion between the Escherichia Coli proU promoter and the gfp reporter gene. Once fused, the gfp reporter gene responds to low osmotic conditions by producing green fluorescence protein. Our goal is to use pProU-gfp to study the periplasmic environment of Gram negative bacteria. In order to do this the plasmid needs to be moved into Bdellovibrio . Then a growth phase cell will report the osmotic conditions of the periplasm by production of green fluorescence protein. The amount of protein can then be measured and the osmotic conditions of the periplasm revealed. Unfortunately the plasmid does not replicate in Bdellovibrio , so a chromosomal integration is required.