Transposon mutagenesis in the bacterial predator Bdellovibrio bacteriovorus.

Abstract

Bdellovibrio is a unique predatory genus of bacteria, which attacks and consumes other gram-negative bacteria. Little is known about the organism, and very few genes have been identified and characterized. In order to develop new tools for further genetic work, a transposon system was tested in Bdellovibrio. The new system used the plasmid pRL27c carrying the transposon Tn5-17. This system was found to have two main advantages: first, that the system worked well in Bdellovibrio, and second, that because of the construction of the transposon, the insertion site as well as the surrounding genomic DNA could be retrieved from the genome and turned into a plasmid for further manipulation and analysis. Seven randomly selected colonies with transposon insertions were chosen, and plasmids containing the transposon site and flanking DNA were isolated and sequenced. Restriction digests of the plasmids strongly implied random insertional events, and the sequence analysis of the flanking DNA has thus far identified five matches from GenBank: a glutamyl-tRNA synthase, a sensory box histidine kinase, a two component hybrid sensory regulator, a conserved hypothetical protein from a methanagen, and a triacylglycerol acyl hydrolase. Further work will include sequencing more insertion sites, screening for null mutations of three observable phenotypes (yellow pigment, protease and amylase activities), and constructing null mutations of genes found through sequencing.