Identification of periplasmically regulated genes in Bdellovibrio bacteriovorus using an IVET approach.
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Bdellovibrio bacteriovorus is a predatory bacterium with two separate life phases. In one, it searches for a suitable Gram negative bacterium to attack and invade. The second phase of life is spent after invasion, within the periplasm of its host, while the predator cell consumes the host. In order to identify genes that are active within this periplasmic cellular compartment, we have modified a pIVET (in vivo expression technology) plasmid so that it can be used with Bdellovibrio. The pIVET plasmid has a suitable restriction site in front of a promoterless chloramphenicol resistance gene. Promoter active DNA, when cloned into this site, results in chloramphenicol resistant transformants. This approach allows us to identify periplasmically regulated genes by inserting fragments of Bdellovibrio chromosomal DNA in front of the promoterless gene and determining whether Bdellovibrio cells carrying the plasmid are resistant to chloramphenicol while they are within the host periplasm. So far, the pIVET plasmid has been modified by in vitro transposon mutagenesis to confer resistance to kanamycin instead of the original ampicillin, to which Bdellovibrio is naturally resistant. Genomic DNA from Bdellovibrio has been successfully cloned into the modified plasmid, called pSVS101, producing various clones with different levels of chloramphenicol resistance. Currently, the products of this experiment are being conjugally introduced into Bdellovibrio for the next stage of this project.