A simple and efficient system for the construction of phoA gene fusions in gram-negative bacteria.

We have developed a two-plasmid system for generating gene fusions between phoA and cloned genes encoding envelope proteins. The vector plasmid carries a temperature-sensitive replication system and can be rescued at high temperature by insertion of an IS1-based transposon carrying the ori region of pBR322 and a phoA gene lacking transcription and translation initiation signals. The vector plasmid also carries the transfer origin of the conjugative plasmid, F, permitting transfer into a suitable recipient strain. We have used this system in the analysis of the bla gene cloned from pBR322.