Mini-Mu-duction as a test for genetic complementation in Escherichia coli

Abstract

In mini-Mu-duction, segments of host DNA bracketed between two copies of an internally deleted Mu phage (a mini-Mu) can be packaged within Mu phage particles. Upon infection of a second host strain, the DNA injected by these particles can insert into the chromosomal DNA in a reaction catalyzed by the phage A gene product (transposase), which is independent of homologous recombination. This results in a partially diploid host strain in which the duplicated host DNA is bracketed by two copies of the mini-Mu phage (Faelen et al., Mol. Gen. Genet. 176:191-197, 1979). The frequency of mini-Mu-duction reported previously was low (10(-8) to 10(-9) per recipient cell) thus limiting its use to rather stable mutational lesions. I have increased the frequency of mini-Mu-duction 10- to 100-fold by use of a helper phage lacking the kil gene and by UV irradiation of the phage stocks. I have also shown that mini-Mu-duction is a reliable complementation assay in rec+ as well as recA recipient strains. This genetic complementation test does not require prior gene localization and (due to the extended host range of phage Mu) should be applicable to many enterobacterial species.