Targeting the Bacillus subtilis genome: An efficient and clean method for gene disruption

A method to disrupt multiple Bacillus subtilis genes is described. A resistance cassette is used to interrupt an amplified target sequence from the B. subtilis chromosome. The cassette is composed of a gene conferring resistance to chloramphenicol (Cm) or spectinomycin (Sp) flanked by two directly oriented β cognate sites (six site) (SCS or SSS, respectively). The linearized construct is used to transform B. subtilis competent cells with selection for Cm or Sp resistance. Transformants with the desired gene disrupted by the SCS or SSS cassette, integrated by a double cross-over event, were confirmed by PCR analysis. A segregationally unstable plasmid-borne β site-specific recombinase is transferred into the background. Protein β catalyzes excision of the intervening sequence between the two six sites leading to a target gene disrupted only by a six site. This site has an internal promoter capable of reading downstream genes. To generate multiple disruptions, the cycle can be repeated many times provided that two six sites are separated by about a 70-kb interval.