A counterselectable pACYC184-based lacZ alpha-complementing plasmid vector with novel multiple cloning sites; construction of chromosomal deletions in Klebsiella pneumoniae.

We have constructed a series of small, chloramphenicol-resistance-encoding, lacZ alpha-complementing vectors with novel multiple cloning sites, based on the pACYC184 replicon. The sacB gene of Bacillus subtilis, which is lethal to Gram- organisms in the presence of sucrose, was cloned into one of these, giving the counterselectable vector pSG335. This was used to substitute a streptomycin-resistance-encoding cassette for the ntrBC genes in the Klebsiella pneumoniae chromosome.