FIG. 2.

Outline of strategy for generating in-frame deletions in genes encoding histidine protein kinases. For each gene to be deleted, a plasmid was constructed by PCR and cloning with approximately 550-bp regions of homology upstream (light gray) or downstream (dark gray) flanking the in-frame construct. In a two-step procedure, deletion strains were isolated by first selecting for kanamycin resistance, followed by galactose counterselection using the galK gene carried on the plasmid and screening for loss of kanamycin resistance. Cells harboring galK die in the presence of galactose. Candidate clones for carrying the in-frame deletion were identified as Gal^r and Kan^S. Clones carrying the in-frame deletion were identified by PCR using the primer pairs EF and GH. For simplicity, the first homologous recombination is shown only for the recombination occurring in the upstream flanking region.