Figure 3

Verification of the insertion/replacement of the rpsL -neo cassette in kdpA by PCR (A) and verification of the kdpA4 phenotype (growth deficiency under K⁺-limitation) (B). A Two positive clones of each recombination step were analyzed by colony PCR. On the left panel, insertion of the rpsL-neo cassette into kdpA was verified, on the two middle and the right panel the replacement of the kdpA4-rpsL- neo cassette by the double-stranded and the two single-stranded kdpA4-fragments (kdpA containing the point mutation G1033A) was confirmed. Below the gels pictures, the respective DNA-fragment used for the recombination is indicated. ds = double-stranded, ss = single-stranded, nt = nucleotides. B Cells were grown overnight in minimal medium containing 10 mM K⁺, washed three times in minimal medium containing 0.02 mM K⁺, diluted, and 2 μl of each suspension containing the indicated number of cells was spotted onto a minimal medium agar plate containing 0.02 mM K⁺ or 10 mM K⁺, respectively. Cells were incubated overnight at 37°C