Figure 2.

Genetic analysis of aqpZ knockout in E. coli. (A) Construction of an aqpZ∷lacZ transcriptional fusion gene on the E. coli MM294-strept^R chromosome. The aqpZ gene is represented by the solid rectangle with the ORF bracketed by vertical dashed lines. A promoterless 3.9-kb lacZ-kan cartridge was inserted into the BsmI site of the aqpZ gene carried by the pZSV suicide plasmid. This yielded a aqpZ∷lacZ-kan fusion gene that was used to replace the aqpZ gene in the wild-type chromosome (MM294 strept^R) by a double allelic recombination. The resulting aqpZ knockout strain, MM1211, contains the intact aqpZ promoter linked to lacZ-kan. (B) Southern analysis of disrupted aqpZ gene. Genomic DNAs from wild-type E. coli strains MC4100 (lane 1), MM294 (lane 2), MM294 strept^R (lane 3), and the aqpZ gene disruption construct MM1211 (lane 4) were cut with BglI and AvaII and electrophoresed into agarose gel. The DNA fragments were transferred to a membrane and hybridized at high stringency with a probe containing the entire coding region of aqpZ labeled with Digoxigenin.