Generation and characterization of gene deletion mutants.

A. Schematic overview of the Pop-In-Pop-Out method for mutant construction. The uracil auxotrophic strain H26 (ΔpyrE2) was transformed with the plasmid carrying the pyrE2 gene and an in frame deletion version of the respective gene with upstream- and downstream sequences. Two possible homologous recombination events (1 and 2) can lead to integration of the plasmid into the genome (Pop-In), which can be selected by the absence of uracil. Pop-Out clones can be selected in the presence of uracil and 5-FOA, leading either to the wild-type (1-1) or to the deletion mutant (1–2). B. Genomic organizations of wild-type, Pop-In and Pop-Out mutants: one example. Schematic overviews of the genomic organizations around HVO_0136 with the wild-type on top, the two possible Pop-In mutants in the middle and the Pop-Out mutant at the bottom. The integrated plasmid is shown above the genome of the Pop-In mutants. Relevant restriction sites are presented as vertical arrows and probes for Southern blot analysis are shown as boxes below the genome organization. C. Southern blot analysis of wild-type and mutants Verification of the wild-type, both Pop-In mutants and two positive deletion Pop-Out mutants of deletion mutant HVO_0136 are shown from left to right. Wild-type and deletion fragments are indicated by their size. D. Verification of the absence of an aIF2α transcript in the deletion mutant The aIF2α and the two aIF2β deletion mutants were cultivated in complex media to mid-exponential growth phase. Cells were harvested, RNA was isolated and transcript levels were analyzed by Northern blot analysis using a probe complementary to the first 360 bp of the aIF2α gene. Samples: 1. H26Δdhfr, 2. H26ΔdhfrΔ0699 (aIF2α), 3. H26ΔdhfrΔ1678 (aIF2α), 4. H26ΔdhfrΔ0699Δ1678 (aIF2α + aIF2β), 5. H26, 6. H26Δ2242.