Fig. 2

Gene replacement via allelic exchange at the hsdR1, hsdR2, hsdR3, xylB, and ptb–buk loci. PCR confirmation of the different double-crossover deletion mutants using external primers annealing to the chromosome upstream and downstream of each deletion cassette. Strains (a) ΔhsdR1. b ΔhsdR1 ΔhsdR2. c ΔhsdR1 ΔhsdR2 ΔhsdR3. d ΔhsdR1 ΔhsdR2 ΔxylB. e ΔhsdR1 ΔhsdR2 Δptb Δbuk. ΔhsdR1: 2141 bp (a, b, c, d, e), WT of hsdR1: 5553 bp (a), catP gene: 622 bp (a, b, c, d, e). ΔhsdR2: 2064 bp (b, c, d, e), WT of hsdR2: 5259 bp (b) ΔhsdR3: 2078 bp (c), WT of hsdR3: 5010 bp (c). ΔxylB: 2081 bp (d), WT of xylB: 3549 bp (d). Δptb Δ buk: 2042 bp (e), and WT of ptb–buk: 4026 bp (e)