Figure 2

Construction of marker-free deletion mutants and overexpression strains of iolG and iolW . (A) Outline of the method for construction of marker-free deletion mutants. (A-I) Positional relationship between target deletion and regions A, B, and C contained in the PCR fragments used for construction of a marker-free deletion. A: upstream sequence; B: downstream sequence; C: sequence for integration of the mazF cassette. (A-II) Recombinant PCR product using fragments A, B, C, and the mazF cassette. (A-III) Integrant of the mazF cassette at the target region via a double crossover at regions A and C. An intrachromosomal single crossover event between the 2 directly repeated regions B results in elimination of the mazF cassette as well as the target region. (A-IV) Final structure of marker-free deletion. (B) Organization of the iolG and iolW overexpression cassettes. (B-I) For iolG overexpression, PCR fragments covering regions A + B (containing PrpsO or PrpoB) + C + D were ligated by recombinant PCR and integrated into the amyE locus by a double crossover event. (B-II) For iolW overexpression, PCR fragments covering regions E + F + G (PrpsO or PrpoB) + H + I were ligated and integrated into the aprE locus for construction of an iolW- overexpressing strain. (B-III) For simultaneous overexpression of iolG and iolW, a PCR fragment covering regions A + B + C + H + D was ligated and integrated into the amyE locus.