Figure 4

Targeted inactivation of polP. (A) Schematic representation for the construction of CY7 mutant. (B) PCR confirmation of the CY7 mutants, as a 1.1-kb aac(3)IV fragment replaced 495-bp of polP, the CY7 mutants give 1.6-kb PCR product, while WT of S. cacaoi produces 1.0-kb product. (C) Bioassay of the metabolites produced by CY7 mutant, the sample of S. cacaoi wild type indicated as (1) was used as positive control, and the samples from CY7 mutants were indicated as (2-4). 35 μl sample (supernatant) was used for all bioassays in this study. (D) HPLC analysis of the metabolites produced by CY7 mutant. Polyoxins authentic standard (ST) and sample from wild-type of S. cacaoi (WT) were used as positive control, and the sample from CY7 mutant was indicated as CY7.