Fig. 1. Scheme of the upp-based counterselection system for P. mendocina NK-01. A and B represent homologous arms flanking the segment to be deleted. In the first step, single-crossover
recombination occurs in either the up- or the downstream region. The resulting strain is sensitive to 5-FU and resistant to tetracycline. In the second step, another recombination event
takes place, in which either the original state is restored or the region of interest is deleted. These strains are now resistant to 5-FU and sensitive to tetracycline. PCR and DNA sequencing
confirmed the chromosomal structures of the mutants. Primers for selection are indicated as p1, p2, and p3 in the diagram and explained in detail in the “Results” section.