Fig. 2

An overview of allelic exchange mutagenesis protocol using pALFI vectors

Mutant allele is first cloned into an all elic exchange vector (pALFI) and transformed into E. coli Jke201 or another suitable donor strain for biparental mating. This step can ideally be completed in two working days. In the second step, the knockout vector is introduced into the Acinetobacter recipient strain by conjugation and screened for single-crossover mutants (positive selection). This step can be completed in 2–3 days. In the final step, single crossovers are transferred onto sucrose plates (negative selection) for mutant screening, which can be completed in 4 working days. Typically, the whole process can be completed in 8–10 working days. A detailed step by step protocol is included as the supplementary material S1 (Created with BioRender.com).