Novel tools for genomic modification and heterologous gene expression in the phylum Planctomycetota

Members of the phylum Planctomycetota possess a plethora of intriguing and hitherto underexplored features including an enlarged periplasmic space, asymmetric cell division ("budding"), and a mostly undiscovered small molecule portfolio. Due to the large phylogenetic distance to frequently used and easily genetically accessible model bacteria, most of the established genetic tools are not readily applicable for the here-investigated bacterial phylum. However, techniques for targeted gene inactivation and the introduction of heterologous genes are crucial to investigate the cell biology in the phylum in greater detail. In this study, the targeted genomic modification of model planctomycetes was achieved by enforcing two types of homologous recombination events: simultaneous double homologous recombination for the deletion of coding regions and insertion-duplication mutagenesis for the introduction of foreign DNA into the chromosome. Upon testing the expression of commonly used fluorescent protein-encoding genes, many of the tested native promoters could not be harnessed for variation of the expression strength. Since also four commonly used inducible gene expression systems did not work in the tested model strain Planctopirus limnophila, a native rhamnose-dependent transcriptional regulator/promoter pair was established as an inducible expression system. The expanded molecular toolbox will allow the future characterization of genome-encoded features in the understudied phylum. KEY POINTS: • Two recombination methods were used for the genetic modification of planctomycetes • Commonly used fluorescent proteins are functional in model planctomycetes • A rhamnose-dependent regulator was turned into an inducible expression system.