Expanding the molecular toolbox for the bacterial phylum Planctomycetota

Strains belonging to the bacterial phylum Planctomycetota show several enigmatic cell biological features including an uncommon form of asymmetric cell division and a cell plan different from that of canonical Gram-negative model bacteria. A detailed analysis of these conspicuous traits is currently limited by the lack of sophisticated molecular biological tools for the genetic manipulation of these non-model microorganisms. Previous studies have reported the transposon-driven introduction of heterologous genes into random loci in the genome and more recently the targeted construction of single gene deletion mutants [1,2]. Still, the genetic potential remains largely untapped and the study of individual gene functions and regulatory networks is not possible without more advanced genetic engineering tools.

We here present data that considerably expands the molecular toolbox applicable for limnic and marine model planctomycetes. Extensive testing of selection marker genes yielded the first strain carrying deletions at multiple genomic loci. While testing different chromosomal locations for the introduction of heterologous genes, the expression of a set of reporter genes encoding fluorescent proteins and the multi-purpose HaloTag was achieved. The characterization of multiple novel members of the phylum in the last two years also yielded detailed information on the occurrence of plasmids. Based on bioinformatic analyses of extrachromosomal elements, we aim to construct replicative shuttle vectors as an episomal expression system based on native and foreign constitutive or inducible promoters.

The line of research will act as a platform for detailed analyses of the planctomycetal cell biology and an exploitation of their (secondary) metabolic repertoire. Genetic and metabolic engineering approaches will help to investigate the ecological role of planctomycetes in inter-species interactions and paves the way for their biotechnological applications.

References

[1] Jogler, C., et al. (2011). Appl. Environ. Microbiol. 77, 5826-5829.

[2] Santana-Molina, C., et al. (2022). Proc. Natl. Acad. Sci. U.S.A. 119, e2210081119.