Genome Editing Method for the Anaerobic Magnetotactic Bacterium Desulfovibrio Magneticus RS-1

Magnetosomes are complex bacterial organelles that serve as model systems for studying bacterial cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related of the genus that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. RS-1 is an anaerobic sulfate-reducing deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified 10 genes in the conserved magnetosome gene island of that are essential for its magnetic phenotype. However, this screen likely missed mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration have not been feasible due to the low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing , a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in and may also be applied to other genetically recalcitrant bacteria. Magnetotactic bacteria (MTB) are a group of organisms that form intracellular nanometer-scale magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membranes, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization and are potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from that of the cubooctahedral-shaped magnetite crystals of the genetically tractable MTB within the Here, we present a method for genome editing in RS-1, a cultured representative of the deeply branching MTB of the class This marks a crucial step in developing as a model for studying diverse mechanisms of magnetic particle formation by MTB.