Abstract
ABSTRACTMagnetosomes are complex bacterial organelles that serve as model systems for studying cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related AlphaproteobacterialMagnetospirillumspp. that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have also 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.Desulfovibrio magneticusRS-1 is an anaerobic sulfate-reducing Deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified ten genes in the conserved magnetosome gene island ofD. magneticusthat are essential for its magnetic phenotype. However, this screen likely missed many interesting 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 has not been feasible due to low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis inD. magneticususing a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of theuppgene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacingkupM,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 inD. magneticusand may also be applied to other genetically recalcitrant bacteria.IMPORTANCEMagnetotactic bacteria (MTB) are a group of organisms that form small, intracellular magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membrane, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization as well as 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 the cubooctahedral-shaped magnetite crystals of the genetically tractable Alphaproteobacteria MTB. Here, we present a method for genome editing in the anaerobic DeltaproteobacteriumDesulfovibrio magneticusRS-1, the first cultured representative of the deeply-branching MTB. This marks a crucial step in developingD. magneticusas a model for studying diverse mechanisms of magnetic particle formation by MTB.
Publisher
Cold Spring Harbor Laboratory