The alphaproteobacterium Magnetospirillum gryphiswaldense biosynthesizes magnetosomes, which consist of membrane-enveloped magnetite crystals aligned in linear chains. As one of only few tractable magnetic bacteria it has emerged as a widely used model for the study of prokaryotic organelle formation and the bioproduction of magnetic nanoparticles. However, analysis and engineering of magnetosome biogenesis has been hampered by the limited toolset for controlled gene expression in this bacterium.
Here, we investigated novel candidate promoters for strong and tunable gene expression. Transcription from a set of putative and known promoters from M. gryphiswaldense and other bacteria was explored in M. gryphiswaldense using the luxABCDE luciferase operon as reporter. The resulting bioluminescence was measured in the absence and presence of cognate repressors. Among all tested promoters, Pcym and cymR, a promoter-repressor system derived from Pseudomonas putida, caused the highest expression of lux genes, thereby exceeding the activity of all currently used constitutive and inducible foreign and endogenous promoters in M. gryphiswaldense such as PmamG, Plac and Ptet. Furthermore, Pcym was tightly repressed in the absence of its inducer cumate, but could be rapidly induced at different cumate concentrations.
To explore cymR/Pcym for the induced expression of single genes, we placed this system in front of mamI, mamE, mamL, mamM, mamO, mamQ, mamB, respectively, all encoding essential magnetosome proteins. Wildtype-like magnetosome formation could be restored in non-magnetic single deletion mutants in the presence of the inducer. By varying the conditions, we are currently exploring the system for the tuned (over)expression of entire accessory magnetosome gene clusters, such as mms6, mamGFDC and mamXYZ. This might enable the tuning of size, shape, and number of magnetosome crystals. In conclusion, this novel genetic tool is expected to be used for functional analysis and engineering of prokaryotic organelle formation, specifically to identify the influential parameters for controlled magnetosome biogenesis in M. gryphiswaldense.