title: A tailored galK counterselection system for efficient markerless gene deletion and chromosomal tagging in Magnetospirillum gryphiswaldense.
abstract: Magnetotactic bacteria have emerged as excellent model systems to study bacterial cell biology, biomineralization, vesicle formation, and protein targeting because of their ability to synthesize single-domain magnetite crystals within unique organelles (magnetosomes). However, only few species are amenable to genetic manipulation, and the limited methods for site-specific mutagenesis are tedious and time-consuming. Here, we report the adaptation and application of a fast and convenient technique for markerless chromosomal manipulation of Magnetospirillum gryphiswaldense using a single antibiotic resistance cassette and galK-based counterselection for marker recycling. We demonstrate the potential of this technique by genomic excision of the phbCAB operon, encoding enzymes for polyhydroxyalkanoate (PHA) synthesis, followed by chromosomal fusion of magnetosome-associated proteins to fluorescent proteins. Because of the absence of interfering PHA particles, these engineered strains are particularly suitable for microscopic analyses of cell biology and magnetosome biosynthesis.
authors.affiliation: Ludwig Maximilian University Munich, Department Biology I, Biocenter, Planegg-Martinsried, Germany Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Planegg-Martinsried, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Raschdorf
authors.affiliation: Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Planegg-Martinsried, Germany Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, the Netherlands.
authors.forename: Jürgen M
authors.initials: JM
authors.lastname: Plitzko
authors.affiliation: Ludwig Maximilian University Munich, Department Biology I, Biocenter, Planegg-Martinsried, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Schüler
authors.affiliation: Ludwig Maximilian University Munich, Department Biology I, Biocenter, Planegg-Martinsried, Germany Frank.Mueller@biologie.uni-muenchen.de.
authors.forename: Frank D
authors.initials: FD
authors.lastname: Müller
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics*; D015169:Colony Count, Microbial; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D023281:Genomics / Q000379:methods; D055944:Magnetosomes / Q000382:microbiology; D042201:Magnetospirillum / Q000235:genetics*; D046529:Microscopy, Electron, Transmission; D016297:Mutagenesis, Site-Directed; D009876:Operon; D054813:Polyhydroxyalkanoates / Q000378:metabolism
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial; D054813:Polyhydroxyalkanoates