title: Scarless Genome Editing and Stable Inducible Expression Vectors for Geobacter sulfurreducens.
abstract: Metal reduction by members of the Geobacteraceae is encoded by multiple gene clusters, and the study of extracellular electron transfer often requires biofilm development on surfaces. Genetic tools that utilize polar antibiotic cassette insertions limit mutant construction and complementation. In addition, unstable plasmids create metabolic burdens that slow growth, and the presence of antibiotics such as kanamycin can interfere with the rate and extent of Geobacter biofilm growth. We report here genetic system improvements for the model anaerobic metal-reducing bacterium Geobacter sulfurreducens. A motile strain of G. sulfurreducens was constructed by precise removal of a transposon interrupting the fgrM flagellar regulator gene using SacB/sucrose counterselection, and Fe(III) citrate reduction was eliminated by deletion of the gene encoding the inner membrane cytochrome imcH. We also show that RK2-based plasmids were maintained in G. sulfurreducens for over 15 generations in the absence of antibiotic selection in contrast to unstable pBBR1 plasmids. Therefore, we engineered a series of new RK2 vectors containing native constitutive Geobacter promoters, and modified one of these promoters for VanR-dependent induction by the small aromatic carboxylic acid vanillate. Inducible plasmids fully complemented ΔimcH mutants for Fe(III) reduction, Mn(IV) oxide reduction, and growth on poised electrodes. A real-time, high-throughput Fe(III) citrate reduction assay is described that can screen numerous G. sulfurreducens strain constructs simultaneously and shows the sensitivity of imcH expression by the vanillate system. These tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.
authors.affiliation: BioTechnology Institute, University of Minnesota-Twin Cities, Saint Paul, Minnesota, USA.
authors.forename: Chi Ho
authors.identifier: http://orcid.org/0000-0002-6596-3436
authors.initials: CH
authors.lastname: Chan
authors.affiliation: BioTechnology Institute, University of Minnesota-Twin Cities, Saint Paul, Minnesota, USA Department of Microbiology, University of Minnesota-Twin Cities, Saint Paul, Minnesota, USA.
authors.forename: Caleb E
authors.initials: CE
authors.lastname: Levar
authors.affiliation: BioTechnology Institute, University of Minnesota-Twin Cities, Saint Paul, Minnesota, USA Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota-Twin Cities, Saint Paul, Minnesota, USA.
authors.forename: Lori
authors.initials: L
authors.lastname: Zacharoff
authors.affiliation: BioTechnology Institute, University of Minnesota-Twin Cities, Saint Paul, Minnesota, USA.
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Badalamenti
authors.affiliation: BioTechnology Institute, University of Minnesota-Twin Cities, Saint Paul, Minnesota, USA Department of Microbiology, University of Minnesota-Twin Cities, Saint Paul, Minnesota, USA dbond@umn.edu.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Bond
mesh_terms: D004579:Electron Transport / Q000235:genetics / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics*; D045302:Geobacter / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics