Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens

Chan, Chi Ho; Levar, Caleb E.; Zacharoff, Lori; Badalamenti, Jonathan P.; Bond, Daniel R.; Loffler, F. E.

doi:10.1128/aem.01967-15

Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens

Journal Article · Fri Aug 07 00:00:00 EDT 2015 · Applied and Environmental Microbiology

DOI:https://doi.org/10.1128/aem.01967-15· OSTI ID:1345183

^[1]; Levar, Caleb E. ^[2]; Zacharoff, Lori ^[2]; Badalamenti, Jonathan P. ^[2]; Bond, Daniel R. ^[2]; Loffler, F. E. ^[2]

Univ. of Minnesota-Twin Cities, Saint Paul, MN (United States); DOE Office of Scientific and Technical Information (OSTI)
Univ. of Minnesota-Twin Cities, Saint Paul, MN (United States)

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. Lastly, these tools will enable more sophisticated genetic studies in G. sulfurreducens without polar insertion effects or need for multiple antibiotics.

View Accepted Manuscript (DOE)

Research Organization:: Univ. of Minnesota-Twin Cities, Saint Paul, MN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)

Grant/Contract Number:: SC0006868

OSTI ID:: 1345183

Journal Information:: Applied and Environmental Microbiology, Journal Name: Applied and Environmental Microbiology Journal Issue: 20 Vol. 81; ISSN 0099-2240

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

References (49)

The proteome of dissimilatory metal-reducing microorganism Geobacter sulfurreducens under various growth conditions Ding, Yan-Huai R.; Hixson, Kim K.; Giometti, Carol S. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1764, Issue 7 https://doi.org/10.1016/j.bbapap.2006.04.017	journal	July 2006
Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes Golitsch, Frederik; Bücking, Clemens; Gescher, Johannes Biosensors and Bioelectronics, Vol. 47 https://doi.org/10.1016/j.bios.2013.03.010	journal	September 2013
Construction of versatile high-level expression vectors for Bartonella henselae and the use of green fluorescent protein as a new expression marker Dehio, Michaela; Knorre, Alexander; Lanz, Christa Gene, Vol. 215, Issue 2 https://doi.org/10.1016/s0378-1119(98)00319-9	journal	July 1998
Laboratory evolution of Geobacter sulfurreducens for enhanced growth on lactate via a single-base-pair substitution in a transcriptional regulator Summers, Zarath M.; Ueki, Toshiyuki; Ismail, Wael The ISME Journal, Vol. 6, Issue 5 https://doi.org/10.1038/ismej.2011.166	journal	November 2011
A Broad Host Range Mobilization System for In Vivo Genetic Engineering Transposon Mutagenesis in Gram Negative Bacteria Simon, R.; Priefer, U.; Pühler, A. Bio/Technology, Vol. 1, Issue 9, p. 784-791 https://doi.org/10.1038/nbt1183-784	journal	November 1983
Characterizing the interplay between multiple levels of organization within bacterial sigma factor regulatory networks Qiu, Yu; Nagarajan, Harish; Embree, Mallory Nature Communications, Vol. 4, Issue 1 https://doi.org/10.1038/ncomms2743	journal	April 2013
Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data Chin, Chen-Shan; Alexander, David H.; Marks, Patrick Nature Methods, Vol. 10, Issue 6 https://doi.org/10.1038/nmeth.2474	journal	May 2013
Principles of genetic circuit design Brophy, Jennifer A. N.; Voigt, Christopher A. Nature Methods, Vol. 11, Issue 5 https://doi.org/10.1038/nmeth.2926	journal	April 2014
Acapsular Staphylococcus aureus with a non-functional agr regains capsule expression after passage through the bloodstream in a bacteremia mouse model Suligoy, Carlos M.; Díaz, Rocío E.; Gehrke, Ana-Katharina Scientific Reports, Vol. 10, Issue 1 https://doi.org/10.1038/s41598-020-70671-1	journal	August 2020
CbiZ, an amidohydrolase enzyme required for salvaging the coenzyme B12 precursor cobinamide in archaea Woodson, J. D.; Escalante-Semerena, J. C. Proceedings of the National Academy of Sciences, Vol. 101, Issue 10 https://doi.org/10.1073/pnas.0305939101	journal	February 2004
Identification of Multicomponent Histidine-Aspartate Phosphorelay System Controlling Flagellar and Motility Gene Expression in Geobacter Species Ueki, Toshiyuki; Leang, Ching; Inoue, Kengo Journal of Biological Chemistry, Vol. 287, Issue 14 https://doi.org/10.1074/jbc.m112.345041	journal	February 2012
A comprehensive set of plasmids for vanillate- and xylose-inducible gene expression in Caulobacter crescentus Thanbichler, M.; Iniesta, A. A.; Shapiro, L. Nucleic Acids Research, Vol. 35, Issue 20 https://doi.org/10.1093/nar/gkm818	journal	November 2007
Model organisms for genetics in the domain Archaea: methanogens, halophiles, Thermococcales and Sulfolobales Leigh, John A.; Albers, Sonja-Verena; Atomi, Haruyuki FEMS Microbiology Reviews, Vol. 35, Issue 4 https://doi.org/10.1111/j.1574-6976.2011.00265.x	journal	July 2011
A genetic system for Geobacter metallireducens: role of the flagellin and pilin in the reduction of Fe(III) oxide: Role of pilin and flagellin in G. metallireducens Tremblay, Pier-Luc; Aklujkar, Muktak; Leang, Ching Environmental Microbiology Reports, Vol. 4, Issue 1 https://doi.org/10.1111/j.1758-2229.2011.00305.x	journal	November 2011
Metagenomic Analysis of Streptomyces lividans Reveals Host-Dependent Functional Expression McMahon, Matthew D.; Guan, Changhui; Handelsman, Jo Applied and Environmental Microbiology, Vol. 78, Issue 10 https://doi.org/10.1128/aem.00044-12	journal	March 2012
Microbial Biofilm Voltammetry: Direct Electrochemical Characterization of Catalytic Electrode-Attached Biofilms Marsili, E.; Rollefson, J. B.; Baron, D. B. Applied and Environmental Microbiology, Vol. 74, Issue 23 https://doi.org/10.1128/aem.00177-08	journal	October 2008
Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate Voordeckers, J. W.; Kim, B. -C.; Izallalen, M. Applied and Environmental Microbiology, Vol. 76, Issue 7 https://doi.org/10.1128/aem.02250-09	journal	February 2010
Organic Matter Mineralization with Reduction of Ferric Iron in Anaerobic Sediments Lovley, Derek R.; Phillips, Elizabeth J. P. Applied and Environmental Microbiology, Vol. 51, Issue 4 https://doi.org/10.1128/aem.51.4.683-689.1986	journal	January 1986
Development of a Genetic System for Geobacter sulfurreducens Coppi, M. V.; Leang, C.; Sandler, S. J. Applied and Environmental Microbiology, Vol. 67, Issue 7, p. 3180-3187 https://doi.org/10.1128/aem.67.7.3180-3187.2001	journal	July 2001
The ars Detoxification System Is Advantageous but Not Required for As(V) Respiration by the Genetically Tractable Shewanella Species Strain ANA-3 Saltikov, C. W.; Cifuentes, A.; Venkateswaran, K. Applied and Environmental Microbiology, Vol. 69, Issue 5 https://doi.org/10.1128/aem.69.5.2800-2809.2003	journal	May 2003
Outer Membrane c-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction in Geobacter sulfurreducens Mehta, T.; Coppi, M. V.; Childers, S. E. Applied and Environmental Microbiology, Vol. 71, Issue 12 https://doi.org/10.1128/aem.71.12.8634-8641.2005	journal	December 2005
Characterization of Metabolism in the Fe(III)-Reducing Organism Geobacter sulfurreducens by Constraint-Based Modeling Mahadevan, R.; Bond, D. R.; Butler, J. E. Applied and Environmental Microbiology, Vol. 72, Issue 2 https://doi.org/10.1128/aem.72.2.1558-1568.2006	journal	February 2006
Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens Rollefson, J. B.; Levar, C. E.; Bond, D. R. Journal of Bacteriology, Vol. 191, Issue 13 https://doi.org/10.1128/jb.00057-09	journal	April 2009
Two Systems for Conditional Gene Expression in Myxococcus xanthus Inducible by Isopropyl- -D-Thiogalactopyranoside or Vanillate Iniesta, A. A.; Garcia-Heras, F.; Abellon-Ruiz, J. Journal of Bacteriology, Vol. 194, Issue 21 https://doi.org/10.1128/jb.01110-12	journal	August 2012
How the xap Locus Put Electrical “Zap” in Geobacter sulfurreducens Biofilms Magnuson, Timothy S. Journal of Bacteriology, Vol. 193, Issue 5 https://doi.org/10.1128/jb.01478-10	journal	March 2011
OmcB, a c-Type Polyheme Cytochrome, Involved in Fe(III) Reduction in Geobacter sulfurreducens Leang, C.; Coppi, M. V.; Lovley, D. R. Journal of Bacteriology, Vol. 185, Issue 7 https://doi.org/10.1128/jb.185.7.2096-2103.2003	journal	April 2003
An Inner Membrane Cytochrome Required Only for Reduction of High Redox Potential Extracellular Electron Acceptors Levar, Caleb E.; Chan, Chi Ho; Mehta-Kolte, Misha G. mBio, Vol. 5, Issue 6 https://doi.org/10.1128/mbio.02034-14	journal	November 2014
Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq Barrick, Jeffrey E.; Colburn, Geoffrey; Deatherage, Daniel E. BMC Genomics, Vol. 15, Issue 1 https://doi.org/10.1186/1471-2164-15-1039	journal	January 2014
Broad-Host-Range cre-lox System for Antibiotic Marker Recycling in Gram-Negative Bacteria Marx, Christopher J.; Lidstrom, Mary E. BioTechniques, Vol. 33, Issue 5 https://doi.org/10.2144/02335rr01	journal	November 2002
Construction of versatile high-level expression vectors for Bartonella henselae and the use of green fluorescent protein as a new expression marker Dehio, Michaela; Knorre, Alexander; Lanz, Christa Gene, Vol. 215, Issue 2 https://doi.org/10.1016/S0378-1119(98)00319-9	journal	July 1998
Identification of Multicomponent Histidine-Aspartate Phosphorelay System Controlling Flagellar and Motility Gene Expression in Geobacter Species Ueki, Toshiyuki; Leang, Ching; Inoue, Kengo Journal of Biological Chemistry, Vol. 287, Issue 14 https://doi.org/10.1074/jbc.M112.345041	journal	February 2012
Differences in Fe(III) reduction in the hyperthermophilic archaeon, Pyrobaculum islandicum , versus mesophilic Fe(III)-reducing bacteria Childers, Susan E.; Lovley, Derek R. FEMS Microbiology Letters, Vol. 195, Issue 2 https://doi.org/10.1111/j.1574-6968.2001.tb10529.x	journal	February 2001
Metagenomic Analysis of Streptomyces lividans Reveals Host-Dependent Functional Expression McMahon, Matthew D.; Guan, Changhui; Handelsman, Jo Applied and Environmental Microbiology, Vol. 78, Issue 10 https://doi.org/10.1128/AEM.00044-12	journal	March 2012
Microbial Biofilm Voltammetry: Direct Electrochemical Characterization of Catalytic Electrode-Attached Biofilms Marsili, E.; Rollefson, J. B.; Baron, D. B. Applied and Environmental Microbiology, Vol. 74, Issue 23 https://doi.org/10.1128/AEM.00177-08	journal	October 2008
Broad-Host-Range Expression Vectors with Tightly Regulated Promoters and Their Use To Examine the Influence of TraR and TraM Expression on Ti Plasmid Quorum Sensing Khan, S. R.; Gaines, J.; Roop, R. M. Applied and Environmental Microbiology, Vol. 74, Issue 16 https://doi.org/10.1128/AEM.01098-08	journal	July 2008
Role of Geobacter sulfurreducens Outer Surface c-Type Cytochromes in Reduction of Soil Humic Acid and Anthraquinone-2,6-Disulfonate Voordeckers, J. W.; Kim, B. -C.; Izallalen, M. Applied and Environmental Microbiology, Vol. 76, Issue 7 https://doi.org/10.1128/AEM.02250-09	journal	February 2010
Organic Matter Mineralization with Reduction of Ferric Iron in Anaerobic Sediments Lovley, Derek R.; Phillips, Elizabeth J. P. Applied and Environmental Microbiology, Vol. 51, Issue 4 https://doi.org/10.1128/AEM.51.4.683-689.1986	journal	January 1986
Development of a Genetic System for Geobacter sulfurreducens Coppi, M. V.; Leang, C.; Sandler, S. J. Applied and Environmental Microbiology, Vol. 67, Issue 7, p. 3180-3187 https://doi.org/10.1128/AEM.67.7.3180-3187.2001	journal	July 2001
The ars Detoxification System Is Advantageous but Not Required for As(V) Respiration by the Genetically Tractable Shewanella Species Strain ANA-3 Saltikov, C. W.; Cifuentes, A.; Venkateswaran, K. Applied and Environmental Microbiology, Vol. 69, Issue 5 https://doi.org/10.1128/AEM.69.5.2800-2809.2003	journal	May 2003
Outer Membrane c-Type Cytochromes Required for Fe(III) and Mn(IV) Oxide Reduction in Geobacter sulfurreducens Mehta, T.; Coppi, M. V.; Childers, S. E. Applied and Environmental Microbiology, Vol. 71, Issue 12 https://doi.org/10.1128/AEM.71.12.8634-8641.2005	journal	December 2005
Characterization of Metabolism in the Fe(III)-Reducing Organism Geobacter sulfurreducens by Constraint-Based Modeling Mahadevan, R.; Bond, D. R.; Butler, J. E. Applied and Environmental Microbiology, Vol. 72, Issue 2 https://doi.org/10.1128/AEM.72.2.1558-1568.2006	journal	February 2006
Identification of Genes Involved in Biofilm Formation and Respiration via Mini-Himar Transposon Mutagenesis of Geobacter sulfurreducens Rollefson, J. B.; Levar, C. E.; Bond, D. R. Journal of Bacteriology, Vol. 191, Issue 13 https://doi.org/10.1128/JB.00057-09	journal	April 2009
Identification of an Extracellular Polysaccharide Network Essential for Cytochrome Anchoring and Biofilm Formation in Geobacter sulfurreducens Rollefson, J. B.; Stephen, C. S.; Tien, M. Journal of Bacteriology, Vol. 193, Issue 5 https://doi.org/10.1128/JB.01092-10	journal	December 2010
Two Systems for Conditional Gene Expression in Myxococcus xanthus Inducible by Isopropyl- -D-Thiogalactopyranoside or Vanillate Iniesta, A. A.; Garcia-Heras, F.; Abellon-Ruiz, J. Journal of Bacteriology, Vol. 194, Issue 21 https://doi.org/10.1128/JB.01110-12	journal	August 2012
Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. Gay, P.; Le Coq, D.; Steinmetz, M. Journal of Bacteriology, Vol. 164, Issue 2 https://doi.org/10.1128/JB.164.2.918-921.1985	journal	January 1985
OmcB, a c-Type Polyheme Cytochrome, Involved in Fe(III) Reduction in Geobacter sulfurreducens Leang, C.; Coppi, M. V.; Lovley, D. R. Journal of Bacteriology, Vol. 185, Issue 7 https://doi.org/10.1128/JB.185.7.2096-2103.2003	journal	April 2003
Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. Gay, P.; Le Coq, D.; Steinmetz, M. Journal of Bacteriology, Vol. 164, Issue 2 https://doi.org/10.1128/jb.164.2.918-921.1985	journal	January 1985
An Inner Membrane Cytochrome Required Only for Reduction of High Redox Potential Extracellular Electron Acceptors Levar, Caleb E.; Chan, Chi Ho; Mehta-Kolte, Misha G. mBio, Vol. 5, Issue 6 https://doi.org/10.1128/mBio.02034-14	journal	November 2014
Pilon: An Integrated Tool for Comprehensive Microbial Variant Detection and Genome Assembly Improvement Walker, Bruce J.; Abeel, Thomas; Shea, Terrance PLoS ONE, Vol. 9, Issue 11 https://doi.org/10.1371/journal.pone.0112963	journal	November 2014

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Genome Scale Mutational Analysis of Geobacter sulfurreducens Reveals Distinct Molecular Mechanisms for Respiration and Sensing of Poised Electrodes versus Fe(III) Oxides Chan, Chi Ho; Levar, Caleb E.; Jiménez-Otero, Fernanda Journal of Bacteriology, Vol. 199, Issue 19 https://doi.org/10.1128/jb.00340-17	journal	July 2017
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Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration Hallberg, Zachary F.; Chan, Chi Ho; Wright, Todd A. eLife, Vol. 8 https://doi.org/10.7554/elife.43959	journal	April 2019
Redox potential as a master variable controlling pathways of metal reduction by Geobacter sulfurreducens Levar, Caleb E.; Hoffman, Colleen L.; Dunshee, Aubrey J. The ISME Journal, Vol. 11, Issue 3 https://doi.org/10.1038/ismej.2016.146	journal	January 2017
Divergent Nrf Family Proteins and MtrCAB Homologs Facilitate Extracellular Electron Transfer in Aeromonas hydrophila Conley, Bridget E.; Intile, Peter J.; Bond, Daniel R. Applied and Environmental Microbiology, Vol. 84, Issue 23 https://doi.org/10.1128/aem.02134-18	journal	September 2018
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