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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)more » 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.« less

Authors:
ORCiD logo [1];  [1];  [1];  [1];  [1];  [1]
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  1. Univ. of Minnesota-Twin Cities, Saint Paul, MN (United States)
Publication Date:
Research Org.:
Univ. of Minnesota-Twin Cities, Saint Paul, MN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1345183
Grant/Contract Number:  
SC0006868
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 81; Journal Issue: 20; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Chan, Chi Ho, Levar, Caleb E., Zacharoff, Lori, Badalamenti, Jonathan P., Bond, Daniel R., and Loffler, F. E. Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens. United States: N. p., 2015. Web. doi:10.1128/aem.01967-15.
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Chan, Chi Ho, Levar, Caleb E., Zacharoff, Lori, Badalamenti, Jonathan P., Bond, Daniel R., & Loffler, F. E. Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens. United States. https://doi.org/10.1128/aem.01967-15
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Chan, Chi Ho, Levar, Caleb E., Zacharoff, Lori, Badalamenti, Jonathan P., Bond, Daniel R., and Loffler, F. E. Fri . "Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens". United States. https://doi.org/10.1128/aem.01967-15. https://www.osti.gov/servlets/purl/1345183.
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@article{osti_1345183,
title = {Scarless genome editing and stable inducible expression vectors for Geobacter sulfurreducens},
author = {Chan, Chi Ho and Levar, Caleb E. and Zacharoff, Lori and Badalamenti, Jonathan P. and Bond, Daniel R. and Loffler, F. E.},
abstractNote = {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.},
doi = {10.1128/aem.01967-15},
journal = {Applied and Environmental Microbiology},
number = 20,
volume = 81,
place = {United States},
year = {Fri Aug 07 00:00:00 EDT 2015},
month = {Fri Aug 07 00:00:00 EDT 2015}
}
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https://doi.org/10.1128/aem.01967-15
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Works referenced in this record:

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Proof of principle for an engineered microbial biosensor based on Shewanella oneidensis outer membrane protein complexes
journal, September 2013

Laboratory evolution of Geobacter sulfurreducens for enhanced growth on lactate via a single-base-pair substitution in a transcriptional regulator
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A Broad Host Range Mobilization System for In Vivo Genetic Engineering Transposon Mutagenesis in Gram Negative Bacteria
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  • Bio/Technology, Vol. 1, Issue 9, p. 784-791
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Characterizing the interplay between multiple levels of organization within bacterial sigma factor regulatory networks
journal, April 2013

  • Qiu, Yu; Nagarajan, Harish; Embree, Mallory
  • Nature Communications, Vol. 4, Issue 1
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Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data
journal, May 2013

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Principles of genetic circuit design
journal, April 2014

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Model organisms for genetics in the domain Archaea: methanogens, halophiles, Thermococcales and Sulfolobales
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Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq
journal, January 2014

  • Barrick, Jeffrey E.; Colburn, Geoffrey; Deatherage, Daniel E.
  • BMC Genomics, Vol. 15, Issue 1
  • DOI: 10.1186/1471-2164-15-1039

Broad-Host-Range cre-lox System for Antibiotic Marker Recycling in Gram-Negative Bacteria
journal, November 2002

  • Marx, Christopher J.; Lidstrom, Mary E.
  • BioTechniques, Vol. 33, Issue 5
  • DOI: 10.2144/02335rr01
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    Works referencing / citing this record:

    Construction of a Geobacter Strain With Exceptional Growth on Cathodes
    journal, July 2018

    Redox potential as a master variable controlling pathways of metal reduction by Geobacter sulfurreducens
    journal, January 2017

    • Levar, Caleb E.; Hoffman, Colleen L.; Dunshee, Aubrey J.
    • The ISME Journal, Vol. 11, Issue 3
    • DOI: 10.1038/ismej.2016.146

    Isolation and Genomic Characterization of ‘Desulfuromonas soudanensis WTL’, a Metal- and Electrode-Respiring Bacterium from Anoxic Deep Subsurface Brine
    journal, June 2016

    • Badalamenti, Jonathan P.; Summers, Zarath M.; Chan, Chi Ho
    • Frontiers in Microbiology, Vol. 7
    • DOI: 10.3389/fmicb.2016.00913

    Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration
    journal, April 2019

    Isolation and Genomic Characterization of Desulfuromonas soudanensis WTL, a Metal- and Electrode-Reducing Bacterium from Anoxic Deep Subsurface Brine
    journal, April 2016

    • Badalamenti, Jonathan P.; Summers, Zarath M.; Chan, Chi Ho
    • Frontiers in Microbiology
    • DOI: 10.1101/047530

    Geobacter sulfurreducens extracellular multiheme cytochrome PgcA facilitates respiration to Fe(III) oxides but not electrodes
    journal, August 2017

    • Zacharoff, Lori; Morrone, Dana; Bond, Daniel R.
    • Frontiers in Microbiology
    • DOI: 10.1101/172775

    cGAMP signaling controls a transient surface-associated lifestyle
    journal, December 2018

    • Hallberg, Zachary F.; Chan, Chi Ho; Wright, Todd A.
    • eLife
    • DOI: 10.1101/495150

    Isolation and Genomic Characterization of ‘Desulfuromonas soudanensis WTL’, a Metal- and Electrode-Respiring Bacterium from Anoxic Deep Subsurface Brine
    journal, June 2016

    • Badalamenti, Jonathan P.; Summers, Zarath M.; Chan, Chi Ho
    • Frontiers in Microbiology, Vol. 7
    • DOI: 10.3389/fmicb.2016.00913

    Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration
    journal, April 2019

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