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Research Article

Identification of Different Putative Outer Membrane Electron Conduits Necessary for Fe(III) Citrate, Fe(III) Oxide, Mn(IV) Oxide, or Electrode Reduction by Geobacter sulfurreducens

Fernanda Jiménez Otero, Chi Ho Chan, Daniel R. Bond
Conrad W. Mullineaux, Editor
Fernanda Jiménez Otero
aBioTechnology Institute, University of Minnesota—Twin Cities, St. Paul, Minnesota, USA
bDepartment of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota—Twin Cities, St. Paul, Minnesota, USA
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  • ORCID record for Fernanda Jiménez Otero
Chi Ho Chan
aBioTechnology Institute, University of Minnesota—Twin Cities, St. Paul, Minnesota, USA
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Daniel R. Bond
aBioTechnology Institute, University of Minnesota—Twin Cities, St. Paul, Minnesota, USA
cDepartment of Plant and Microbial Biology, University of Minnesota—Twin Cities, St. Paul, Minnesota, USA
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Conrad W. Mullineaux
Queen Mary University of London
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DOI: 10.1128/JB.00347-18
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  • FIG 1
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    FIG 1

    The outer membrane electron conduit gene clusters of G. sulfurreducens. (A) Genetic organization and predicted features of operons containing putative outer membrane conduits. Deletion constructs are indicated by dashed lines. (B) Identity matrix from amino acid sequence alignment of each cytochrome or β-barrel component using ClustalΩ.

  • FIG 2
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    FIG 2

    OmcBC or ExtABCD is sufficient during Fe(III) citrate reduction; deletion of all clusters eliminates Fe(III) citrate reduction. Shown is growth using 55 mM Fe(III) citrate as an electron acceptor by mutants with single conduit cluster deletions (A), triple mutants lacking all but one cytochrome conduit, as well as the Δ5 strain lacking all five cytochrome conduits (B), mutants in an ΔomcBC background (C), and Δ5 mutants expressing omcB or extABCD or carrying an empty expression vector as control (D). All experiments were conducted in triplicate, and curves are averages ± standard deviations (SDs) from ≥3 replicates.

  • FIG 3
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    FIG 3

    No single outer membrane cluster is essential but all are necessary for wild-type levels of electron transfer to Fe(III) and Mn(IV) oxides. Shown is growth of single-cluster-deletion mutants and triple mutants lacking all but one cytochrome conduit cluster, as well as the Δ5 mutant lacking all clusters utilizing 70 mM Fe(III) oxide (A and B) or 20 mM Mn(IV) oxide (C and D) as the terminal electron acceptor. All experiments were conducted in triplicate, and curves are averages ± SD from ≥3 replicates.

  • FIG 4
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    FIG 4

    OmcBC and ExtEFG have additive roles in Fe(III) and Mn(IV) oxide reduction. Shown is reduction of 70 mM Fe(III) oxide (A) or 20 mM Mn(IV) oxide (B) by the ΔomcBC strain and additional deletions in an ΔomcBC background. All experiments were conducted in triplicate, and curves are averages ± SD from ≥3 replicates.

  • FIG 5
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    FIG 5

    Partial complementation by single conduit clusters supports the hypothesis that multiple conduit complexes are necessary for wild-type levels of metal oxide reduction. Shown is reduction of 70 mM Fe(III) oxide (A) or 20 mM Mn(IV) oxide (B) by the Δ5 mutant expressing extABCD or the omcB cluster compared to the empty vector control. All experiments were conducted in triplicate, and curves are averages ± SD from ≥3 replicates.

  • FIG 6
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    FIG 6

    Only the ExtABCD conduit cluster is necessary for electrode reduction. Shown is current density produced by single- (A) and multiple (B)-cluster-deletion mutants on graphite electrodes poised at +0.24 V versus SHE. All mutants were grown in at least two separate experiments, and curves are representative of results from ≥3 independent replicates per experiment. Similar results were obtained at lower (−0.1 V versus SHE) redox potentials.

  • FIG 7
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    FIG 7

    Effect of kanamycin on final current density and comparison of ExtABCD and OmcBC complementation. (A) Final current density of wild-type G. sulfurreducens compared to the wild type carrying an empty vector in the presence of increasing kanamycin concentrations. (B) Current density produced by the Δ5 strain plus either extABCD or omcB cluster-containing vectors in the presence of 5 μg/ml residual kanamycin. Wild-type and Δ5 strains carrying the empty vector were used as controls. All experiments were conducted in duplicate, and curves are representative of results from ≥3 replicates per experiment.

  • FIG 8
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    FIG 8

    Transcriptomic analysis comparing fumarate versus electrode growth for extABCD+ and wild-type strains. (A) Comparison of expression levels of wild-type exponentially growing cells under fumarate- and electrode-respiring conditions, showing no significant up- or downregulation of ext clusters (orange triangles) or most other known electron transfer proteins (red circles). Dark and light gray dotted lines represent thresholds of 4 and 2 log2, respectively. (B) RPKM and log2 change of open reading frames with largest expression changes as well as genes studied in this work (for additional data, see Table S2). (C and D) Comparison of the transcriptomes of wild-type and extABCD+ cells exponentially growing using fumarate (C) or electrode poised at +240 mV (D) as the terminal electron acceptor, showing no changes to electron transfer proteins due to deletion of omBC, extEFG, and extHIJKL clusters. Averages of biological replicate samples are shown.

  • FIG 9
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    FIG 9

    Cytochrome conduit conservation across the order Desulfuromonodales. Shown is a representation of cytochrome conduit clusters from the Desulfuromonodales with homologs to either OmcBC (A), ExtABCD (B), ExtEFG (C), or ExtHIJKL (D). Complete clusters with all components sharing >40% identity to the corresponding G. sulfurreducens cytochrome conduit are indicated in boxes to the left of each gene cluster. Clusters in which one or more proteins are replaced by a new element with <40% identity are listed on the right side of each gene cluster. Numbers with proteins indicate the percent identity to the G. sulfurreducens version. Red arrows, putative outer membrane products with a predicted lipid attachment site; yellow arrows, predicted periplasmic components; green arrows, predicted outer membrane anchor components. Superscript letters a to d indicate the following: a, OmcBC homologs in these gene clusters also encode Hox hydrogenase complexes; b, gene clusters have contiguous extBCD loci but extA is not in the vicinity, as extA was found in separate parts of the genome for some of those organisms (see Table S2 in the supplemental material); c, the gene cluster has additional lipoprotein decaheme c-type cytochrome upstream of extE; d, lipid attachment sites corresponding to ExtJL could not be found, but there is an additional small lipoprotein encoded within the gene cluster. For ExtHIJKL clusters, homologs depicted above extH are found in gene clusters containing only extI, whereas homologs depicted below extH are found in gene clusters containing full extHIJKL loci. Upstream and on the opposite strand to all gene clusters homologous to extHIJKL there is a transcription regulator of the LysR family, except where marked by superscript letter e, where there is no transcriptional regulator in that region, and superscript letter f, where there are transcriptional regulators of the TetR family instead.

Tables

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  • TABLE 1

    Comparative performance of G. sulfurreducens strains lacking one cluster or containing only one clustera

    Substrate% of wild type growth
    ΔomcBCΔextABCDΔextEFGΔextHIJKLomcBC+extABCD+extEFG+extHIJKL+Δ5
    Fe(III) citrate61.2 ± 10.5105 ± 6.662.5 ± 4.966.3 ± 2.5101.1 ± 8.499.2 ± 11.322.5 ± 2.423.8 ± 6.40.1 ± 0.6
    FeIII) oxide68.9 ± 8.483.3 ± 12.187.5 ± 14.995.8 ± 24.978.8 ± 3.929.2 ± 2.660.4 ± 9.552.1 ± 3.70.1 ± 0.3
    Mn(IV) oxide94.5 ± 6.495.1 ± 2.899.6 ± 3.497.9 ± 6.183.3 ± 14.126.7 ± 5.986.8 ± 6.575.6 ± 7.31.7 ± 0.9
    Electrode76.5 ± 16.520.9 ± 6.0104.8 ± 2.186.3 ± 15.328.3 ± 5.2137.9 ± 9.521.2 ± 6.525.9 ± 4.221.9 ± 4.4
    • ↵a Growth of single cytochrome conduit deletion mutants and mutants lacking all clusters except one, averaged from eight biological replicates or more and represented as percentage of wild-type growth. Averages and standard deviations are represented.

  • TABLE 2

    Strains and plasmids used in this study

    Strains or plasmidDescription or relevant genotypeReference or source
    Geobacter sulfurreducens strains
        DB1279ΔGSU2731–GSU2739 (ΔomcBC)46
        DB1280ΔGSU2645–GSU2642 (ΔextABCD)46
        DB1281ΔGSU2940–GSU2936 (ΔextHIJKL)46
        DB1282ΔGSU2724–GSU2726 (ΔextEFG)46
        DB1487ΔGSU2731–GSU2739 ΔGSU2645–GSU2642 (ΔomcBC ΔextABCD)This study
        DB1488ΔGSU2731–GSU2739 ΔGSU2724–GSU2726 (ΔomcBC ΔextEFG)This study
        DB1289ΔGSU2731–GSU2739 ΔGSU2940–GSU2936 (ΔomcBC ΔextHIJKL)This study
        DB1489ΔGSU2645–GSU2642 ΔGSU2724–GSU2726 (ΔextABCD ΔextEFG)This study
        DB1490ΔGSU2645–GSU2642 ΔGSU2940–GSU2936 (ΔextABCD ΔextHIJKL)This study
        DB1290ΔGSU2731–GSU2739 ΔGSU2940–GSU2936 ΔGSU2724–GSU2726 (extABCD+)This study
        DB1291ΔGSU2731–GSU2739 ΔGSU2645–GSU2642 ΔGSU2936–GSU2940 (extEFG+)This study
        DB1491ΔGSU2731–GSU2739 ΔGSU2645–GSU2642 ΔGSU2726–GSU2724 (extHIJKL+)This study
        DB1492ΔGSU2645–GSU2642 ΔGSU2726–GSU2724 ΔGSU2940–GSU2936 (omcBC+)This study
        DB1493ΔGSU2731–GSU2739 ΔGSU2645–GSU2642 ΔGSU2726–GSU2724 ΔGSU2940–GSU2936 (Δ5)This study
    Escherichia coli S17-1recA pro hsdR RP4-2-Tc::Mu-Km::Tn783
    Plasmids
        pK18mobsacB83
        pRK2-Geo256
        pDomcBCFlanking regions of omcBC in pK18mobsacBThis study
        pDextABCDFlanking regions of extABCD in pK18mobsacBThis study
        pDextEFGFlanking regions of extEFG in pK18mobsacBThis study
        pDextHIJKLFlanking regions of extHIJKL in pK18mobsacBThis study
        pomcBombB-omaB-omcB in pRK2-Geo2This study
        pextABCDextABCD in pRK2-Geo2This study

Additional Files

  • Figures
  • Tables
  • Supplemental material

    • Supplemental file 1 -

      Fig. S1 (Deletion of second clusters in ΔextABCD background results in residual activity) and S2 (ΔextABCD and Δ5 suppressor strains do not arise after 150 h) and Table S1 (Primers)

      PDF, 688K

    • Supplemental file 2 -

      Table S2 (RNAseq raw reads)

      XLSX, 446K

    • Supplemental file 3 -

      Table S3 (List of conduit cluster homolog locus tags)

      XLSX, 20K

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Identification of Different Putative Outer Membrane Electron Conduits Necessary for Fe(III) Citrate, Fe(III) Oxide, Mn(IV) Oxide, or Electrode Reduction by Geobacter sulfurreducens
Fernanda Jiménez Otero, Chi Ho Chan, Daniel R. Bond
Journal of Bacteriology Sep 2018, 200 (19) e00347-18; DOI: 10.1128/JB.00347-18

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Identification of Different Putative Outer Membrane Electron Conduits Necessary for Fe(III) Citrate, Fe(III) Oxide, Mn(IV) Oxide, or Electrode Reduction by Geobacter sulfurreducens
Fernanda Jiménez Otero, Chi Ho Chan, Daniel R. Bond
Journal of Bacteriology Sep 2018, 200 (19) e00347-18; DOI: 10.1128/JB.00347-18
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KEYWORDS

Geobacter
extracellular electron transfer
multiheme cytochrome
c-type cytochrome conduit
cytochrome electron conduit
multiheme c-type cytochomes

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