f nucleatum  (ATCC)


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    Structured Review

    ATCC f nucleatum
    Growth of F. <t>nucleatum</t> 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.
    F Nucleatum, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria"

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.15.5330-5340.2005

    Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.
    Figure Legend Snippet: Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.

    Techniques Used:

    RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin
    Figure Legend Snippet: RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

    DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC
    Figure Legend Snippet: DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC

    Techniques Used: Dot Blot

    I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes
    Figure Legend Snippet: I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes

    Techniques Used: Western Blot, Staining, SDS Page

    Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are
    Figure Legend Snippet: Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are

    Techniques Used: Sequencing

    Construction and screening of F. nucleatum 12230 cosmid library.
    Figure Legend Snippet: Construction and screening of F. nucleatum 12230 cosmid library.

    Techniques Used:

    Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the
    Figure Legend Snippet: Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the

    Techniques Used: Mutagenesis

    Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.
    Figure Legend Snippet: Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.

    Techniques Used:

    2) Product Images from "Diverse Toll-Like Receptors Mediate Cytokine Production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in Macrophages"

    Article Title: Diverse Toll-Like Receptors Mediate Cytokine Production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in Macrophages

    Journal:

    doi: 10.1128/IAI.01226-13

    Cytokines production by WT and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. BMDMs from WT and MyD88-deficient mice were infected with F. nucleatum or A. actinomycetemcomitans for 6 h, and, as indicated, the
    Figure Legend Snippet: Cytokines production by WT and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. BMDMs from WT and MyD88-deficient mice were infected with F. nucleatum or A. actinomycetemcomitans for 6 h, and, as indicated, the

    Techniques Used: Infection, Mouse Assay

    Production of IL-6 and TNF-α in WT and TLR2/4- and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. WT and TLR2/4-, and MyD88-deficient BMDMs were infected with F. nucleatum and A. actinomycetemcomitans
    Figure Legend Snippet: Production of IL-6 and TNF-α in WT and TLR2/4- and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. WT and TLR2/4-, and MyD88-deficient BMDMs were infected with F. nucleatum and A. actinomycetemcomitans

    Techniques Used: Infection

    Effect of NF-κB and MAPKs on F. nucleatum - and A. actinomycetemcomitans -induced production of cytokines by macrophages. WT BMDMs were pretreated with various doses of each inhibitor 2 h before infection. The cells were then infected with F. nucleatum
    Figure Legend Snippet: Effect of NF-κB and MAPKs on F. nucleatum - and A. actinomycetemcomitans -induced production of cytokines by macrophages. WT BMDMs were pretreated with various doses of each inhibitor 2 h before infection. The cells were then infected with F. nucleatum

    Techniques Used: Infection

    Effect of endosomal TLRs and bacterial DNA on cytokine production by macrophages in response to F. nucleatum or A. actinomycetemcomitans infection. TLR2/4-deficient BMDMs were pretreated with chloroquine (CLQ) 2 h before infection. The cells were then
    Figure Legend Snippet: Effect of endosomal TLRs and bacterial DNA on cytokine production by macrophages in response to F. nucleatum or A. actinomycetemcomitans infection. TLR2/4-deficient BMDMs were pretreated with chloroquine (CLQ) 2 h before infection. The cells were then

    Techniques Used: Infection

    Production of IL-6 and TNF-α by WT and TLR2-, TLR4-, and TLR2/4-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans . BMDMs were infected with F. nucleatum or A. actinomycetemcomitans at the indicated MOI. At 6 (A to D) or
    Figure Legend Snippet: Production of IL-6 and TNF-α by WT and TLR2-, TLR4-, and TLR2/4-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans . BMDMs were infected with F. nucleatum or A. actinomycetemcomitans at the indicated MOI. At 6 (A to D) or

    Techniques Used: Infection

    NF-κB and MAPK activation in WT and TLR2/4-, and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. Cells were infected with F. nucleatum or A. actinomycetemcomitans at an MOI of 1/100, and cellular protein
    Figure Legend Snippet: NF-κB and MAPK activation in WT and TLR2/4-, and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. Cells were infected with F. nucleatum or A. actinomycetemcomitans at an MOI of 1/100, and cellular protein

    Techniques Used: Activation Assay, Infection

    3) Product Images from "Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria"

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.15.5330-5340.2005

    Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.
    Figure Legend Snippet: Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.

    Techniques Used:

    RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin
    Figure Legend Snippet: RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

    DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC
    Figure Legend Snippet: DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC

    Techniques Used: Dot Blot

    I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes
    Figure Legend Snippet: I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes

    Techniques Used: Western Blot, Staining, SDS Page

    Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are
    Figure Legend Snippet: Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are

    Techniques Used: Sequencing

    Construction and screening of F. nucleatum 12230 cosmid library.
    Figure Legend Snippet: Construction and screening of F. nucleatum 12230 cosmid library.

    Techniques Used:

    Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the
    Figure Legend Snippet: Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the

    Techniques Used: Mutagenesis

    Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.
    Figure Legend Snippet: Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.

    Techniques Used:

    4) Product Images from "Tea polyphenols inhibit the activation of NF-κB and the secretion of cytokines and matrix metalloproteinases by macrophages stimulated with Fusobacterium nucleatum"

    Article Title: Tea polyphenols inhibit the activation of NF-κB and the secretion of cytokines and matrix metalloproteinases by macrophages stimulated with Fusobacterium nucleatum

    Journal: Scientific Reports

    doi: 10.1038/srep34520

    Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of TNF-α by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p
    Figure Legend Snippet: Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of TNF-α by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p

    Techniques Used: Positive Control

    Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of sTREM-1 by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p
    Figure Legend Snippet: Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of sTREM-1 by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p

    Techniques Used: Positive Control

    Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of IL-1β by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p
    Figure Legend Snippet: Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of IL-1β by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p

    Techniques Used: Positive Control

    Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of CXCL8 by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p
    Figure Legend Snippet: Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of CXCL8 by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p

    Techniques Used: Positive Control

    Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of IL-6 by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p
    Figure Legend Snippet: Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of IL-6 by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p

    Techniques Used: Positive Control

    Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of MMP-3 by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p
    Figure Legend Snippet: Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of MMP-3 by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p

    Techniques Used: Positive Control

    Effect of the green tea extract, EGCG, black tea extract, and theaflavins on F. nucleatum -mediated activation of the NF-κB signaling pathway using the U937-3xκB cell model. A value of 100% was assigned to the activation obtained with F. nucleatum at an MOI of 100 (-----) in the absence of tea polyphenols. The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p
    Figure Legend Snippet: Effect of the green tea extract, EGCG, black tea extract, and theaflavins on F. nucleatum -mediated activation of the NF-κB signaling pathway using the U937-3xκB cell model. A value of 100% was assigned to the activation obtained with F. nucleatum at an MOI of 100 (-----) in the absence of tea polyphenols. The commercial inhibitor BAY-11-7082 (INH; 5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p

    Techniques Used: Activation Assay, Positive Control

    Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of MMP-9 by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p
    Figure Legend Snippet: Effect of the green tea extract, EGCG, black tea extract, and theaflavins on the secretion of MMP-9 by macrophages stimulated with F. nucleatum at an MOI of 100 (-----). The commercial inhibitor BAY-11-7082 (5 μg/ml) was used as a positive control. Results are expressed as the means ± SD of triplicate assays from three independent experiments. (*) Significant decrease ( p

    Techniques Used: Positive Control

    5) Product Images from "Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria"

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.15.5330-5340.2005

    Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.
    Figure Legend Snippet: Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.

    Techniques Used:

    RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin
    Figure Legend Snippet: RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

    DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC
    Figure Legend Snippet: DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC

    Techniques Used: Dot Blot

    I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes
    Figure Legend Snippet: I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes

    Techniques Used: Western Blot, Staining, SDS Page

    Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are
    Figure Legend Snippet: Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are

    Techniques Used: Sequencing

    Construction and screening of F. nucleatum 12230 cosmid library.
    Figure Legend Snippet: Construction and screening of F. nucleatum 12230 cosmid library.

    Techniques Used:

    Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the
    Figure Legend Snippet: Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the

    Techniques Used: Mutagenesis

    Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.
    Figure Legend Snippet: Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.

    Techniques Used:

    6) Product Images from "Diverse Toll-Like Receptors Mediate Cytokine Production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in Macrophages"

    Article Title: Diverse Toll-Like Receptors Mediate Cytokine Production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in Macrophages

    Journal:

    doi: 10.1128/IAI.01226-13

    Cytokines production by WT and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. BMDMs from WT and MyD88-deficient mice were infected with F. nucleatum or A. actinomycetemcomitans for 6 h, and, as indicated, the
    Figure Legend Snippet: Cytokines production by WT and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. BMDMs from WT and MyD88-deficient mice were infected with F. nucleatum or A. actinomycetemcomitans for 6 h, and, as indicated, the

    Techniques Used: Infection, Mouse Assay

    Production of IL-6 and TNF-α in WT and TLR2/4- and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. WT and TLR2/4-, and MyD88-deficient BMDMs were infected with F. nucleatum and A. actinomycetemcomitans
    Figure Legend Snippet: Production of IL-6 and TNF-α in WT and TLR2/4- and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. WT and TLR2/4-, and MyD88-deficient BMDMs were infected with F. nucleatum and A. actinomycetemcomitans

    Techniques Used: Infection

    Effect of NF-κB and MAPKs on F. nucleatum - and A. actinomycetemcomitans -induced production of cytokines by macrophages. WT BMDMs were pretreated with various doses of each inhibitor 2 h before infection. The cells were then infected with F. nucleatum
    Figure Legend Snippet: Effect of NF-κB and MAPKs on F. nucleatum - and A. actinomycetemcomitans -induced production of cytokines by macrophages. WT BMDMs were pretreated with various doses of each inhibitor 2 h before infection. The cells were then infected with F. nucleatum

    Techniques Used: Infection

    Effect of endosomal TLRs and bacterial DNA on cytokine production by macrophages in response to F. nucleatum or A. actinomycetemcomitans infection. TLR2/4-deficient BMDMs were pretreated with chloroquine (CLQ) 2 h before infection. The cells were then
    Figure Legend Snippet: Effect of endosomal TLRs and bacterial DNA on cytokine production by macrophages in response to F. nucleatum or A. actinomycetemcomitans infection. TLR2/4-deficient BMDMs were pretreated with chloroquine (CLQ) 2 h before infection. The cells were then

    Techniques Used: Infection

    Production of IL-6 and TNF-α by WT and TLR2-, TLR4-, and TLR2/4-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans . BMDMs were infected with F. nucleatum or A. actinomycetemcomitans at the indicated MOI. At 6 (A to D) or
    Figure Legend Snippet: Production of IL-6 and TNF-α by WT and TLR2-, TLR4-, and TLR2/4-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans . BMDMs were infected with F. nucleatum or A. actinomycetemcomitans at the indicated MOI. At 6 (A to D) or

    Techniques Used: Infection

    NF-κB and MAPK activation in WT and TLR2/4-, and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. Cells were infected with F. nucleatum or A. actinomycetemcomitans at an MOI of 1/100, and cellular protein
    Figure Legend Snippet: NF-κB and MAPK activation in WT and TLR2/4-, and MyD88-deficient BMDMs in response to F. nucleatum and A. actinomycetemcomitans infection. Cells were infected with F. nucleatum or A. actinomycetemcomitans at an MOI of 1/100, and cellular protein

    Techniques Used: Activation Assay, Infection

    7) Product Images from "Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria"

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.15.5330-5340.2005

    Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.
    Figure Legend Snippet: Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.

    Techniques Used:

    RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin
    Figure Legend Snippet: RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

    DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC
    Figure Legend Snippet: DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC

    Techniques Used: Dot Blot

    I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes
    Figure Legend Snippet: I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes

    Techniques Used: Western Blot, Staining, SDS Page

    Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are
    Figure Legend Snippet: Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are

    Techniques Used: Sequencing

    Construction and screening of F. nucleatum 12230 cosmid library.
    Figure Legend Snippet: Construction and screening of F. nucleatum 12230 cosmid library.

    Techniques Used:

    Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the
    Figure Legend Snippet: Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the

    Techniques Used: Mutagenesis

    Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.
    Figure Legend Snippet: Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.

    Techniques Used:

    8) Product Images from "Autoaggregation Response of Fusobacterium nucleatum "

    Article Title: Autoaggregation Response of Fusobacterium nucleatum

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00916-09

    Phase-contrast images of F. nucleatum cells used for microarray. Phase-contrast images of the same cultures used for RNA extraction and microarray were taken just prior to RNA extraction. The total magnification is ×1,000. The medium compositions
    Figure Legend Snippet: Phase-contrast images of F. nucleatum cells used for microarray. Phase-contrast images of the same cultures used for RNA extraction and microarray were taken just prior to RNA extraction. The total magnification is ×1,000. The medium compositions

    Techniques Used: Microarray, RNA Extraction

    Effect of 50 mM coaggregation inhibitors on autoaggregation. F. nucleatum cells were incubated in SDM containing 25% saliva and several different coaggregation inhibitors. The samples from left to right are as follows: water, lactose, galactose,
    Figure Legend Snippet: Effect of 50 mM coaggregation inhibitors on autoaggregation. F. nucleatum cells were incubated in SDM containing 25% saliva and several different coaggregation inhibitors. The samples from left to right are as follows: water, lactose, galactose,

    Techniques Used: Incubation

    Effect of saliva concentration on autoaggregation. F. nucleatum cells were incubated in SDM containing a range of saliva concentrations and measured for OD every 10 min. The saliva concentrations are as follows: squares, 0%; triangles, 10%;
    Figure Legend Snippet: Effect of saliva concentration on autoaggregation. F. nucleatum cells were incubated in SDM containing a range of saliva concentrations and measured for OD every 10 min. The saliva concentrations are as follows: squares, 0%; triangles, 10%;

    Techniques Used: Concentration Assay, Incubation

    Effect of lysine concentration on autoaggregation. F. nucleatum cells were incubated in SDM containing a range of lysine concentrations and measured for OD every 10 min. The lysine concentrations are as follows: squares, 0 mM; triangles, 6.25 mM; asterisks,
    Figure Legend Snippet: Effect of lysine concentration on autoaggregation. F. nucleatum cells were incubated in SDM containing a range of lysine concentrations and measured for OD every 10 min. The lysine concentrations are as follows: squares, 0 mM; triangles, 6.25 mM; asterisks,

    Techniques Used: Concentration Assay, Incubation

    9) Product Images from "Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria"

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.15.5330-5340.2005

    Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.
    Figure Legend Snippet: Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.

    Techniques Used:

    RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin
    Figure Legend Snippet: RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

    DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC
    Figure Legend Snippet: DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC

    Techniques Used: Dot Blot

    I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes
    Figure Legend Snippet: I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes

    Techniques Used: Western Blot, Staining, SDS Page

    Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are
    Figure Legend Snippet: Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are

    Techniques Used: Sequencing

    Construction and screening of F. nucleatum 12230 cosmid library.
    Figure Legend Snippet: Construction and screening of F. nucleatum 12230 cosmid library.

    Techniques Used:

    Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the
    Figure Legend Snippet: Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the

    Techniques Used: Mutagenesis

    Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.
    Figure Legend Snippet: Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.

    Techniques Used:

    10) Product Images from "Proteomics of Fusobacterium nucleatum within a model developing oral microbial community"

    Article Title: Proteomics of Fusobacterium nucleatum within a model developing oral microbial community

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.204

    FnPgSg versus FnSgEnergy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with S. gordonii .
    Figure Legend Snippet: FnPgSg versus FnSgEnergy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with S. gordonii .

    Techniques Used:

    FnSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Streptococcus gordonii comparison to F. nucleatum with Porphyromonas gingivalis .
    Figure Legend Snippet: FnSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Streptococcus gordonii comparison to F. nucleatum with Porphyromonas gingivalis .

    Techniques Used:

    FnSg versus Fn energy metabolism and end products. Proteins catalyzing each step are shown by their Fusobacterium nucleatum FN number. Red numbers indicate increased levels in the first condition compared to the second condition, green decreased levels, yellow no statistical change, and black undetected in at least one of the conditions.
    Figure Legend Snippet: FnSg versus Fn energy metabolism and end products. Proteins catalyzing each step are shown by their Fusobacterium nucleatum FN number. Red numbers indicate increased levels in the first condition compared to the second condition, green decreased levels, yellow no statistical change, and black undetected in at least one of the conditions.

    Techniques Used:

    FnPgSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with P. gingivalis .
    Figure Legend Snippet: FnPgSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with P. gingivalis .

    Techniques Used:

    FnPgSg versus Fn energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum .
    Figure Legend Snippet: FnPgSg versus Fn energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum .

    Techniques Used:

    FnPg versus Fn energy metabolism and end products. The diagram shows a schematic of the glycolysis and pentose phosphate pathways for Fn including the end products of the metabolism, formate, acetate, l -lactate, and butanoate, for the Fusobacterium nucleatum with Porphyromonas gingivalis sample compared to F. nucleatum . Labels and color coding as described for Figure 1 .
    Figure Legend Snippet: FnPg versus Fn energy metabolism and end products. The diagram shows a schematic of the glycolysis and pentose phosphate pathways for Fn including the end products of the metabolism, formate, acetate, l -lactate, and butanoate, for the Fusobacterium nucleatum with Porphyromonas gingivalis sample compared to F. nucleatum . Labels and color coding as described for Figure 1 .

    Techniques Used:

    Electron micrographs. Thin-section transmission electron micrographs after 18 h in a model biofilm of (A) Fusobacterium nucleatum alone, (B) F. nucleatum with Porphyromonas gingivalis , (C) F. nucleatum with Streptococcus gordonii , or (D) F. nucleatum with both P. gingivalis and S. gordonii . Magnification: ×49,000.
    Figure Legend Snippet: Electron micrographs. Thin-section transmission electron micrographs after 18 h in a model biofilm of (A) Fusobacterium nucleatum alone, (B) F. nucleatum with Porphyromonas gingivalis , (C) F. nucleatum with Streptococcus gordonii , or (D) F. nucleatum with both P. gingivalis and S. gordonii . Magnification: ×49,000.

    Techniques Used: Transmission Assay

    11) Product Images from "Proteomics of Fusobacterium nucleatum within a model developing oral microbial community"

    Article Title: Proteomics of Fusobacterium nucleatum within a model developing oral microbial community

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.204

    FnPgSg versus FnSgEnergy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with S. gordonii .
    Figure Legend Snippet: FnPgSg versus FnSgEnergy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with S. gordonii .

    Techniques Used:

    FnSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Streptococcus gordonii comparison to F. nucleatum with Porphyromonas gingivalis .
    Figure Legend Snippet: FnSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Streptococcus gordonii comparison to F. nucleatum with Porphyromonas gingivalis .

    Techniques Used:

    FnSg versus Fn energy metabolism and end products. Proteins catalyzing each step are shown by their Fusobacterium nucleatum FN number. Red numbers indicate increased levels in the first condition compared to the second condition, green decreased levels, yellow no statistical change, and black undetected in at least one of the conditions.
    Figure Legend Snippet: FnSg versus Fn energy metabolism and end products. Proteins catalyzing each step are shown by their Fusobacterium nucleatum FN number. Red numbers indicate increased levels in the first condition compared to the second condition, green decreased levels, yellow no statistical change, and black undetected in at least one of the conditions.

    Techniques Used:

    FnPgSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with P. gingivalis .
    Figure Legend Snippet: FnPgSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with P. gingivalis .

    Techniques Used:

    FnPgSg versus Fn energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum .
    Figure Legend Snippet: FnPgSg versus Fn energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum .

    Techniques Used:

    FnPg versus Fn energy metabolism and end products. The diagram shows a schematic of the glycolysis and pentose phosphate pathways for Fn including the end products of the metabolism, formate, acetate, l -lactate, and butanoate, for the Fusobacterium nucleatum with Porphyromonas gingivalis sample compared to F. nucleatum . Labels and color coding as described for Figure 1 .
    Figure Legend Snippet: FnPg versus Fn energy metabolism and end products. The diagram shows a schematic of the glycolysis and pentose phosphate pathways for Fn including the end products of the metabolism, formate, acetate, l -lactate, and butanoate, for the Fusobacterium nucleatum with Porphyromonas gingivalis sample compared to F. nucleatum . Labels and color coding as described for Figure 1 .

    Techniques Used:

    Electron micrographs. Thin-section transmission electron micrographs after 18 h in a model biofilm of (A) Fusobacterium nucleatum alone, (B) F. nucleatum with Porphyromonas gingivalis , (C) F. nucleatum with Streptococcus gordonii , or (D) F. nucleatum with both P. gingivalis and S. gordonii . Magnification: ×49,000.
    Figure Legend Snippet: Electron micrographs. Thin-section transmission electron micrographs after 18 h in a model biofilm of (A) Fusobacterium nucleatum alone, (B) F. nucleatum with Porphyromonas gingivalis , (C) F. nucleatum with Streptococcus gordonii , or (D) F. nucleatum with both P. gingivalis and S. gordonii . Magnification: ×49,000.

    Techniques Used: Transmission Assay

    12) Product Images from "Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria"

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.15.5330-5340.2005

    Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.
    Figure Legend Snippet: Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.

    Techniques Used:

    RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin
    Figure Legend Snippet: RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

    DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC
    Figure Legend Snippet: DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC

    Techniques Used: Dot Blot

    I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes
    Figure Legend Snippet: I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes

    Techniques Used: Western Blot, Staining, SDS Page

    Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are
    Figure Legend Snippet: Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are

    Techniques Used: Sequencing

    Construction and screening of F. nucleatum 12230 cosmid library.
    Figure Legend Snippet: Construction and screening of F. nucleatum 12230 cosmid library.

    Techniques Used:

    Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the
    Figure Legend Snippet: Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the

    Techniques Used: Mutagenesis

    Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.
    Figure Legend Snippet: Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.

    Techniques Used:

    13) Product Images from "Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria"

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.15.5330-5340.2005

    Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.
    Figure Legend Snippet: Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.

    Techniques Used:

    RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin
    Figure Legend Snippet: RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

    DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC
    Figure Legend Snippet: DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC

    Techniques Used: Dot Blot

    I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes
    Figure Legend Snippet: I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes

    Techniques Used: Western Blot, Staining, SDS Page

    Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are
    Figure Legend Snippet: Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are

    Techniques Used: Sequencing

    Construction and screening of F. nucleatum 12230 cosmid library.
    Figure Legend Snippet: Construction and screening of F. nucleatum 12230 cosmid library.

    Techniques Used:

    Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the
    Figure Legend Snippet: Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the

    Techniques Used: Mutagenesis

    Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.
    Figure Legend Snippet: Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.

    Techniques Used:

    14) Product Images from "Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria"

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.15.5330-5340.2005

    Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.
    Figure Legend Snippet: Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.

    Techniques Used:

    RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin
    Figure Legend Snippet: RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

    DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC
    Figure Legend Snippet: DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC

    Techniques Used: Dot Blot

    I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes
    Figure Legend Snippet: I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes

    Techniques Used: Western Blot, Staining, SDS Page

    Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are
    Figure Legend Snippet: Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are

    Techniques Used: Sequencing

    Construction and screening of F. nucleatum 12230 cosmid library.
    Figure Legend Snippet: Construction and screening of F. nucleatum 12230 cosmid library.

    Techniques Used:

    Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the
    Figure Legend Snippet: Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the

    Techniques Used: Mutagenesis

    Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.
    Figure Legend Snippet: Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.

    Techniques Used:

    15) Product Images from "Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria"

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.15.5330-5340.2005

    Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.
    Figure Legend Snippet: Growth of F. nucleatum 12230 (solid triangles and solid line) and F. nucleatum 12230-US1 (open squares and dashed line) in Columbia broth. OD 600, optical density at 600 nm.

    Techniques Used:

    RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin
    Figure Legend Snippet: RT-PCR and Northern blot analyses of F. nucleatum 12230 and F. nucleatum 12230-US1. A. Schematic diagram showing locations of primers used for RT-PCR. The 2.4-kb fadA -containing fragment from F. nucleatum 12230 is presented as solid lines. The hairpin

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

    DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC
    Figure Legend Snippet: DNA dot blot analysis of the fadA gene in different Fusobacterium species. 1, F. gonidiaformans DUMC CF65-1; 2, F. gonidiaformans DUMC CF63-1; 3, F. mortiferum ATCC 25557; 4, F. naviforme DUMC CF108-1; 5, F. nucleatum ATCC 10953; 6, F. nucleatum ATCC

    Techniques Used: Dot Blot

    I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes
    Figure Legend Snippet: I and II. Identification of F. nucleatum adhesins by far-Western analysis. a. F. nucleatum 12230 (I) or 40P (II) components stained with Coomassie blue following 12% SDS-PAGE. b. F. nucleatum 12230 (I) or 40P (II) components immobilized on PVDF membranes

    Techniques Used: Western Blot, Staining, SDS Page

    Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are
    Figure Legend Snippet: Amino acid sequence alignment of FadA and its paralogues. Highlighted in gray are the identical residues shared among FadA proteins. The sequences of two FadA paralogues, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, are

    Techniques Used: Sequencing

    Construction and screening of F. nucleatum 12230 cosmid library.
    Figure Legend Snippet: Construction and screening of F. nucleatum 12230 cosmid library.

    Techniques Used:

    Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the
    Figure Legend Snippet: Inactivation of the fadA gene of F. nucleatum 12230. A. Schematic diagram of construction of the Δ fadA :: erm mutant by double-crossover allelic exchange. The erythromycin resistance cassette ermF-ermAM was inserted between bp 71 and 365 of the

    Techniques Used: Mutagenesis

    Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.
    Figure Legend Snippet: Attachment of F. nucleatum 12230 and F. nucleatum 12230-US1 to KB (hatched bars) and CHO (open bars) cells. The levels of attachment are means and standard deviations from three separate experiments, each performed in triplicate.

    Techniques Used:

    16) Product Images from "A novel vaccine targeting Fusobacterium nucleatum against abscesses and halitosis"

    Article Title: A novel vaccine targeting Fusobacterium nucleatum against abscesses and halitosis

    Journal:

    doi: 10.1016/j.vaccine.2008.12.058

    In vivo protective immunity. A gum pocket model with abscesses and swollen tissues was created, as described in and Section 2. After inoculation with live F. nucleatum (4 × 10 8 CFU) for 3 days, the change in the volume of gum swelling
    Figure Legend Snippet: In vivo protective immunity. A gum pocket model with abscesses and swollen tissues was created, as described in and Section 2. After inoculation with live F. nucleatum (4 × 10 8 CFU) for 3 days, the change in the volume of gum swelling

    Techniques Used: In Vivo

    The production of antibody (IgG) to F. nucleatum. For immunization, cultured F. nucleatum and S. mutans were irradiated by UV at a total energy of 7000 J/m 2 . F. nucleatum lysate (20 µg) was separated by 10% SDS-PAGE and then subjected to Western
    Figure Legend Snippet: The production of antibody (IgG) to F. nucleatum. For immunization, cultured F. nucleatum and S. mutans were irradiated by UV at a total energy of 7000 J/m 2 . F. nucleatum lysate (20 µg) was separated by 10% SDS-PAGE and then subjected to Western

    Techniques Used: Cell Culture, Irradiation, SDS Page, Western Blot

    An abscess induced by the injection of live F. nucleatum into the gums of lower incisors of ICR mice. One hundred micro liter of PBS (A, C and E) or F. nucleatum (4 × 10 8 CFU in PBS) (B, D and F) was inoculated into a mouse oral cavity for 3 days,
    Figure Legend Snippet: An abscess induced by the injection of live F. nucleatum into the gums of lower incisors of ICR mice. One hundred micro liter of PBS (A, C and E) or F. nucleatum (4 × 10 8 CFU in PBS) (B, D and F) was inoculated into a mouse oral cavity for 3 days,

    Techniques Used: Injection, Mouse Assay

    Detection of VSC production and biofilm formation of F. nucleatum . F. nucleatum (4 × 10 9 CFU/well) was cultured on a 6-well nonpyrogenic polystyrene plate for 6, 12, 24 and 36 h. (A) After removing the media, each well containing attached bacteria
    Figure Legend Snippet: Detection of VSC production and biofilm formation of F. nucleatum . F. nucleatum (4 × 10 9 CFU/well) was cultured on a 6-well nonpyrogenic polystyrene plate for 6, 12, 24 and 36 h. (A) After removing the media, each well containing attached bacteria

    Techniques Used: Cell Culture

    Inhibition of VSC production and biofilm formation by anti- F. nucleatum serum. F. nucleatum (4 × 10 9 CFU) was pre-incubated with 2.5% (v/v) anti- F. nucleatum (Anti-N) or anti- S. mutans (Anti-M) sera without activated complements for 2 h. Afterwards,
    Figure Legend Snippet: Inhibition of VSC production and biofilm formation by anti- F. nucleatum serum. F. nucleatum (4 × 10 9 CFU) was pre-incubated with 2.5% (v/v) anti- F. nucleatum (Anti-N) or anti- S. mutans (Anti-M) sera without activated complements for 2 h. Afterwards,

    Techniques Used: Inhibition, Incubation

    17) Product Images from "A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif"

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001596

    Protective effect of Na + and Li + against DCCD inhibition of the purified F. nucleatum ATP synthase. The purified protein (120–140 µg) was incubated at 25°C for 20 min in the presence of either 400 µM DCCD or 400 µM DCCD and 50 mM NaCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , (A) at pH 7.5, (B) at pH 9, and (C) at pH 6.5. (D) NaCl was substituted by either LiCl or KCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , pH 7.5. 100% enzyme activity corresponds to 1–2 units/mg of protein. The values plotted are representative of at least two to three individual experiments; the statistical variance was less than 20%.
    Figure Legend Snippet: Protective effect of Na + and Li + against DCCD inhibition of the purified F. nucleatum ATP synthase. The purified protein (120–140 µg) was incubated at 25°C for 20 min in the presence of either 400 µM DCCD or 400 µM DCCD and 50 mM NaCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , (A) at pH 7.5, (B) at pH 9, and (C) at pH 6.5. (D) NaCl was substituted by either LiCl or KCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , pH 7.5. 100% enzyme activity corresponds to 1–2 units/mg of protein. The values plotted are representative of at least two to three individual experiments; the statistical variance was less than 20%.

    Techniques Used: Inhibition, Purification, Incubation, Activity Assay

    Likelihood of protonation of the Na + -loaded binding sites in the F. nucleatum c-ring, and most probable configuration. (A) Energetics of proton transfer between Glu65 and Glu32 (models A and B in Figure 1 ), with a Na + concurrently bound, based on Hartree-Fock quantum-mechanical calculations for a reduced model of the binding site. The potential energy values plotted correspond to the optimized geometries in each case. The geometry and potential energy of the transition state are also provided. (B) Calculated free-energy gain associated with the protonation of Glu32, relative to a side-chain analog in solution (propionate); the formation energy of the chemical bond is omitted, as this is expected to be constant. Calculated values are translated into a pK a scale, by setting the free-energy value for propionate to its known pK a value, i.e., 4.9. The protonation of Glu32 is studied either in the Na + -bound, closed conformation of the c-ring binding sites ( Figure 1A ); or in an open, hydrated conformation ( Figure 9 ). In both cases, to the values calculated by FEP/MD (light grey) we added a correction calculated with Poisson electrostatic theory on account of the membrane polarizability (dark grey).
    Figure Legend Snippet: Likelihood of protonation of the Na + -loaded binding sites in the F. nucleatum c-ring, and most probable configuration. (A) Energetics of proton transfer between Glu65 and Glu32 (models A and B in Figure 1 ), with a Na + concurrently bound, based on Hartree-Fock quantum-mechanical calculations for a reduced model of the binding site. The potential energy values plotted correspond to the optimized geometries in each case. The geometry and potential energy of the transition state are also provided. (B) Calculated free-energy gain associated with the protonation of Glu32, relative to a side-chain analog in solution (propionate); the formation energy of the chemical bond is omitted, as this is expected to be constant. Calculated values are translated into a pK a scale, by setting the free-energy value for propionate to its known pK a value, i.e., 4.9. The protonation of Glu32 is studied either in the Na + -bound, closed conformation of the c-ring binding sites ( Figure 1A ); or in an open, hydrated conformation ( Figure 9 ). In both cases, to the values calculated by FEP/MD (light grey) we added a correction calculated with Poisson electrostatic theory on account of the membrane polarizability (dark grey).

    Techniques Used: Binding Assay

    Alternative models of the ion-binding sites in the F. nucleatum c-ring, with Na + bound. The ion-coordinating groups in the protein are indicated with dashed lines. Hydrogen atoms in non-polar groups are omitted for clarity. (A) Model with Glu32 protonated in cis and Glu65 in the deprotonated state (model A), predicted to be the most likely (see Results). (B) Model with Glu32 deprotonated and Glu65 protonated (model B). In this model, the proton bound to Glu65 was initially modeled in cis , as this is the most probable geometry for the isolated side-chain. However, within 250 ps of the start of the simulation, the carboxyl group of Glu65 switched to trans . (C) Model with Glu32 and Glu65 in the deprotonated state (model C). During the simulation of this model, the electrostatic repulsion between Glu32 and Glu65 caused the latter to displace outwards, away from the Na + ion, which becomes coordinated by Glu32 in a bi-dentate configuration. Note the bound water reorients and no longer contributes to Na + coordination.
    Figure Legend Snippet: Alternative models of the ion-binding sites in the F. nucleatum c-ring, with Na + bound. The ion-coordinating groups in the protein are indicated with dashed lines. Hydrogen atoms in non-polar groups are omitted for clarity. (A) Model with Glu32 protonated in cis and Glu65 in the deprotonated state (model A), predicted to be the most likely (see Results). (B) Model with Glu32 deprotonated and Glu65 protonated (model B). In this model, the proton bound to Glu65 was initially modeled in cis , as this is the most probable geometry for the isolated side-chain. However, within 250 ps of the start of the simulation, the carboxyl group of Glu65 switched to trans . (C) Model with Glu32 and Glu65 in the deprotonated state (model C). During the simulation of this model, the electrostatic repulsion between Glu32 and Glu65 caused the latter to displace outwards, away from the Na + ion, which becomes coordinated by Glu32 in a bi-dentate configuration. Note the bound water reorients and no longer contributes to Na + coordination.

    Techniques Used: Binding Assay, Isolation

    Kinetics of NCD-4 modification of detergent-solubilized c-rings from F. nucleatum . 100 µM NCD-4 was added to a sample of purified F. nucleatum c 11 ring in MES buffer (pH 5.7) and 1.5% (w/v) n-octyl-β-D-glycoside. A continuous increase in fluorescence was measured upon reaction of NCD-4 with Glu65. Addition of 15 mM NaCl precluded further increase in the fluorescence. An extended control measurement, with no NaCl added, is shown in Figure S12 .
    Figure Legend Snippet: Kinetics of NCD-4 modification of detergent-solubilized c-rings from F. nucleatum . 100 µM NCD-4 was added to a sample of purified F. nucleatum c 11 ring in MES buffer (pH 5.7) and 1.5% (w/v) n-octyl-β-D-glycoside. A continuous increase in fluorescence was measured upon reaction of NCD-4 with Glu65. Addition of 15 mM NaCl precluded further increase in the fluorescence. An extended control measurement, with no NaCl added, is shown in Figure S12 .

    Techniques Used: Modification, Purification, Fluorescence

    Calculated ion selectivity of the F. nucleatum c-ring. (A) Predicted structure of the ion binding sites in the absence of Na + , with two H + bound. Hydrogen atoms in non-polar groups are omitted for clarity. Note the binding site harbors two H + , one bound to Glu32 and another to Glu65 (both in cis ). Two alternative configurations, one of which lacks a bound water molecule, are also shown in Figure S3 . (B) Calculated free-energy of selectivity for H + vs. Na + of the F. nucleatum c-ring binding sites, relative to other c-rings previously characterized. The Na + state is that rendered in Figure 1A ; note the bound water molecule. Two possible H + -bound states are considered, in which the water molecule is either preserved (+w), as in Figure 3A , or removed (−w), as in Figure S3B .
    Figure Legend Snippet: Calculated ion selectivity of the F. nucleatum c-ring. (A) Predicted structure of the ion binding sites in the absence of Na + , with two H + bound. Hydrogen atoms in non-polar groups are omitted for clarity. Note the binding site harbors two H + , one bound to Glu32 and another to Glu65 (both in cis ). Two alternative configurations, one of which lacks a bound water molecule, are also shown in Figure S3 . (B) Calculated free-energy of selectivity for H + vs. Na + of the F. nucleatum c-ring binding sites, relative to other c-rings previously characterized. The Na + state is that rendered in Figure 1A ; note the bound water molecule. Two possible H + -bound states are considered, in which the water molecule is either preserved (+w), as in Figure 3A , or removed (−w), as in Figure S3B .

    Techniques Used: Binding Assay

    ATP-driven proton translocation in E. coli and F. nucleatum inverted membranes vesicles. Proton translocation was determined at 37°C by the quenching of acridine orange (AO) fluorescence. The reaction was initiated by the addition of 1.25 mM ATP and terminated with 30 µM CCCP at the times indicated by arrows. (A) E. coli native membranes in pH 7.5 buffer. (B) F. nucleatum native membranes in pH 6 buffer with 1.25 mM Na + -ATP; (C) Same as (B), with 10 mM NaCl added to the buffer.
    Figure Legend Snippet: ATP-driven proton translocation in E. coli and F. nucleatum inverted membranes vesicles. Proton translocation was determined at 37°C by the quenching of acridine orange (AO) fluorescence. The reaction was initiated by the addition of 1.25 mM ATP and terminated with 30 µM CCCP at the times indicated by arrows. (A) E. coli native membranes in pH 7.5 buffer. (B) F. nucleatum native membranes in pH 6 buffer with 1.25 mM Na + -ATP; (C) Same as (B), with 10 mM NaCl added to the buffer.

    Techniques Used: Translocation Assay, Fluorescence

    Catalytic activity of the purified F 1 F o -ATP synthase from F. nucleatum . (A) Activation of the F. nucleatum ATP synthase by Na + , Li + , and K + ions (Na + , •; Li + , ▪; and K + , ▴). (B) Activation of the ATP synthase by Na + ions at pH 7.5 (•) and pH 9.0 (▪). The ATP hydrolysis activity was determined using the ATP-regenerating assay (120–140 µg protein), at 37°C. Activity in units/mg of protein (1 unit = 1 µmol ATP hydrolysed/min). 100% of activity corresponds 1–2 units/mg at pH 7.5 and 0.6–0.72 units/mg at pH 9.0. The assay mixture contained 50 mM MOPS, 2 mM MgCl 2 (pH 7.5) in (A) and 50 mM MES-MOPS-Tris, 2 mM MgCl 2 in (B). The values plotted are representative of at least two biological replicates; the statistical variance was less than 20%.
    Figure Legend Snippet: Catalytic activity of the purified F 1 F o -ATP synthase from F. nucleatum . (A) Activation of the F. nucleatum ATP synthase by Na + , Li + , and K + ions (Na + , •; Li + , ▪; and K + , ▴). (B) Activation of the ATP synthase by Na + ions at pH 7.5 (•) and pH 9.0 (▪). The ATP hydrolysis activity was determined using the ATP-regenerating assay (120–140 µg protein), at 37°C. Activity in units/mg of protein (1 unit = 1 µmol ATP hydrolysed/min). 100% of activity corresponds 1–2 units/mg at pH 7.5 and 0.6–0.72 units/mg at pH 9.0. The assay mixture contained 50 mM MOPS, 2 mM MgCl 2 (pH 7.5) in (A) and 50 mM MES-MOPS-Tris, 2 mM MgCl 2 in (B). The values plotted are representative of at least two biological replicates; the statistical variance was less than 20%.

    Techniques Used: Activity Assay, Purification, Activation Assay

    Simulation model of the open-state of the Na + -binding site in the F. nucleatum c-ring. The c-ring is shown in cartoon format (yellow), viewed from the membrane plane. Lipid molecules in the surrounding membrane bilayer are omitted for clarity. A crevice of water molecules (transparent red spheres) was modeled on the cytoplasmic side of the protein-lipid interface, so as to mimic the hydrated environment at the interface of the c-ring and subunit a, in the complete enzyme. A guanidinium ion (GND + ) was added to the crevice, to mimic a conserved Arg side-chain in the fourth transmembrane helix of subunit a. Key side-chains in the open binding site, namely Glu65, Glu32, Ser66, and Tyr70, are shown as sticks (non-polar hydrogen atoms are omitted); these form an interaction network in the closed state ( Figure 8 ), which in this open state is largely disrupted. Glu65 projects out of the binding site and interacts with the guanidinium ion; Glu32, however, is highly likely to remain protonated ( Figure 2B ).
    Figure Legend Snippet: Simulation model of the open-state of the Na + -binding site in the F. nucleatum c-ring. The c-ring is shown in cartoon format (yellow), viewed from the membrane plane. Lipid molecules in the surrounding membrane bilayer are omitted for clarity. A crevice of water molecules (transparent red spheres) was modeled on the cytoplasmic side of the protein-lipid interface, so as to mimic the hydrated environment at the interface of the c-ring and subunit a, in the complete enzyme. A guanidinium ion (GND + ) was added to the crevice, to mimic a conserved Arg side-chain in the fourth transmembrane helix of subunit a. Key side-chains in the open binding site, namely Glu65, Glu32, Ser66, and Tyr70, are shown as sticks (non-polar hydrogen atoms are omitted); these form an interaction network in the closed state ( Figure 8 ), which in this open state is largely disrupted. Glu65 projects out of the binding site and interacts with the guanidinium ion; Glu32, however, is highly likely to remain protonated ( Figure 2B ).

    Techniques Used: Binding Assay

    The Na + -binding site of the F. nucleatum c 11 ring at pH 5.3 and 8.7. (A) A functional Na + -binding site consists of two adjacent c-subunits (in yellow and green). Residues discussed in the text are indicated. (B) Close-up of the Na + -binding site viewed from the membrane plane, from a crystal grown at pH 5.3 and 100 mM NaOAc; the resolution of the data is 2.2 Å. The Na + and a structural water molecule are shown in purple and red spheres, respectively. The H-bond network and ion-protein interactions are indicated by dashed lines. Residues involved in Na + binding are labeled. (C) Close-up of the Na + binding site from a crystal grown at pH 8.7 and 100 mM buffer, viewed as in (B); the resolution is 2.6 Å. Electron density maps (2F obs -F calc , blue mesh) are shown at 1.9σ. In both cases Na + ions can be discerned in the ion-binding sites, and consistently the key Glu65 side-chain is observed in the ion-locked conformation, while Glu32 is protonated.
    Figure Legend Snippet: The Na + -binding site of the F. nucleatum c 11 ring at pH 5.3 and 8.7. (A) A functional Na + -binding site consists of two adjacent c-subunits (in yellow and green). Residues discussed in the text are indicated. (B) Close-up of the Na + -binding site viewed from the membrane plane, from a crystal grown at pH 5.3 and 100 mM NaOAc; the resolution of the data is 2.2 Å. The Na + and a structural water molecule are shown in purple and red spheres, respectively. The H-bond network and ion-protein interactions are indicated by dashed lines. Residues involved in Na + binding are labeled. (C) Close-up of the Na + binding site from a crystal grown at pH 8.7 and 100 mM buffer, viewed as in (B); the resolution is 2.6 Å. Electron density maps (2F obs -F calc , blue mesh) are shown at 1.9σ. In both cases Na + ions can be discerned in the ion-binding sites, and consistently the key Glu65 side-chain is observed in the ion-locked conformation, while Glu32 is protonated.

    Techniques Used: Binding Assay, Functional Assay, Labeling

    Crystal structure of the c 11 ring of the F. nucleatum ATP synthase at 2.2 Å resolution. The pH of crystallization buffer was 5.3, and included 100 mM Na + . (A) Side view of the c-ring along the membrane plane. The c-subunits are displayed in different colors in ribbon representation. The transmembrane region, 35 Å in width, is indicated with grey bars. (B) View of the c-ring from the cytoplasm, along the perpendicular to the membrane. The sodium ion and the water molecule bound to each of the 11 ion-binding sites (purple and red spheres, respectively), are shown by F obs -F calc omit electron-density maps at 3.2σ (blue meshes).
    Figure Legend Snippet: Crystal structure of the c 11 ring of the F. nucleatum ATP synthase at 2.2 Å resolution. The pH of crystallization buffer was 5.3, and included 100 mM Na + . (A) Side view of the c-ring along the membrane plane. The c-subunits are displayed in different colors in ribbon representation. The transmembrane region, 35 Å in width, is indicated with grey bars. (B) View of the c-ring from the cytoplasm, along the perpendicular to the membrane. The sodium ion and the water molecule bound to each of the 11 ion-binding sites (purple and red spheres, respectively), are shown by F obs -F calc omit electron-density maps at 3.2σ (blue meshes).

    Techniques Used: Crystallization Assay, Binding Assay

    18) Product Images from "A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif"

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001596

    Protective effect of Na + and Li + against DCCD inhibition of the purified F. nucleatum ATP synthase. The purified protein (120–140 µg) was incubated at 25°C for 20 min in the presence of either 400 µM DCCD or 400 µM DCCD and 50 mM NaCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , (A) at pH 7.5, (B) at pH 9, and (C) at pH 6.5. (D) NaCl was substituted by either LiCl or KCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , pH 7.5. 100% enzyme activity corresponds to 1–2 units/mg of protein. The values plotted are representative of at least two to three individual experiments; the statistical variance was less than 20%.
    Figure Legend Snippet: Protective effect of Na + and Li + against DCCD inhibition of the purified F. nucleatum ATP synthase. The purified protein (120–140 µg) was incubated at 25°C for 20 min in the presence of either 400 µM DCCD or 400 µM DCCD and 50 mM NaCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , (A) at pH 7.5, (B) at pH 9, and (C) at pH 6.5. (D) NaCl was substituted by either LiCl or KCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , pH 7.5. 100% enzyme activity corresponds to 1–2 units/mg of protein. The values plotted are representative of at least two to three individual experiments; the statistical variance was less than 20%.

    Techniques Used: Inhibition, Purification, Incubation, Activity Assay

    Likelihood of protonation of the Na + -loaded binding sites in the F. nucleatum c-ring, and most probable configuration. (A) Energetics of proton transfer between Glu65 and Glu32 (models A and B in Figure 1 ), with a Na + concurrently bound, based on Hartree-Fock quantum-mechanical calculations for a reduced model of the binding site. The potential energy values plotted correspond to the optimized geometries in each case. The geometry and potential energy of the transition state are also provided. (B) Calculated free-energy gain associated with the protonation of Glu32, relative to a side-chain analog in solution (propionate); the formation energy of the chemical bond is omitted, as this is expected to be constant. Calculated values are translated into a pK a scale, by setting the free-energy value for propionate to its known pK a value, i.e., 4.9. The protonation of Glu32 is studied either in the Na + -bound, closed conformation of the c-ring binding sites ( Figure 1A ); or in an open, hydrated conformation ( Figure 9 ). In both cases, to the values calculated by FEP/MD (light grey) we added a correction calculated with Poisson electrostatic theory on account of the membrane polarizability (dark grey).
    Figure Legend Snippet: Likelihood of protonation of the Na + -loaded binding sites in the F. nucleatum c-ring, and most probable configuration. (A) Energetics of proton transfer between Glu65 and Glu32 (models A and B in Figure 1 ), with a Na + concurrently bound, based on Hartree-Fock quantum-mechanical calculations for a reduced model of the binding site. The potential energy values plotted correspond to the optimized geometries in each case. The geometry and potential energy of the transition state are also provided. (B) Calculated free-energy gain associated with the protonation of Glu32, relative to a side-chain analog in solution (propionate); the formation energy of the chemical bond is omitted, as this is expected to be constant. Calculated values are translated into a pK a scale, by setting the free-energy value for propionate to its known pK a value, i.e., 4.9. The protonation of Glu32 is studied either in the Na + -bound, closed conformation of the c-ring binding sites ( Figure 1A ); or in an open, hydrated conformation ( Figure 9 ). In both cases, to the values calculated by FEP/MD (light grey) we added a correction calculated with Poisson electrostatic theory on account of the membrane polarizability (dark grey).

    Techniques Used: Binding Assay

    Alternative models of the ion-binding sites in the F. nucleatum c-ring, with Na + bound. The ion-coordinating groups in the protein are indicated with dashed lines. Hydrogen atoms in non-polar groups are omitted for clarity. (A) Model with Glu32 protonated in cis and Glu65 in the deprotonated state (model A), predicted to be the most likely (see Results). (B) Model with Glu32 deprotonated and Glu65 protonated (model B). In this model, the proton bound to Glu65 was initially modeled in cis , as this is the most probable geometry for the isolated side-chain. However, within 250 ps of the start of the simulation, the carboxyl group of Glu65 switched to trans . (C) Model with Glu32 and Glu65 in the deprotonated state (model C). During the simulation of this model, the electrostatic repulsion between Glu32 and Glu65 caused the latter to displace outwards, away from the Na + ion, which becomes coordinated by Glu32 in a bi-dentate configuration. Note the bound water reorients and no longer contributes to Na + coordination.
    Figure Legend Snippet: Alternative models of the ion-binding sites in the F. nucleatum c-ring, with Na + bound. The ion-coordinating groups in the protein are indicated with dashed lines. Hydrogen atoms in non-polar groups are omitted for clarity. (A) Model with Glu32 protonated in cis and Glu65 in the deprotonated state (model A), predicted to be the most likely (see Results). (B) Model with Glu32 deprotonated and Glu65 protonated (model B). In this model, the proton bound to Glu65 was initially modeled in cis , as this is the most probable geometry for the isolated side-chain. However, within 250 ps of the start of the simulation, the carboxyl group of Glu65 switched to trans . (C) Model with Glu32 and Glu65 in the deprotonated state (model C). During the simulation of this model, the electrostatic repulsion between Glu32 and Glu65 caused the latter to displace outwards, away from the Na + ion, which becomes coordinated by Glu32 in a bi-dentate configuration. Note the bound water reorients and no longer contributes to Na + coordination.

    Techniques Used: Binding Assay, Isolation

    Kinetics of NCD-4 modification of detergent-solubilized c-rings from F. nucleatum . 100 µM NCD-4 was added to a sample of purified F. nucleatum c 11 ring in MES buffer (pH 5.7) and 1.5% (w/v) n-octyl-β-D-glycoside. A continuous increase in fluorescence was measured upon reaction of NCD-4 with Glu65. Addition of 15 mM NaCl precluded further increase in the fluorescence. An extended control measurement, with no NaCl added, is shown in Figure S12 .
    Figure Legend Snippet: Kinetics of NCD-4 modification of detergent-solubilized c-rings from F. nucleatum . 100 µM NCD-4 was added to a sample of purified F. nucleatum c 11 ring in MES buffer (pH 5.7) and 1.5% (w/v) n-octyl-β-D-glycoside. A continuous increase in fluorescence was measured upon reaction of NCD-4 with Glu65. Addition of 15 mM NaCl precluded further increase in the fluorescence. An extended control measurement, with no NaCl added, is shown in Figure S12 .

    Techniques Used: Modification, Purification, Fluorescence

    Calculated ion selectivity of the F. nucleatum c-ring. (A) Predicted structure of the ion binding sites in the absence of Na + , with two H + bound. Hydrogen atoms in non-polar groups are omitted for clarity. Note the binding site harbors two H + , one bound to Glu32 and another to Glu65 (both in cis ). Two alternative configurations, one of which lacks a bound water molecule, are also shown in Figure S3 . (B) Calculated free-energy of selectivity for H + vs. Na + of the F. nucleatum c-ring binding sites, relative to other c-rings previously characterized. The Na + state is that rendered in Figure 1A ; note the bound water molecule. Two possible H + -bound states are considered, in which the water molecule is either preserved (+w), as in Figure 3A , or removed (−w), as in Figure S3B .
    Figure Legend Snippet: Calculated ion selectivity of the F. nucleatum c-ring. (A) Predicted structure of the ion binding sites in the absence of Na + , with two H + bound. Hydrogen atoms in non-polar groups are omitted for clarity. Note the binding site harbors two H + , one bound to Glu32 and another to Glu65 (both in cis ). Two alternative configurations, one of which lacks a bound water molecule, are also shown in Figure S3 . (B) Calculated free-energy of selectivity for H + vs. Na + of the F. nucleatum c-ring binding sites, relative to other c-rings previously characterized. The Na + state is that rendered in Figure 1A ; note the bound water molecule. Two possible H + -bound states are considered, in which the water molecule is either preserved (+w), as in Figure 3A , or removed (−w), as in Figure S3B .

    Techniques Used: Binding Assay

    ATP-driven proton translocation in E. coli and F. nucleatum inverted membranes vesicles. Proton translocation was determined at 37°C by the quenching of acridine orange (AO) fluorescence. The reaction was initiated by the addition of 1.25 mM ATP and terminated with 30 µM CCCP at the times indicated by arrows. (A) E. coli native membranes in pH 7.5 buffer. (B) F. nucleatum native membranes in pH 6 buffer with 1.25 mM Na + -ATP; (C) Same as (B), with 10 mM NaCl added to the buffer.
    Figure Legend Snippet: ATP-driven proton translocation in E. coli and F. nucleatum inverted membranes vesicles. Proton translocation was determined at 37°C by the quenching of acridine orange (AO) fluorescence. The reaction was initiated by the addition of 1.25 mM ATP and terminated with 30 µM CCCP at the times indicated by arrows. (A) E. coli native membranes in pH 7.5 buffer. (B) F. nucleatum native membranes in pH 6 buffer with 1.25 mM Na + -ATP; (C) Same as (B), with 10 mM NaCl added to the buffer.

    Techniques Used: Translocation Assay, Fluorescence

    Catalytic activity of the purified F 1 F o -ATP synthase from F. nucleatum . (A) Activation of the F. nucleatum ATP synthase by Na + , Li + , and K + ions (Na + , •; Li + , ▪; and K + , ▴). (B) Activation of the ATP synthase by Na + ions at pH 7.5 (•) and pH 9.0 (▪). The ATP hydrolysis activity was determined using the ATP-regenerating assay (120–140 µg protein), at 37°C. Activity in units/mg of protein (1 unit = 1 µmol ATP hydrolysed/min). 100% of activity corresponds 1–2 units/mg at pH 7.5 and 0.6–0.72 units/mg at pH 9.0. The assay mixture contained 50 mM MOPS, 2 mM MgCl 2 (pH 7.5) in (A) and 50 mM MES-MOPS-Tris, 2 mM MgCl 2 in (B). The values plotted are representative of at least two biological replicates; the statistical variance was less than 20%.
    Figure Legend Snippet: Catalytic activity of the purified F 1 F o -ATP synthase from F. nucleatum . (A) Activation of the F. nucleatum ATP synthase by Na + , Li + , and K + ions (Na + , •; Li + , ▪; and K + , ▴). (B) Activation of the ATP synthase by Na + ions at pH 7.5 (•) and pH 9.0 (▪). The ATP hydrolysis activity was determined using the ATP-regenerating assay (120–140 µg protein), at 37°C. Activity in units/mg of protein (1 unit = 1 µmol ATP hydrolysed/min). 100% of activity corresponds 1–2 units/mg at pH 7.5 and 0.6–0.72 units/mg at pH 9.0. The assay mixture contained 50 mM MOPS, 2 mM MgCl 2 (pH 7.5) in (A) and 50 mM MES-MOPS-Tris, 2 mM MgCl 2 in (B). The values plotted are representative of at least two biological replicates; the statistical variance was less than 20%.

    Techniques Used: Activity Assay, Purification, Activation Assay

    Simulation model of the open-state of the Na + -binding site in the F. nucleatum c-ring. The c-ring is shown in cartoon format (yellow), viewed from the membrane plane. Lipid molecules in the surrounding membrane bilayer are omitted for clarity. A crevice of water molecules (transparent red spheres) was modeled on the cytoplasmic side of the protein-lipid interface, so as to mimic the hydrated environment at the interface of the c-ring and subunit a, in the complete enzyme. A guanidinium ion (GND + ) was added to the crevice, to mimic a conserved Arg side-chain in the fourth transmembrane helix of subunit a. Key side-chains in the open binding site, namely Glu65, Glu32, Ser66, and Tyr70, are shown as sticks (non-polar hydrogen atoms are omitted); these form an interaction network in the closed state ( Figure 8 ), which in this open state is largely disrupted. Glu65 projects out of the binding site and interacts with the guanidinium ion; Glu32, however, is highly likely to remain protonated ( Figure 2B ).
    Figure Legend Snippet: Simulation model of the open-state of the Na + -binding site in the F. nucleatum c-ring. The c-ring is shown in cartoon format (yellow), viewed from the membrane plane. Lipid molecules in the surrounding membrane bilayer are omitted for clarity. A crevice of water molecules (transparent red spheres) was modeled on the cytoplasmic side of the protein-lipid interface, so as to mimic the hydrated environment at the interface of the c-ring and subunit a, in the complete enzyme. A guanidinium ion (GND + ) was added to the crevice, to mimic a conserved Arg side-chain in the fourth transmembrane helix of subunit a. Key side-chains in the open binding site, namely Glu65, Glu32, Ser66, and Tyr70, are shown as sticks (non-polar hydrogen atoms are omitted); these form an interaction network in the closed state ( Figure 8 ), which in this open state is largely disrupted. Glu65 projects out of the binding site and interacts with the guanidinium ion; Glu32, however, is highly likely to remain protonated ( Figure 2B ).

    Techniques Used: Binding Assay

    The Na + -binding site of the F. nucleatum c 11 ring at pH 5.3 and 8.7. (A) A functional Na + -binding site consists of two adjacent c-subunits (in yellow and green). Residues discussed in the text are indicated. (B) Close-up of the Na + -binding site viewed from the membrane plane, from a crystal grown at pH 5.3 and 100 mM NaOAc; the resolution of the data is 2.2 Å. The Na + and a structural water molecule are shown in purple and red spheres, respectively. The H-bond network and ion-protein interactions are indicated by dashed lines. Residues involved in Na + binding are labeled. (C) Close-up of the Na + binding site from a crystal grown at pH 8.7 and 100 mM buffer, viewed as in (B); the resolution is 2.6 Å. Electron density maps (2F obs -F calc , blue mesh) are shown at 1.9σ. In both cases Na + ions can be discerned in the ion-binding sites, and consistently the key Glu65 side-chain is observed in the ion-locked conformation, while Glu32 is protonated.
    Figure Legend Snippet: The Na + -binding site of the F. nucleatum c 11 ring at pH 5.3 and 8.7. (A) A functional Na + -binding site consists of two adjacent c-subunits (in yellow and green). Residues discussed in the text are indicated. (B) Close-up of the Na + -binding site viewed from the membrane plane, from a crystal grown at pH 5.3 and 100 mM NaOAc; the resolution of the data is 2.2 Å. The Na + and a structural water molecule are shown in purple and red spheres, respectively. The H-bond network and ion-protein interactions are indicated by dashed lines. Residues involved in Na + binding are labeled. (C) Close-up of the Na + binding site from a crystal grown at pH 8.7 and 100 mM buffer, viewed as in (B); the resolution is 2.6 Å. Electron density maps (2F obs -F calc , blue mesh) are shown at 1.9σ. In both cases Na + ions can be discerned in the ion-binding sites, and consistently the key Glu65 side-chain is observed in the ion-locked conformation, while Glu32 is protonated.

    Techniques Used: Binding Assay, Functional Assay, Labeling

    Crystal structure of the c 11 ring of the F. nucleatum ATP synthase at 2.2 Å resolution. The pH of crystallization buffer was 5.3, and included 100 mM Na + . (A) Side view of the c-ring along the membrane plane. The c-subunits are displayed in different colors in ribbon representation. The transmembrane region, 35 Å in width, is indicated with grey bars. (B) View of the c-ring from the cytoplasm, along the perpendicular to the membrane. The sodium ion and the water molecule bound to each of the 11 ion-binding sites (purple and red spheres, respectively), are shown by F obs -F calc omit electron-density maps at 3.2σ (blue meshes).
    Figure Legend Snippet: Crystal structure of the c 11 ring of the F. nucleatum ATP synthase at 2.2 Å resolution. The pH of crystallization buffer was 5.3, and included 100 mM Na + . (A) Side view of the c-ring along the membrane plane. The c-subunits are displayed in different colors in ribbon representation. The transmembrane region, 35 Å in width, is indicated with grey bars. (B) View of the c-ring from the cytoplasm, along the perpendicular to the membrane. The sodium ion and the water molecule bound to each of the 11 ion-binding sites (purple and red spheres, respectively), are shown by F obs -F calc omit electron-density maps at 3.2σ (blue meshes).

    Techniques Used: Crystallization Assay, Binding Assay

    19) Product Images from "Proteomics of Fusobacterium nucleatum within a model developing oral microbial community"

    Article Title: Proteomics of Fusobacterium nucleatum within a model developing oral microbial community

    Journal: MicrobiologyOpen

    doi: 10.1002/mbo3.204

    FnPgSg versus FnSgEnergy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with S. gordonii .
    Figure Legend Snippet: FnPgSg versus FnSgEnergy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with S. gordonii .

    Techniques Used:

    FnSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Streptococcus gordonii comparison to F. nucleatum with Porphyromonas gingivalis .
    Figure Legend Snippet: FnSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Streptococcus gordonii comparison to F. nucleatum with Porphyromonas gingivalis .

    Techniques Used:

    FnSg versus Fn energy metabolism and end products. Proteins catalyzing each step are shown by their Fusobacterium nucleatum FN number. Red numbers indicate increased levels in the first condition compared to the second condition, green decreased levels, yellow no statistical change, and black undetected in at least one of the conditions.
    Figure Legend Snippet: FnSg versus Fn energy metabolism and end products. Proteins catalyzing each step are shown by their Fusobacterium nucleatum FN number. Red numbers indicate increased levels in the first condition compared to the second condition, green decreased levels, yellow no statistical change, and black undetected in at least one of the conditions.

    Techniques Used:

    FnPgSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with P. gingivalis .
    Figure Legend Snippet: FnPgSg versus FnPg energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum with P. gingivalis .

    Techniques Used:

    FnPgSg versus Fn energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum .
    Figure Legend Snippet: FnPgSg versus Fn energy metabolism and end products. Labels and color coding as described for Figure 1 , for the Fusobacterium nucleatum with Porphyromonas gingivalis and Streptococcus gordonii comparison to F. nucleatum .

    Techniques Used:

    FnPg versus Fn energy metabolism and end products. The diagram shows a schematic of the glycolysis and pentose phosphate pathways for Fn including the end products of the metabolism, formate, acetate, l -lactate, and butanoate, for the Fusobacterium nucleatum with Porphyromonas gingivalis sample compared to F. nucleatum . Labels and color coding as described for Figure 1 .
    Figure Legend Snippet: FnPg versus Fn energy metabolism and end products. The diagram shows a schematic of the glycolysis and pentose phosphate pathways for Fn including the end products of the metabolism, formate, acetate, l -lactate, and butanoate, for the Fusobacterium nucleatum with Porphyromonas gingivalis sample compared to F. nucleatum . Labels and color coding as described for Figure 1 .

    Techniques Used:

    Electron micrographs. Thin-section transmission electron micrographs after 18 h in a model biofilm of (A) Fusobacterium nucleatum alone, (B) F. nucleatum with Porphyromonas gingivalis , (C) F. nucleatum with Streptococcus gordonii , or (D) F. nucleatum with both P. gingivalis and S. gordonii . Magnification: ×49,000.
    Figure Legend Snippet: Electron micrographs. Thin-section transmission electron micrographs after 18 h in a model biofilm of (A) Fusobacterium nucleatum alone, (B) F. nucleatum with Porphyromonas gingivalis , (C) F. nucleatum with Streptococcus gordonii , or (D) F. nucleatum with both P. gingivalis and S. gordonii . Magnification: ×49,000.

    Techniques Used: Transmission Assay

    20) Product Images from "Identification and Characterization of Fusolisin, the Fusobacterium nucleatum Autotransporter Serine Protease"

    Article Title: Identification and Characterization of Fusolisin, the Fusobacterium nucleatum Autotransporter Serine Protease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111329

    Fu-S-P activity correlates with the number of F. nucleatum cells. Fu-S-P (0.03 mM) was incubated for 2 hrs with increasing numbers of washed F. nucleatum cells. Relative Fluorescent Units (RFU) were determined as described in Materials and Methods. No activity was observed with boiled cells.
    Figure Legend Snippet: Fu-S-P activity correlates with the number of F. nucleatum cells. Fu-S-P (0.03 mM) was incubated for 2 hrs with increasing numbers of washed F. nucleatum cells. Relative Fluorescent Units (RFU) were determined as described in Materials and Methods. No activity was observed with boiled cells.

    Techniques Used: Activity Assay, Incubation

    PMSF inhibits growth of F. nucleatum but not of E. coli . (A) Growth of F. nucleatum 12230 (black line) is inhibited by PMSF (solid green line), but this inhibition is relieved by P. gingivalis supernatant (SN Pg) containing PMSF-resistant cysteine proteases (broken green line). (B) Growth of E. coli is not affected by PMSF, ruling out PMSF toxicity. *P
    Figure Legend Snippet: PMSF inhibits growth of F. nucleatum but not of E. coli . (A) Growth of F. nucleatum 12230 (black line) is inhibited by PMSF (solid green line), but this inhibition is relieved by P. gingivalis supernatant (SN Pg) containing PMSF-resistant cysteine proteases (broken green line). (B) Growth of E. coli is not affected by PMSF, ruling out PMSF toxicity. *P

    Techniques Used: Inhibition

    Identification of the fusobacterial serine protease. Amino acid sequences of the putative serine protease open reading frames FN1426 (Fsp25586) (A), and FNV0835 (Fsp49256) (B). Red highlight indicates sequences identified by mass spectrometry of the 99 kDa serine protease of F. nucleatum ATCC 25586 (A), and of the 55 kDa serine protease of F. nucleatum ATCC 49256 (B).
    Figure Legend Snippet: Identification of the fusobacterial serine protease. Amino acid sequences of the putative serine protease open reading frames FN1426 (Fsp25586) (A), and FNV0835 (Fsp49256) (B). Red highlight indicates sequences identified by mass spectrometry of the 99 kDa serine protease of F. nucleatum ATCC 25586 (A), and of the 55 kDa serine protease of F. nucleatum ATCC 49256 (B).

    Techniques Used: Mass Spectrometry

    PMSF inhibits the proteolytic activity of F. nucleatum . A, F. nucleatum FDC 364. B, F. nucleatum ATCC 25586. C, F. nucleatum 12230. D, F. nucleatum ATCC 23726. M, Molecular weight markers. Presented data are of representative zymograms.
    Figure Legend Snippet: PMSF inhibits the proteolytic activity of F. nucleatum . A, F. nucleatum FDC 364. B, F. nucleatum ATCC 25586. C, F. nucleatum 12230. D, F. nucleatum ATCC 23726. M, Molecular weight markers. Presented data are of representative zymograms.

    Techniques Used: Activity Assay, Molecular Weight

    Self-restriction of Fsp25586 is not efficient. Zymogram analysis of cell culture supernatant prepared from F. nucleatum ATCC 23726 carrying the pHS30 vector (A), or the pHSPROT plasmid expressing Fsp25586 (B).
    Figure Legend Snippet: Self-restriction of Fsp25586 is not efficient. Zymogram analysis of cell culture supernatant prepared from F. nucleatum ATCC 23726 carrying the pHS30 vector (A), or the pHSPROT plasmid expressing Fsp25586 (B).

    Techniques Used: Cell Culture, Plasmid Preparation, Expressing

    Protease profiles of F. nucleatum growth medium supernatants on fibrinogen containing zymograms. M, Molecular weight markers. A, F. nucleatum ATCC 49256. B, F. nucleatum FDC 364. C, F. nucleatum ATCC 10953. D, F. nucleatum ATCC 25586. E, F. nucleatum ATCC 23726. F, F. nucleatum 12230. Arrows indicate proteolytic bands. Presented data are of representative zymograms.
    Figure Legend Snippet: Protease profiles of F. nucleatum growth medium supernatants on fibrinogen containing zymograms. M, Molecular weight markers. A, F. nucleatum ATCC 49256. B, F. nucleatum FDC 364. C, F. nucleatum ATCC 10953. D, F. nucleatum ATCC 25586. E, F. nucleatum ATCC 23726. F, F. nucleatum 12230. Arrows indicate proteolytic bands. Presented data are of representative zymograms.

    Techniques Used: Molecular Weight

    Related Articles

    Modification:

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif
    Article Snippet: The theoretical mass of the c-monomer is 8,862.94 Da (unformylated) and 8,890.94 Da (formylated); the theoretical mass of the formylated, single-oxidized c-monomer is 8,906.94 Da, and 8,922.94 Da for the double-oxidized form. (TIF) Click here for additional data file. .. Figure S12 Long-term kinetics of NCD-4 modification of detergent-solubilized c-rings from F. nucleatum , without the addition of Na+ . .. A 27 µg c-ring sample in 0.5 M MES buffer pH 5.7 containing 1.5% (w/v) n-octyl-β-D-glycoside was monitored in a fluorescence spectrophotometer (λex = 342 nm, λem = 452 nm) at selected time points for a total of 120 min.

    Concentration Assay:

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif
    Article Snippet: The values plotted are the mean of three biological replicates and their standard errors. (TIF) Click here for additional data file. .. Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ). .. Controls were grown with an equivalent volume of ethanol (•).

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif
    Article Snippet: The water molecule that coordinates the bound Na+ is either preserved—as in the model shown in —or removed. (A) Alternate configuration of the site when a water molecule is preserved in the H+ bound state, similarly populated to that shown in . (B) Predicted structure of the site when the H+ state does not include the water molecule included in the Na+ state. (TIF) Click here for additional data file. .. Figure S4 Effect of ionophores on the growth of F. nucleatum ATCC 25586 in batch culture. (A) CCCP was added to a final concentration (f.c.) of 50 µM (○), 100 µM (□) and 200 µM (Δ). (B) 2,4-dinitrophenol (DNP) was added to a f.c. of 50 µM (○), 100 µM (□) and 200 µM (Δ). (C) 3,3′,4′,5-tetrachlorosalicylanilide (TCS) was added to a f.c. of 2 µM (○), 5 µM (□), and 10 µM (Δ). (D) Monensin was added to a f.c. of 1 µM (○), 5 µM (□), and 10 µM (Δ). (E) Amiloride was added to a f.c. of 50 µM (○), 100 µM (□), and 200 µM (Δ). (F) 5-(N -ethyl-N -isopropyl)amiloride (EIPA) was added to a f.c. of 50 µM (○), 100 µM (□), and 200 µM (Δ). .. Controls were grown with an equivalent volume of ethanol (•).

    Isolation:

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif
    Article Snippet: A molecular weight marker (M, PageRuler Unstained Protein Ladder, Fermentas) in kDa is given on the left. (TIF) Click here for additional data file. .. Figure S11 Determination of the c-subunit mass from the isolated c-ring of F. nucleatum by MALDI-MS. .. The theoretical mass of the c-monomer is 8,862.94 Da (unformylated) and 8,890.94 Da (formylated); the theoretical mass of the formylated, single-oxidized c-monomer is 8,906.94 Da, and 8,922.94 Da for the double-oxidized form. (TIF) Click here for additional data file.

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria
    Article Snippet: Therefore, fadA may be used as a marker for identification of orally related fusobacteria. .. The conservation of fadA in F. nucleatum isolated from intrauterine infections and its absence in the vaginal species F. gonidiaformans and F. naviforme further support the hypothesis that intrauterine F. nucleatum originates from the oral cavity rather than the vaginal tract ( ). .. BLAST searches also identified two paralogues of FadA, FN1529 from F. nucleatum ATCC 25586 and FNV2159 from F. nucleatum ATCC 49256, which share 31% identity with FadA and 98% identity with each other.

    Cell Culture:

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria
    Article Snippet: P. gingivalis (ATCC (American Type Culture Collection, Manassas, VA) 33277) was cultured under anaerobic conditions (85% N2 , 10% H2 , and 5% CO2 ) at 37 °C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin, and 1 mg of menadione per liter. .. F. nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37 °C under anaerobic conditions.

    Purification:

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif
    Article Snippet: The bands for the c11 ring and the β-subunit overlap. (TIF) Click here for additional data file. .. Figure S8 Catalytic activity of the purified F1 Fo -ATP synthase from F. nucleatum . (A) Hydrolytic activity of the ATP synthase in membranes, soluble fraction (DDM extracted) and purified protein over time (all stored at 4°C). (B) Effect of ATP on hydrolytic activity of the purified enzyme at 37°C. (C) Effect of MgCl2 on hydrolytic activity at 37°C. .. The ATPase activity was determined using the ATP regenerating assay (120–140 µg protein) in (A), (B), and the Pi assay (60–70 µg protein) in (C).

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif
    Article Snippet: Values are representative of two to three separate experiments; the statistical variance was less than 20%. (TIF) Click here for additional data file. .. Figure S10 Silver-stained SDS-PAGE of the purified c11 ring from F. nucleatum . .. Lane 1, 1 µg of c11 ring; lane 2, 1 µg of c-ring precipitated with 15% (w/v) trichloroacetic acid.

    Dot Blot:

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria
    Article Snippet: In addition, a paralogue (i.e., homologue on the same chromosome), FN1529, is present in F. nucleatum ATCC 25586, sharing 31% identity with FadA (Fig. ). .. More recently, the genome sequence of another strain, F. nucleatum subsp. vincentii ATCC 49256, became available ( ).

    Polymerase Chain Reaction:

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria
    Article Snippet: In addition, a paralogue (i.e., homologue on the same chromosome), FN1529, is present in F. nucleatum ATCC 25586, sharing 31% identity with FadA (Fig. ). .. More recently, the genome sequence of another strain, F. nucleatum subsp. vincentii ATCC 49256, became available ( ).

    Incubation:

    Article Title: A novel vaccine targeting Fusobacterium nucleatum against abscesses and halitosis
    Article Snippet: The result strongly validates the in vivo protective immunity of an inactivated F. nucleatum -based vaccine in the prevention of abscess progression. .. To verify if inactivated F. nucleatum -based vaccines elicit neutralizing antibodies against VSC production and biofilm formation, F. nucleatum (4 × 109 CFU) was incubated with serum obtained from the F. nucleatum - or S. mutans -immunized mice. .. VSCs ( ) and biofilms ( ) were detected on lead acetate-contained agar plates as described in Section 2.

    other:

    Article Title: Tea polyphenols inhibit the activation of NF-κB and the secretion of cytokines and matrix metalloproteinases by macrophages stimulated with Fusobacterium nucleatum
    Article Snippet: F. nucleatum ATCC 25586 was grown anaerobically (80% N2 , 10% CO2 , 10% H2 ) for 24 h at 37 °C in Todd-Hewitt broth (THB; Becton, Dickinson and Company, USA) supplemented with 0.001% hemin and 0.0001% vitamin K.

    Article Title: Tea polyphenols inhibit the activation of NF-κB and the secretion of cytokines and matrix metalloproteinases by macrophages stimulated with Fusobacterium nucleatum
    Article Snippet: The bacterial suspension was prepared from an overnight culture of F. nucleatum .

    Activity Assay:

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif
    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ). .. Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif
    Article Snippet: The bands for the c11 ring and the β-subunit overlap. (TIF) Click here for additional data file. .. Figure S8 Catalytic activity of the purified F1 Fo -ATP synthase from F. nucleatum . (A) Hydrolytic activity of the ATP synthase in membranes, soluble fraction (DDM extracted) and purified protein over time (all stored at 4°C). (B) Effect of ATP on hydrolytic activity of the purified enzyme at 37°C. (C) Effect of MgCl2 on hydrolytic activity at 37°C. .. The ATPase activity was determined using the ATP regenerating assay (120–140 µg protein) in (A), (B), and the Pi assay (60–70 µg protein) in (C).

    Infection:

    Article Title: Tea polyphenols inhibit the activation of NF-κB and the secretion of cytokines and matrix metalloproteinases by macrophages stimulated with Fusobacterium nucleatum
    Article Snippet: To investigate the effect of tea polyphenols on F. nucleatum -induced NF-κB activation, U937 3xκB-LUC cells (106 cells/ml) were pre-incubated with the compounds (non-cytotoxic concentrations; in RPMI containing 1% FBS) for 30 min in the wells of a black wall, black bottom 96-well microplate (Greiner Bio-One North America Inc., USA). .. They were then stimulated for 6 h with F. nucleatum at a multiplicity of infection (MOI) of 100. .. The bacterial suspension was prepared from an overnight culture of F. nucleatum .

    Sequencing:

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria
    Article Snippet: DNA sequence analysis showed that FadA was highly conserved among the fadA -positive species (Fig. ). .. Following identification of the fadA gene, the genomic sequence of F. nucleatum ATCC 25586 became available ( ). .. A BLAST search showed that the 2.4-kb fragment containing the fadA gene was highly conserved between F. nucleatum 12230 and ATCC 25586 (data not shown).

    Article Title: Identification and Characterization of a Novel Adhesin Unique to Oral Fusobacteria
    Article Snippet: Following identification of the fadA gene, the genomic sequence of F. nucleatum ATCC 25586 became available ( ). .. In addition, a paralogue (i.e., homologue on the same chromosome), FN1529, is present in F. nucleatum ATCC 25586, sharing 31% identity with FadA (Fig. ).

    Mouse Assay:

    Article Title: A novel vaccine targeting Fusobacterium nucleatum against abscesses and halitosis
    Article Snippet: The result strongly validates the in vivo protective immunity of an inactivated F. nucleatum -based vaccine in the prevention of abscess progression. .. To verify if inactivated F. nucleatum -based vaccines elicit neutralizing antibodies against VSC production and biofilm formation, F. nucleatum (4 × 109 CFU) was incubated with serum obtained from the F. nucleatum - or S. mutans -immunized mice. .. VSCs ( ) and biofilms ( ) were detected on lead acetate-contained agar plates as described in Section 2.

    SDS Page:

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif
    Article Snippet: Values are representative of two to three separate experiments; the statistical variance was less than 20%. (TIF) Click here for additional data file. .. Figure S10 Silver-stained SDS-PAGE of the purified c11 ring from F. nucleatum . .. Lane 1, 1 µg of c11 ring; lane 2, 1 µg of c-ring precipitated with 15% (w/v) trichloroacetic acid.

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif
    Article Snippet: The arrow indicates the time point at which the NCD-4 labeling of the c-ring was stopped in the experiment reported in , by addition of 15 mM NaCl. (TIF) Click here for additional data file. .. Figure S13 SDS-PAGE of the c-ring and the c-monomer from F. nucleatum after reaction with NCD-4. .. The NCD-4 modified F. nucleatum c11 ring was loaded on a 13.2% SDS-polyacrylamide gel.

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    ATCC f nucleatum
    mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium <t>nucleatum</t> (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P
    F Nucleatum, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: mRNA expression of innate immune markers human β-defensin 2 (hBD-2) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m) or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of hBD2 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of hBD2 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Article Snippet: F. nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37 °C under anaerobic conditions.

    Techniques: Expressing, Histone Deacetylase Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CTL Assay

    mRNA expression of innate immune markers CC chemokine ligand 20 (CCL20) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of ( b ) CCL20 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of CCL20 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: mRNA expression of innate immune markers CC chemokine ligand 20 (CCL20) are increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Gene expression of ( b ) CCL20 was evaluated by quantitative real-time PCR (QRT-PCR) compared with unstimulated control after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Controls include unstimulated control, bacteria-alone treatment, and various inhibitors alone as indicated. No significant changes were found in the gene expression of CCL20 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Data are expressed as means of fold change±s.e.m. from three donors evaluated in duplicate. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Article Snippet: F. nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37 °C under anaerobic conditions.

    Techniques: Expressing, Histone Deacetylase Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CTL Assay

    Differential decreased mRNA expression of HDAC1, HDAC2 and DNMT1 in gingival epithelial cells in response to various doses of oral bacteria. mRNA expression of ( a ) DNA methyltransferase (DNMT1), ( b ) histone deacetylase 1 (HDAC1), and ( c ) histone deacetylase 2 (HDAC2) are differentially decreased in gingival epithelial cells (GECs) in response to various doses of Porphyromonas gingivalis vs. Fusobacterium nucleatum . GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, 100:1, and 200:1 for 24 h. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the unstimulated control after normalization with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase ( GAPDH ). The data are derived from three different cell donors tested in duplicate. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: Differential decreased mRNA expression of HDAC1, HDAC2 and DNMT1 in gingival epithelial cells in response to various doses of oral bacteria. mRNA expression of ( a ) DNA methyltransferase (DNMT1), ( b ) histone deacetylase 1 (HDAC1), and ( c ) histone deacetylase 2 (HDAC2) are differentially decreased in gingival epithelial cells (GECs) in response to various doses of Porphyromonas gingivalis vs. Fusobacterium nucleatum . GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, 100:1, and 200:1 for 24 h. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the unstimulated control after normalization with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase ( GAPDH ). The data are derived from three different cell donors tested in duplicate. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Article Snippet: F. nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37 °C under anaerobic conditions.

    Techniques: Expressing, Histone Deacetylase Assay, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Derivative Assay, CTL Assay

    Protein levels of histone H3 methylated at Lys4 were evaluated with PathScan enzyme-linked immunosorbent assay (ELISA). Gingival epithelial cells (GECs) were exposed to Porphyromonas gingivalis or Fusobacterium nucleatum for 24 h at multiplicity of infection (MOI) of 100:1, and then nuclear protein was extracted followed by sonication. The H3 tri-methylated at Lys4 was captured by coated antibody after incubation with cell lysates, and histone H3 protein level was quantified according to the absorbance readings at 450 nm. Protein expression was expressed as the ratio of absorbance readings normalized to relative protein amount. The data are average from three different donor cells tested with standard error deviation. The asterisks indicate the significant difference vs. the respective unstimulated control (** P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: Protein levels of histone H3 methylated at Lys4 were evaluated with PathScan enzyme-linked immunosorbent assay (ELISA). Gingival epithelial cells (GECs) were exposed to Porphyromonas gingivalis or Fusobacterium nucleatum for 24 h at multiplicity of infection (MOI) of 100:1, and then nuclear protein was extracted followed by sonication. The H3 tri-methylated at Lys4 was captured by coated antibody after incubation with cell lysates, and histone H3 protein level was quantified according to the absorbance readings at 450 nm. Protein expression was expressed as the ratio of absorbance readings normalized to relative protein amount. The data are average from three different donor cells tested with standard error deviation. The asterisks indicate the significant difference vs. the respective unstimulated control (** P

    Article Snippet: F. nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37 °C under anaerobic conditions.

    Techniques: Methylation, Enzyme-linked Immunosorbent Assay, Infection, Sonication, Incubation, Expressing

    Protein levels of histone deacetylases 1 and 2 (HDAC1 and HDAC2) and DNA methyltransferase (DNMT1) are differentially expressed in gingival epithelial cells (GECs) in response to Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn). GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 100:1 for 24 h. Nuclear proteins were extracted, denatured at 70 °C for 10 min, and separated by NuPAGE electrophoresis system. Nuclear extracts of Hela cells probed with individual primary antibody were used as positive controls. The data are derived from two different cell donors tested in duplicate.

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: Protein levels of histone deacetylases 1 and 2 (HDAC1 and HDAC2) and DNA methyltransferase (DNMT1) are differentially expressed in gingival epithelial cells (GECs) in response to Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn). GECs were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 100:1 for 24 h. Nuclear proteins were extracted, denatured at 70 °C for 10 min, and separated by NuPAGE electrophoresis system. Nuclear extracts of Hela cells probed with individual primary antibody were used as positive controls. The data are derived from two different cell donors tested in duplicate.

    Article Snippet: F. nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37 °C under anaerobic conditions.

    Techniques: Infection, Electrophoresis, Derivative Assay

    Differential mRNA expression of HDAC1, HDAC2 and DNMT1 in human TERT cells in response to oral bacteria. Differential mRNA expression of ( a ) histone deacetylases 1 and 2 (HDAC1 and HDAC2) and ( b ) DNA methyltransferase (DNMT1) in human TERT cells in response to Porphyromonas gingivalis vs. Fusobacterium nucleatum . Human TERT cells were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, and 100:1 for 4 or 24 h. Unstimulated cells at 4 and 24 h served as controls. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the corresponding unstimulated controls (4 and 24 h) after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The experiment was repeated twice using TERT cells. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: Differential mRNA expression of HDAC1, HDAC2 and DNMT1 in human TERT cells in response to oral bacteria. Differential mRNA expression of ( a ) histone deacetylases 1 and 2 (HDAC1 and HDAC2) and ( b ) DNA methyltransferase (DNMT1) in human TERT cells in response to Porphyromonas gingivalis vs. Fusobacterium nucleatum . Human TERT cells were stimulated with P. gingivalis (Pg) or F. nucleatum (Fn) at multiplicities of infection (MOIs) of 10:1, 50:1, and 100:1 for 4 or 24 h. Unstimulated cells at 4 and 24 h served as controls. Changes in mRNA expression were evaluated by quantitative real-time PCR (QRT-PCR) and results are expressed as fold change in gene expression compared with the corresponding unstimulated controls (4 and 24 h) after normalization with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The experiment was repeated twice using TERT cells. Error bars indicate s.e.m. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Article Snippet: F. nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37 °C under anaerobic conditions.

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CTL Assay

    Interleukin-8 (IL-8) secretion is increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Secretion of IL-8 in response to various oral bacteria is evaluated by enzyme-linked immunosorbent assay (ELISA). Cell-free supernatant was collected and the amount of IL-8 secreted is shown in pg ml –1 . Unstimulated cells (UN) are used as controls in each experiment. Data from duplicates with cells from three different donors are shown. No significant changes were found in the secretion of IL-8 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Journal: Mucosal Immunology

    Article Title: Epigenetic regulation of human ?-defensin 2 and CC chemokine ligand 20 expression in gingival epithelial cells in response to oral bacteria

    doi: 10.1038/mi.2010.83

    Figure Lengend Snippet: Interleukin-8 (IL-8) secretion is increased when histone deacetylase (HDAC) and DNA methyltransferase (DNMT) are inhibited. Gingival epithelial cells (GECs) were pretreated with trichostatin A (TSA; 9 and 45 m), sodium butyrate (SB; 0.5 and 2.0 m), or 5′-azacytidine (AZA; 1 and 10 μ) for 4 h, and subsequently exposed to Porphyromonas gingivalis (multiplicity of infection (MOI) 100:1) or Fusobacterium nucleatum (MOI 100:1) for 16 h. Secretion of IL-8 in response to various oral bacteria is evaluated by enzyme-linked immunosorbent assay (ELISA). Cell-free supernatant was collected and the amount of IL-8 secreted is shown in pg ml –1 . Unstimulated cells (UN) are used as controls in each experiment. Data from duplicates with cells from three different donors are shown. No significant changes were found in the secretion of IL-8 in GECs treated with inhibitor only: ( a ) SB, ( b ) TSA, ( c ) SB+TSA, and ( d ) AZA compared with unstimulated control. Asterisks indicate statistically significant difference compared with unstimulated control (Ctl) (* P

    Article Snippet: F. nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37 °C under anaerobic conditions.

    Techniques: Histone Deacetylase Assay, Infection, Enzyme-linked Immunosorbent Assay, CTL Assay

    Protective effect of Na + and Li + against DCCD inhibition of the purified F. nucleatum ATP synthase. The purified protein (120–140 µg) was incubated at 25°C for 20 min in the presence of either 400 µM DCCD or 400 µM DCCD and 50 mM NaCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , (A) at pH 7.5, (B) at pH 9, and (C) at pH 6.5. (D) NaCl was substituted by either LiCl or KCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , pH 7.5. 100% enzyme activity corresponds to 1–2 units/mg of protein. The values plotted are representative of at least two to three individual experiments; the statistical variance was less than 20%.

    Journal: PLoS Biology

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    doi: 10.1371/journal.pbio.1001596

    Figure Lengend Snippet: Protective effect of Na + and Li + against DCCD inhibition of the purified F. nucleatum ATP synthase. The purified protein (120–140 µg) was incubated at 25°C for 20 min in the presence of either 400 µM DCCD or 400 µM DCCD and 50 mM NaCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , (A) at pH 7.5, (B) at pH 9, and (C) at pH 6.5. (D) NaCl was substituted by either LiCl or KCl in 50 mM MES-MOPS-Tris, 2 mM MgCl 2 , pH 7.5. 100% enzyme activity corresponds to 1–2 units/mg of protein. The values plotted are representative of at least two to three individual experiments; the statistical variance was less than 20%.

    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Techniques: Inhibition, Purification, Incubation, Activity Assay

    Likelihood of protonation of the Na + -loaded binding sites in the F. nucleatum c-ring, and most probable configuration. (A) Energetics of proton transfer between Glu65 and Glu32 (models A and B in Figure 1 ), with a Na + concurrently bound, based on Hartree-Fock quantum-mechanical calculations for a reduced model of the binding site. The potential energy values plotted correspond to the optimized geometries in each case. The geometry and potential energy of the transition state are also provided. (B) Calculated free-energy gain associated with the protonation of Glu32, relative to a side-chain analog in solution (propionate); the formation energy of the chemical bond is omitted, as this is expected to be constant. Calculated values are translated into a pK a scale, by setting the free-energy value for propionate to its known pK a value, i.e., 4.9. The protonation of Glu32 is studied either in the Na + -bound, closed conformation of the c-ring binding sites ( Figure 1A ); or in an open, hydrated conformation ( Figure 9 ). In both cases, to the values calculated by FEP/MD (light grey) we added a correction calculated with Poisson electrostatic theory on account of the membrane polarizability (dark grey).

    Journal: PLoS Biology

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    doi: 10.1371/journal.pbio.1001596

    Figure Lengend Snippet: Likelihood of protonation of the Na + -loaded binding sites in the F. nucleatum c-ring, and most probable configuration. (A) Energetics of proton transfer between Glu65 and Glu32 (models A and B in Figure 1 ), with a Na + concurrently bound, based on Hartree-Fock quantum-mechanical calculations for a reduced model of the binding site. The potential energy values plotted correspond to the optimized geometries in each case. The geometry and potential energy of the transition state are also provided. (B) Calculated free-energy gain associated with the protonation of Glu32, relative to a side-chain analog in solution (propionate); the formation energy of the chemical bond is omitted, as this is expected to be constant. Calculated values are translated into a pK a scale, by setting the free-energy value for propionate to its known pK a value, i.e., 4.9. The protonation of Glu32 is studied either in the Na + -bound, closed conformation of the c-ring binding sites ( Figure 1A ); or in an open, hydrated conformation ( Figure 9 ). In both cases, to the values calculated by FEP/MD (light grey) we added a correction calculated with Poisson electrostatic theory on account of the membrane polarizability (dark grey).

    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Techniques: Binding Assay

    Alternative models of the ion-binding sites in the F. nucleatum c-ring, with Na + bound. The ion-coordinating groups in the protein are indicated with dashed lines. Hydrogen atoms in non-polar groups are omitted for clarity. (A) Model with Glu32 protonated in cis and Glu65 in the deprotonated state (model A), predicted to be the most likely (see Results). (B) Model with Glu32 deprotonated and Glu65 protonated (model B). In this model, the proton bound to Glu65 was initially modeled in cis , as this is the most probable geometry for the isolated side-chain. However, within 250 ps of the start of the simulation, the carboxyl group of Glu65 switched to trans . (C) Model with Glu32 and Glu65 in the deprotonated state (model C). During the simulation of this model, the electrostatic repulsion between Glu32 and Glu65 caused the latter to displace outwards, away from the Na + ion, which becomes coordinated by Glu32 in a bi-dentate configuration. Note the bound water reorients and no longer contributes to Na + coordination.

    Journal: PLoS Biology

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    doi: 10.1371/journal.pbio.1001596

    Figure Lengend Snippet: Alternative models of the ion-binding sites in the F. nucleatum c-ring, with Na + bound. The ion-coordinating groups in the protein are indicated with dashed lines. Hydrogen atoms in non-polar groups are omitted for clarity. (A) Model with Glu32 protonated in cis and Glu65 in the deprotonated state (model A), predicted to be the most likely (see Results). (B) Model with Glu32 deprotonated and Glu65 protonated (model B). In this model, the proton bound to Glu65 was initially modeled in cis , as this is the most probable geometry for the isolated side-chain. However, within 250 ps of the start of the simulation, the carboxyl group of Glu65 switched to trans . (C) Model with Glu32 and Glu65 in the deprotonated state (model C). During the simulation of this model, the electrostatic repulsion between Glu32 and Glu65 caused the latter to displace outwards, away from the Na + ion, which becomes coordinated by Glu32 in a bi-dentate configuration. Note the bound water reorients and no longer contributes to Na + coordination.

    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Techniques: Binding Assay, Isolation

    Kinetics of NCD-4 modification of detergent-solubilized c-rings from F. nucleatum . 100 µM NCD-4 was added to a sample of purified F. nucleatum c 11 ring in MES buffer (pH 5.7) and 1.5% (w/v) n-octyl-β-D-glycoside. A continuous increase in fluorescence was measured upon reaction of NCD-4 with Glu65. Addition of 15 mM NaCl precluded further increase in the fluorescence. An extended control measurement, with no NaCl added, is shown in Figure S12 .

    Journal: PLoS Biology

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    doi: 10.1371/journal.pbio.1001596

    Figure Lengend Snippet: Kinetics of NCD-4 modification of detergent-solubilized c-rings from F. nucleatum . 100 µM NCD-4 was added to a sample of purified F. nucleatum c 11 ring in MES buffer (pH 5.7) and 1.5% (w/v) n-octyl-β-D-glycoside. A continuous increase in fluorescence was measured upon reaction of NCD-4 with Glu65. Addition of 15 mM NaCl precluded further increase in the fluorescence. An extended control measurement, with no NaCl added, is shown in Figure S12 .

    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Techniques: Modification, Purification, Fluorescence

    Calculated ion selectivity of the F. nucleatum c-ring. (A) Predicted structure of the ion binding sites in the absence of Na + , with two H + bound. Hydrogen atoms in non-polar groups are omitted for clarity. Note the binding site harbors two H + , one bound to Glu32 and another to Glu65 (both in cis ). Two alternative configurations, one of which lacks a bound water molecule, are also shown in Figure S3 . (B) Calculated free-energy of selectivity for H + vs. Na + of the F. nucleatum c-ring binding sites, relative to other c-rings previously characterized. The Na + state is that rendered in Figure 1A ; note the bound water molecule. Two possible H + -bound states are considered, in which the water molecule is either preserved (+w), as in Figure 3A , or removed (−w), as in Figure S3B .

    Journal: PLoS Biology

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    doi: 10.1371/journal.pbio.1001596

    Figure Lengend Snippet: Calculated ion selectivity of the F. nucleatum c-ring. (A) Predicted structure of the ion binding sites in the absence of Na + , with two H + bound. Hydrogen atoms in non-polar groups are omitted for clarity. Note the binding site harbors two H + , one bound to Glu32 and another to Glu65 (both in cis ). Two alternative configurations, one of which lacks a bound water molecule, are also shown in Figure S3 . (B) Calculated free-energy of selectivity for H + vs. Na + of the F. nucleatum c-ring binding sites, relative to other c-rings previously characterized. The Na + state is that rendered in Figure 1A ; note the bound water molecule. Two possible H + -bound states are considered, in which the water molecule is either preserved (+w), as in Figure 3A , or removed (−w), as in Figure S3B .

    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Techniques: Binding Assay

    ATP-driven proton translocation in E. coli and F. nucleatum inverted membranes vesicles. Proton translocation was determined at 37°C by the quenching of acridine orange (AO) fluorescence. The reaction was initiated by the addition of 1.25 mM ATP and terminated with 30 µM CCCP at the times indicated by arrows. (A) E. coli native membranes in pH 7.5 buffer. (B) F. nucleatum native membranes in pH 6 buffer with 1.25 mM Na + -ATP; (C) Same as (B), with 10 mM NaCl added to the buffer.

    Journal: PLoS Biology

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    doi: 10.1371/journal.pbio.1001596

    Figure Lengend Snippet: ATP-driven proton translocation in E. coli and F. nucleatum inverted membranes vesicles. Proton translocation was determined at 37°C by the quenching of acridine orange (AO) fluorescence. The reaction was initiated by the addition of 1.25 mM ATP and terminated with 30 µM CCCP at the times indicated by arrows. (A) E. coli native membranes in pH 7.5 buffer. (B) F. nucleatum native membranes in pH 6 buffer with 1.25 mM Na + -ATP; (C) Same as (B), with 10 mM NaCl added to the buffer.

    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Techniques: Translocation Assay, Fluorescence

    Catalytic activity of the purified F 1 F o -ATP synthase from F. nucleatum . (A) Activation of the F. nucleatum ATP synthase by Na + , Li + , and K + ions (Na + , •; Li + , ▪; and K + , ▴). (B) Activation of the ATP synthase by Na + ions at pH 7.5 (•) and pH 9.0 (▪). The ATP hydrolysis activity was determined using the ATP-regenerating assay (120–140 µg protein), at 37°C. Activity in units/mg of protein (1 unit = 1 µmol ATP hydrolysed/min). 100% of activity corresponds 1–2 units/mg at pH 7.5 and 0.6–0.72 units/mg at pH 9.0. The assay mixture contained 50 mM MOPS, 2 mM MgCl 2 (pH 7.5) in (A) and 50 mM MES-MOPS-Tris, 2 mM MgCl 2 in (B). The values plotted are representative of at least two biological replicates; the statistical variance was less than 20%.

    Journal: PLoS Biology

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    doi: 10.1371/journal.pbio.1001596

    Figure Lengend Snippet: Catalytic activity of the purified F 1 F o -ATP synthase from F. nucleatum . (A) Activation of the F. nucleatum ATP synthase by Na + , Li + , and K + ions (Na + , •; Li + , ▪; and K + , ▴). (B) Activation of the ATP synthase by Na + ions at pH 7.5 (•) and pH 9.0 (▪). The ATP hydrolysis activity was determined using the ATP-regenerating assay (120–140 µg protein), at 37°C. Activity in units/mg of protein (1 unit = 1 µmol ATP hydrolysed/min). 100% of activity corresponds 1–2 units/mg at pH 7.5 and 0.6–0.72 units/mg at pH 9.0. The assay mixture contained 50 mM MOPS, 2 mM MgCl 2 (pH 7.5) in (A) and 50 mM MES-MOPS-Tris, 2 mM MgCl 2 in (B). The values plotted are representative of at least two biological replicates; the statistical variance was less than 20%.

    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Techniques: Activity Assay, Purification, Activation Assay

    Simulation model of the open-state of the Na + -binding site in the F. nucleatum c-ring. The c-ring is shown in cartoon format (yellow), viewed from the membrane plane. Lipid molecules in the surrounding membrane bilayer are omitted for clarity. A crevice of water molecules (transparent red spheres) was modeled on the cytoplasmic side of the protein-lipid interface, so as to mimic the hydrated environment at the interface of the c-ring and subunit a, in the complete enzyme. A guanidinium ion (GND + ) was added to the crevice, to mimic a conserved Arg side-chain in the fourth transmembrane helix of subunit a. Key side-chains in the open binding site, namely Glu65, Glu32, Ser66, and Tyr70, are shown as sticks (non-polar hydrogen atoms are omitted); these form an interaction network in the closed state ( Figure 8 ), which in this open state is largely disrupted. Glu65 projects out of the binding site and interacts with the guanidinium ion; Glu32, however, is highly likely to remain protonated ( Figure 2B ).

    Journal: PLoS Biology

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    doi: 10.1371/journal.pbio.1001596

    Figure Lengend Snippet: Simulation model of the open-state of the Na + -binding site in the F. nucleatum c-ring. The c-ring is shown in cartoon format (yellow), viewed from the membrane plane. Lipid molecules in the surrounding membrane bilayer are omitted for clarity. A crevice of water molecules (transparent red spheres) was modeled on the cytoplasmic side of the protein-lipid interface, so as to mimic the hydrated environment at the interface of the c-ring and subunit a, in the complete enzyme. A guanidinium ion (GND + ) was added to the crevice, to mimic a conserved Arg side-chain in the fourth transmembrane helix of subunit a. Key side-chains in the open binding site, namely Glu65, Glu32, Ser66, and Tyr70, are shown as sticks (non-polar hydrogen atoms are omitted); these form an interaction network in the closed state ( Figure 8 ), which in this open state is largely disrupted. Glu65 projects out of the binding site and interacts with the guanidinium ion; Glu32, however, is highly likely to remain protonated ( Figure 2B ).

    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Techniques: Binding Assay

    The Na + -binding site of the F. nucleatum c 11 ring at pH 5.3 and 8.7. (A) A functional Na + -binding site consists of two adjacent c-subunits (in yellow and green). Residues discussed in the text are indicated. (B) Close-up of the Na + -binding site viewed from the membrane plane, from a crystal grown at pH 5.3 and 100 mM NaOAc; the resolution of the data is 2.2 Å. The Na + and a structural water molecule are shown in purple and red spheres, respectively. The H-bond network and ion-protein interactions are indicated by dashed lines. Residues involved in Na + binding are labeled. (C) Close-up of the Na + binding site from a crystal grown at pH 8.7 and 100 mM buffer, viewed as in (B); the resolution is 2.6 Å. Electron density maps (2F obs -F calc , blue mesh) are shown at 1.9σ. In both cases Na + ions can be discerned in the ion-binding sites, and consistently the key Glu65 side-chain is observed in the ion-locked conformation, while Glu32 is protonated.

    Journal: PLoS Biology

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    doi: 10.1371/journal.pbio.1001596

    Figure Lengend Snippet: The Na + -binding site of the F. nucleatum c 11 ring at pH 5.3 and 8.7. (A) A functional Na + -binding site consists of two adjacent c-subunits (in yellow and green). Residues discussed in the text are indicated. (B) Close-up of the Na + -binding site viewed from the membrane plane, from a crystal grown at pH 5.3 and 100 mM NaOAc; the resolution of the data is 2.2 Å. The Na + and a structural water molecule are shown in purple and red spheres, respectively. The H-bond network and ion-protein interactions are indicated by dashed lines. Residues involved in Na + binding are labeled. (C) Close-up of the Na + binding site from a crystal grown at pH 8.7 and 100 mM buffer, viewed as in (B); the resolution is 2.6 Å. Electron density maps (2F obs -F calc , blue mesh) are shown at 1.9σ. In both cases Na + ions can be discerned in the ion-binding sites, and consistently the key Glu65 side-chain is observed in the ion-locked conformation, while Glu32 is protonated.

    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Techniques: Binding Assay, Functional Assay, Labeling

    Crystal structure of the c 11 ring of the F. nucleatum ATP synthase at 2.2 Å resolution. The pH of crystallization buffer was 5.3, and included 100 mM Na + . (A) Side view of the c-ring along the membrane plane. The c-subunits are displayed in different colors in ribbon representation. The transmembrane region, 35 Å in width, is indicated with grey bars. (B) View of the c-ring from the cytoplasm, along the perpendicular to the membrane. The sodium ion and the water molecule bound to each of the 11 ion-binding sites (purple and red spheres, respectively), are shown by F obs -F calc omit electron-density maps at 3.2σ (blue meshes).

    Journal: PLoS Biology

    Article Title: A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

    doi: 10.1371/journal.pbio.1001596

    Figure Lengend Snippet: Crystal structure of the c 11 ring of the F. nucleatum ATP synthase at 2.2 Å resolution. The pH of crystallization buffer was 5.3, and included 100 mM Na + . (A) Side view of the c-ring along the membrane plane. The c-subunits are displayed in different colors in ribbon representation. The transmembrane region, 35 Å in width, is indicated with grey bars. (B) View of the c-ring from the cytoplasm, along the perpendicular to the membrane. The sodium ion and the water molecule bound to each of the 11 ion-binding sites (purple and red spheres, respectively), are shown by F obs -F calc omit electron-density maps at 3.2σ (blue meshes).

    Article Snippet: Figure S5 Effect of ATP synthase inhibitors on the growth of F. nucleatum ATCC 25586 in batch culture. (A) DCCD was added to a final concentration of 100 µM (○), 200 µM (□), and 400 µM (Δ). (B) Tributyltin chloride (TBT-Cl) was added to a final concentration of 25 µM (○), 50 µM (□), and 150 µM (Δ).

    Techniques: Crystallization Assay, Binding Assay