f nucleatum strain atcc 23726  (ATCC)


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    ATCC f nucleatum strain atcc 23726
    F Nucleatum Strain Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    f nucleatum strain atcc 23726  (ATCC)


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    ATCC f nucleatum strain atcc 23726
    Role of <t>Fusobacterium</t> <t>nucleatum</t> (F. nucleatum) in adverse pregnancy outcomes (APO). ( A ) Interaction between Fusobacterium adhesion A (FadA), with vascular endothelial cadherin (VE-Cadherin) to internalize F. nucleatum in endothelial cells for bacterial dissemination, leading to increased inflammatory cytokines causing chorioamnionitis and preterm birth. ( B ) Fusobacterium apoptosis-inducing protein 2 (Fap2) binds with D-galactose-β (1–3)-N-acetyl-D-galactosamine (Gal-GalNAc) on endothelial cells to localize into placenta by suppressing TIGIT mediated activation of T cells and natural killer (NK) cells. ( C ) Lipopolysaccharide (LPS) interacts with TLR 4 on endothelial cells to activate the NF-κB pathway, leading to an inflammatory cytokine storm causing placental inflammation.
    F Nucleatum Strain Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Fusobacterium nucleatum : An Overview of Evidence, Demi-Decadal Trends, and Its Role in Adverse Pregnancy Outcomes and Various Gynecological Diseases, including Cancers"

    Article Title: Fusobacterium nucleatum : An Overview of Evidence, Demi-Decadal Trends, and Its Role in Adverse Pregnancy Outcomes and Various Gynecological Diseases, including Cancers

    Journal: Cells

    doi: 10.3390/cells13080717

    Role of Fusobacterium nucleatum (F. nucleatum) in adverse pregnancy outcomes (APO). ( A ) Interaction between Fusobacterium adhesion A (FadA), with vascular endothelial cadherin (VE-Cadherin) to internalize F. nucleatum in endothelial cells for bacterial dissemination, leading to increased inflammatory cytokines causing chorioamnionitis and preterm birth. ( B ) Fusobacterium apoptosis-inducing protein 2 (Fap2) binds with D-galactose-β (1–3)-N-acetyl-D-galactosamine (Gal-GalNAc) on endothelial cells to localize into placenta by suppressing TIGIT mediated activation of T cells and natural killer (NK) cells. ( C ) Lipopolysaccharide (LPS) interacts with TLR 4 on endothelial cells to activate the NF-κB pathway, leading to an inflammatory cytokine storm causing placental inflammation.
    Figure Legend Snippet: Role of Fusobacterium nucleatum (F. nucleatum) in adverse pregnancy outcomes (APO). ( A ) Interaction between Fusobacterium adhesion A (FadA), with vascular endothelial cadherin (VE-Cadherin) to internalize F. nucleatum in endothelial cells for bacterial dissemination, leading to increased inflammatory cytokines causing chorioamnionitis and preterm birth. ( B ) Fusobacterium apoptosis-inducing protein 2 (Fap2) binds with D-galactose-β (1–3)-N-acetyl-D-galactosamine (Gal-GalNAc) on endothelial cells to localize into placenta by suppressing TIGIT mediated activation of T cells and natural killer (NK) cells. ( C ) Lipopolysaccharide (LPS) interacts with TLR 4 on endothelial cells to activate the NF-κB pathway, leading to an inflammatory cytokine storm causing placental inflammation.

    Techniques Used: Activation Assay

    Role of Fusobacterium nucleatum (F. nucleatum) in gynecological diseases (GD). ( A ) F. nucleatum increases TAGLN expression in endometrial fibroblasts to convert them into endometriosis lesion-forming myofibroblast cells. ( B ) Schematic representation displaying the interdependent beneficial relationship between F. nucleatum and vaginal bacteria, ultimately leading to bacterial vaginosis.
    Figure Legend Snippet: Role of Fusobacterium nucleatum (F. nucleatum) in gynecological diseases (GD). ( A ) F. nucleatum increases TAGLN expression in endometrial fibroblasts to convert them into endometriosis lesion-forming myofibroblast cells. ( B ) Schematic representation displaying the interdependent beneficial relationship between F. nucleatum and vaginal bacteria, ultimately leading to bacterial vaginosis.

    Techniques Used: Expressing, Bacteria

    Role of Fusobacterium nucleatum ( F. nucleatum ) in gynecological cancers (GC). ( A ) Fusobacterium apoptosis-inducing protein 2 (Fap2) binds with D-galactose-β (1–3)-N-acetyl-D-galactosamine (Gal-GalNAc) on cancer cells to induce metastasis from primary tumor sites to other organs. ( B ) Lipopolysaccharide (LPS) on F. nucleatum activated TLR4/MyD88 pathway to induce chemoresistance in cancer cells. ( C ) Interaction between Fusobacterium adhesion A (FadA), with epithelial cadherin (E Cadherin) on cancer cells to increase b-catenin expression and induce cancer cell proliferation. ( D ) Reactive oxygen species (ROS) and extracellular vesicles secreted from F. nucleatum induce DNA damage in cancer cells to acquire further mutation and genomic instability.
    Figure Legend Snippet: Role of Fusobacterium nucleatum ( F. nucleatum ) in gynecological cancers (GC). ( A ) Fusobacterium apoptosis-inducing protein 2 (Fap2) binds with D-galactose-β (1–3)-N-acetyl-D-galactosamine (Gal-GalNAc) on cancer cells to induce metastasis from primary tumor sites to other organs. ( B ) Lipopolysaccharide (LPS) on F. nucleatum activated TLR4/MyD88 pathway to induce chemoresistance in cancer cells. ( C ) Interaction between Fusobacterium adhesion A (FadA), with epithelial cadherin (E Cadherin) on cancer cells to increase b-catenin expression and induce cancer cell proliferation. ( D ) Reactive oxygen species (ROS) and extracellular vesicles secreted from F. nucleatum induce DNA damage in cancer cells to acquire further mutation and genomic instability.

    Techniques Used: Expressing, Mutagenesis

    f nucleatum subsp nucleatum strains atcc 23726  (ATCC)


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    ATCC f nucleatum subsp nucleatum strains atcc 23726
    F Nucleatum Subsp Nucleatum Strains Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    f nucleatum subsp nucleatum strains atcc 23726  (ATCC)


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    ATCC f nucleatum subsp nucleatum strains atcc 23726
    F Nucleatum Subsp Nucleatum Strains Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    f nucleatum strain atcc 23726  (ATCC)


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    ATCC f nucleatum strain atcc 23726
    Construction and characterization of CRISPRi system in Fusobacterium nucleatum. (A) Mechanism of CRISPRi. The nuclease deficient (dCas9) binds single-guide RNA (sgRNA) to target the gene of interest via a 20-nt base-paring region. Instead of cleaving DNA, dCas9 obstructs RNA polymerase (RNAP) movement and inhibits transcription elongation, suppressing the target gene’s expression. (B) pZP4C construction blueprint for CRISPRi. pZP4C is derived from the E. coli-F. nucleatum shuttle vector pCWU6 and is aimed at ftsX. pCWU6 underwent SacI/HindIII digestion and was conjoined with two PCR fragments (Pfdx-E-dcas9 and PrpsJFP-sgRNA) through Gibson assembly, resulting in pZP4C. Within pZP4C, the dCas9’s expression is governed by Pfdx and a theophylline-responsive riboswitch E (indicated “E”), both at the transcriptional and translation stages. The sgRNA is driven by a constitutive active PrpsJ promoter from Fusobacterium periodonticum and can be replaced using the MscI and NotI restriction sites. Key features include using Pfdx-E from pREcas1 and the dCas9 and sgRNA element from pIA33, replication regions (oriFn, repA, and oriEc), and the catP, the chloramphenicol acetyltransferase gene. (C) ftsX gene suppression via CRISPRi. The wild-type F. nucleatum strain with pZP4C was cultivated in a TSPC medium under varying theophylline concentrations (0, 0.1, and 2 mM) for 22 hours. Induction of dcas9 expression by theophylline results in cells setting at the tube’s base after overnight growth, leaving a transparent supernatant characteristic similar to the ftsX deletion phenotype. (D) Western blot analysis validates ftsX silencing. Cell samples, including a wild-type control without inducer, were run on SDS-PAGE and immunoblotted. Antibodies for FtsX (α-FtsX) and FomA (α-FomA) were used, with FomA as the loading control. Molecular mass markers (in kilodaltons) are labeled on the left of the blot. (E) Silencing FtsX alters fusobacterial cell morphology. A phase-contrast microscopy recorded cell shapes in panel B. Bars, 2 µm.
    F Nucleatum Strain Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum"

    Article Title: Use of CRISPR interference for efficient and rapid gene inactivation in Fusobacterium nucleatum

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/aem.01665-23

    Construction and characterization of CRISPRi system in Fusobacterium nucleatum. (A) Mechanism of CRISPRi. The nuclease deficient (dCas9) binds single-guide RNA (sgRNA) to target the gene of interest via a 20-nt base-paring region. Instead of cleaving DNA, dCas9 obstructs RNA polymerase (RNAP) movement and inhibits transcription elongation, suppressing the target gene’s expression. (B) pZP4C construction blueprint for CRISPRi. pZP4C is derived from the E. coli-F. nucleatum shuttle vector pCWU6 and is aimed at ftsX. pCWU6 underwent SacI/HindIII digestion and was conjoined with two PCR fragments (Pfdx-E-dcas9 and PrpsJFP-sgRNA) through Gibson assembly, resulting in pZP4C. Within pZP4C, the dCas9’s expression is governed by Pfdx and a theophylline-responsive riboswitch E (indicated “E”), both at the transcriptional and translation stages. The sgRNA is driven by a constitutive active PrpsJ promoter from Fusobacterium periodonticum and can be replaced using the MscI and NotI restriction sites. Key features include using Pfdx-E from pREcas1 and the dCas9 and sgRNA element from pIA33, replication regions (oriFn, repA, and oriEc), and the catP, the chloramphenicol acetyltransferase gene. (C) ftsX gene suppression via CRISPRi. The wild-type F. nucleatum strain with pZP4C was cultivated in a TSPC medium under varying theophylline concentrations (0, 0.1, and 2 mM) for 22 hours. Induction of dcas9 expression by theophylline results in cells setting at the tube’s base after overnight growth, leaving a transparent supernatant characteristic similar to the ftsX deletion phenotype. (D) Western blot analysis validates ftsX silencing. Cell samples, including a wild-type control without inducer, were run on SDS-PAGE and immunoblotted. Antibodies for FtsX (α-FtsX) and FomA (α-FomA) were used, with FomA as the loading control. Molecular mass markers (in kilodaltons) are labeled on the left of the blot. (E) Silencing FtsX alters fusobacterial cell morphology. A phase-contrast microscopy recorded cell shapes in panel B. Bars, 2 µm.
    Figure Legend Snippet: Construction and characterization of CRISPRi system in Fusobacterium nucleatum. (A) Mechanism of CRISPRi. The nuclease deficient (dCas9) binds single-guide RNA (sgRNA) to target the gene of interest via a 20-nt base-paring region. Instead of cleaving DNA, dCas9 obstructs RNA polymerase (RNAP) movement and inhibits transcription elongation, suppressing the target gene’s expression. (B) pZP4C construction blueprint for CRISPRi. pZP4C is derived from the E. coli-F. nucleatum shuttle vector pCWU6 and is aimed at ftsX. pCWU6 underwent SacI/HindIII digestion and was conjoined with two PCR fragments (Pfdx-E-dcas9 and PrpsJFP-sgRNA) through Gibson assembly, resulting in pZP4C. Within pZP4C, the dCas9’s expression is governed by Pfdx and a theophylline-responsive riboswitch E (indicated “E”), both at the transcriptional and translation stages. The sgRNA is driven by a constitutive active PrpsJ promoter from Fusobacterium periodonticum and can be replaced using the MscI and NotI restriction sites. Key features include using Pfdx-E from pREcas1 and the dCas9 and sgRNA element from pIA33, replication regions (oriFn, repA, and oriEc), and the catP, the chloramphenicol acetyltransferase gene. (C) ftsX gene suppression via CRISPRi. The wild-type F. nucleatum strain with pZP4C was cultivated in a TSPC medium under varying theophylline concentrations (0, 0.1, and 2 mM) for 22 hours. Induction of dcas9 expression by theophylline results in cells setting at the tube’s base after overnight growth, leaving a transparent supernatant characteristic similar to the ftsX deletion phenotype. (D) Western blot analysis validates ftsX silencing. Cell samples, including a wild-type control without inducer, were run on SDS-PAGE and immunoblotted. Antibodies for FtsX (α-FtsX) and FomA (α-FomA) were used, with FomA as the loading control. Molecular mass markers (in kilodaltons) are labeled on the left of the blot. (E) Silencing FtsX alters fusobacterial cell morphology. A phase-contrast microscopy recorded cell shapes in panel B. Bars, 2 µm.

    Techniques Used: Expressing, Derivative Assay, Plasmid Preparation, Western Blot, SDS Page, Labeling, Microscopy

    f nucleatum strain atcc 23726 genome  (ATCC)


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    ATCC f nucleatum strain atcc 23726 genome
    F Nucleatum Strain Atcc 23726 Genome, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    f nucleatum strain atcc 23726  (ATCC)


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    ATCC f nucleatum strain atcc 23726
    F Nucleatum Strain Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    f nucleatum strains atcc 23726  (ATCC)


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    ATCC f nucleatum strains atcc 23726
    (A) Schematic of Type 4 pili proteins identified in F. nucleatum and their proposed assembly based on the characterization of Type 4 pili in other bacteria. * Stars on proteins in the schematic indicate that Fusobacterium do not contain these genes. (B) Identification of Type 4 pilus genes in seven species of Fusobacterium that covers 8 strains. (C) Chromosomal location of pilus genes in the strain F. nucleatum subsp. nucleatum 23736. (D) Analysis of the pilQ genes in F. nucleatum 23726 and F. nucleatum 25586, showing a frame shift creating an inactive pilus protein in F. nucleatum 25586. Fu: Fusobacterium ulcerans; Fv: Fusobacterium varium; Fm: Fusobacterium mortiferum; Fn: Fusobacterium nucleatum; Fp: Fusobacterium periodonticum; Fne: Fusobacterium necrophorum; Fg: Fusobacterium gonidiaformans .
    F Nucleatum Strains Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Type 4 pili mediated natural competence in Fusobacterium nucleatum"

    Article Title: Type 4 pili mediated natural competence in Fusobacterium nucleatum

    Journal: bioRxiv

    doi: 10.1101/2023.06.09.544391

    (A) Schematic of Type 4 pili proteins identified in F. nucleatum and their proposed assembly based on the characterization of Type 4 pili in other bacteria. * Stars on proteins in the schematic indicate that Fusobacterium do not contain these genes. (B) Identification of Type 4 pilus genes in seven species of Fusobacterium that covers 8 strains. (C) Chromosomal location of pilus genes in the strain F. nucleatum subsp. nucleatum 23736. (D) Analysis of the pilQ genes in F. nucleatum 23726 and F. nucleatum 25586, showing a frame shift creating an inactive pilus protein in F. nucleatum 25586. Fu: Fusobacterium ulcerans; Fv: Fusobacterium varium; Fm: Fusobacterium mortiferum; Fn: Fusobacterium nucleatum; Fp: Fusobacterium periodonticum; Fne: Fusobacterium necrophorum; Fg: Fusobacterium gonidiaformans .
    Figure Legend Snippet: (A) Schematic of Type 4 pili proteins identified in F. nucleatum and their proposed assembly based on the characterization of Type 4 pili in other bacteria. * Stars on proteins in the schematic indicate that Fusobacterium do not contain these genes. (B) Identification of Type 4 pilus genes in seven species of Fusobacterium that covers 8 strains. (C) Chromosomal location of pilus genes in the strain F. nucleatum subsp. nucleatum 23736. (D) Analysis of the pilQ genes in F. nucleatum 23726 and F. nucleatum 25586, showing a frame shift creating an inactive pilus protein in F. nucleatum 25586. Fu: Fusobacterium ulcerans; Fv: Fusobacterium varium; Fm: Fusobacterium mortiferum; Fn: Fusobacterium nucleatum; Fp: Fusobacterium periodonticum; Fne: Fusobacterium necrophorum; Fg: Fusobacterium gonidiaformans .

    Techniques Used:

    f nucleatum atcc 23726 strain  (ATCC)


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    ATCC f nucleatum atcc 23726 strain
    F Nucleatum Atcc 23726 Strain, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    f nucleatum strain atcc 23726 genome  (ATCC)


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    ATCC f nucleatum strain atcc 23726 genome
    (A) Schematic diagram of assembling and working mode of the xylose-inducible system developed in this study to regulate reporter gene expression. This system was constructed by combining xylose repressor gene xylR , its operator sequence xylO , and the strong constitutive promoter P xylAB , the divergent promoter to drive the expression of Luciola Red luciferase ( luc ) gene reporter. The XylR is expressed constitutively from P xylR . It binds to the XylR operator (schematically depicted as a black) and blocks luc expression from the P xylAB promoter in the absence of xylose. XylR is released from the operator with xylose, initiating the luc expression. (B) The intergenic promoter region of the divergently transcribed xylR and luc in this xylose-inducible system. The translation initiation codon and the ribosomal bind site (RBS) are in bold red. The -10 and -35 sequences are shown as underlined sequences. The xylO element is highlighted in the box with dashed lines. Palindromic sequences in xylO are indicated by arrows. (C) Schematic diagram of assembling and working mode of the xylose and riboswitch-based dual inducible system developed in this study. The dual control system contains xylR , xylO , P xylAB, and a theophylline-responsive synthetical riboswitch E unit. The riboswitch is inserted to replace the RBS in the xylose-inducible system (A) . The reporter luc gene expression is initially repressed by XylR and is only activated in the presence of xylose. In the latter case, the luc gene is transcribed, but the mRNA is not translated in the absence of the riboswitch signal theophylline (RBS is hidden in a step-loop structure in riboswitch). In the presence of theophylline, the riboswitch changes its conformation and exposes the RBS so the ribosome can bind, resulting in synthesizing the luciferase protein. (D) The intergenic promoter region of the divergently transcribed xylR and luc in this dual inducible system. The linker region (underlined) and riboswitch E sequence were positioned as indicated in Figure (C). (E) Genetic maps of the xylose and riboswitch-based dual inducible expression reporter plasmid pBCG06 and the xylose-inducible alone system expression plasmid pBCG06a. Both plasmids are derivatives of E. coli-F. nucleatum shuttle vector pCWU6. Feature depicted: The xylR-PxylAB is from the vector pXCH, a xylose-inducible gene expression vector for Clostridium perfringens ; luc encodes a Luciola Red luciferase protein that is codon optimized for expression in low-GC bacteria. oriFn and repA , the replication region; oriEc , replication region of the E. coli plasmid pBR322; catP , the chloramphenicol acetyltransferase gene, conferring resistance to thiamphenicol in F. nucleatum or chloramphenicol in E. coli ; KanR is kanamycin resistance cassette from pJRD215. (F) Tunable induction of luciferase protein from P xylAB and the riboswitch control with different inducer(s) concentrations. The overnight cultures of F. nucleatum strain <t>ATCC</t> <t>23726</t> carrying pCWU6 (empty vector, EV), pBCG06, or pBCG06a were diluted 1:20 into TSPC. When cultures grew to OD600 ∼0.65, the inducers were added with various concentrations, as indicated. A tested culture received 20 mM glucose to assess its impact on the inducible system. After 2 hours of further incubation, 100 µl aliquots of each culture were taken out for luciferase assay. Luciferase activity (RLU) was normalized with cell density (OD 600 ). Each culture’s mean RLU/OD 600 was indicated above the corresponding column. The inserted graph showed the OD 600 value of the cultures when the luciferase assay was performed. Data represent the means of standard deviations of the results of triplicate biological experiments.
    F Nucleatum Strain Atcc 23726 Genome, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Development of a Xylose-Inducible Promoter and Riboswitch Combination System for Manipulating Gene Expression in Fusobacterium nucleatum"

    Article Title: Development of a Xylose-Inducible Promoter and Riboswitch Combination System for Manipulating Gene Expression in Fusobacterium nucleatum

    Journal: bioRxiv

    doi: 10.1101/2023.04.24.538132

    (A) Schematic diagram of assembling and working mode of the xylose-inducible system developed in this study to regulate reporter gene expression. This system was constructed by combining xylose repressor gene xylR , its operator sequence xylO , and the strong constitutive promoter P xylAB , the divergent promoter to drive the expression of Luciola Red luciferase ( luc ) gene reporter. The XylR is expressed constitutively from P xylR . It binds to the XylR operator (schematically depicted as a black) and blocks luc expression from the P xylAB promoter in the absence of xylose. XylR is released from the operator with xylose, initiating the luc expression. (B) The intergenic promoter region of the divergently transcribed xylR and luc in this xylose-inducible system. The translation initiation codon and the ribosomal bind site (RBS) are in bold red. The -10 and -35 sequences are shown as underlined sequences. The xylO element is highlighted in the box with dashed lines. Palindromic sequences in xylO are indicated by arrows. (C) Schematic diagram of assembling and working mode of the xylose and riboswitch-based dual inducible system developed in this study. The dual control system contains xylR , xylO , P xylAB, and a theophylline-responsive synthetical riboswitch E unit. The riboswitch is inserted to replace the RBS in the xylose-inducible system (A) . The reporter luc gene expression is initially repressed by XylR and is only activated in the presence of xylose. In the latter case, the luc gene is transcribed, but the mRNA is not translated in the absence of the riboswitch signal theophylline (RBS is hidden in a step-loop structure in riboswitch). In the presence of theophylline, the riboswitch changes its conformation and exposes the RBS so the ribosome can bind, resulting in synthesizing the luciferase protein. (D) The intergenic promoter region of the divergently transcribed xylR and luc in this dual inducible system. The linker region (underlined) and riboswitch E sequence were positioned as indicated in Figure (C). (E) Genetic maps of the xylose and riboswitch-based dual inducible expression reporter plasmid pBCG06 and the xylose-inducible alone system expression plasmid pBCG06a. Both plasmids are derivatives of E. coli-F. nucleatum shuttle vector pCWU6. Feature depicted: The xylR-PxylAB is from the vector pXCH, a xylose-inducible gene expression vector for Clostridium perfringens ; luc encodes a Luciola Red luciferase protein that is codon optimized for expression in low-GC bacteria. oriFn and repA , the replication region; oriEc , replication region of the E. coli plasmid pBR322; catP , the chloramphenicol acetyltransferase gene, conferring resistance to thiamphenicol in F. nucleatum or chloramphenicol in E. coli ; KanR is kanamycin resistance cassette from pJRD215. (F) Tunable induction of luciferase protein from P xylAB and the riboswitch control with different inducer(s) concentrations. The overnight cultures of F. nucleatum strain ATCC 23726 carrying pCWU6 (empty vector, EV), pBCG06, or pBCG06a were diluted 1:20 into TSPC. When cultures grew to OD600 ∼0.65, the inducers were added with various concentrations, as indicated. A tested culture received 20 mM glucose to assess its impact on the inducible system. After 2 hours of further incubation, 100 µl aliquots of each culture were taken out for luciferase assay. Luciferase activity (RLU) was normalized with cell density (OD 600 ). Each culture’s mean RLU/OD 600 was indicated above the corresponding column. The inserted graph showed the OD 600 value of the cultures when the luciferase assay was performed. Data represent the means of standard deviations of the results of triplicate biological experiments.
    Figure Legend Snippet: (A) Schematic diagram of assembling and working mode of the xylose-inducible system developed in this study to regulate reporter gene expression. This system was constructed by combining xylose repressor gene xylR , its operator sequence xylO , and the strong constitutive promoter P xylAB , the divergent promoter to drive the expression of Luciola Red luciferase ( luc ) gene reporter. The XylR is expressed constitutively from P xylR . It binds to the XylR operator (schematically depicted as a black) and blocks luc expression from the P xylAB promoter in the absence of xylose. XylR is released from the operator with xylose, initiating the luc expression. (B) The intergenic promoter region of the divergently transcribed xylR and luc in this xylose-inducible system. The translation initiation codon and the ribosomal bind site (RBS) are in bold red. The -10 and -35 sequences are shown as underlined sequences. The xylO element is highlighted in the box with dashed lines. Palindromic sequences in xylO are indicated by arrows. (C) Schematic diagram of assembling and working mode of the xylose and riboswitch-based dual inducible system developed in this study. The dual control system contains xylR , xylO , P xylAB, and a theophylline-responsive synthetical riboswitch E unit. The riboswitch is inserted to replace the RBS in the xylose-inducible system (A) . The reporter luc gene expression is initially repressed by XylR and is only activated in the presence of xylose. In the latter case, the luc gene is transcribed, but the mRNA is not translated in the absence of the riboswitch signal theophylline (RBS is hidden in a step-loop structure in riboswitch). In the presence of theophylline, the riboswitch changes its conformation and exposes the RBS so the ribosome can bind, resulting in synthesizing the luciferase protein. (D) The intergenic promoter region of the divergently transcribed xylR and luc in this dual inducible system. The linker region (underlined) and riboswitch E sequence were positioned as indicated in Figure (C). (E) Genetic maps of the xylose and riboswitch-based dual inducible expression reporter plasmid pBCG06 and the xylose-inducible alone system expression plasmid pBCG06a. Both plasmids are derivatives of E. coli-F. nucleatum shuttle vector pCWU6. Feature depicted: The xylR-PxylAB is from the vector pXCH, a xylose-inducible gene expression vector for Clostridium perfringens ; luc encodes a Luciola Red luciferase protein that is codon optimized for expression in low-GC bacteria. oriFn and repA , the replication region; oriEc , replication region of the E. coli plasmid pBR322; catP , the chloramphenicol acetyltransferase gene, conferring resistance to thiamphenicol in F. nucleatum or chloramphenicol in E. coli ; KanR is kanamycin resistance cassette from pJRD215. (F) Tunable induction of luciferase protein from P xylAB and the riboswitch control with different inducer(s) concentrations. The overnight cultures of F. nucleatum strain ATCC 23726 carrying pCWU6 (empty vector, EV), pBCG06, or pBCG06a were diluted 1:20 into TSPC. When cultures grew to OD600 ∼0.65, the inducers were added with various concentrations, as indicated. A tested culture received 20 mM glucose to assess its impact on the inducible system. After 2 hours of further incubation, 100 µl aliquots of each culture were taken out for luciferase assay. Luciferase activity (RLU) was normalized with cell density (OD 600 ). Each culture’s mean RLU/OD 600 was indicated above the corresponding column. The inserted graph showed the OD 600 value of the cultures when the luciferase assay was performed. Data represent the means of standard deviations of the results of triplicate biological experiments.

    Techniques Used: Expressing, Construct, Sequencing, Luciferase, Plasmid Preparation, Incubation, Activity Assay

    (A) The effect of inducer(s) on the growth of conditional lepB mutants in the liquid media. F. nucleatum strains (wild-type: strain ATCC 23726 harboring an empty vector pCWU6 (red triangle); lepB conditional mutant: Δ lepB pAN01) were grown statically under anaerobic conditions on TSPC media with various doses of inducers (xylose and theophylline). Turbidity was monitored at an optical density of 600 nm. Data represented the means of standard deviation of the results of triplicate biological experiments. (B) The effects of inducer(s) on the growth of conditional lepB mutants in solid media. Ten-fold serial dilutions of overnight cultures of the wild-type 23726 and the conditional Δ lepB strains were spotted on agar plates with and without inducer(s) xylose and theophylline and cultivated in an anaerobic chamber at 37°C. Cell growth was recorded after three days of incubation. This experiment was repeated three times, and one representative experiment was shown. (C) LepB depletion creates short filaments and alters outer membrane structures. The lepB-depleted and lepB-inducing cells were immobilized on carbon-coated nickel grids and stained with 0.1% uranyl acetate before viewing with a transmission electron microscope. Enlarged areas of the top panels are shown in the bottom panels. Bars, 1 µm. (D) The altered cell surface of conditional lepB mutant was revealed by scanning electron microscopy. The cells from lepB depletion or lepB induction were immobilized on a silicon wafer and fixed with 2.5% glutaraldehyde before viewing by a scanning electron microscope. Enlarge areas of the top panel are shown in the below panels. Bars, 1 µm. (E) Comparison of lepB conditional mutant strain umorphology. Cells were grown under two distinct conditions with inducers (0.1% xylose/0.2 mM theophylline and 0.5% xylose/1.0 mM theophylline). Microscopic examination was conducted after cells reached the stationary phase to assess morphological differences. (F) The quantitative coaggregation assay was performed by mixing wild-type F. nucleatum , radD , and lepB mutants with an equal number of A. oris cells in the coaggregation buffer (CAB). The mixture was allowed to aggregate for 20 mins before measuring the OD 600 absorbance. Measurements were taken both before and after the aggregation process. The presented data represent the average of three independent experiments. Statistical analysis was performed using Student’s t -test with GraphPad Prism software.
    Figure Legend Snippet: (A) The effect of inducer(s) on the growth of conditional lepB mutants in the liquid media. F. nucleatum strains (wild-type: strain ATCC 23726 harboring an empty vector pCWU6 (red triangle); lepB conditional mutant: Δ lepB pAN01) were grown statically under anaerobic conditions on TSPC media with various doses of inducers (xylose and theophylline). Turbidity was monitored at an optical density of 600 nm. Data represented the means of standard deviation of the results of triplicate biological experiments. (B) The effects of inducer(s) on the growth of conditional lepB mutants in solid media. Ten-fold serial dilutions of overnight cultures of the wild-type 23726 and the conditional Δ lepB strains were spotted on agar plates with and without inducer(s) xylose and theophylline and cultivated in an anaerobic chamber at 37°C. Cell growth was recorded after three days of incubation. This experiment was repeated three times, and one representative experiment was shown. (C) LepB depletion creates short filaments and alters outer membrane structures. The lepB-depleted and lepB-inducing cells were immobilized on carbon-coated nickel grids and stained with 0.1% uranyl acetate before viewing with a transmission electron microscope. Enlarged areas of the top panels are shown in the bottom panels. Bars, 1 µm. (D) The altered cell surface of conditional lepB mutant was revealed by scanning electron microscopy. The cells from lepB depletion or lepB induction were immobilized on a silicon wafer and fixed with 2.5% glutaraldehyde before viewing by a scanning electron microscope. Enlarge areas of the top panel are shown in the below panels. Bars, 1 µm. (E) Comparison of lepB conditional mutant strain umorphology. Cells were grown under two distinct conditions with inducers (0.1% xylose/0.2 mM theophylline and 0.5% xylose/1.0 mM theophylline). Microscopic examination was conducted after cells reached the stationary phase to assess morphological differences. (F) The quantitative coaggregation assay was performed by mixing wild-type F. nucleatum , radD , and lepB mutants with an equal number of A. oris cells in the coaggregation buffer (CAB). The mixture was allowed to aggregate for 20 mins before measuring the OD 600 absorbance. Measurements were taken both before and after the aggregation process. The presented data represent the average of three independent experiments. Statistical analysis was performed using Student’s t -test with GraphPad Prism software.

    Techniques Used: Plasmid Preparation, Mutagenesis, Standard Deviation, Incubation, Staining, Transmission Assay, Microscopy, Electron Microscopy, Software

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    ATCC f nucleatum strain atcc 23726
    F Nucleatum Strain Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC f nucleatum subsp nucleatum strains atcc 23726
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    ATCC f nucleatum strain atcc 23726 genome
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    ATCC f nucleatum strains atcc 23726
    (A) Schematic of Type 4 pili proteins identified in F. nucleatum and their proposed assembly based on the characterization of Type 4 pili in other bacteria. * Stars on proteins in the schematic indicate that Fusobacterium do not contain these genes. (B) Identification of Type 4 pilus genes in seven species of Fusobacterium that covers 8 strains. (C) Chromosomal location of pilus genes in the strain F. nucleatum subsp. nucleatum 23736. (D) Analysis of the pilQ genes in F. nucleatum 23726 and F. nucleatum 25586, showing a frame shift creating an inactive pilus protein in F. nucleatum 25586. Fu: Fusobacterium ulcerans; Fv: Fusobacterium varium; Fm: Fusobacterium mortiferum; Fn: Fusobacterium nucleatum; Fp: Fusobacterium periodonticum; Fne: Fusobacterium necrophorum; Fg: Fusobacterium gonidiaformans .
    F Nucleatum Strains Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC f nucleatum atcc 23726 strain
    (A) Schematic of Type 4 pili proteins identified in F. nucleatum and their proposed assembly based on the characterization of Type 4 pili in other bacteria. * Stars on proteins in the schematic indicate that Fusobacterium do not contain these genes. (B) Identification of Type 4 pilus genes in seven species of Fusobacterium that covers 8 strains. (C) Chromosomal location of pilus genes in the strain F. nucleatum subsp. nucleatum 23736. (D) Analysis of the pilQ genes in F. nucleatum 23726 and F. nucleatum 25586, showing a frame shift creating an inactive pilus protein in F. nucleatum 25586. Fu: Fusobacterium ulcerans; Fv: Fusobacterium varium; Fm: Fusobacterium mortiferum; Fn: Fusobacterium nucleatum; Fp: Fusobacterium periodonticum; Fne: Fusobacterium necrophorum; Fg: Fusobacterium gonidiaformans .
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    (A) Schematic of Type 4 pili proteins identified in F. nucleatum and their proposed assembly based on the characterization of Type 4 pili in other bacteria. * Stars on proteins in the schematic indicate that Fusobacterium do not contain these genes. (B) Identification of Type 4 pilus genes in seven species of Fusobacterium that covers 8 strains. (C) Chromosomal location of pilus genes in the strain F. nucleatum subsp. nucleatum 23736. (D) Analysis of the pilQ genes in F. nucleatum 23726 and F. nucleatum 25586, showing a frame shift creating an inactive pilus protein in F. nucleatum 25586. Fu: Fusobacterium ulcerans; Fv: Fusobacterium varium; Fm: Fusobacterium mortiferum; Fn: Fusobacterium nucleatum; Fp: Fusobacterium periodonticum; Fne: Fusobacterium necrophorum; Fg: Fusobacterium gonidiaformans .

    Journal: bioRxiv

    Article Title: Type 4 pili mediated natural competence in Fusobacterium nucleatum

    doi: 10.1101/2023.06.09.544391

    Figure Lengend Snippet: (A) Schematic of Type 4 pili proteins identified in F. nucleatum and their proposed assembly based on the characterization of Type 4 pili in other bacteria. * Stars on proteins in the schematic indicate that Fusobacterium do not contain these genes. (B) Identification of Type 4 pilus genes in seven species of Fusobacterium that covers 8 strains. (C) Chromosomal location of pilus genes in the strain F. nucleatum subsp. nucleatum 23736. (D) Analysis of the pilQ genes in F. nucleatum 23726 and F. nucleatum 25586, showing a frame shift creating an inactive pilus protein in F. nucleatum 25586. Fu: Fusobacterium ulcerans; Fv: Fusobacterium varium; Fm: Fusobacterium mortiferum; Fn: Fusobacterium nucleatum; Fp: Fusobacterium periodonticum; Fne: Fusobacterium necrophorum; Fg: Fusobacterium gonidiaformans .

    Article Snippet: F. nucleatum strains ATCC 23726 and 25586 were cultured on solid agar plates made with Columbia Broth (Gibco) substituted with hemin (5 μg/mL) and menadione (0.5 μg/mL) (CBHK) under anaerobic conditions (90% N 2 , 5% H 2 , 5% CO 2 ) at 37 °C.

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