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: 99/100, 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 .

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    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

    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: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    f nucleatum wild type strain atcc 23726  (ATCC)


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    ATCC f nucleatum wild type strain atcc 23726
    The qualitative and quantitative coaggregation assay for E. faecalis ( Ef ) and F. nucleatum ( Fn ) . (a) Coaggregation assay to screen eight different F. nucleatum ATCC23726 outer-membrane protein mutants for defective coaggregation phenotype with E. faecalis OG1RF. The arrow points to the fap2 mutant with binding deficiency. (b) Coaggregation assay to screen sugars and amino acids for their inhibitory efforts on interaction between E. faecalis and F. nucleatum . The arrow points to galactose with inhibitory effort. (c) Microscopic observation of coaggregation between E. faecalis and F. nucleatum under different conditions: a. E. faecalis alone; b. F. nucleatum alone; c. E. faecalis and F. nucleatum ; d. E. faecalis and F. nucleatum fap2 . For each interacting pair, the pictures of 10 random views were taken and 1 representative image was shown. 100X magnification. The scale bar is 10 µm. (d) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between <t>wild</t> <t>type</t> E. faecalis ( Ef WT)/ E. faecalis clinical isolate ( Ef DX1) and wild type F. nucleatum ( Fn WT) and its outer-membrane protein mutants. (e) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between E. faecalis and F. nucleatum in the presence of selected sugars and amino acids, which are D-galactose (Gal), D-Mannose (Man), L-arginine (Arg), L-leucine (Leu) and L-glutamic acid (Glu). All co-aggregation assays have 120 min incubation time. The data presented are based on experiments conducted in triplicate.
    F Nucleatum Wild Type Strain Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Antagonistic interaction between two key endodontic pathogens Enterococcus faecalis and Fusobacterium nucleatum"

    Article Title: Antagonistic interaction between two key endodontic pathogens Enterococcus faecalis and Fusobacterium nucleatum

    Journal: Journal of Oral Microbiology

    doi: 10.1080/20002297.2022.2149448

    The qualitative and quantitative coaggregation assay for E. faecalis ( Ef ) and F. nucleatum ( Fn ) . (a) Coaggregation assay to screen eight different F. nucleatum ATCC23726 outer-membrane protein mutants for defective coaggregation phenotype with E. faecalis OG1RF. The arrow points to the fap2 mutant with binding deficiency. (b) Coaggregation assay to screen sugars and amino acids for their inhibitory efforts on interaction between E. faecalis and F. nucleatum . The arrow points to galactose with inhibitory effort. (c) Microscopic observation of coaggregation between E. faecalis and F. nucleatum under different conditions: a. E. faecalis alone; b. F. nucleatum alone; c. E. faecalis and F. nucleatum ; d. E. faecalis and F. nucleatum fap2 . For each interacting pair, the pictures of 10 random views were taken and 1 representative image was shown. 100X magnification. The scale bar is 10 µm. (d) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between wild type E. faecalis ( Ef WT)/ E. faecalis clinical isolate ( Ef DX1) and wild type F. nucleatum ( Fn WT) and its outer-membrane protein mutants. (e) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between E. faecalis and F. nucleatum in the presence of selected sugars and amino acids, which are D-galactose (Gal), D-Mannose (Man), L-arginine (Arg), L-leucine (Leu) and L-glutamic acid (Glu). All co-aggregation assays have 120 min incubation time. The data presented are based on experiments conducted in triplicate.
    Figure Legend Snippet: The qualitative and quantitative coaggregation assay for E. faecalis ( Ef ) and F. nucleatum ( Fn ) . (a) Coaggregation assay to screen eight different F. nucleatum ATCC23726 outer-membrane protein mutants for defective coaggregation phenotype with E. faecalis OG1RF. The arrow points to the fap2 mutant with binding deficiency. (b) Coaggregation assay to screen sugars and amino acids for their inhibitory efforts on interaction between E. faecalis and F. nucleatum . The arrow points to galactose with inhibitory effort. (c) Microscopic observation of coaggregation between E. faecalis and F. nucleatum under different conditions: a. E. faecalis alone; b. F. nucleatum alone; c. E. faecalis and F. nucleatum ; d. E. faecalis and F. nucleatum fap2 . For each interacting pair, the pictures of 10 random views were taken and 1 representative image was shown. 100X magnification. The scale bar is 10 µm. (d) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between wild type E. faecalis ( Ef WT)/ E. faecalis clinical isolate ( Ef DX1) and wild type F. nucleatum ( Fn WT) and its outer-membrane protein mutants. (e) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between E. faecalis and F. nucleatum in the presence of selected sugars and amino acids, which are D-galactose (Gal), D-Mannose (Man), L-arginine (Arg), L-leucine (Leu) and L-glutamic acid (Glu). All co-aggregation assays have 120 min incubation time. The data presented are based on experiments conducted in triplicate.

    Techniques Used: Mutagenesis, Binding Assay, Incubation

    The acidic environment generated by E. faecalis inhibits F. nucleatum growth . (a) pH challenging assay. F. nucleatum ATCC 23726 viability was tested under different pH conditions in Columbia broth. The data presented are based on experiments conducted in triplicates. WT, wild type. (b) Competition assay under buffered pH condition. Quantitative analysis of the competition effects between E. faecalis ( Ef ) and F. nucleatum ( Fn ) under buffered pH condition. Data are expressed as relative CFU (%) compared with the Fn alone CFU at each timepoint as 100%. Graph shows means and SD of readings from two individual experiments performed in triplicate. Data represent the means and standard deviation of at least three independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001. WT, wild type.
    Figure Legend Snippet: The acidic environment generated by E. faecalis inhibits F. nucleatum growth . (a) pH challenging assay. F. nucleatum ATCC 23726 viability was tested under different pH conditions in Columbia broth. The data presented are based on experiments conducted in triplicates. WT, wild type. (b) Competition assay under buffered pH condition. Quantitative analysis of the competition effects between E. faecalis ( Ef ) and F. nucleatum ( Fn ) under buffered pH condition. Data are expressed as relative CFU (%) compared with the Fn alone CFU at each timepoint as 100%. Graph shows means and SD of readings from two individual experiments performed in triplicate. Data represent the means and standard deviation of at least three independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001. WT, wild type.

    Techniques Used: Generated, Competitive Binding Assay, Standard Deviation

    strain f nucleatum atcc 23726 urogentical tract isolate  (ATCC)


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    ATCC strain f nucleatum atcc 23726 urogentical tract isolate
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    plasmid description reference strain f nucleatum atcc 23726 urogentical tract isolate  (ATCC)


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    ATCC plasmid description reference strain f nucleatum atcc 23726 urogentical tract isolate
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    ATCC f nucleatum atcc 23726 strain
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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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    ATCC f nucleatum strain atcc 23726 genome
    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 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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    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 wild type strain atcc 23726
    The qualitative and quantitative coaggregation assay for E. faecalis ( Ef ) and F. nucleatum ( Fn ) . (a) Coaggregation assay to screen eight different F. nucleatum ATCC23726 outer-membrane protein mutants for defective coaggregation phenotype with E. faecalis OG1RF. The arrow points to the fap2 mutant with binding deficiency. (b) Coaggregation assay to screen sugars and amino acids for their inhibitory efforts on interaction between E. faecalis and F. nucleatum . The arrow points to galactose with inhibitory effort. (c) Microscopic observation of coaggregation between E. faecalis and F. nucleatum under different conditions: a. E. faecalis alone; b. F. nucleatum alone; c. E. faecalis and F. nucleatum ; d. E. faecalis and F. nucleatum fap2 . For each interacting pair, the pictures of 10 random views were taken and 1 representative image was shown. 100X magnification. The scale bar is 10 µm. (d) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between <t>wild</t> <t>type</t> E. faecalis ( Ef WT)/ E. faecalis clinical isolate ( Ef DX1) and wild type F. nucleatum ( Fn WT) and its outer-membrane protein mutants. (e) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between E. faecalis and F. nucleatum in the presence of selected sugars and amino acids, which are D-galactose (Gal), D-Mannose (Man), L-arginine (Arg), L-leucine (Leu) and L-glutamic acid (Glu). All co-aggregation assays have 120 min incubation time. The data presented are based on experiments conducted in triplicate.
    F Nucleatum Wild Type Strain Atcc 23726, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC strain f nucleatum atcc 23726 urogentical tract isolate
    The qualitative and quantitative coaggregation assay for E. faecalis ( Ef ) and F. nucleatum ( Fn ) . (a) Coaggregation assay to screen eight different F. nucleatum ATCC23726 outer-membrane protein mutants for defective coaggregation phenotype with E. faecalis OG1RF. The arrow points to the fap2 mutant with binding deficiency. (b) Coaggregation assay to screen sugars and amino acids for their inhibitory efforts on interaction between E. faecalis and F. nucleatum . The arrow points to galactose with inhibitory effort. (c) Microscopic observation of coaggregation between E. faecalis and F. nucleatum under different conditions: a. E. faecalis alone; b. F. nucleatum alone; c. E. faecalis and F. nucleatum ; d. E. faecalis and F. nucleatum fap2 . For each interacting pair, the pictures of 10 random views were taken and 1 representative image was shown. 100X magnification. The scale bar is 10 µm. (d) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between <t>wild</t> <t>type</t> E. faecalis ( Ef WT)/ E. faecalis clinical isolate ( Ef DX1) and wild type F. nucleatum ( Fn WT) and its outer-membrane protein mutants. (e) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between E. faecalis and F. nucleatum in the presence of selected sugars and amino acids, which are D-galactose (Gal), D-Mannose (Man), L-arginine (Arg), L-leucine (Leu) and L-glutamic acid (Glu). All co-aggregation assays have 120 min incubation time. The data presented are based on experiments conducted in triplicate.
    Strain F Nucleatum Atcc 23726 Urogentical Tract Isolate, 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 plasmid description reference strain f nucleatum atcc 23726 urogentical tract isolate
    The qualitative and quantitative coaggregation assay for E. faecalis ( Ef ) and F. nucleatum ( Fn ) . (a) Coaggregation assay to screen eight different F. nucleatum ATCC23726 outer-membrane protein mutants for defective coaggregation phenotype with E. faecalis OG1RF. The arrow points to the fap2 mutant with binding deficiency. (b) Coaggregation assay to screen sugars and amino acids for their inhibitory efforts on interaction between E. faecalis and F. nucleatum . The arrow points to galactose with inhibitory effort. (c) Microscopic observation of coaggregation between E. faecalis and F. nucleatum under different conditions: a. E. faecalis alone; b. F. nucleatum alone; c. E. faecalis and F. nucleatum ; d. E. faecalis and F. nucleatum fap2 . For each interacting pair, the pictures of 10 random views were taken and 1 representative image was shown. 100X magnification. The scale bar is 10 µm. (d) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between <t>wild</t> <t>type</t> E. faecalis ( Ef WT)/ E. faecalis clinical isolate ( Ef DX1) and wild type F. nucleatum ( Fn WT) and its outer-membrane protein mutants. (e) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between E. faecalis and F. nucleatum in the presence of selected sugars and amino acids, which are D-galactose (Gal), D-Mannose (Man), L-arginine (Arg), L-leucine (Leu) and L-glutamic acid (Glu). All co-aggregation assays have 120 min incubation time. The data presented are based on experiments conducted in triplicate.
    Plasmid Description Reference Strain F Nucleatum Atcc 23726 Urogentical Tract Isolate, 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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    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.

    Journal: Applied and Environmental Microbiology

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

    doi: 10.1128/aem.01665-23

    Figure Lengend 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.

    Article Snippet: These set it apart from the F. nucleatum strain ATCC 23726, which is sourced from the human urogenital tract.

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

    (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.

    Techniques:

    The qualitative and quantitative coaggregation assay for E. faecalis ( Ef ) and F. nucleatum ( Fn ) . (a) Coaggregation assay to screen eight different F. nucleatum ATCC23726 outer-membrane protein mutants for defective coaggregation phenotype with E. faecalis OG1RF. The arrow points to the fap2 mutant with binding deficiency. (b) Coaggregation assay to screen sugars and amino acids for their inhibitory efforts on interaction between E. faecalis and F. nucleatum . The arrow points to galactose with inhibitory effort. (c) Microscopic observation of coaggregation between E. faecalis and F. nucleatum under different conditions: a. E. faecalis alone; b. F. nucleatum alone; c. E. faecalis and F. nucleatum ; d. E. faecalis and F. nucleatum fap2 . For each interacting pair, the pictures of 10 random views were taken and 1 representative image was shown. 100X magnification. The scale bar is 10 µm. (d) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between wild type E. faecalis ( Ef WT)/ E. faecalis clinical isolate ( Ef DX1) and wild type F. nucleatum ( Fn WT) and its outer-membrane protein mutants. (e) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between E. faecalis and F. nucleatum in the presence of selected sugars and amino acids, which are D-galactose (Gal), D-Mannose (Man), L-arginine (Arg), L-leucine (Leu) and L-glutamic acid (Glu). All co-aggregation assays have 120 min incubation time. The data presented are based on experiments conducted in triplicate.

    Journal: Journal of Oral Microbiology

    Article Title: Antagonistic interaction between two key endodontic pathogens Enterococcus faecalis and Fusobacterium nucleatum

    doi: 10.1080/20002297.2022.2149448

    Figure Lengend Snippet: The qualitative and quantitative coaggregation assay for E. faecalis ( Ef ) and F. nucleatum ( Fn ) . (a) Coaggregation assay to screen eight different F. nucleatum ATCC23726 outer-membrane protein mutants for defective coaggregation phenotype with E. faecalis OG1RF. The arrow points to the fap2 mutant with binding deficiency. (b) Coaggregation assay to screen sugars and amino acids for their inhibitory efforts on interaction between E. faecalis and F. nucleatum . The arrow points to galactose with inhibitory effort. (c) Microscopic observation of coaggregation between E. faecalis and F. nucleatum under different conditions: a. E. faecalis alone; b. F. nucleatum alone; c. E. faecalis and F. nucleatum ; d. E. faecalis and F. nucleatum fap2 . For each interacting pair, the pictures of 10 random views were taken and 1 representative image was shown. 100X magnification. The scale bar is 10 µm. (d) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between wild type E. faecalis ( Ef WT)/ E. faecalis clinical isolate ( Ef DX1) and wild type F. nucleatum ( Fn WT) and its outer-membrane protein mutants. (e) Spectrophotometric coaggregation assay to quantitatively measure coaggregation between E. faecalis and F. nucleatum in the presence of selected sugars and amino acids, which are D-galactose (Gal), D-Mannose (Man), L-arginine (Arg), L-leucine (Leu) and L-glutamic acid (Glu). All co-aggregation assays have 120 min incubation time. The data presented are based on experiments conducted in triplicate.

    Article Snippet: F. nucleatum wild type strain ATCC 23726 and its mutant derivatives defective in outer membrane autotransporter proteins, including Fn 1449( fap2 ), Fn 1526( radD ), Fn 2058( aim1 ), Fn 2047, Fn 0254, Fn 1554, Fn 1253( aid1 ), and Fn 1893 [ ], were maintained under anaerobic conditions (10% H 2 , 10% CO 2 , 80% N 2 ) at 37°C on either Columbia agar supplemented with 5% sheep blood or in Columbia Broth (CB; BD Difco, Detroit, MI, USA).

    Techniques: Mutagenesis, Binding Assay, Incubation

    The acidic environment generated by E. faecalis inhibits F. nucleatum growth . (a) pH challenging assay. F. nucleatum ATCC 23726 viability was tested under different pH conditions in Columbia broth. The data presented are based on experiments conducted in triplicates. WT, wild type. (b) Competition assay under buffered pH condition. Quantitative analysis of the competition effects between E. faecalis ( Ef ) and F. nucleatum ( Fn ) under buffered pH condition. Data are expressed as relative CFU (%) compared with the Fn alone CFU at each timepoint as 100%. Graph shows means and SD of readings from two individual experiments performed in triplicate. Data represent the means and standard deviation of at least three independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001. WT, wild type.

    Journal: Journal of Oral Microbiology

    Article Title: Antagonistic interaction between two key endodontic pathogens Enterococcus faecalis and Fusobacterium nucleatum

    doi: 10.1080/20002297.2022.2149448

    Figure Lengend Snippet: The acidic environment generated by E. faecalis inhibits F. nucleatum growth . (a) pH challenging assay. F. nucleatum ATCC 23726 viability was tested under different pH conditions in Columbia broth. The data presented are based on experiments conducted in triplicates. WT, wild type. (b) Competition assay under buffered pH condition. Quantitative analysis of the competition effects between E. faecalis ( Ef ) and F. nucleatum ( Fn ) under buffered pH condition. Data are expressed as relative CFU (%) compared with the Fn alone CFU at each timepoint as 100%. Graph shows means and SD of readings from two individual experiments performed in triplicate. Data represent the means and standard deviation of at least three independent experiments. *P < 0.05; ** P < 0.01; *** P < 0.001. WT, wild type.

    Article Snippet: F. nucleatum wild type strain ATCC 23726 and its mutant derivatives defective in outer membrane autotransporter proteins, including Fn 1449( fap2 ), Fn 1526( radD ), Fn 2058( aim1 ), Fn 2047, Fn 0254, Fn 1554, Fn 1253( aid1 ), and Fn 1893 [ ], were maintained under anaerobic conditions (10% H 2 , 10% CO 2 , 80% N 2 ) at 37°C on either Columbia agar supplemented with 5% sheep blood or in Columbia Broth (CB; BD Difco, Detroit, MI, USA).

    Techniques: Generated, Competitive Binding Assay, Standard Deviation