Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Phylogenetic relationship of representative F. nucleatum strains used in this study, grouped by subspecies—subsp. nucleatum (FNN; ATCC 25586, ATCC 23726, CTI-2), subsp. vincentii (FNV; 3_1_27, ATCC 49256, ATCC 51190), subsp. animalis (FNA; 7_1, F0401, ATCC 51191), and subsp. polymorphum (FNP; ATCC 10953, 12230). The phylogenetic tree was constructed based on znpA gene using the maximum-likelihood method implemented in DNAMAN Version 10 (Lynnon Biosoft). Fusobacterium periodonticum ATCC 33693 (FP) was included as an outgroup. (B) Schematic of the chromosomal region between uraA and pepF showing subspecies-specific presence of luxS . luxS is absent from FNN and FNV at this locus, present as an intact gene in FNA (between uraA and pepF ), and disrupted in FNP by insertion of an IS200-family element. The corresponding region from F. periodonticum is shown for comparison. Arrows indicate gene orientation; uraA (gray), pepF (black), luxS (blue), IS200 insertion (magenta), and the adjacent gene ( ddpA , orange) are indicated. (C) AI-2 activity in cell-free culture supernatants was measured using the Vibrio harveyi BB170 bioluminescence reporter assay. Supernatants from FNN, FNV, and FNP strains showed signals at or near background levels, whereas all tested FNA strains and F. periodonticum generated robust reporter induction. E. coli wild type (WT) and its Δ luxS mutant served as positive and negative controls, respectively. Data are presented as relative fluorescence units (RFU; mean ± SD) from three independent experiments (each assayed in technical triplicate); the y-axis includes a break to display both low- and high-signal samples.

Article Snippet: FNN ATCC 23726 wild-type and luxS insertion strains, as well as FNA strain 7_1 and its Δ luxS mutant, were grown overnight in TSPC broth under anaerobic conditions at 37°C.

Techniques: Construct, Comparison, Activity Assay, Reporter Assay, Generated, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic of the uraA–pepF chromosomal locus in FNN ATCC 23726 before (WT) and after in-frame insertion of the FNA 7_1 luxS gene (WT:: luxS 7_1 ) between uraA and pepF . (B) PCR confirming correct chromosomal insertion of luxS 7_1 in ATCC 23726 (WT band versus the larger amplicon from WT:: luxS 7_1). (C) AI-2 activity measured by the V. harveyi BB170 bioluminescence reporter assay. WT ATCC 23726 and WT ATCC 25586 showed background-level signals, whereas ATCC 23726 carrying the chromosomal luxS 7_1 insertion (WT:: luxS 7_1 ) and ATCC 25586 expressing luxS 7_1 from a shuttle plasmid (p luxS 7_1 ) produced robust AI-2 signals comparable to the E. coli WT positive control. The E. coli Δ luxS strain served as a negative control. (D) Growth analysis showing that expression of luxS 7_1 in ATCC 23726 (WT:: luxS 7_1 ) or ATCC 25586 (p luxS 7_1 )) did not significantly alter final culture density relative to the corresponding WT strains (n.s., Student’s t test). (E) Representative crystal violet–stained monospecies biofilms demonstrating no obvious difference in biofilm biomass between WT and luxS 7_1 -expressing derivatives of ATCC 23726 and ATCC 25586 after anaerobic growth in TSPC for 72 h.

Article Snippet: FNN ATCC 23726 wild-type and luxS insertion strains, as well as FNA strain 7_1 and its Δ luxS mutant, were grown overnight in TSPC broth under anaerobic conditions at 37°C.

Techniques: Amplification, Activity Assay, Reporter Assay, Expressing, Plasmid Preparation, Produced, Positive Control, Negative Control, Staining

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Time-course analysis of extracellular AI-2 activity during growth of FNA strain 7_1. AI-2 levels were measured using the V. harveyi BB170 reporter assay, and culture density (OD 600 ) was recorded at each time point. FNN ATCC 23726, which lacks luxS , served as a negative control. Data represent the mean ± SD from three independent experiments. (B) Volcano plot showing differential gene expression between Δ luxS and WT 7_1 at OD 600 ≈ 0.8, as determined by RNA-seq analysis. Genes meeting the threshold of |log₂(fold change)| ≥ 1 and p ≤ 0.05 are highlighted. (C) Volcano plot showing differential gene expression between Δ luxS and WT 7_1 at OD 600 ≈ 1.2. Few genes met the differential expression threshold, and fold changes were modest in magnitude. (D) Volcano plot of RNA-seq analysis comparing FNN ATCC 23726 treated with synthetic AI-2 (DPD) versus untreated controls. Only a small number of genes showed ≥2-fold changes, indicating that exogenous AI-2 does not induce a coordinated transcriptional response in this subspecies.

Article Snippet: FNN ATCC 23726 wild-type and luxS insertion strains, as well as FNA strain 7_1 and its Δ luxS mutant, were grown overnight in TSPC broth under anaerobic conditions at 37°C.

Techniques: Activity Assay, Reporter Assay, Negative Control, Gene Expression, RNA Sequencing, Quantitative Proteomics

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic representation of the activated methyl cycle (AMC) illustrating methyl group transfer and methionine recycling. MetK (S-adenosylmethionine synthetase) converts methionine to S-adenosylmethionine (SAM), the universal methyl donor. Following methyl transfer by S-adenosylmethionine-dependent methyltransferases (SDMs), SAM is converted to S-adenosylhomocysteine (SAH). In the LuxS-dependent pathway, SAH is processed by Pfs (5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase) to form S-ribosylhomocysteine (SRH), which is subsequently cleaved by LuxS to generate homocysteine and the AI-2 precursor DPD. In alternative pathways found in other bacteria, SAH can be directly converted to homocysteine by SahH (S-adenosylhomocysteine hydrolase). (B) Schematic diagram of the metK deletion construct (pCM-galK-Δ metK ) used to generate an in-frame chromosomal deletion in FNN ATCC 23726. Approximately 1.5 kb of upstream and downstream homologous regions flank the deleted metK coding sequence to facilitate double-crossover recombination. ( C) PCR screening of more than 100 counterselected colonies following allelic exchange showed retention of the wild-type metK allele, with no Δ metK mutants recovered, indicating that metK is essential under the tested conditions. Representative PCR results from 10 independent colonies are shown. (D) Strategy for the construction of a conditional metK mutant. Because metK is essential, chromosomal deletion was performed in the presence of a plasmid-borne copy of metK expressed under the control of a theophylline- inducible riboswitch, allowing complementation in trans. (E) PCR confirmation of successful chromosomal deletion of metK in the presence of plasmid-mediated complementation, demonstrating that deletion is possible only when metK expression is provided in trans. (F) Growth analysis of the conditional metK mutant showing strict dependence on theophylline for viability. Bacterial growth exhibited a dose-dependent response to the inducer, and no growth was observed in its absence, confirming that metK is essential for survival in F. nucleatum . (G) Transmission electron microscopy (TEM) of the conditional Δ metK strain. Cells grown in the presence of 3 mM theophylline displayed normal morphology comparable to wild type. In contrast, depletion of metK (no inducer; cells precultured with 2 mM theophylline and then grown for 12 h without inducer) resulted in pronounced morphological abnormalities, including curved cells ( H1 ), surface-associated tubular-like structures ( H2 ), and marked cell elongation (H3; enlarged view shown).

Article Snippet: FNN ATCC 23726 wild-type and luxS insertion strains, as well as FNA strain 7_1 and its Δ luxS mutant, were grown overnight in TSPC broth under anaerobic conditions at 37°C.

Techniques: Bacteria, Construct, Sequencing, Mutagenesis, Plasmid Preparation, Control, Expressing, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Phylogenetic relationship of representative F. nucleatum strains used in this study, grouped by subspecies—subsp. nucleatum (FNN; ATCC 25586, ATCC 23726, CTI-2), subsp. vincentii (FNV; 3_1_27, ATCC 49256, ATCC 51190), subsp. animalis (FNA; 7_1, F0401, ATCC 51191), and subsp. polymorphum (FNP; ATCC 10953, 12230). The phylogenetic tree was constructed based on znpA gene using the maximum-likelihood method implemented in DNAMAN Version 10 (Lynnon Biosoft). Fusobacterium periodonticum ATCC 33693 (FP) was included as an outgroup. (B) Schematic of the chromosomal region between uraA and pepF showing subspecies-specific presence of luxS . luxS is absent from FNN and FNV at this locus, present as an intact gene in FNA (between uraA and pepF ), and disrupted in FNP by insertion of an IS200-family element. The corresponding region from F. periodonticum is shown for comparison. Arrows indicate gene orientation; uraA (gray), pepF (black), luxS (blue), IS200 insertion (magenta), and the adjacent gene ( ddpA , orange) are indicated. (C) AI-2 activity in cell-free culture supernatants was measured using the Vibrio harveyi BB170 bioluminescence reporter assay. Supernatants from FNN, FNV, and FNP strains showed signals at or near background levels, whereas all tested FNA strains and F. periodonticum generated robust reporter induction. E. coli wild type (WT) and its Δ luxS mutant served as positive and negative controls, respectively. Data are presented as relative fluorescence units (RFU; mean ± SD) from three independent experiments (each assayed in technical triplicate); the y-axis includes a break to display both low- and high-signal samples.

Article Snippet: A previously described galK -based counterselection strategy was adapted to generate a non-polar, in-frame metK deletion in FNN ATCC 23726( ).

Techniques: Construct, Comparison, Activity Assay, Reporter Assay, Generated, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic of the uraA–pepF chromosomal locus in FNN ATCC 23726 before (WT) and after in-frame insertion of the FNA 7_1 luxS gene (WT:: luxS 7_1 ) between uraA and pepF . (B) PCR confirming correct chromosomal insertion of luxS 7_1 in ATCC 23726 (WT band versus the larger amplicon from WT:: luxS 7_1). (C) AI-2 activity measured by the V. harveyi BB170 bioluminescence reporter assay. WT ATCC 23726 and WT ATCC 25586 showed background-level signals, whereas ATCC 23726 carrying the chromosomal luxS 7_1 insertion (WT:: luxS 7_1 ) and ATCC 25586 expressing luxS 7_1 from a shuttle plasmid (p luxS 7_1 ) produced robust AI-2 signals comparable to the E. coli WT positive control. The E. coli Δ luxS strain served as a negative control. (D) Growth analysis showing that expression of luxS 7_1 in ATCC 23726 (WT:: luxS 7_1 ) or ATCC 25586 (p luxS 7_1 )) did not significantly alter final culture density relative to the corresponding WT strains (n.s., Student’s t test). (E) Representative crystal violet–stained monospecies biofilms demonstrating no obvious difference in biofilm biomass between WT and luxS 7_1 -expressing derivatives of ATCC 23726 and ATCC 25586 after anaerobic growth in TSPC for 72 h.

Article Snippet: A previously described galK -based counterselection strategy was adapted to generate a non-polar, in-frame metK deletion in FNN ATCC 23726( ).

Techniques: Amplification, Activity Assay, Reporter Assay, Expressing, Plasmid Preparation, Produced, Positive Control, Negative Control, Staining

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Time-course analysis of extracellular AI-2 activity during growth of FNA strain 7_1. AI-2 levels were measured using the V. harveyi BB170 reporter assay, and culture density (OD 600 ) was recorded at each time point. FNN ATCC 23726, which lacks luxS , served as a negative control. Data represent the mean ± SD from three independent experiments. (B) Volcano plot showing differential gene expression between Δ luxS and WT 7_1 at OD 600 ≈ 0.8, as determined by RNA-seq analysis. Genes meeting the threshold of |log₂(fold change)| ≥ 1 and p ≤ 0.05 are highlighted. (C) Volcano plot showing differential gene expression between Δ luxS and WT 7_1 at OD 600 ≈ 1.2. Few genes met the differential expression threshold, and fold changes were modest in magnitude. (D) Volcano plot of RNA-seq analysis comparing FNN ATCC 23726 treated with synthetic AI-2 (DPD) versus untreated controls. Only a small number of genes showed ≥2-fold changes, indicating that exogenous AI-2 does not induce a coordinated transcriptional response in this subspecies.

Article Snippet: A previously described galK -based counterselection strategy was adapted to generate a non-polar, in-frame metK deletion in FNN ATCC 23726( ).

Techniques: Activity Assay, Reporter Assay, Negative Control, Gene Expression, RNA Sequencing, Quantitative Proteomics

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic representation of the activated methyl cycle (AMC) illustrating methyl group transfer and methionine recycling. MetK (S-adenosylmethionine synthetase) converts methionine to S-adenosylmethionine (SAM), the universal methyl donor. Following methyl transfer by S-adenosylmethionine-dependent methyltransferases (SDMs), SAM is converted to S-adenosylhomocysteine (SAH). In the LuxS-dependent pathway, SAH is processed by Pfs (5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase) to form S-ribosylhomocysteine (SRH), which is subsequently cleaved by LuxS to generate homocysteine and the AI-2 precursor DPD. In alternative pathways found in other bacteria, SAH can be directly converted to homocysteine by SahH (S-adenosylhomocysteine hydrolase). (B) Schematic diagram of the metK deletion construct (pCM-galK-Δ metK ) used to generate an in-frame chromosomal deletion in FNN ATCC 23726. Approximately 1.5 kb of upstream and downstream homologous regions flank the deleted metK coding sequence to facilitate double-crossover recombination. ( C) PCR screening of more than 100 counterselected colonies following allelic exchange showed retention of the wild-type metK allele, with no Δ metK mutants recovered, indicating that metK is essential under the tested conditions. Representative PCR results from 10 independent colonies are shown. (D) Strategy for the construction of a conditional metK mutant. Because metK is essential, chromosomal deletion was performed in the presence of a plasmid-borne copy of metK expressed under the control of a theophylline- inducible riboswitch, allowing complementation in trans. (E) PCR confirmation of successful chromosomal deletion of metK in the presence of plasmid-mediated complementation, demonstrating that deletion is possible only when metK expression is provided in trans. (F) Growth analysis of the conditional metK mutant showing strict dependence on theophylline for viability. Bacterial growth exhibited a dose-dependent response to the inducer, and no growth was observed in its absence, confirming that metK is essential for survival in F. nucleatum . (G) Transmission electron microscopy (TEM) of the conditional Δ metK strain. Cells grown in the presence of 3 mM theophylline displayed normal morphology comparable to wild type. In contrast, depletion of metK (no inducer; cells precultured with 2 mM theophylline and then grown for 12 h without inducer) resulted in pronounced morphological abnormalities, including curved cells ( H1 ), surface-associated tubular-like structures ( H2 ), and marked cell elongation (H3; enlarged view shown).

Article Snippet: A previously described galK -based counterselection strategy was adapted to generate a non-polar, in-frame metK deletion in FNN ATCC 23726( ).

Techniques: Bacteria, Construct, Sequencing, Mutagenesis, Plasmid Preparation, Control, Expressing, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Phylogenetic relationship of representative F. nucleatum strains used in this study, grouped by subspecies—subsp. nucleatum (FNN; ATCC 25586, ATCC 23726, CTI-2), subsp. vincentii (FNV; 3_1_27, ATCC 49256, ATCC 51190), subsp. animalis (FNA; 7_1, F0401, ATCC 51191), and subsp. polymorphum (FNP; ATCC 10953, 12230). The phylogenetic tree was constructed based on znpA gene using the maximum-likelihood method implemented in DNAMAN Version 10 (Lynnon Biosoft). Fusobacterium periodonticum ATCC 33693 (FP) was included as an outgroup. (B) Schematic of the chromosomal region between uraA and pepF showing subspecies-specific presence of luxS . luxS is absent from FNN and FNV at this locus, present as an intact gene in FNA (between uraA and pepF ), and disrupted in FNP by insertion of an IS200-family element. The corresponding region from F. periodonticum is shown for comparison. Arrows indicate gene orientation; uraA (gray), pepF (black), luxS (blue), IS200 insertion (magenta), and the adjacent gene ( ddpA , orange) are indicated. (C) AI-2 activity in cell-free culture supernatants was measured using the Vibrio harveyi BB170 bioluminescence reporter assay. Supernatants from FNN, FNV, and FNP strains showed signals at or near background levels, whereas all tested FNA strains and F. periodonticum generated robust reporter induction. E. coli wild type (WT) and its Δ luxS mutant served as positive and negative controls, respectively. Data are presented as relative fluorescence units (RFU; mean ± SD) from three independent experiments (each assayed in technical triplicate); the y-axis includes a break to display both low- and high-signal samples.

Article Snippet: The plasmid pBCG02-luxS7_1 was introduced into electrocompetent FNN ATCC 23726 by electroporation( ).

Techniques: Construct, Comparison, Activity Assay, Reporter Assay, Generated, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic of the uraA–pepF chromosomal locus in FNN ATCC 23726 before (WT) and after in-frame insertion of the FNA 7_1 luxS gene (WT:: luxS 7_1 ) between uraA and pepF . (B) PCR confirming correct chromosomal insertion of luxS 7_1 in ATCC 23726 (WT band versus the larger amplicon from WT:: luxS 7_1). (C) AI-2 activity measured by the V. harveyi BB170 bioluminescence reporter assay. WT ATCC 23726 and WT ATCC 25586 showed background-level signals, whereas ATCC 23726 carrying the chromosomal luxS 7_1 insertion (WT:: luxS 7_1 ) and ATCC 25586 expressing luxS 7_1 from a shuttle plasmid (p luxS 7_1 ) produced robust AI-2 signals comparable to the E. coli WT positive control. The E. coli Δ luxS strain served as a negative control. (D) Growth analysis showing that expression of luxS 7_1 in ATCC 23726 (WT:: luxS 7_1 ) or ATCC 25586 (p luxS 7_1 )) did not significantly alter final culture density relative to the corresponding WT strains (n.s., Student’s t test). (E) Representative crystal violet–stained monospecies biofilms demonstrating no obvious difference in biofilm biomass between WT and luxS 7_1 -expressing derivatives of ATCC 23726 and ATCC 25586 after anaerobic growth in TSPC for 72 h.

Article Snippet: The plasmid pBCG02-luxS7_1 was introduced into electrocompetent FNN ATCC 23726 by electroporation( ).

Techniques: Amplification, Activity Assay, Reporter Assay, Expressing, Plasmid Preparation, Produced, Positive Control, Negative Control, Staining

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Time-course analysis of extracellular AI-2 activity during growth of FNA strain 7_1. AI-2 levels were measured using the V. harveyi BB170 reporter assay, and culture density (OD 600 ) was recorded at each time point. FNN ATCC 23726, which lacks luxS , served as a negative control. Data represent the mean ± SD from three independent experiments. (B) Volcano plot showing differential gene expression between Δ luxS and WT 7_1 at OD 600 ≈ 0.8, as determined by RNA-seq analysis. Genes meeting the threshold of |log₂(fold change)| ≥ 1 and p ≤ 0.05 are highlighted. (C) Volcano plot showing differential gene expression between Δ luxS and WT 7_1 at OD 600 ≈ 1.2. Few genes met the differential expression threshold, and fold changes were modest in magnitude. (D) Volcano plot of RNA-seq analysis comparing FNN ATCC 23726 treated with synthetic AI-2 (DPD) versus untreated controls. Only a small number of genes showed ≥2-fold changes, indicating that exogenous AI-2 does not induce a coordinated transcriptional response in this subspecies.

Article Snippet: The plasmid pBCG02-luxS7_1 was introduced into electrocompetent FNN ATCC 23726 by electroporation( ).

Techniques: Activity Assay, Reporter Assay, Negative Control, Gene Expression, RNA Sequencing, Quantitative Proteomics

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic representation of the activated methyl cycle (AMC) illustrating methyl group transfer and methionine recycling. MetK (S-adenosylmethionine synthetase) converts methionine to S-adenosylmethionine (SAM), the universal methyl donor. Following methyl transfer by S-adenosylmethionine-dependent methyltransferases (SDMs), SAM is converted to S-adenosylhomocysteine (SAH). In the LuxS-dependent pathway, SAH is processed by Pfs (5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase) to form S-ribosylhomocysteine (SRH), which is subsequently cleaved by LuxS to generate homocysteine and the AI-2 precursor DPD. In alternative pathways found in other bacteria, SAH can be directly converted to homocysteine by SahH (S-adenosylhomocysteine hydrolase). (B) Schematic diagram of the metK deletion construct (pCM-galK-Δ metK ) used to generate an in-frame chromosomal deletion in FNN ATCC 23726. Approximately 1.5 kb of upstream and downstream homologous regions flank the deleted metK coding sequence to facilitate double-crossover recombination. ( C) PCR screening of more than 100 counterselected colonies following allelic exchange showed retention of the wild-type metK allele, with no Δ metK mutants recovered, indicating that metK is essential under the tested conditions. Representative PCR results from 10 independent colonies are shown. (D) Strategy for the construction of a conditional metK mutant. Because metK is essential, chromosomal deletion was performed in the presence of a plasmid-borne copy of metK expressed under the control of a theophylline- inducible riboswitch, allowing complementation in trans. (E) PCR confirmation of successful chromosomal deletion of metK in the presence of plasmid-mediated complementation, demonstrating that deletion is possible only when metK expression is provided in trans. (F) Growth analysis of the conditional metK mutant showing strict dependence on theophylline for viability. Bacterial growth exhibited a dose-dependent response to the inducer, and no growth was observed in its absence, confirming that metK is essential for survival in F. nucleatum . (G) Transmission electron microscopy (TEM) of the conditional Δ metK strain. Cells grown in the presence of 3 mM theophylline displayed normal morphology comparable to wild type. In contrast, depletion of metK (no inducer; cells precultured with 2 mM theophylline and then grown for 12 h without inducer) resulted in pronounced morphological abnormalities, including curved cells ( H1 ), surface-associated tubular-like structures ( H2 ), and marked cell elongation (H3; enlarged view shown).

Article Snippet: The plasmid pBCG02-luxS7_1 was introduced into electrocompetent FNN ATCC 23726 by electroporation( ).

Techniques: Bacteria, Construct, Sequencing, Mutagenesis, Plasmid Preparation, Control, Expressing, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Phylogenetic relationship of representative F. nucleatum strains used in this study, grouped by subspecies—subsp. nucleatum (FNN; ATCC 25586, ATCC 23726, CTI-2), subsp. vincentii (FNV; 3_1_27, ATCC 49256, ATCC 51190), subsp. animalis (FNA; 7_1, F0401, ATCC 51191), and subsp. polymorphum (FNP; ATCC 10953, 12230). The phylogenetic tree was constructed based on znpA gene using the maximum-likelihood method implemented in DNAMAN Version 10 (Lynnon Biosoft). Fusobacterium periodonticum ATCC 33693 (FP) was included as an outgroup. (B) Schematic of the chromosomal region between uraA and pepF showing subspecies-specific presence of luxS . luxS is absent from FNN and FNV at this locus, present as an intact gene in FNA (between uraA and pepF ), and disrupted in FNP by insertion of an IS200-family element. The corresponding region from F. periodonticum is shown for comparison. Arrows indicate gene orientation; uraA (gray), pepF (black), luxS (blue), IS200 insertion (magenta), and the adjacent gene ( ddpA , orange) are indicated. (C) AI-2 activity in cell-free culture supernatants was measured using the Vibrio harveyi BB170 bioluminescence reporter assay. Supernatants from FNN, FNV, and FNP strains showed signals at or near background levels, whereas all tested FNA strains and F. periodonticum generated robust reporter induction. E. coli wild type (WT) and its Δ luxS mutant served as positive and negative controls, respectively. Data are presented as relative fluorescence units (RFU; mean ± SD) from three independent experiments (each assayed in technical triplicate); the y-axis includes a break to display both low- and high-signal samples.

Article Snippet: To determine whether AMC activity is essential in F. nucleatum, we attempted to delete metK in the genetically tractable FNN strain ATCC 23726.

Techniques: Construct, Comparison, Activity Assay, Reporter Assay, Generated, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic of the uraA–pepF chromosomal locus in FNN ATCC 23726 before (WT) and after in-frame insertion of the FNA 7_1 luxS gene (WT:: luxS 7_1 ) between uraA and pepF . (B) PCR confirming correct chromosomal insertion of luxS 7_1 in ATCC 23726 (WT band versus the larger amplicon from WT:: luxS 7_1). (C) AI-2 activity measured by the V. harveyi BB170 bioluminescence reporter assay. WT ATCC 23726 and WT ATCC 25586 showed background-level signals, whereas ATCC 23726 carrying the chromosomal luxS 7_1 insertion (WT:: luxS 7_1 ) and ATCC 25586 expressing luxS 7_1 from a shuttle plasmid (p luxS 7_1 ) produced robust AI-2 signals comparable to the E. coli WT positive control. The E. coli Δ luxS strain served as a negative control. (D) Growth analysis showing that expression of luxS 7_1 in ATCC 23726 (WT:: luxS 7_1 ) or ATCC 25586 (p luxS 7_1 )) did not significantly alter final culture density relative to the corresponding WT strains (n.s., Student’s t test). (E) Representative crystal violet–stained monospecies biofilms demonstrating no obvious difference in biofilm biomass between WT and luxS 7_1 -expressing derivatives of ATCC 23726 and ATCC 25586 after anaerobic growth in TSPC for 72 h.

Article Snippet: To determine whether AMC activity is essential in F. nucleatum, we attempted to delete metK in the genetically tractable FNN strain ATCC 23726.

Techniques: Amplification, Activity Assay, Reporter Assay, Expressing, Plasmid Preparation, Produced, Positive Control, Negative Control, Staining

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Time-course analysis of extracellular AI-2 activity during growth of FNA strain 7_1. AI-2 levels were measured using the V. harveyi BB170 reporter assay, and culture density (OD 600 ) was recorded at each time point. FNN ATCC 23726, which lacks luxS , served as a negative control. Data represent the mean ± SD from three independent experiments. (B) Volcano plot showing differential gene expression between Δ luxS and WT 7_1 at OD 600 ≈ 0.8, as determined by RNA-seq analysis. Genes meeting the threshold of |log₂(fold change)| ≥ 1 and p ≤ 0.05 are highlighted. (C) Volcano plot showing differential gene expression between Δ luxS and WT 7_1 at OD 600 ≈ 1.2. Few genes met the differential expression threshold, and fold changes were modest in magnitude. (D) Volcano plot of RNA-seq analysis comparing FNN ATCC 23726 treated with synthetic AI-2 (DPD) versus untreated controls. Only a small number of genes showed ≥2-fold changes, indicating that exogenous AI-2 does not induce a coordinated transcriptional response in this subspecies.

Article Snippet: To determine whether AMC activity is essential in F. nucleatum, we attempted to delete metK in the genetically tractable FNN strain ATCC 23726.

Techniques: Activity Assay, Reporter Assay, Negative Control, Gene Expression, RNA Sequencing, Quantitative Proteomics

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic representation of the activated methyl cycle (AMC) illustrating methyl group transfer and methionine recycling. MetK (S-adenosylmethionine synthetase) converts methionine to S-adenosylmethionine (SAM), the universal methyl donor. Following methyl transfer by S-adenosylmethionine-dependent methyltransferases (SDMs), SAM is converted to S-adenosylhomocysteine (SAH). In the LuxS-dependent pathway, SAH is processed by Pfs (5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase) to form S-ribosylhomocysteine (SRH), which is subsequently cleaved by LuxS to generate homocysteine and the AI-2 precursor DPD. In alternative pathways found in other bacteria, SAH can be directly converted to homocysteine by SahH (S-adenosylhomocysteine hydrolase). (B) Schematic diagram of the metK deletion construct (pCM-galK-Δ metK ) used to generate an in-frame chromosomal deletion in FNN ATCC 23726. Approximately 1.5 kb of upstream and downstream homologous regions flank the deleted metK coding sequence to facilitate double-crossover recombination. ( C) PCR screening of more than 100 counterselected colonies following allelic exchange showed retention of the wild-type metK allele, with no Δ metK mutants recovered, indicating that metK is essential under the tested conditions. Representative PCR results from 10 independent colonies are shown. (D) Strategy for the construction of a conditional metK mutant. Because metK is essential, chromosomal deletion was performed in the presence of a plasmid-borne copy of metK expressed under the control of a theophylline- inducible riboswitch, allowing complementation in trans. (E) PCR confirmation of successful chromosomal deletion of metK in the presence of plasmid-mediated complementation, demonstrating that deletion is possible only when metK expression is provided in trans. (F) Growth analysis of the conditional metK mutant showing strict dependence on theophylline for viability. Bacterial growth exhibited a dose-dependent response to the inducer, and no growth was observed in its absence, confirming that metK is essential for survival in F. nucleatum . (G) Transmission electron microscopy (TEM) of the conditional Δ metK strain. Cells grown in the presence of 3 mM theophylline displayed normal morphology comparable to wild type. In contrast, depletion of metK (no inducer; cells precultured with 2 mM theophylline and then grown for 12 h without inducer) resulted in pronounced morphological abnormalities, including curved cells ( H1 ), surface-associated tubular-like structures ( H2 ), and marked cell elongation (H3; enlarged view shown).

Article Snippet: To determine whether AMC activity is essential in F. nucleatum, we attempted to delete metK in the genetically tractable FNN strain ATCC 23726.

Techniques: Bacteria, Construct, Sequencing, Mutagenesis, Plasmid Preparation, Control, Expressing, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Phylogenetic relationship of representative F. nucleatum strains used in this study, grouped by subspecies—subsp. nucleatum (FNN; ATCC 25586, ATCC 23726, CTI-2), subsp. vincentii (FNV; 3_1_27, ATCC 49256, ATCC 51190), subsp. animalis (FNA; 7_1, F0401, ATCC 51191), and subsp. polymorphum (FNP; ATCC 10953, 12230). The phylogenetic tree was constructed based on znpA gene using the maximum-likelihood method implemented in DNAMAN Version 10 (Lynnon Biosoft). Fusobacterium periodonticum ATCC 33693 (FP) was included as an outgroup. (B) Schematic of the chromosomal region between uraA and pepF showing subspecies-specific presence of luxS . luxS is absent from FNN and FNV at this locus, present as an intact gene in FNA (between uraA and pepF ), and disrupted in FNP by insertion of an IS200-family element. The corresponding region from F. periodonticum is shown for comparison. Arrows indicate gene orientation; uraA (gray), pepF (black), luxS (blue), IS200 insertion (magenta), and the adjacent gene ( ddpA , orange) are indicated. (C) AI-2 activity in cell-free culture supernatants was measured using the Vibrio harveyi BB170 bioluminescence reporter assay. Supernatants from FNN, FNV, and FNP strains showed signals at or near background levels, whereas all tested FNA strains and F. periodonticum generated robust reporter induction. E. coli wild type (WT) and its Δ luxS mutant served as positive and negative controls, respectively. Data are presented as relative fluorescence units (RFU; mean ± SD) from three independent experiments (each assayed in technical triplicate); the y-axis includes a break to display both low- and high-signal samples.

Article Snippet: FNN strain ATCC 23726 was treated with 30 μM synthetic DPD, a concentration that induces V. harveyi BB170 reporter activity comparable to the peak AI-2 levels detected in late-log–phase FNA culture supernatants (Fig. S2).

Techniques: Construct, Comparison, Activity Assay, Reporter Assay, Generated, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic of the uraA–pepF chromosomal locus in FNN ATCC 23726 before (WT) and after in-frame insertion of the FNA 7_1 luxS gene (WT:: luxS 7_1 ) between uraA and pepF . (B) PCR confirming correct chromosomal insertion of luxS 7_1 in ATCC 23726 (WT band versus the larger amplicon from WT:: luxS 7_1). (C) AI-2 activity measured by the V. harveyi BB170 bioluminescence reporter assay. WT ATCC 23726 and WT ATCC 25586 showed background-level signals, whereas ATCC 23726 carrying the chromosomal luxS 7_1 insertion (WT:: luxS 7_1 ) and ATCC 25586 expressing luxS 7_1 from a shuttle plasmid (p luxS 7_1 ) produced robust AI-2 signals comparable to the E. coli WT positive control. The E. coli Δ luxS strain served as a negative control. (D) Growth analysis showing that expression of luxS 7_1 in ATCC 23726 (WT:: luxS 7_1 ) or ATCC 25586 (p luxS 7_1 )) did not significantly alter final culture density relative to the corresponding WT strains (n.s., Student’s t test). (E) Representative crystal violet–stained monospecies biofilms demonstrating no obvious difference in biofilm biomass between WT and luxS 7_1 -expressing derivatives of ATCC 23726 and ATCC 25586 after anaerobic growth in TSPC for 72 h.

Article Snippet: FNN strain ATCC 23726 was treated with 30 μM synthetic DPD, a concentration that induces V. harveyi BB170 reporter activity comparable to the peak AI-2 levels detected in late-log–phase FNA culture supernatants (Fig. S2).

Techniques: Amplification, Activity Assay, Reporter Assay, Expressing, Plasmid Preparation, Produced, Positive Control, Negative Control, Staining

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Time-course analysis of extracellular AI-2 activity during growth of FNA strain 7_1. AI-2 levels were measured using the V. harveyi BB170 reporter assay, and culture density (OD 600 ) was recorded at each time point. FNN ATCC 23726, which lacks luxS , served as a negative control. Data represent the mean ± SD from three independent experiments. (B) Volcano plot showing differential gene expression between Δ luxS and WT 7_1 at OD 600 ≈ 0.8, as determined by RNA-seq analysis. Genes meeting the threshold of |log₂(fold change)| ≥ 1 and p ≤ 0.05 are highlighted. (C) Volcano plot showing differential gene expression between Δ luxS and WT 7_1 at OD 600 ≈ 1.2. Few genes met the differential expression threshold, and fold changes were modest in magnitude. (D) Volcano plot of RNA-seq analysis comparing FNN ATCC 23726 treated with synthetic AI-2 (DPD) versus untreated controls. Only a small number of genes showed ≥2-fold changes, indicating that exogenous AI-2 does not induce a coordinated transcriptional response in this subspecies.

Article Snippet: FNN strain ATCC 23726 was treated with 30 μM synthetic DPD, a concentration that induces V. harveyi BB170 reporter activity comparable to the peak AI-2 levels detected in late-log–phase FNA culture supernatants (Fig. S2).

Techniques: Activity Assay, Reporter Assay, Negative Control, Gene Expression, RNA Sequencing, Quantitative Proteomics

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic representation of the activated methyl cycle (AMC) illustrating methyl group transfer and methionine recycling. MetK (S-adenosylmethionine synthetase) converts methionine to S-adenosylmethionine (SAM), the universal methyl donor. Following methyl transfer by S-adenosylmethionine-dependent methyltransferases (SDMs), SAM is converted to S-adenosylhomocysteine (SAH). In the LuxS-dependent pathway, SAH is processed by Pfs (5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase) to form S-ribosylhomocysteine (SRH), which is subsequently cleaved by LuxS to generate homocysteine and the AI-2 precursor DPD. In alternative pathways found in other bacteria, SAH can be directly converted to homocysteine by SahH (S-adenosylhomocysteine hydrolase). (B) Schematic diagram of the metK deletion construct (pCM-galK-Δ metK ) used to generate an in-frame chromosomal deletion in FNN ATCC 23726. Approximately 1.5 kb of upstream and downstream homologous regions flank the deleted metK coding sequence to facilitate double-crossover recombination. ( C) PCR screening of more than 100 counterselected colonies following allelic exchange showed retention of the wild-type metK allele, with no Δ metK mutants recovered, indicating that metK is essential under the tested conditions. Representative PCR results from 10 independent colonies are shown. (D) Strategy for the construction of a conditional metK mutant. Because metK is essential, chromosomal deletion was performed in the presence of a plasmid-borne copy of metK expressed under the control of a theophylline- inducible riboswitch, allowing complementation in trans. (E) PCR confirmation of successful chromosomal deletion of metK in the presence of plasmid-mediated complementation, demonstrating that deletion is possible only when metK expression is provided in trans. (F) Growth analysis of the conditional metK mutant showing strict dependence on theophylline for viability. Bacterial growth exhibited a dose-dependent response to the inducer, and no growth was observed in its absence, confirming that metK is essential for survival in F. nucleatum . (G) Transmission electron microscopy (TEM) of the conditional Δ metK strain. Cells grown in the presence of 3 mM theophylline displayed normal morphology comparable to wild type. In contrast, depletion of metK (no inducer; cells precultured with 2 mM theophylline and then grown for 12 h without inducer) resulted in pronounced morphological abnormalities, including curved cells ( H1 ), surface-associated tubular-like structures ( H2 ), and marked cell elongation (H3; enlarged view shown).

Article Snippet: FNN strain ATCC 23726 was treated with 30 μM synthetic DPD, a concentration that induces V. harveyi BB170 reporter activity comparable to the peak AI-2 levels detected in late-log–phase FNA culture supernatants (Fig. S2).

Techniques: Bacteria, Construct, Sequencing, Mutagenesis, Plasmid Preparation, Control, Expressing, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Phylogenetic relationship of representative F. nucleatum strains used in this study, grouped by subspecies—subsp. nucleatum (FNN; ATCC 25586, ATCC 23726, CTI-2), subsp. vincentii (FNV; 3_1_27, ATCC 49256, ATCC 51190), subsp. animalis (FNA; 7_1, F0401, ATCC 51191), and subsp. polymorphum (FNP; ATCC 10953, 12230). The phylogenetic tree was constructed based on znpA gene using the maximum-likelihood method implemented in DNAMAN Version 10 (Lynnon Biosoft). Fusobacterium periodonticum ATCC 33693 (FP) was included as an outgroup. (B) Schematic of the chromosomal region between uraA and pepF showing subspecies-specific presence of luxS . luxS is absent from FNN and FNV at this locus, present as an intact gene in FNA (between uraA and pepF ), and disrupted in FNP by insertion of an IS200-family element. The corresponding region from F. periodonticum is shown for comparison. Arrows indicate gene orientation; uraA (gray), pepF (black), luxS (blue), IS200 insertion (magenta), and the adjacent gene ( ddpA , orange) are indicated. (C) AI-2 activity in cell-free culture supernatants was measured using the Vibrio harveyi BB170 bioluminescence reporter assay. Supernatants from FNN, FNV, and FNP strains showed signals at or near background levels, whereas all tested FNA strains and F. periodonticum generated robust reporter induction. E. coli wild type (WT) and its Δ luxS mutant served as positive and negative controls, respectively. Data are presented as relative fluorescence units (RFU; mean ± SD) from three independent experiments (each assayed in technical triplicate); the y-axis includes a break to display both low- and high-signal samples.

Article Snippet: To generate a markerless Δ luxS mutant in FNA strain 7_1, the deletion plasmid pBCG10-Δ luxS was first introduced into E. coli SZU604, which expresses three F. nucleatum methyltransferases derived from FNN ATCC 25586( ).

Techniques: Construct, Comparison, Activity Assay, Reporter Assay, Generated, Mutagenesis, Fluorescence

Journal: bioRxiv

Article Title: AI-2 Production in Fusobacterium nucleatum Is Subspecies-Specific and Uncoupled from Quorum Sensing

doi: 10.64898/2026.03.02.709096

Figure Lengend Snippet: (A) Schematic of the uraA–pepF chromosomal locus in FNN ATCC 23726 before (WT) and after in-frame insertion of the FNA 7_1 luxS gene (WT:: luxS 7_1 ) between uraA and pepF . (B) PCR confirming correct chromosomal insertion of luxS 7_1 in ATCC 23726 (WT band versus the larger amplicon from WT:: luxS 7_1). (C) AI-2 activity measured by the V. harveyi BB170 bioluminescence reporter assay. WT ATCC 23726 and WT ATCC 25586 showed background-level signals, whereas ATCC 23726 carrying the chromosomal luxS 7_1 insertion (WT:: luxS 7_1 ) and ATCC 25586 expressing luxS 7_1 from a shuttle plasmid (p luxS 7_1 ) produced robust AI-2 signals comparable to the E. coli WT positive control. The E. coli Δ luxS strain served as a negative control. (D) Growth analysis showing that expression of luxS 7_1 in ATCC 23726 (WT:: luxS 7_1 ) or ATCC 25586 (p luxS 7_1 )) did not significantly alter final culture density relative to the corresponding WT strains (n.s., Student’s t test). (E) Representative crystal violet–stained monospecies biofilms demonstrating no obvious difference in biofilm biomass between WT and luxS 7_1 -expressing derivatives of ATCC 23726 and ATCC 25586 after anaerobic growth in TSPC for 72 h.

Article Snippet: To generate a markerless Δ luxS mutant in FNA strain 7_1, the deletion plasmid pBCG10-Δ luxS was first introduced into E. coli SZU604, which expresses three F. nucleatum methyltransferases derived from FNN ATCC 25586( ).

Techniques: Amplification, Activity Assay, Reporter Assay, Expressing, Plasmid Preparation, Produced, Positive Control, Negative Control, Staining