f. nucleatum wt  (Envigo)

Journal: The Journal of Biological Chemistry

Article Title: Discovery, characterization, and structure of a cofactor-independent histidine racemase from the oral pathogen Fusobacterium nucleatum

doi: 10.1016/j.jbc.2024.107896

Figure Lengend Snippet: Fusobacterium nucleatum ATCC 25586 DapF is a histidine racemase enzyme, and substrate turnover is faster with histidine than with lanthionine. A , D 2 O 1 H-NMR assay of F. nucleatum ATCC 25586 HisR with L,L-lanthionine. Solutions of 5 mM L,L-lanthionine in D 2 O buffer were prepared and analyzed with a 600 MHz NMR spectrometer. ∼10 μg of HisR enzyme diluted in D 2 O buffer was added, and spectra were collected again after 24 h. The small triplets at 3.94 and 2.98 ppm are from 2-mercaptoethanol, which was added to experiments longer than 8 h to keep HisR reduced. B , D 2 O 1 H-NMR assay of F. nucleatum ATCC 25586 HisR with L-histidine. Solutions of 5 mM L-histidine in D 2 O buffer were prepared and analyzed with a 600 MHz NMR spectrometer. ∼10 μg of HisR enzyme diluted in D 2 O buffer was added, and spectra were collected again after 15 min. C , circular dichroism monitoring of HisR enzyme (5 μg) activity with 30 mM solutions of either lanthionine or histidine. The CD signal was recorded at 212 nm, and the average integrated CD signal in mdeg was recorded every five seconds. Experiments were performed in duplicate and CD signals displayed are the average between the two trials. D , proposed enzyme mechanism of F. nucleatum HisR, based on the general mechanism of cofactor-independent (PLP-independent) racemase enzymes. ATCC, American Type Culture Collection; DapF, diaminopimelic acid epimerase; HisR, histidine racemase; PLP, pyridoxal 5′-phosphate.

Article Snippet: A , comparison of growth of F. nucleatum ATCC 23726 WT and ΔhisR .

Techniques: Circular Dichroism, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Discovery, characterization, and structure of a cofactor-independent histidine racemase from the oral pathogen Fusobacterium nucleatum

doi: 10.1016/j.jbc.2024.107896

Figure Lengend Snippet: Relative enzyme activity of Fusobacterium nucleatum ATCC 25586 HisR active site mutants compared to the WT enzyme. A , circular dichroism of HisR (67 μg/ml) or its mutants with L-histidine (30 mM). B , circular dichroism of HisR (67 μg/ml) or its mutants with D-histidine (30 mM). ATCC, American Type Culture Collection; HisR, histidine racemase.

Article Snippet: A , comparison of growth of F. nucleatum ATCC 23726 WT and ΔhisR .

Techniques: Activity Assay, Circular Dichroism

Journal: The Journal of Biological Chemistry

Article Title: Discovery, characterization, and structure of a cofactor-independent histidine racemase from the oral pathogen Fusobacterium nucleatum

doi: 10.1016/j.jbc.2024.107896

Figure Lengend Snippet: Crystal structures of C67S (PDB 9CR1 ) and C209S (PDB 9CR6 ) HisR mutant enzymes from Fusobacterium nucleatum ATCC 25586 at 2.5 Å. A , overlay of C67S ( green ) and C209S ( pink ) HisR crystal structures, with backbone r.m.s.d. of 0.493 Å by aligning α-carbons of 260 residues. The left panel shows an overlay of homodimers formed by HisR C67S and C209S, with the dimerization interface shown in the middle of the structure. The middle panel shows an overlay of monomer units of C67S and C209S HisR enzymes. The active site of each enzyme is occupied by a sulfate (C67S) or phosphate (C209S) ion. The right panel shows an overlay of active site residues of C67S and C209S HisR enzymes, with catalytic cysteines/serines indicated. B , N-terminal ( purple ) and C-terminal ( orange ) domains of HisR. The left panel shows a color-coded representation of the two domains, while the right panels show a structural alignment of the two domains. C , the electrostatic surface map of C67S HisR shows a negatively charged active site. The left panel shows the complete electrostatic surface map of C67S HisR, indicating that the active site pocket displays an overall positive charge. The pocket can be identified in the middle of the structure via presence of the sulfate ion ( sticks ) situated between the two catalytic residues. The middle panel shows a partially transparent electrostatic surface map indicating the position of the active site residues, which are situated in the center of the structure at the ends of helices 2 (residue 67) and 4 (residue 209), and on either side of the sulfate ion. The right panel shows a close up view of the active site with its electrostatic surface map. ATCC, American Type Culture Collection; HisR, histidine racemase; PDB, protein data bank.

Article Snippet: A , comparison of growth of F. nucleatum ATCC 23726 WT and ΔhisR .

Techniques: Mutagenesis, Residue

Journal: The Journal of Biological Chemistry

Article Title: Discovery, characterization, and structure of a cofactor-independent histidine racemase from the oral pathogen Fusobacterium nucleatum

doi: 10.1016/j.jbc.2024.107896

Figure Lengend Snippet: Comparison of HisR from Fusobacterium nucleatum ATCC 25586 with CntK from Staphylococcus aureus , currently the only other reported cofactor-independent histidine racemase. A , overlay of crystal structures from F. nucleatum ATCC 25586 HisR C67S mutant (PDB 9CR1 , green ) with S. aureus Mu50 CntK C72S mutant (PDB 6JIW , blue ). The overall r.m.s.d. (α-C) of the two structures is 1.412 Å. Both structures contain a sulfate ion situated in the active site pocket situated between the two catalytic residues. The residues labeled in green are based on the HisR protein sequence. B , active site residues flanking catalytic cysteine 209 in HisR ( green ), overlaid with active site residues of CntK flanking cysteine 211 ( blue ). The residues labeled in green are based on the HisR protein sequence. C , active site residues flanking mutated serine 67 in HisR ( green ), overlaid with active site residues of CntK flanking serine 72 ( blue ). The residues labeled in green are based on the HisR protein sequence. D , sequence similarity network of HisR from F. nucleatum ATCC 25586. The network (617 nodes and 18,197 edges; representative nodes shown based on 100% identity) was constructed with a threshold sequence identity for drawing edges of ∼40% (alignment score 55), minimum length 250 residues, and maximum length 308 residues. Cluster 2 contains primarily proteins from Staphylococcus sp. , while cluster 3 contains primarily proteins from Fusobacterium sp. CntK from S. aureus Mu50 and HisR from F. nucleatum ATCC 25586 are indicated as black ellipses in respective clusters. E , genome neighborhood diagrams for Staphylococcus aureus Mu50 CntK and F. nucleatum ATCC 25586 HisR. Histidine racemase genes are indicated with green boxes . The complete list of genes upstream and downstream of both histidine racemase genes are described in Table 3 . Here, only the S. aureus cnt biosynthetic gene cluster is labeled, while for F. nucleatum only the genes that appear in the genome neighborhood network ( Table 4 ) with >80% cooccurrence with HisR in cluster 3 are indicated. ATCC, American Type Culture Collection; HisR, histidine racemase; PDB, Protein Data Bank.

Article Snippet: A , comparison of growth of F. nucleatum ATCC 23726 WT and ΔhisR .

Techniques: Comparison, Mutagenesis, Labeling, Sequencing, Construct

Journal: The Journal of Biological Chemistry

Article Title: Discovery, characterization, and structure of a cofactor-independent histidine racemase from the oral pathogen Fusobacterium nucleatum

doi: 10.1016/j.jbc.2024.107896

Figure Lengend Snippet: Annotation of genes from upstream to downstream (±10) of hisR and cntK in F. nucleatum ATCC 25586 and Staphylococcus aureus Mu50

Article Snippet: A , comparison of growth of F. nucleatum ATCC 23726 WT and ΔhisR .

Techniques: Modification

Journal: The Journal of Biological Chemistry

Article Title: Discovery, characterization, and structure of a cofactor-independent histidine racemase from the oral pathogen Fusobacterium nucleatum

doi: 10.1016/j.jbc.2024.107896

Figure Lengend Snippet: Growth curves of WT and ΔhisR strains of Fusobacterium nucleatum ATCC 23726. A , comparison of growth of F. nucleatum ATCC 23726 WT and ΔhisR . B , comparison of growth of F. nucleatum ATCC 23726 WT and ΔhisR in the presence of 1.5 mM D- or L-histidine. C , comparison of growth of F. nucleatum ATCC 23726 WT and ΔhisR in regular or metal ion-depleted media. ATCC, American Type Culture Collection; HisR, histidine racemase.

Article Snippet: A , comparison of growth of F. nucleatum ATCC 23726 WT and ΔhisR .

Techniques: Comparison

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A) Heat map showing percent identity of NanA homologs in sequenced F. nucleatum strains to the sialic acid lyase of E. coli MG1655. Homologs showed high similarity to amino acid sequence of lyase from F. nucleatum 23726 . (B) Amount of sialic acid remaining in the growth medium 24-48 hours post inoculation of different F. nucleatum strains (as indicated), relative to un-inoculated media control. All bacteria were grown in medium supplemented with free sialic acid (Neu5Ac, 100 μM). Error bars shown are standard deviation of the mean, from three or more experiments.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Sequencing, Control, Bacteria, Standard Deviation

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A-B) E. coli MG1655 ΔnanA was complemented with empty vector, E. coli nanA or putative nanA from F. nucleatum 23726. F.n. = F. nucleatum ; E.c. = E. coli . (A) Growth was assessed in minimal media with sialic acid by measuring absorbance at 600 nm. (B) Lysates of E. coli nanA mutant with empty vector or the complemented strain were incubated with Neu5Ac and its disappearance was monitored over time by DMB-HPLC. (C) Concentration of sialic acid (Neu5Ac) remaining in the medium 24 h post inoculation. Sialic acid consumption by F. nucleatum 23726 wild-type (WT) and ΩsiaT was studied in complete growth medium supplemented with free sialic acid. Data shown is representative of two independent experiments. Error bars show standard deviation from the mean value. (D) Growth of F. nucleatum ( F. nuc.) WT and ΩsiaT in low carbohydrate (carb) medium with or without supplementation with the indicated carbohydrates. Data shown is representative of two independent experiments. (E) Cell associated sialidase activity of anaerobically cultured G. vaginalis JCP8151B and F. nucleatum ATCC23726 strains was analyzed using fluorogenic 4MU-Neu5Ac substrate. (F) F. nucleatum was grown anaerobically in media that was either untreated or exposed to purified sialidase from Arthrobacter ureafaciens ( A.u.) or G. vaginalis ( G.v.). Total and free sialic acid content was measured at 0 and 48 h post inoculation. Bound sialic acids (Bound = Total - Free) are inaccessible to F. nucleatum , except in the presence of exogenous sialidase. Data shown are representative of at least two technical replicates per condition performed in two or more independent experiments. Error bars represent standard deviation from the mean value. See also , and .

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Plasmid Preparation, Mutagenesis, Incubation, Concentration Assay, Standard Deviation, Activity Assay, Cell Culture, Purification

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: Sialidase-producers in the vaginal microbial community release free sialic acids (red diamonds) from host glyco-conjugates, which may be accessed by F. nucleatum , which does not produce sialidase. SiaT = sialic acid transporter; NanA = N -acetylneuraminate lyase; More information about F. nucleatum genes shown in sialic acid catabolic gene cluster, and the enzymes they encode, can be found in .

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques:

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet:

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques:

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A) Schematic showing integration of plasmid containing siaT insert (pLR25) into the F. nucleatum chromosome. Positions of forward (Fwd) and reverse (Rev) primers used for confirming integration of plasmid are also indicated. (B) Agarose gel image with the expected PCR product confirming integration of plasmid into the siaT locus.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Plasmid Preparation, Agarose Gel Electrophoresis

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: Relative amount of bound (bound = total-free) and free sialic acid remaining in the medium at 0 and 48 hours post inoculation. F. nucleatum was grown anaerobically in media that was either untreated or exposed to purified sialidase from Arthrobacter ureafaciens ( A.u. ) or P. bivia (P.b.) ATCC 29303. Bound sialic acids are inaccessible to F. nucleatum except in the presence of exogenous sialidase. Error bars represent standard deviation of the mean value.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Purification, Standard Deviation

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: Mouse vaginal microbial communities-collection and amplification. Step 1 - *Collection of mouse vaginal washes. Washes were pooled from mice co-housed in the same cage. A portion of vaginal wash pools was cultured overnight anaerobically (referred to as “microbiota pools”) to amplify the vaginal bacteria and frozen for subsequent use. Remaining pooled material was stored at −20°C. Step 2 - On the day of the experiment, frozen microbiota pools were used to recover mouse vaginal bacteria by streaking out on Columbia blood plates in anaerobic chamber and incubating for 24 h at 37°C. Colonies from these plates were re-suspended in liquid media either (a) for DNA extraction, or (b) for co-culture experiments with F. nucleatum . *Publication of this animal image was approved by IACUC.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Amplification, Cell Culture, Bacteria, DNA Extraction, Co-Culture Assay

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: Heat map showing the log10 abundance of different OTUs in the microbiota pools of Jackson (Jax, 1-4) mice and Envigo (Envi, 5-8) collected before estrogenization (BE), after estrogenization (AE), and at 1 day post inoculation (dpi) with F. nucleatum . Microbiome analysis was done on uncultured and cultured vaginal washes pooled from 5 mice in the same cage. Each column represents one microbiota pool = 1 cage = 5 mice, total = 4 microbiota pools per vendor per condition. OTUs were assigned using the UNOISE algorithm. Data was clustered using hierarchical Euclidean clustering in the R project for statistical computing.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A) Heat map shows abundance of OTUs in uncultured pooled vaginal specimens (1 pool = 1 cage = 5 mice) collected 1-day post inoculation with F. nucleatum . OTUs were assigned using the UPARSE-OTU algorithm. Data was clustered using hierarchical Euclidean clustering. (B, C) F. nucleatum titers in vaginal wash collected at indicated time points post inoculation (B) in mice from Envigo, and (C) in mice from Jackson. ** P < 0.01, Mann-Whitney. (D-G) Course of C57BL/6 mice (Envigo and Jackson) colonization with the F. nucleatum wild-type (WT) and ΩsiaT mutant. Number of mice colonized in percent ( y axis) was monitored on day 1 and every 2 days for 38 days (x axis). Statistically significance assessed by Log Rank test, * P < 0.05. (D) Comparison of WT vs. ΩsiaT colonization in Jackson mice. (E) Comparison of WT vs. ΩsiaT colonization in Envigo mice. (F) Comparison of WT colonization in Envigo vs. Jackson mice. (G) Comparison of ΩsiaT colonization in Envigo vs. Jackson mice. For Kaplan-Meier analysis, mice were considered cleared when no cfu were detected in undiluted wash at 2 consecutive time points. The graphs represent combined data from two experiments performed separately. OTU = operational taxonomic unit, hpi = hours post inoculation. See also .

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: MANN-WHITNEY, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A, B) F. nucleatum titers in vaginal wash collected from individual mice. (A) WT and ΩsiaT titers in mice from Envigo facility from 1 to 8 dpi. (B) WT and ΩsiaT titers in mice from Jackson facility from 1 to 8 dpi. Data is combined from 2 independent experiments, dpi = days post inoculation. Each experiment had 10 mice per group.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques:

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A-B) Sialidase activity in vaginal washes from individual animals purchased from Envigo, estrogenized, and inoculated with E. coli , or F. nucleatum from 1 to 8 dpi. (C) Sialidase activity (after estrogenization) in vaginal wash pooled specimens (1 microbiota pool = 1 cage = 5 mice) from Jackson and Envigo mice, cultured with or without F. nucleatum (WT or ΩsiaT ). No sialidase activity was observed in cultured vaginal microbial communities from Jackson mice, both with and without F. nucleatum . Data shown here is combined from 2 independent experiments.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Activity Assay, Cell Culture

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A) Sialidase activity in vaginal washes from 1 to 8 dpi from individual animals purchased from Envigo, estrogenized, and inoculated with either WT or ΩsiaT F. nucleatum . Data at later time points were compared to day 1 values using Friedman test, along with correction for multiple planned comparisons using Dunn’s test. (B) Same experiment and data as shown in A but analyzed to compare between WT or ΩsiaT- inoculated animals at each time point using the Mann-Whitney test. Data in A and B represent combined data from two independent experiments. Data points with negative values were set to 0.001 to represent them on the log scale. (C) Sialidase activity in microbiota pools from Envigo mice. Communities were cultured in the presence or absence of F. nucleatum ( F. nuc. ) WT or ΩsiaT . Each “microbiota pool” consists of a cultured vaginal community from pooled vaginal wash of 4-5 co-housed mice. Wilcoxon paired-sign rank test was used for pairwise comparison of sialidase activity in each cultured microbiome compared to the identical microbiome cultured in the presence of F. nucleatum . Data shown is combined from 4 independent biological replicates with 7-8 technical replicates for each microbiota pool. (D) Same experimental data as in C . Data shown is combined from all microbiota pools for each group and analyzed to allow for comparison of the relative sialidase boost between no added F. nucleatum ( F.n. ) versus addition of WT or ΩsiaT . A statistical comparison between the two groups was performed using Wilcoxon matched-pairs signed rank test. Data shown is combined from 3 independent biological replicates. Line in the bar indicates mean value. On all graphs *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. See also .

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Activity Assay, MANN-WHITNEY, Cell Culture, Comparison

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: Human vaginal microbial communities-collection and amplification. Step 1 - Anaerobic and aerobic vaginal swabs were collected on the same day from each participant. Aerobic swabs were eluted in sodium acetate buffer (pH 5.5) and sialidase activity was checked in the swab eluates using fluorogenic 4MU-Neu5Ac substrate. Anaerobic swabs were eluted in 2X NYCIII media (in an anaerobic chamber) and the communities were “fresh frozen”, without any amplification / overnight culture , by mixing with cryoprotectant and storing at −80°C. Step 2 - On the day of the experiment - fresh frozen anaerobic vaginal communities, from women who had detectable sialidase activity in their aerobic swab eluates, were thawed at 4°C and diluted 4-fold in NYCIII media (in an anaerobic chamber). The diluted communities were used for co-culture experiments with F. nucleatum .

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Amplification, Activity Assay, Co-Culture Assay

Journal: bioRxiv

Article Title: Glycan cross-feeding drives mutualism between Fusobacterium and the vaginal microbiota

doi: 10.1101/463349

Figure Lengend Snippet: (A) Human vaginal communities were cultivated anaerobically in Columbia media in the presence or absence of added F. nucleatum . Sialidase activity was measured following anaerobic culture. Communities from 21 individual women were used. Data are combined from 2 independent experiments. A statistical comparison between the two groups was performed using Wilcoxon matched-pairs signed rank test. Negative values were set to 0.0018 (lowest positive value) to depict them on the log scale. (B-C) G. vaginalis ( G.v. ) was co-cultivated anaerobically in Columbia media in the presence or absence of F. nucleatum ( F.n. ), followed by measurement of sialidase activity (B) and viable titers of G. vaginalis (colony forming units, C). Note that G. vaginalis was not detectable under these conditions in the absence of F. nucleatum. In this case, G. vaginalis levels were plotted at one half the limit of detection (LOD=200 CFU/mL). Heat map data is representative of two independent experiments. CFU data is combined from two independent experiments, each with 3 technical replicates each. On all graphs ***P<0.001, ****P<0.0001.

Article Snippet: The prolonged colonization of WT F. nucleatum in Envigo mice suggests that the ability to take up and catabolize sialic acid sustains F. nucleatum vaginal colonization in the sialidase-positive environment.

Techniques: Activity Assay, Comparison