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    ATCC t forsythia atcc 43037
    (A) Coomassie Brilliant Blue staining of crude cell extracts from T. forsythia <t>ATCC</t> 43037 wild-type and glycosyltransferase-deficient mutants after separation on a 7.5% SDS-PA gel. The S-layer glycoproteins (labeled TfsA and TfsB) are indicated and the downshifts resulting from glycan truncation can be seen in the mutants. S-layer glycoprotein bands were further processed for MS analyses. PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a protein molecular weight marker. (B) Western-blots probed with α-TfsA and α-TfsB antiserum for confirmation of the identity of S-layer glycoproteins. Glycoproteins from all glycosyltransferase-deficient mutants (Δ gtfSMILE ) experienced a downshift resulting from glycan truncation, whereas the reconstituted strains (denoted with +) regained wild-type migration, indicating the presence of the complete mature glycan, proving successful recombination. (C, i–vi) ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia wild-type and mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z ). O -Glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications.
    T Forsythia Atcc 43037, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    ATCC t forsythia 92a2
    (A) Coomassie Brilliant Blue staining of crude cell extracts from T. forsythia <t>ATCC</t> 43037 wild-type and glycosyltransferase-deficient mutants after separation on a 7.5% SDS-PA gel. The S-layer glycoproteins (labeled TfsA and TfsB) are indicated and the downshifts resulting from glycan truncation can be seen in the mutants. S-layer glycoprotein bands were further processed for MS analyses. PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a protein molecular weight marker. (B) Western-blots probed with α-TfsA and α-TfsB antiserum for confirmation of the identity of S-layer glycoproteins. Glycoproteins from all glycosyltransferase-deficient mutants (Δ gtfSMILE ) experienced a downshift resulting from glycan truncation, whereas the reconstituted strains (denoted with +) regained wild-type migration, indicating the presence of the complete mature glycan, proving successful recombination. (C, i–vi) ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia wild-type and mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z ). O -Glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications.
    T Forsythia 92a2, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ATCC t forsythia atcc 700198
    (A) Coomassie Brilliant Blue staining of crude cell extracts from T. forsythia <t>ATCC</t> 43037 wild-type and glycosyltransferase-deficient mutants after separation on a 7.5% SDS-PA gel. The S-layer glycoproteins (labeled TfsA and TfsB) are indicated and the downshifts resulting from glycan truncation can be seen in the mutants. S-layer glycoprotein bands were further processed for MS analyses. PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a protein molecular weight marker. (B) Western-blots probed with α-TfsA and α-TfsB antiserum for confirmation of the identity of S-layer glycoproteins. Glycoproteins from all glycosyltransferase-deficient mutants (Δ gtfSMILE ) experienced a downshift resulting from glycan truncation, whereas the reconstituted strains (denoted with +) regained wild-type migration, indicating the presence of the complete mature glycan, proving successful recombination. (C, i–vi) ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia wild-type and mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z ). O -Glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications.
    T Forsythia Atcc 700198, supplied by ATCC, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (A) Coomassie Brilliant Blue staining of crude cell extracts from T. forsythia ATCC 43037 wild-type and glycosyltransferase-deficient mutants after separation on a 7.5% SDS-PA gel. The S-layer glycoproteins (labeled TfsA and TfsB) are indicated and the downshifts resulting from glycan truncation can be seen in the mutants. S-layer glycoprotein bands were further processed for MS analyses. PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a protein molecular weight marker. (B) Western-blots probed with α-TfsA and α-TfsB antiserum for confirmation of the identity of S-layer glycoproteins. Glycoproteins from all glycosyltransferase-deficient mutants (Δ gtfSMILE ) experienced a downshift resulting from glycan truncation, whereas the reconstituted strains (denoted with +) regained wild-type migration, indicating the presence of the complete mature glycan, proving successful recombination. (C, i–vi) ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia wild-type and mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z ). O -Glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications.

    Journal: Frontiers in Microbiology

    Article Title: A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

    doi: 10.3389/fmicb.2018.02008

    Figure Lengend Snippet: (A) Coomassie Brilliant Blue staining of crude cell extracts from T. forsythia ATCC 43037 wild-type and glycosyltransferase-deficient mutants after separation on a 7.5% SDS-PA gel. The S-layer glycoproteins (labeled TfsA and TfsB) are indicated and the downshifts resulting from glycan truncation can be seen in the mutants. S-layer glycoprotein bands were further processed for MS analyses. PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a protein molecular weight marker. (B) Western-blots probed with α-TfsA and α-TfsB antiserum for confirmation of the identity of S-layer glycoproteins. Glycoproteins from all glycosyltransferase-deficient mutants (Δ gtfSMILE ) experienced a downshift resulting from glycan truncation, whereas the reconstituted strains (denoted with +) regained wild-type migration, indicating the presence of the complete mature glycan, proving successful recombination. (C, i–vi) ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia wild-type and mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z ). O -Glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications.

    Article Snippet: To investigate the biosynthesis of the T. forsythia O -glycan, in a previous study, parts of an S-layer protein O -glycosylation gene locus were identified in the T. forsythia ATCC 43037 genome, and clustering of highly homologous genes was observed in different Bacteroidetes species ( ).

    Techniques: Staining, Labeling, Mass Spectrometry, Molecular Weight, Marker, Western Blot, Migration

    Alignment of protein O- glycosylation gene clusters from different T. forsythia strains showing comparable sizes and gene organizations (drawn to scale). Genes showing sequence identity > 50% and sequence coverage > 50% between strains appear in the same color. The major difference in the analyzed strains are for genes synthesizing either CMP-Pse ( pseBCFHGI ; light green colors; strains ATCC 43037 and UB20) or CMP-Leg ( legBCHIGF, ptmE ; dark green colors; strains FDC 92A2, UB4, KS16, UB22). Genes encoding Gtfs ( gtfSMILE ; blue color), Mtfs ( mtfJOY ; yellow color) and carbohydrate modifying enzymes ( asnB, wecC, wecB ; gray color) show high sequence homology between analyzed strains. Genomes of all strains synthesizing CMP-Leg encode an additional putative Mtf gene ( mtfX ), which does not share sequence homology to other Mtfs located within the cluster. In strain UB22, mtfJ is not predicted and for strain 3313 only five out of seven genes needed for the synthesis of CMP-Leg are predicted confidently. Due to low homology, isolate Tannerella sp. HOT-286 (phylotype BU063) could not be aligned with the other T. forsythia strains; for that isolate, the genomic area between a wzx -like gene and the gtfE gene is shown for comparison. P, Pse transferase; L, Leg transferase; HP, hypothetical protein; the star symbol ( ∗ ) indicates a transposable element; genes written in bold letters were investigated in detail in course of this study.

    Journal: Frontiers in Microbiology

    Article Title: A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

    doi: 10.3389/fmicb.2018.02008

    Figure Lengend Snippet: Alignment of protein O- glycosylation gene clusters from different T. forsythia strains showing comparable sizes and gene organizations (drawn to scale). Genes showing sequence identity > 50% and sequence coverage > 50% between strains appear in the same color. The major difference in the analyzed strains are for genes synthesizing either CMP-Pse ( pseBCFHGI ; light green colors; strains ATCC 43037 and UB20) or CMP-Leg ( legBCHIGF, ptmE ; dark green colors; strains FDC 92A2, UB4, KS16, UB22). Genes encoding Gtfs ( gtfSMILE ; blue color), Mtfs ( mtfJOY ; yellow color) and carbohydrate modifying enzymes ( asnB, wecC, wecB ; gray color) show high sequence homology between analyzed strains. Genomes of all strains synthesizing CMP-Leg encode an additional putative Mtf gene ( mtfX ), which does not share sequence homology to other Mtfs located within the cluster. In strain UB22, mtfJ is not predicted and for strain 3313 only five out of seven genes needed for the synthesis of CMP-Leg are predicted confidently. Due to low homology, isolate Tannerella sp. HOT-286 (phylotype BU063) could not be aligned with the other T. forsythia strains; for that isolate, the genomic area between a wzx -like gene and the gtfE gene is shown for comparison. P, Pse transferase; L, Leg transferase; HP, hypothetical protein; the star symbol ( ∗ ) indicates a transposable element; genes written in bold letters were investigated in detail in course of this study.

    Article Snippet: To investigate the biosynthesis of the T. forsythia O -glycan, in a previous study, parts of an S-layer protein O -glycosylation gene locus were identified in the T. forsythia ATCC 43037 genome, and clustering of highly homologous genes was observed in different Bacteroidetes species ( ).

    Techniques: Sequencing

    ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia ATCC 43037 methyltransferase knock-out mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z ). Other O -glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications. The lack of methyl modifications is indicated by a red circle in the symbolic O -glycan structure representation.

    Journal: Frontiers in Microbiology

    Article Title: A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

    doi: 10.3389/fmicb.2018.02008

    Figure Lengend Snippet: ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia ATCC 43037 methyltransferase knock-out mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z ). Other O -glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications. The lack of methyl modifications is indicated by a red circle in the symbolic O -glycan structure representation.

    Article Snippet: To investigate the biosynthesis of the T. forsythia O -glycan, in a previous study, parts of an S-layer protein O -glycosylation gene locus were identified in the T. forsythia ATCC 43037 genome, and clustering of highly homologous genes was observed in different Bacteroidetes species ( ).

    Techniques: Mass Spectrometry, Knock-Out

    Model for the biosynthesis of the species-specific portion of the T. forsythia ATCC 43037 O -glycan. Upon synthesis of the pentasaccharide core on an undP lipid carrier, the first carbohydrate residue of the species-specific glycan is a Fuc residue conferred by GtfE. The glycan is elongated with a Gal residue which is transferred by GtfL and methylated by MtfY. The assembly of the three sugar branch, consisting of a ManNAcA residue (transferred by GtfI), a ManNAcCONH 2 residue (GtfM), which is methylated by either MtfJ or MtfO, and a Pse5Am7Gra residue (transferred via GtfS), completes the synthesis of the decasaccharide.

    Journal: Frontiers in Microbiology

    Article Title: A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

    doi: 10.3389/fmicb.2018.02008

    Figure Lengend Snippet: Model for the biosynthesis of the species-specific portion of the T. forsythia ATCC 43037 O -glycan. Upon synthesis of the pentasaccharide core on an undP lipid carrier, the first carbohydrate residue of the species-specific glycan is a Fuc residue conferred by GtfE. The glycan is elongated with a Gal residue which is transferred by GtfL and methylated by MtfY. The assembly of the three sugar branch, consisting of a ManNAcA residue (transferred by GtfI), a ManNAcCONH 2 residue (GtfM), which is methylated by either MtfJ or MtfO, and a Pse5Am7Gra residue (transferred via GtfS), completes the synthesis of the decasaccharide.

    Article Snippet: To investigate the biosynthesis of the T. forsythia O -glycan, in a previous study, parts of an S-layer protein O -glycosylation gene locus were identified in the T. forsythia ATCC 43037 genome, and clustering of highly homologous genes was observed in different Bacteroidetes species ( ).

    Techniques: Methylation

    (A) Coomassie Brilliant Blue staining of crude cell extracts from T. forsythia ATCC 43037 wild-type and glycosyltransferase-deficient mutants after separation on a 7.5% SDS-PA gel. The S-layer glycoproteins (labeled TfsA and TfsB) are indicated and the downshifts resulting from glycan truncation can be seen in the mutants. S-layer glycoprotein bands were further processed for MS analyses. PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a protein molecular weight marker. (B) Western-blots probed with α-TfsA and α-TfsB antiserum for confirmation of the identity of S-layer glycoproteins. Glycoproteins from all glycosyltransferase-deficient mutants (Δ gtfSMILE ) experienced a downshift resulting from glycan truncation, whereas the reconstituted strains (denoted with +) regained wild-type migration, indicating the presence of the complete mature glycan, proving successful recombination. (C, i–vi) ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia wild-type and mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z values) are shown in SNFG representations ( Varki et al., 2015 ). O -Glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications.

    Journal: Frontiers in Microbiology

    Article Title: A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

    doi: 10.3389/fmicb.2018.02008

    Figure Lengend Snippet: (A) Coomassie Brilliant Blue staining of crude cell extracts from T. forsythia ATCC 43037 wild-type and glycosyltransferase-deficient mutants after separation on a 7.5% SDS-PA gel. The S-layer glycoproteins (labeled TfsA and TfsB) are indicated and the downshifts resulting from glycan truncation can be seen in the mutants. S-layer glycoprotein bands were further processed for MS analyses. PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a protein molecular weight marker. (B) Western-blots probed with α-TfsA and α-TfsB antiserum for confirmation of the identity of S-layer glycoproteins. Glycoproteins from all glycosyltransferase-deficient mutants (Δ gtfSMILE ) experienced a downshift resulting from glycan truncation, whereas the reconstituted strains (denoted with +) regained wild-type migration, indicating the presence of the complete mature glycan, proving successful recombination. (C, i–vi) ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia wild-type and mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z values) are shown in SNFG representations ( Varki et al., 2015 ). O -Glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications.

    Article Snippet: Transcription Analysis of the Protein O -Glycosylation Gene Cluster To analyze whether the genes encoded by the protein O- glycosylation gene cluster are transcriptionally linked, total RNA from T. forsythia ATCC 43037 cells was extracted and co-transcription of the relevant genes was analyzed using RT-PCR as outlined in Supplementary Figure .

    Techniques: Staining, Labeling, Mass Spectrometry, Molecular Weight, Marker, Western Blot, Migration

    Alignment of protein O- glycosylation gene clusters from different T. forsythia strains showing comparable sizes and gene organizations (drawn to scale). Genes showing sequence identity > 50% and sequence coverage > 50% between strains appear in the same color. The major difference in the analyzed strains are for genes synthesizing either CMP-Pse ( pseBCFHGI ; light green colors; strains ATCC 43037 and UB20) or CMP-Leg ( legBCHIGF, ptmE ; dark green colors; strains FDC 92A2, UB4, KS16, UB22). Genes encoding Gtfs ( gtfSMILE ; blue color), Mtfs ( mtfJOY ; yellow color) and carbohydrate modifying enzymes ( asnB, wecC, wecB ; gray color) show high sequence homology between analyzed strains. Genomes of all strains synthesizing CMP-Leg encode an additional putative Mtf gene ( mtfX ), which does not share sequence homology to other Mtfs located within the cluster. In strain UB22, mtfJ is not predicted and for strain 3313 only five out of seven genes needed for the synthesis of CMP-Leg are predicted confidently. Due to low homology, isolate Tannerella sp. HOT-286 (phylotype BU063) could not be aligned with the other T. forsythia strains; for that isolate, the genomic area between a wzx -like gene and the gtfE gene is shown for comparison. P, Pse transferase; L, Leg transferase; HP, hypothetical protein; the star symbol ( ∗ ) indicates a transposable element; genes written in bold letters were investigated in detail in course of this study.

    Journal: Frontiers in Microbiology

    Article Title: A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

    doi: 10.3389/fmicb.2018.02008

    Figure Lengend Snippet: Alignment of protein O- glycosylation gene clusters from different T. forsythia strains showing comparable sizes and gene organizations (drawn to scale). Genes showing sequence identity > 50% and sequence coverage > 50% between strains appear in the same color. The major difference in the analyzed strains are for genes synthesizing either CMP-Pse ( pseBCFHGI ; light green colors; strains ATCC 43037 and UB20) or CMP-Leg ( legBCHIGF, ptmE ; dark green colors; strains FDC 92A2, UB4, KS16, UB22). Genes encoding Gtfs ( gtfSMILE ; blue color), Mtfs ( mtfJOY ; yellow color) and carbohydrate modifying enzymes ( asnB, wecC, wecB ; gray color) show high sequence homology between analyzed strains. Genomes of all strains synthesizing CMP-Leg encode an additional putative Mtf gene ( mtfX ), which does not share sequence homology to other Mtfs located within the cluster. In strain UB22, mtfJ is not predicted and for strain 3313 only five out of seven genes needed for the synthesis of CMP-Leg are predicted confidently. Due to low homology, isolate Tannerella sp. HOT-286 (phylotype BU063) could not be aligned with the other T. forsythia strains; for that isolate, the genomic area between a wzx -like gene and the gtfE gene is shown for comparison. P, Pse transferase; L, Leg transferase; HP, hypothetical protein; the star symbol ( ∗ ) indicates a transposable element; genes written in bold letters were investigated in detail in course of this study.

    Article Snippet: Transcription Analysis of the Protein O -Glycosylation Gene Cluster To analyze whether the genes encoded by the protein O- glycosylation gene cluster are transcriptionally linked, total RNA from T. forsythia ATCC 43037 cells was extracted and co-transcription of the relevant genes was analyzed using RT-PCR as outlined in Supplementary Figure .

    Techniques: Sequencing

    ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia ATCC 43037 methyltransferase knock-out mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z values) are shown in SNFG representation ( Varki et al., 2015 ). Other O -glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications. The lack of methyl modifications is indicated by a red circle in the symbolic O -glycan structure representation.

    Journal: Frontiers in Microbiology

    Article Title: A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

    doi: 10.3389/fmicb.2018.02008

    Figure Lengend Snippet: ESI-MS sum spectra of β-eliminated TfsB O -glycans from T. forsythia ATCC 43037 methyltransferase knock-out mutants. The glycan structures of the signals corresponding to the largest mass (bold m / z values) are shown in SNFG representation ( Varki et al., 2015 ). Other O -glycan signals detected for the respective mutants were assigned based on the m / z mass differences corresponding to the loss of individual sugar units and/or modifications. The lack of methyl modifications is indicated by a red circle in the symbolic O -glycan structure representation.

    Article Snippet: Transcription Analysis of the Protein O -Glycosylation Gene Cluster To analyze whether the genes encoded by the protein O- glycosylation gene cluster are transcriptionally linked, total RNA from T. forsythia ATCC 43037 cells was extracted and co-transcription of the relevant genes was analyzed using RT-PCR as outlined in Supplementary Figure .

    Techniques: Mass Spectrometry, Knock-Out

    (A) Scheme of the T. forsythia ATCC 43037 S-layer O -glycan structure. Monosaccharide symbols are shown according to the Symbol Nomenclature for Glycans (SNFG) ( Varki et al., 2015 ). Please note that the position of the branching Fuc remained unclear ( Posch et al., 2011 ) until it was determined in the course of this study to be on the reducing-end Gal. (B) Scheme of the 27-kb protein O -glycosylation gene cluster of T. forsythia ATCC 43037. Wzx (black), flippase; pseBCFHGI (green), CMP-Pse biosynthesis genes; gtfSMILE (blue), Gtf genes; mtfJOY (yellow), Mtf genes; asnB (putative asparagine synthetase B), wecC (UDP- N -acetyl- D -mannosamine dehydrogenase) and wecB (UDP- N- acetylglucosamine 2-epimerase) (purple); hypothetical proteins, HP (gray). Genes are not drawn to scale.

    Journal: Frontiers in Microbiology

    Article Title: A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

    doi: 10.3389/fmicb.2018.02008

    Figure Lengend Snippet: (A) Scheme of the T. forsythia ATCC 43037 S-layer O -glycan structure. Monosaccharide symbols are shown according to the Symbol Nomenclature for Glycans (SNFG) ( Varki et al., 2015 ). Please note that the position of the branching Fuc remained unclear ( Posch et al., 2011 ) until it was determined in the course of this study to be on the reducing-end Gal. (B) Scheme of the 27-kb protein O -glycosylation gene cluster of T. forsythia ATCC 43037. Wzx (black), flippase; pseBCFHGI (green), CMP-Pse biosynthesis genes; gtfSMILE (blue), Gtf genes; mtfJOY (yellow), Mtf genes; asnB (putative asparagine synthetase B), wecC (UDP- N -acetyl- D -mannosamine dehydrogenase) and wecB (UDP- N- acetylglucosamine 2-epimerase) (purple); hypothetical proteins, HP (gray). Genes are not drawn to scale.

    Article Snippet: Transcription Analysis of the Protein O -Glycosylation Gene Cluster To analyze whether the genes encoded by the protein O- glycosylation gene cluster are transcriptionally linked, total RNA from T. forsythia ATCC 43037 cells was extracted and co-transcription of the relevant genes was analyzed using RT-PCR as outlined in Supplementary Figure .

    Techniques:

    Model for the biosynthesis of the species-specific portion of the T. forsythia ATCC 43037 O -glycan. Upon synthesis of the pentasaccharide core on an undP lipid carrier, the first carbohydrate residue of the species-specific glycan is a Fuc residue conferred by GtfE. The glycan is elongated with a Gal residue which is transferred by GtfL and methylated by MtfY. The assembly of the three sugar branch, consisting of a ManNAcA residue (transferred by GtfI), a ManNAcCONH 2 residue (GtfM), which is methylated by either MtfJ or MtfO, and a Pse5Am7Gra residue (transferred via GtfS), completes the synthesis of the decasaccharide.

    Journal: Frontiers in Microbiology

    Article Title: A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

    doi: 10.3389/fmicb.2018.02008

    Figure Lengend Snippet: Model for the biosynthesis of the species-specific portion of the T. forsythia ATCC 43037 O -glycan. Upon synthesis of the pentasaccharide core on an undP lipid carrier, the first carbohydrate residue of the species-specific glycan is a Fuc residue conferred by GtfE. The glycan is elongated with a Gal residue which is transferred by GtfL and methylated by MtfY. The assembly of the three sugar branch, consisting of a ManNAcA residue (transferred by GtfI), a ManNAcCONH 2 residue (GtfM), which is methylated by either MtfJ or MtfO, and a Pse5Am7Gra residue (transferred via GtfS), completes the synthesis of the decasaccharide.

    Article Snippet: Transcription Analysis of the Protein O -Glycosylation Gene Cluster To analyze whether the genes encoded by the protein O- glycosylation gene cluster are transcriptionally linked, total RNA from T. forsythia ATCC 43037 cells was extracted and co-transcription of the relevant genes was analyzed using RT-PCR as outlined in Supplementary Figure .

    Techniques: Methylation

    Dual fluorescence in situ hybridization staining of Tannerella forsythia and Campylobacter rectus for biofilms harboring ATCC 43037 wild‐type (A), UB 4 wild‐type (B), and ATCC 43037 ∆tfs AB (C). Red/yellow: T. forsythia , cyan: C. rectus; green: non‐hybridized cells ( DNA staining YoPro‐1+Sytox). Scale bars 20 μm (A, B) and 15 μm (C)

    Journal: Molecular Oral Microbiology

    Article Title: Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms. Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms

    doi: 10.1111/omi.12182

    Figure Lengend Snippet: Dual fluorescence in situ hybridization staining of Tannerella forsythia and Campylobacter rectus for biofilms harboring ATCC 43037 wild‐type (A), UB 4 wild‐type (B), and ATCC 43037 ∆tfs AB (C). Red/yellow: T. forsythia , cyan: C. rectus; green: non‐hybridized cells ( DNA staining YoPro‐1+Sytox). Scale bars 20 μm (A, B) and 15 μm (C)

    Article Snippet: Based on the analysis of planktonic and monospecies biofilm growth, we employed in this study the subgingival “Zurich biofilm model” to investigate how the T. forsythia wild‐type strains ATCC 43037 and UB4 and defined cell surface mutants thereof perform in a multispecies consortium.

    Techniques: Fluorescence, In Situ Hybridization, Staining

    Fluorescence in situ hybridization staining of biofilms harboring Tannerella forsythia ATCC 43037 mutants (A) ∆pseC , (B) ∆wecC , and (C) ∆tfs AB . Red: T. forsythia , cyan: Porphyromonas gingivalis , green: non‐hybridized cells ( DNA staining YoPro‐1+Sytox). Scale bars 20 μm (A) and 10 μm (B, C)

    Journal: Molecular Oral Microbiology

    Article Title: Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms. Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms

    doi: 10.1111/omi.12182

    Figure Lengend Snippet: Fluorescence in situ hybridization staining of biofilms harboring Tannerella forsythia ATCC 43037 mutants (A) ∆pseC , (B) ∆wecC , and (C) ∆tfs AB . Red: T. forsythia , cyan: Porphyromonas gingivalis , green: non‐hybridized cells ( DNA staining YoPro‐1+Sytox). Scale bars 20 μm (A) and 10 μm (B, C)

    Article Snippet: Based on the analysis of planktonic and monospecies biofilm growth, we employed in this study the subgingival “Zurich biofilm model” to investigate how the T. forsythia wild‐type strains ATCC 43037 and UB4 and defined cell surface mutants thereof perform in a multispecies consortium.

    Techniques: Fluorescence, In Situ Hybridization, Staining

    Box plots showing cell numbers of all species determined by quantitative real‐time PCR for biofilms with Tannerella forsythia ATCC 43037 wild‐type or mutants (∆pseC , ∆wecC , ∆tfs AB , ∆pseC comp ) (A) and UB 4 wild‐type or mutants (∆legC , ∆legC comp ), respectively (B). Data derived from three independent experiments were plotted on a logarithmic scale. Asterisk (*) indicates significant differences ( P ≤.05) between the groups

    Journal: Molecular Oral Microbiology

    Article Title: Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms. Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms

    doi: 10.1111/omi.12182

    Figure Lengend Snippet: Box plots showing cell numbers of all species determined by quantitative real‐time PCR for biofilms with Tannerella forsythia ATCC 43037 wild‐type or mutants (∆pseC , ∆wecC , ∆tfs AB , ∆pseC comp ) (A) and UB 4 wild‐type or mutants (∆legC , ∆legC comp ), respectively (B). Data derived from three independent experiments were plotted on a logarithmic scale. Asterisk (*) indicates significant differences ( P ≤.05) between the groups

    Article Snippet: Based on the analysis of planktonic and monospecies biofilm growth, we employed in this study the subgingival “Zurich biofilm model” to investigate how the T. forsythia wild‐type strains ATCC 43037 and UB4 and defined cell surface mutants thereof perform in a multispecies consortium.

    Techniques: Real-time Polymerase Chain Reaction, Derivative Assay

    Comparison of 10‐species biofilms with two Tannerella forsythia wild‐type strains. (A) Whiskers boxplots (5th to 95th centile) show bacterial numbers determined by quantitative real‐time PCR from three independent experiments. Asterisk (*) indicates a statistically significant difference ( P ≤.05) between groups. The two groups represent biofilms with either T. forsythia ATCC 43037 wild‐type or T. forsythia UB 4 wild‐type. (B, C) Fluorescence in situ hybridization stainings of fixed biofilms showing the localization of ATCC 43037 wild‐type (B) and UB 4 wild‐type (C). Red/yellow: T. forsythia; cyan: Porphyromonas gingivalis , green: non‐hybridized cells ( DNA staining YoPro‐1+Sytox). Here a representative area for one disk each is shown with a top view in the left panel and a side view with the biofilm–disk interface directed towards the top view; scale bars 5 μm (B) and 10 μm (C)

    Journal: Molecular Oral Microbiology

    Article Title: Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms. Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms

    doi: 10.1111/omi.12182

    Figure Lengend Snippet: Comparison of 10‐species biofilms with two Tannerella forsythia wild‐type strains. (A) Whiskers boxplots (5th to 95th centile) show bacterial numbers determined by quantitative real‐time PCR from three independent experiments. Asterisk (*) indicates a statistically significant difference ( P ≤.05) between groups. The two groups represent biofilms with either T. forsythia ATCC 43037 wild‐type or T. forsythia UB 4 wild‐type. (B, C) Fluorescence in situ hybridization stainings of fixed biofilms showing the localization of ATCC 43037 wild‐type (B) and UB 4 wild‐type (C). Red/yellow: T. forsythia; cyan: Porphyromonas gingivalis , green: non‐hybridized cells ( DNA staining YoPro‐1+Sytox). Here a representative area for one disk each is shown with a top view in the left panel and a side view with the biofilm–disk interface directed towards the top view; scale bars 5 μm (B) and 10 μm (C)

    Article Snippet: Based on the analysis of planktonic and monospecies biofilm growth, we employed in this study the subgingival “Zurich biofilm model” to investigate how the T. forsythia wild‐type strains ATCC 43037 and UB4 and defined cell surface mutants thereof perform in a multispecies consortium.

    Techniques: Real-time Polymerase Chain Reaction, Fluorescence, In Situ Hybridization, Staining

    Monospecies biofilm formation of Tannerella forsythia wild‐type and mutant strains. (A) Biofilm formation of T. forsythia ATCC 43037 wild‐type compared with its mutants ATCC 43037 Δ pseC , Δ wecC , Δ tfs AB and the complemented mutant Δ pseC comp . (B) Biofilm formation of T. forsythia UB 4 wild‐type compared with its mutant UB 4 Δ legC and the complemented mutant Δ legC comp . Mean values ± SD of four independent experiments with three replicates, each, are shown. Asterisks (**) indicate significant differences between samples as determined by the unpaired Student's t ‐test ( P ≤.01)

    Journal: Molecular Oral Microbiology

    Article Title: Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms. Behavior of two Tannerella forsythia strains and their cell surface mutants in multispecies oral biofilms

    doi: 10.1111/omi.12182

    Figure Lengend Snippet: Monospecies biofilm formation of Tannerella forsythia wild‐type and mutant strains. (A) Biofilm formation of T. forsythia ATCC 43037 wild‐type compared with its mutants ATCC 43037 Δ pseC , Δ wecC , Δ tfs AB and the complemented mutant Δ pseC comp . (B) Biofilm formation of T. forsythia UB 4 wild‐type compared with its mutant UB 4 Δ legC and the complemented mutant Δ legC comp . Mean values ± SD of four independent experiments with three replicates, each, are shown. Asterisks (**) indicate significant differences between samples as determined by the unpaired Student's t ‐test ( P ≤.01)

    Article Snippet: Based on the analysis of planktonic and monospecies biofilm growth, we employed in this study the subgingival “Zurich biofilm model” to investigate how the T. forsythia wild‐type strains ATCC 43037 and UB4 and defined cell surface mutants thereof perform in a multispecies consortium.

    Techniques: Mutagenesis

    Amplification plots of genomic DNA from lysed cells. Serial dilutions of genomic DNA from F. nucleatum ATCC 10953 (A) or T. forsythensis ATCC 43037 (B) are shown. The log-transformed relative fluorescence [ΔRn (log)] was monitored as the increase in reporter dye intensity relative to that of the passive internal reference dye. The threshold fluorescence, or the level at which the threshold cycle was determined, is shown. The standard curves were generated from the amplification plots in the insets (correlation coefficients, 0.999 for F. nucleatum and 0.994 for T. forsythensis ). Ct is the cycle number at which the threshold fluorescence was reached.

    Journal: Journal of Clinical Microbiology

    Article Title: Quantitative Microbiological Study of Subgingival Plaque by Real-Time PCR Shows Correlation between Levels of Tannerella forsythensis and Fusobacterium spp.

    doi: 10.1128/JCM.42.5.2255-2257.2004

    Figure Lengend Snippet: Amplification plots of genomic DNA from lysed cells. Serial dilutions of genomic DNA from F. nucleatum ATCC 10953 (A) or T. forsythensis ATCC 43037 (B) are shown. The log-transformed relative fluorescence [ΔRn (log)] was monitored as the increase in reporter dye intensity relative to that of the passive internal reference dye. The threshold fluorescence, or the level at which the threshold cycle was determined, is shown. The standard curves were generated from the amplification plots in the insets (correlation coefficients, 0.999 for F. nucleatum and 0.994 for T. forsythensis ). Ct is the cycle number at which the threshold fluorescence was reached.

    Article Snippet: Standard curves for each organism were plotted for each primer-probe set with the Ct values obtained by amplifying successive 10-fold dilutions of a known concentration of DNA extracted from bacterial cells containing 1.2 × 106 CFU (230 ng/μl) for F. nucleatum ATCC 10953 and 4.2 × 105 CFU (370 ng/μl) for T. forsythensis ATCC 43037 (Fig. ).

    Techniques: Amplification, Transformation Assay, Fluorescence, Generated

    Effects of protein O -glycosylation on T. forsythia immunogenicity. (A) Secretion of inflammatory cytokines by human DCs upon stimulation with T. forsythia wild-type (WT) and glycosyltransferase-deficient mutants ( T. forsythia Δ gtfE , Δ gtfI , and Δ gtfS ) as measured in culture supernatants by ProcartaPlex Multiplex Immunoassay. (B,C) T cell-priming upon APC stimulation with T. forsythia wild-type and glycosyltransferase-deficient mutants was assessed by culturing PBMCs. (B) T cell activation was measured via expression of CD25 by flow cytometry. Cells were pre-gated for live CD3 + cells, T cell proliferation was assessed after 8 days by CFSE dilution,. (C) CD4 + T cell differentiation was assessed by expression of signature transcription factors for Th17 (RORγT) and Treg (FoxP3) cells as measured by flow cytometry. All data are presented as mean ± SEM of triplicate determinations. One representative out of three (A) and two (B,C) , respectively, independent experiments is shown. Statistically significant differences in (A) are indicated as ∗ p

    Journal: Frontiers in Microbiology

    Article Title: A General Protein O-Glycosylation Gene Cluster Encodes the Species-Specific Glycan of the Oral Pathogen Tannerella forsythia: O-Glycan Biosynthesis and Immunological Implications

    doi: 10.3389/fmicb.2018.02008

    Figure Lengend Snippet: Effects of protein O -glycosylation on T. forsythia immunogenicity. (A) Secretion of inflammatory cytokines by human DCs upon stimulation with T. forsythia wild-type (WT) and glycosyltransferase-deficient mutants ( T. forsythia Δ gtfE , Δ gtfI , and Δ gtfS ) as measured in culture supernatants by ProcartaPlex Multiplex Immunoassay. (B,C) T cell-priming upon APC stimulation with T. forsythia wild-type and glycosyltransferase-deficient mutants was assessed by culturing PBMCs. (B) T cell activation was measured via expression of CD25 by flow cytometry. Cells were pre-gated for live CD3 + cells, T cell proliferation was assessed after 8 days by CFSE dilution,. (C) CD4 + T cell differentiation was assessed by expression of signature transcription factors for Th17 (RORγT) and Treg (FoxP3) cells as measured by flow cytometry. All data are presented as mean ± SEM of triplicate determinations. One representative out of three (A) and two (B,C) , respectively, independent experiments is shown. Statistically significant differences in (A) are indicated as ∗ p

    Article Snippet: This implicates that a previously described three-gene “exopolysaccharide synthesis operon” spanning Tanf_01280 to Tanf_01290 ( ) is part of the contiguous transcription unit of the T. forsythia ATCC 43037 protein O- glycosylation gene cluster.

    Techniques: Multiplex Assay, Activation Assay, Expressing, Flow Cytometry, Cytometry, Cell Differentiation