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The genetic organization of C. hepaticus <t>HV10</t> <t>T</t> protein N -glycosylation locus . Open reading frames predicted to be involved in sugar biosynthesis are illustrated in green, flippase in orange, glycosyltransferases in yellow, oligosaccharyltransferase in black, and pglG in white, as it has no known function in the C. jejuni N- glycosylation pathway . The red arrow represents the predicted putative promoter of galE .
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The genetic organization of C. hepaticus <t>HV10</t> T protein N -glycosylation locus . Open reading frames predicted to be involved in sugar biosynthesis are illustrated in green, flippase in orange, glycosyltransferases in yellow, oligosaccharyltransferase in black, and pglG in white, as it has no known function in the C. jejuni N- glycosylation pathway . The red arrow represents the predicted putative promoter of galE .
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Image Search Results


The genetic organization of C. hepaticus HV10 T protein N -glycosylation locus . Open reading frames predicted to be involved in sugar biosynthesis are illustrated in green, flippase in orange, glycosyltransferases in yellow, oligosaccharyltransferase in black, and pglG in white, as it has no known function in the C. jejuni N- glycosylation pathway . The red arrow represents the predicted putative promoter of galE .

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: The genetic organization of C. hepaticus HV10 T protein N -glycosylation locus . Open reading frames predicted to be involved in sugar biosynthesis are illustrated in green, flippase in orange, glycosyltransferases in yellow, oligosaccharyltransferase in black, and pglG in white, as it has no known function in the C. jejuni N- glycosylation pathway . The red arrow represents the predicted putative promoter of galE .

Article Snippet: A 96-well High Bind Microplate (Corning) was coated with 50 µL of whole cell lysates containing 200 µg/mL% protein diluted in 0.5 M carbonate buffer from C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan M7, C. hepaticus HV10 T ∆ pglB::kan M7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan M7(pJBM5.2), STM1 (negative control), and 0.5 M carbonate buffer as a blank control.

Techniques:

Efficiency of electro-transformation of C. hepaticus and C. bilis strains with shuttle vector pJBM3 isolated from E. coli NEB 5-alpha or  C. hepaticus HV10 T  <xref ref-type= a " width="100%" height="100%">

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: Efficiency of electro-transformation of C. hepaticus and C. bilis strains with shuttle vector pJBM3 isolated from E. coli NEB 5-alpha or C. hepaticus HV10 T a

Article Snippet: A 96-well High Bind Microplate (Corning) was coated with 50 µL of whole cell lysates containing 200 µg/mL% protein diluted in 0.5 M carbonate buffer from C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan M7, C. hepaticus HV10 T ∆ pglB::kan M7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan M7(pJBM5.2), STM1 (negative control), and 0.5 M carbonate buffer as a blank control.

Techniques: Plasmid Preparation, Isolation, Transformation Assay

Schematic diagram of the construction of pglB mutants by homologous recombination. Relevant chromosomal genes are shown. The diagram illustrates the process by which the wild-type (WT) pglB gene from C. hepaticus HV10 T was deleted by replacement with the aph(3’)-IIIa on pCH_ pglB _SV by a double crossover recombination event utilizing 1,103 and 1,120 bp homologous left and right flanking regions (blue) of pglB, respectively. The primers used to screen for double crossover events are provided in purple on the C. hepaticus HV10 T ∆ pglB::kan chromosome.

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: Schematic diagram of the construction of pglB mutants by homologous recombination. Relevant chromosomal genes are shown. The diagram illustrates the process by which the wild-type (WT) pglB gene from C. hepaticus HV10 T was deleted by replacement with the aph(3’)-IIIa on pCH_ pglB _SV by a double crossover recombination event utilizing 1,103 and 1,120 bp homologous left and right flanking regions (blue) of pglB, respectively. The primers used to screen for double crossover events are provided in purple on the C. hepaticus HV10 T ∆ pglB::kan chromosome.

Article Snippet: A 96-well High Bind Microplate (Corning) was coated with 50 µL of whole cell lysates containing 200 µg/mL% protein diluted in 0.5 M carbonate buffer from C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan M7, C. hepaticus HV10 T ∆ pglB::kan M7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan M7(pJBM5.2), STM1 (negative control), and 0.5 M carbonate buffer as a blank control.

Techniques: Homologous Recombination

The effect of pglB mutagenesis on SBA binding profiles to N- glycans present in C. hepaticus HV10 T ∆ pglB::kan mutants. Mutant 3 was independently derived from mutants 7, 8, 9, and 10. ( A ) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and ( B ) SBA lectin blotting of C. hepaticus HV10 T (WT) and C. hepaticus HV10 T ∆ pglB::kan mutants of whole cell lysates containing 35–40 μg of protein. Whole cell lysates were separated by 8%–16% SDS-PAGE and either developed with SimplyBlue SafeStain or transferred to PVDF membranes for lectin blotting.

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: The effect of pglB mutagenesis on SBA binding profiles to N- glycans present in C. hepaticus HV10 T ∆ pglB::kan mutants. Mutant 3 was independently derived from mutants 7, 8, 9, and 10. ( A ) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and ( B ) SBA lectin blotting of C. hepaticus HV10 T (WT) and C. hepaticus HV10 T ∆ pglB::kan mutants of whole cell lysates containing 35–40 μg of protein. Whole cell lysates were separated by 8%–16% SDS-PAGE and either developed with SimplyBlue SafeStain or transferred to PVDF membranes for lectin blotting.

Article Snippet: A 96-well High Bind Microplate (Corning) was coated with 50 µL of whole cell lysates containing 200 µg/mL% protein diluted in 0.5 M carbonate buffer from C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan M7, C. hepaticus HV10 T ∆ pglB::kan M7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan M7(pJBM5.2), STM1 (negative control), and 0.5 M carbonate buffer as a blank control.

Techniques: Mutagenesis, Binding Assay, Derivative Assay, Polyacrylamide Gel Electrophoresis, SDS Page

Plasmid map of shuttle vector pJBM5.2 used to complement C. hepaticus HV10 T ∆ pglB::kan mutant 7. Genetic symbols represent: Rep, replication initiation protein; HP, hypothetical protein; Mob, mobilization protein; OriV, Campylobacter origin of replication; ampR, Ampicillin resistance gene; ori, E.coli origin of replication from pJBM5; galE putative promoter; pglB, pglA, pglC, pglD, pglE, and pglF from C. hepaticus HV10 T ; and tet(O ), tetracycline resistance gene from C. hepaticus 84B. The position of the primers used to construct pJBM5.2 by Gibson assembly is shown in purple.

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: Plasmid map of shuttle vector pJBM5.2 used to complement C. hepaticus HV10 T ∆ pglB::kan mutant 7. Genetic symbols represent: Rep, replication initiation protein; HP, hypothetical protein; Mob, mobilization protein; OriV, Campylobacter origin of replication; ampR, Ampicillin resistance gene; ori, E.coli origin of replication from pJBM5; galE putative promoter; pglB, pglA, pglC, pglD, pglE, and pglF from C. hepaticus HV10 T ; and tet(O ), tetracycline resistance gene from C. hepaticus 84B. The position of the primers used to construct pJBM5.2 by Gibson assembly is shown in purple.

Article Snippet: A 96-well High Bind Microplate (Corning) was coated with 50 µL of whole cell lysates containing 200 µg/mL% protein diluted in 0.5 M carbonate buffer from C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan M7, C. hepaticus HV10 T ∆ pglB::kan M7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan M7(pJBM5.2), STM1 (negative control), and 0.5 M carbonate buffer as a blank control.

Techniques: Plasmid Preparation, Mutagenesis, Construct

Complementation of C. hepaticus HV10 T ∆ pglB::kan mutant 7 in trans with pJBM5.2, expressing the pgl locus genes including and downstream of pglB and excluding pglG . ( A ) SBA lectin blot binding profiles to N- glycans present in C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan mutant 7, C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM4) (empty vector control), and two clones from separate isogenic complements (clones 1 and 2 were derived independently of clones 3 and 4) of C. hepaticus HV10 T ∆ pglB::kan mutant 7 clones expressing functional pgl genes encoded on pJBM5.2. Whole cell lysates contained 27 µg of protein and were separated by 4%–20% SDS-PAGE and either developed with SimplyBlue SafeStain or transferred to PVDF membranes for lectin blotting. The corresponding SDS-PAGE gel to demonstrate equal loading of proteins across samples is provided in Fig. S8. ( B ) Quantitative analysis of SBA binding to GalNAc present in whole cell lysates. ELISA results represent SBA binding to whole cell lysates of C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan mutant 7, C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM5.2), and Salmonella Typhimurium 82/6915 ∆ aroA (STM1) (negative control for N -glycosylation) measured at OD 450 . Gray bars represent the standard deviation of n = 4 technical replicates for each strain. Statistical differences between groups were analyzed using a one-way ANOVA with Tukey’s post hoc analysis and are indicated as: ns, no statistically significant difference ( P -value > 0.05); and **** indicate statistically significant differences with P -values < 0.0001. For all Tukey’s multiple comparisons, see Table S1.

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: Complementation of C. hepaticus HV10 T ∆ pglB::kan mutant 7 in trans with pJBM5.2, expressing the pgl locus genes including and downstream of pglB and excluding pglG . ( A ) SBA lectin blot binding profiles to N- glycans present in C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan mutant 7, C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM4) (empty vector control), and two clones from separate isogenic complements (clones 1 and 2 were derived independently of clones 3 and 4) of C. hepaticus HV10 T ∆ pglB::kan mutant 7 clones expressing functional pgl genes encoded on pJBM5.2. Whole cell lysates contained 27 µg of protein and were separated by 4%–20% SDS-PAGE and either developed with SimplyBlue SafeStain or transferred to PVDF membranes for lectin blotting. The corresponding SDS-PAGE gel to demonstrate equal loading of proteins across samples is provided in Fig. S8. ( B ) Quantitative analysis of SBA binding to GalNAc present in whole cell lysates. ELISA results represent SBA binding to whole cell lysates of C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan mutant 7, C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM5.2), and Salmonella Typhimurium 82/6915 ∆ aroA (STM1) (negative control for N -glycosylation) measured at OD 450 . Gray bars represent the standard deviation of n = 4 technical replicates for each strain. Statistical differences between groups were analyzed using a one-way ANOVA with Tukey’s post hoc analysis and are indicated as: ns, no statistically significant difference ( P -value > 0.05); and **** indicate statistically significant differences with P -values < 0.0001. For all Tukey’s multiple comparisons, see Table S1.

Article Snippet: A 96-well High Bind Microplate (Corning) was coated with 50 µL of whole cell lysates containing 200 µg/mL% protein diluted in 0.5 M carbonate buffer from C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan M7, C. hepaticus HV10 T ∆ pglB::kan M7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan M7(pJBM5.2), STM1 (negative control), and 0.5 M carbonate buffer as a blank control.

Techniques: Mutagenesis, Expressing, Binding Assay, Plasmid Preparation, Control, Clone Assay, Derivative Assay, Functional Assay, SDS Page, Enzyme-linked Immunosorbent Assay, Negative Control, Standard Deviation

Bacterial strains and plasmids used in this study

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: A 96-well High Bind Microplate (Corning) was coated with 50 µL of whole cell lysates containing 200 µg/mL% protein diluted in 0.5 M carbonate buffer from C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan M7, C. hepaticus HV10 T ∆ pglB::kan M7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan M7(pJBM5.2), STM1 (negative control), and 0.5 M carbonate buffer as a blank control.

Techniques: Plasmid Preparation, Mutagenesis, Clone Assay, Isolation

The genetic organization of C. hepaticus HV10 T protein N -glycosylation locus . Open reading frames predicted to be involved in sugar biosynthesis are illustrated in green, flippase in orange, glycosyltransferases in yellow, oligosaccharyltransferase in black, and pglG in white, as it has no known function in the C. jejuni N- glycosylation pathway . The red arrow represents the predicted putative promoter of galE .

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: The genetic organization of C. hepaticus HV10 T protein N -glycosylation locus . Open reading frames predicted to be involved in sugar biosynthesis are illustrated in green, flippase in orange, glycosyltransferases in yellow, oligosaccharyltransferase in black, and pglG in white, as it has no known function in the C. jejuni N- glycosylation pathway . The red arrow represents the predicted putative promoter of galE .

Article Snippet: Genomic DNA was isolated from C. hepaticus HV10 T WT and mutant strains, grown for 24 h using a Monarch Genomic DNA Purification Kit (NEB) according to the manufacturer’s instructions.

Techniques:

Efficiency of electro-transformation of C. hepaticus and C. bilis strains with shuttle vector pJBM3 isolated from E. coli NEB 5-alpha or  C. hepaticus HV10 T  <xref ref-type= a " width="100%" height="100%">

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: Efficiency of electro-transformation of C. hepaticus and C. bilis strains with shuttle vector pJBM3 isolated from E. coli NEB 5-alpha or C. hepaticus HV10 T a

Article Snippet: Genomic DNA was isolated from C. hepaticus HV10 T WT and mutant strains, grown for 24 h using a Monarch Genomic DNA Purification Kit (NEB) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Isolation, Transformation Assay

Schematic diagram of the construction of pglB mutants by homologous recombination. Relevant chromosomal genes are shown. The diagram illustrates the process by which the wild-type (WT) pglB gene from C. hepaticus HV10 T was deleted by replacement with the aph(3’)-IIIa on pCH_ pglB _SV by a double crossover recombination event utilizing 1,103 and 1,120 bp homologous left and right flanking regions (blue) of pglB, respectively. The primers used to screen for double crossover events are provided in purple on the C. hepaticus HV10 T ∆ pglB::kan chromosome.

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: Schematic diagram of the construction of pglB mutants by homologous recombination. Relevant chromosomal genes are shown. The diagram illustrates the process by which the wild-type (WT) pglB gene from C. hepaticus HV10 T was deleted by replacement with the aph(3’)-IIIa on pCH_ pglB _SV by a double crossover recombination event utilizing 1,103 and 1,120 bp homologous left and right flanking regions (blue) of pglB, respectively. The primers used to screen for double crossover events are provided in purple on the C. hepaticus HV10 T ∆ pglB::kan chromosome.

Article Snippet: Genomic DNA was isolated from C. hepaticus HV10 T WT and mutant strains, grown for 24 h using a Monarch Genomic DNA Purification Kit (NEB) according to the manufacturer’s instructions.

Techniques: Homologous Recombination

The effect of pglB mutagenesis on SBA binding profiles to N- glycans present in C. hepaticus HV10 T ∆ pglB::kan mutants. Mutant 3 was independently derived from mutants 7, 8, 9, and 10. ( A ) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and ( B ) SBA lectin blotting of C. hepaticus HV10 T (WT) and C. hepaticus HV10 T ∆ pglB::kan mutants of whole cell lysates containing 35–40 μg of protein. Whole cell lysates were separated by 8%–16% SDS-PAGE and either developed with SimplyBlue SafeStain or transferred to PVDF membranes for lectin blotting.

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: The effect of pglB mutagenesis on SBA binding profiles to N- glycans present in C. hepaticus HV10 T ∆ pglB::kan mutants. Mutant 3 was independently derived from mutants 7, 8, 9, and 10. ( A ) Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and ( B ) SBA lectin blotting of C. hepaticus HV10 T (WT) and C. hepaticus HV10 T ∆ pglB::kan mutants of whole cell lysates containing 35–40 μg of protein. Whole cell lysates were separated by 8%–16% SDS-PAGE and either developed with SimplyBlue SafeStain or transferred to PVDF membranes for lectin blotting.

Article Snippet: Genomic DNA was isolated from C. hepaticus HV10 T WT and mutant strains, grown for 24 h using a Monarch Genomic DNA Purification Kit (NEB) according to the manufacturer’s instructions.

Techniques: Mutagenesis, Binding Assay, Derivative Assay, Polyacrylamide Gel Electrophoresis, SDS Page

Plasmid map of shuttle vector pJBM5.2 used to complement C. hepaticus HV10 T ∆ pglB::kan mutant 7. Genetic symbols represent: Rep, replication initiation protein; HP, hypothetical protein; Mob, mobilization protein; OriV, Campylobacter origin of replication; ampR, Ampicillin resistance gene; ori, E.coli origin of replication from pJBM5; galE putative promoter; pglB, pglA, pglC, pglD, pglE, and pglF from C. hepaticus HV10 T ; and tet(O ), tetracycline resistance gene from C. hepaticus 84B. The position of the primers used to construct pJBM5.2 by Gibson assembly is shown in purple.

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: Plasmid map of shuttle vector pJBM5.2 used to complement C. hepaticus HV10 T ∆ pglB::kan mutant 7. Genetic symbols represent: Rep, replication initiation protein; HP, hypothetical protein; Mob, mobilization protein; OriV, Campylobacter origin of replication; ampR, Ampicillin resistance gene; ori, E.coli origin of replication from pJBM5; galE putative promoter; pglB, pglA, pglC, pglD, pglE, and pglF from C. hepaticus HV10 T ; and tet(O ), tetracycline resistance gene from C. hepaticus 84B. The position of the primers used to construct pJBM5.2 by Gibson assembly is shown in purple.

Article Snippet: Genomic DNA was isolated from C. hepaticus HV10 T WT and mutant strains, grown for 24 h using a Monarch Genomic DNA Purification Kit (NEB) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Mutagenesis, Construct

Complementation of C. hepaticus HV10 T ∆ pglB::kan mutant 7 in trans with pJBM5.2, expressing the pgl locus genes including and downstream of pglB and excluding pglG . ( A ) SBA lectin blot binding profiles to N- glycans present in C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan mutant 7, C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM4) (empty vector control), and two clones from separate isogenic complements (clones 1 and 2 were derived independently of clones 3 and 4) of C. hepaticus HV10 T ∆ pglB::kan mutant 7 clones expressing functional pgl genes encoded on pJBM5.2. Whole cell lysates contained 27 µg of protein and were separated by 4%–20% SDS-PAGE and either developed with SimplyBlue SafeStain or transferred to PVDF membranes for lectin blotting. The corresponding SDS-PAGE gel to demonstrate equal loading of proteins across samples is provided in Fig. S8. ( B ) Quantitative analysis of SBA binding to GalNAc present in whole cell lysates. ELISA results represent SBA binding to whole cell lysates of C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan mutant 7, C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM5.2), and Salmonella Typhimurium 82/6915 ∆ aroA (STM1) (negative control for N -glycosylation) measured at OD 450 . Gray bars represent the standard deviation of n = 4 technical replicates for each strain. Statistical differences between groups were analyzed using a one-way ANOVA with Tukey’s post hoc analysis and are indicated as: ns, no statistically significant difference ( P -value > 0.05); and **** indicate statistically significant differences with P -values < 0.0001. For all Tukey’s multiple comparisons, see Table S1.

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: Complementation of C. hepaticus HV10 T ∆ pglB::kan mutant 7 in trans with pJBM5.2, expressing the pgl locus genes including and downstream of pglB and excluding pglG . ( A ) SBA lectin blot binding profiles to N- glycans present in C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan mutant 7, C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM4) (empty vector control), and two clones from separate isogenic complements (clones 1 and 2 were derived independently of clones 3 and 4) of C. hepaticus HV10 T ∆ pglB::kan mutant 7 clones expressing functional pgl genes encoded on pJBM5.2. Whole cell lysates contained 27 µg of protein and were separated by 4%–20% SDS-PAGE and either developed with SimplyBlue SafeStain or transferred to PVDF membranes for lectin blotting. The corresponding SDS-PAGE gel to demonstrate equal loading of proteins across samples is provided in Fig. S8. ( B ) Quantitative analysis of SBA binding to GalNAc present in whole cell lysates. ELISA results represent SBA binding to whole cell lysates of C. hepaticus HV10 T , C. hepaticus HV10 T ∆ pglB::kan mutant 7, C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM4), C. hepaticus HV10 T ∆ pglB::kan mutant 7(pJBM5.2), and Salmonella Typhimurium 82/6915 ∆ aroA (STM1) (negative control for N -glycosylation) measured at OD 450 . Gray bars represent the standard deviation of n = 4 technical replicates for each strain. Statistical differences between groups were analyzed using a one-way ANOVA with Tukey’s post hoc analysis and are indicated as: ns, no statistically significant difference ( P -value > 0.05); and **** indicate statistically significant differences with P -values < 0.0001. For all Tukey’s multiple comparisons, see Table S1.

Article Snippet: Genomic DNA was isolated from C. hepaticus HV10 T WT and mutant strains, grown for 24 h using a Monarch Genomic DNA Purification Kit (NEB) according to the manufacturer’s instructions.

Techniques: Mutagenesis, Expressing, Binding Assay, Plasmid Preparation, Control, Clone Assay, Derivative Assay, Functional Assay, SDS Page, Enzyme-linked Immunosorbent Assay, Negative Control, Standard Deviation

Bacterial strains and plasmids used in this study

Journal: mBio

Article Title: Development of tools for the genetic manipulation of Campylobacter and their application to the N- glycosylation system of Campylobacter hepaticus, an emerging pathogen of poultry

doi: 10.1128/mbio.01101-24

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: Genomic DNA was isolated from C. hepaticus HV10 T WT and mutant strains, grown for 24 h using a Monarch Genomic DNA Purification Kit (NEB) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Mutagenesis, Clone Assay, Isolation