A Bacteriophage-Acquired O-Antigen Polymerase (Wzyβ) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα

Veronique L. Taylor, Jesse F. J. Hoage, Sandra Wingaard Thrane, Steven M. Huszczynski, Lars Jelsbak, Joseph S. Lam

    Research output: Contribution to journalJournal articleResearchpeer-review

    164 Downloads (Pure)

    Abstract

    Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and β, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a β-polymerase (Wzyβ). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyβ was determined using a dual phoA-lacZα reporter system wherein random 3′ gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphatase activity to β-galactosidase activity. The topology of Wzyβ developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg254, Arg270, Arg272, and His300 were found to be essential for Wzyβ function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzyβ knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce β-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzyβ under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and β-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyβ utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion.
    Original languageEnglish
    Article number393
    JournalFrontiers in Microbiology
    Volume7
    Number of pages15
    ISSN1664-302X
    DOIs
    Publication statusPublished - 2016

    Bibliographical note

    © 2016 Taylor, Hoage, Thrane, Huszczynski, Jelsbak and Lam. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    Keywords

    • Pseudomonas aeruginosa
    • Lipopolysaccharide
    • Bacteriophage
    • Serotype
    • Polymerase
    • Glycosyltransferase
    • O-antigen biosynthesis

    Cite this

    Taylor, Veronique L. ; Hoage, Jesse F. J. ; Thrane, Sandra Wingaard ; Huszczynski, Steven M. ; Jelsbak, Lars ; Lam, Joseph S. / A Bacteriophage-Acquired O-Antigen Polymerase (Wzyβ) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα. In: Frontiers in Microbiology. 2016 ; Vol. 7.
    @article{5ce11776d0be40d9840891e67796a865,
    title = "A Bacteriophage-Acquired O-Antigen Polymerase (Wzyβ) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα",
    abstract = "Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and β, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a β-polymerase (Wzyβ). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyβ was determined using a dual phoA-lacZα reporter system wherein random 3′ gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphatase activity to β-galactosidase activity. The topology of Wzyβ developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg254, Arg270, Arg272, and His300 were found to be essential for Wzyβ function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzyβ knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce β-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzyβ under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and β-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyβ utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion.",
    keywords = "Pseudomonas aeruginosa, Lipopolysaccharide, Bacteriophage, Serotype, Polymerase, Glycosyltransferase, O-antigen biosynthesis",
    author = "Taylor, {Veronique L.} and Hoage, {Jesse F. J.} and Thrane, {Sandra Wingaard} and Huszczynski, {Steven M.} and Lars Jelsbak and Lam, {Joseph S.}",
    note = "{\circledC} 2016 Taylor, Hoage, Thrane, Huszczynski, Jelsbak and Lam. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.",
    year = "2016",
    doi = "10.3389/fmicb.2016.00393",
    language = "English",
    volume = "7",
    journal = "Frontiers in Microbiology",
    issn = "1664-302X",
    publisher = "Frontiers Media S.A.",

    }

    A Bacteriophage-Acquired O-Antigen Polymerase (Wzyβ) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα. / Taylor, Veronique L.; Hoage, Jesse F. J.; Thrane, Sandra Wingaard; Huszczynski, Steven M.; Jelsbak, Lars; Lam, Joseph S.

    In: Frontiers in Microbiology, Vol. 7, 393, 2016.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - A Bacteriophage-Acquired O-Antigen Polymerase (Wzyβ) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα

    AU - Taylor, Veronique L.

    AU - Hoage, Jesse F. J.

    AU - Thrane, Sandra Wingaard

    AU - Huszczynski, Steven M.

    AU - Jelsbak, Lars

    AU - Lam, Joseph S.

    N1 - © 2016 Taylor, Hoage, Thrane, Huszczynski, Jelsbak and Lam. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    PY - 2016

    Y1 - 2016

    N2 - Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and β, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a β-polymerase (Wzyβ). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyβ was determined using a dual phoA-lacZα reporter system wherein random 3′ gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphatase activity to β-galactosidase activity. The topology of Wzyβ developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg254, Arg270, Arg272, and His300 were found to be essential for Wzyβ function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzyβ knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce β-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzyβ under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and β-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyβ utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion.

    AB - Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and β, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a β-polymerase (Wzyβ). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyβ was determined using a dual phoA-lacZα reporter system wherein random 3′ gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphatase activity to β-galactosidase activity. The topology of Wzyβ developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg254, Arg270, Arg272, and His300 were found to be essential for Wzyβ function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzyβ knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce β-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzyβ under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and β-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyβ utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion.

    KW - Pseudomonas aeruginosa

    KW - Lipopolysaccharide

    KW - Bacteriophage

    KW - Serotype

    KW - Polymerase

    KW - Glycosyltransferase

    KW - O-antigen biosynthesis

    U2 - 10.3389/fmicb.2016.00393

    DO - 10.3389/fmicb.2016.00393

    M3 - Journal article

    VL - 7

    JO - Frontiers in Microbiology

    JF - Frontiers in Microbiology

    SN - 1664-302X

    M1 - 393

    ER -