Expression of the bifunctional Bacillus subtilis TatAd protein in Escherichia coli reveals distinct TatA/B-family and TatB-specific domains

James P Barnett, Janna Lawrence, Sharon Mendel, Colin Robinson

    Research output: Contribution to journalArticle

    8 Citations (Scopus)

    Abstract

    In the Tat protein export pathway of Gram-negative bacteria, TatA and TatB are homologous proteins that carry out distinct and essential functions in separate sub-complexes. In contrast, Gram-positive Tat systems usually lack TatB and the TatA protein is bifunctional. We have used a mutagenesis approach to delineate TatA/B-type domains in the bifunctional TatAd protein from Bacillus subtilis. This involved expression of mutated TatAd variants in Escherichia coli and tests to determine whether the variants could function as TatA or TatB by complementing E. coli tatA and/or tatB mutants. We show that mutations in the C-terminal half of the transmembrane span and the subsequent FGP 'hinge' motif are critical for TatAd function with its partner TatCd subunit, and the same determinants are required for complementation of either tatA or tatB mutants in Escherichia coli. This is thus a critical domain in both TatA and TatB proteins. In contrast, substitution of a series of residues at the N-terminus specifically blocks the ability of TatAd to substitute for E. coli TatB. The results point to the presence of a universally conserved domain in the TatA/B-family, together with a separate N-terminal domain that is linked to the TatB-type function in Gram-negative bacteria.

    LanguageEnglish
    Pages583-594
    Number of pages12
    JournalArchives of Microbiology
    Volume193
    Issue number8
    DOIs
    Publication statusPublished - Aug 2011

    Fingerprint

    Escherichia coli Proteins
    Bacilli
    Bacillus subtilis
    Escherichia coli
    Gram-Negative Bacteria
    Bacteria
    Proteins
    tat Gene Products
    Mutagenesis
    Hinges
    Substitution reactions
    Mutation

    Keywords

    • Amino Acid Motifs
    • Amino Acid Sequence
    • Amino Acid Substitution
    • Bacillus subtilis
    • Bacterial Proteins
    • Escherichia coli
    • Genetic Complementation Test
    • Membrane Transport Proteins
    • Molecular Sequence Data
    • Mutagenesis, Site-Directed
    • Protein Interaction Domains and Motifs
    • Protein Transport
    • Sequence Alignment
    • Journal Article
    • Research Support, Non-U.S. Gov't

    Cite this

    Expression of the bifunctional Bacillus subtilis TatAd protein in Escherichia coli reveals distinct TatA/B-family and TatB-specific domains. / Barnett, James P; Lawrence, Janna; Mendel, Sharon; Robinson, Colin.

    In: Archives of Microbiology, Vol. 193, No. 8, 08.2011, p. 583-594.

    Research output: Contribution to journalArticle

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    abstract = "In the Tat protein export pathway of Gram-negative bacteria, TatA and TatB are homologous proteins that carry out distinct and essential functions in separate sub-complexes. In contrast, Gram-positive Tat systems usually lack TatB and the TatA protein is bifunctional. We have used a mutagenesis approach to delineate TatA/B-type domains in the bifunctional TatAd protein from Bacillus subtilis. This involved expression of mutated TatAd variants in Escherichia coli and tests to determine whether the variants could function as TatA or TatB by complementing E. coli tatA and/or tatB mutants. We show that mutations in the C-terminal half of the transmembrane span and the subsequent FGP 'hinge' motif are critical for TatAd function with its partner TatCd subunit, and the same determinants are required for complementation of either tatA or tatB mutants in Escherichia coli. This is thus a critical domain in both TatA and TatB proteins. In contrast, substitution of a series of residues at the N-terminus specifically blocks the ability of TatAd to substitute for E. coli TatB. The results point to the presence of a universally conserved domain in the TatA/B-family, together with a separate N-terminal domain that is linked to the TatB-type function in Gram-negative bacteria.",
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    AB - In the Tat protein export pathway of Gram-negative bacteria, TatA and TatB are homologous proteins that carry out distinct and essential functions in separate sub-complexes. In contrast, Gram-positive Tat systems usually lack TatB and the TatA protein is bifunctional. We have used a mutagenesis approach to delineate TatA/B-type domains in the bifunctional TatAd protein from Bacillus subtilis. This involved expression of mutated TatAd variants in Escherichia coli and tests to determine whether the variants could function as TatA or TatB by complementing E. coli tatA and/or tatB mutants. We show that mutations in the C-terminal half of the transmembrane span and the subsequent FGP 'hinge' motif are critical for TatAd function with its partner TatCd subunit, and the same determinants are required for complementation of either tatA or tatB mutants in Escherichia coli. This is thus a critical domain in both TatA and TatB proteins. In contrast, substitution of a series of residues at the N-terminus specifically blocks the ability of TatAd to substitute for E. coli TatB. The results point to the presence of a universally conserved domain in the TatA/B-family, together with a separate N-terminal domain that is linked to the TatB-type function in Gram-negative bacteria.

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