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Microbial Cell Biology

Substrate Specificity Classes and the Recognition Signal for Salmonella Type III Flagellar Export

Takanori Hirano, Tohru Minamino, Keiichi Namba, Robert M. Macnab
Takanori Hirano
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114
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Tohru Minamino
Protonic NanoMachine Project, ERATO, JST, Seika, Kyoto 619-0237
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Keiichi Namba
Protonic NanoMachine Project, ERATO, JST, Seika, Kyoto 619-0237Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan
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Robert M. Macnab
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114
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  • For correspondence: robert.macnab@yale.edu
DOI: 10.1128/JB.185.8.2485-2492.2003
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  • FIG. 1.
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    FIG. 1.

    Coomassie-stained gels of the culture supernatants of SJW1353 (flgE) (A) and SJW2177 (flgK) (B) mutants carrying pTrc99AFF4-based plasmids producing various flagellar proteins under induction with 1 mM IPTG. The proteins produced are identified above each lane, and their positions are indicated by arrowheads. V, vector alone. Molecular mass markers (in kilodaltons) are shown to the left.

  • FIG. 2.
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    FIG. 2.

    Multicopy effects of rod-type proteins. The results of assays for determination of the swarming ability of the wild-type strain SJW1103 transformed with pET22b-, pTrc99AFF4-, and pTrc99AET-based plasmids are shown. The individual panels correspond to plasmids encoding the proteins shown at the right. V, vector alone. (In the third column, pTrc99A, not pTrc99AET, was used). Cells were inoculated onto soft tryptone agar plates containing 0.1 mM IPTG and incubated for 6 h at 30°C. Bottom panel: intracellular levels of FlgG produced in the same hosts as in the upper panels. A fixed number of cells was serially diluted at 1/1, 1/3, and 1/10 through to 1/300 and subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with polyclonal anti-FlgG antibody.

  • FIG. 3.
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    FIG. 3.

    Immunoblotting (using anti-FlgD antibody) of the culture supernatants from SJW1353 (flgE) cells transformed with pTrc99AET-based plasmids and grown at 37°C in the presence (+) and absence (−) of 0.1 mM IPTG. The proteins produced by the various plasmids are shown above each lane. V, vector alone (pTrc99A). The positions of FlgD are indicated by arrowheads. Molecular mass markers (in kilodaltons) are shown to the left.

  • FIG. 4.
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    FIG. 4.

    Immunoblotting (using anti-FlgG [A and B], anti-FlgD [C and D], and anti-FlgL [E and F] antibodies) of the cells and culture supernatants of SJW1353 (flgE) carrying pMM202 (FlgG) (A and B), SJW1353 (flgE) carrying pRCD1 (FlgD) (C and D), and SJW2177 (flgK) carrying pMM1903 (FlgL) (E and F), respectively, in the presence (+) and absence (−) of 100 μg of spectinomycin/ml. The positions of FlgG, FlgD, and FlgL are indicated by arrowheads. The high-molecular-mass bands in the left panels of Fig. 4A, B, and C represent either aggregates or a cross-reacting species. Molecular mass markers (in kilodaltons) are shown to the left.

  • FIG. 5.
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    FIG. 5.

    Anti-FliC immunoblot of the cells and culture supernatants from SJW2177 (flgK) in the presence (+) and absence (−) of spectinomycin. The positions of flagellin (FliC) are indicated by arrowheads. Molecular mass marker (in kilodaltons) is shown to the left.

Tables

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  • TABLE 1.

    Strains and plasmids used in this study

    Strains and plasmidsRelevant characteristicsSource or reference
    Salmonella
        JR501Conversion of E. coli-derived plasmids to compatibility with Salmonella35
        SJW1103Wild type for motility and chemotaxis41
        SJW1353flgE33
        SJW2177flgK13
    Plasmids
        pET22bCloning vectorNovagen
        pTrc99ACloning vectorPharmacia
        pTrc99AFF4Cloning vector32
        pTrc99AETCloning vector (see Materials and Methods)This study
        pMM202pTrc99AFF4/FlgGThis study
        pMM204pET19b/His-FlgG31
        pMM501pET19b/His-FlgJThis study
        pMM504pTrc99AFF4/FlgJThis study
        pMM507pET22b/FlgJ11
        pMM711pET19b/His-FlgD29
        pMM1001pTrc99AFF4/FliE31
        pMM1002pET19b/His-FliE30
        pMM1101pTrc99AFF4/FlgB31
        pMM1102pET19b/His-FlgB30
        pMM1201pTrc99AFF4/FlgCThis study
        pMM1202pET19b/His-FlgC31
        pMM1301pTrc99AFF4/FlgFThis study
        pMM1302pET19b/His-FlgF31
        pMM1901pET19b/His-FlgL30
        pMM1903pTrc99AFF4/FlgLThis study
        pRCD1pTrc99A/FlgDThis study
        pTH090pTrc99AET/FlgJThis study
        pTH130pET22b/FliEThis study
        pTH190pTrc99AET/FliEThis study
        pTH230pET22b/FlgBThis study
        pTH290pTrc99AET/FlgBThis study
        pTH330pET22b/FlgCThis study
        pTH390pTrc99AET/FlgCThis study
        pTH430pET22b/FlgGThis study
        pTH490pTrc99AET/FlgGThis study
        pTH530pET22b/FlgFThis study
        pTH590pTrc99AET/FlgFThis study
        pTH730pET22b/FlgDThis study
        pTH790pTrc99AET/FlgDThis study
        pTH1230pET22b/FlgLThis study
        pTH1290pTrc99AET/FlgLThis study
  • TABLE 2.

    Primers used in this study

    Primer namePrimer sequencea
    SFLGG-NdeAGGC ATA TGA TGA TCA GGT CAT TAT GGA
    SFLGG-BamB2GCC GGT GGC GGA TCC CCC ACC GGC
    SFLGJ-NdeA1GGA AAT CAT CAT ATG ATC GGA GAC
    SFLGJ-BamB1AGC GAC TAC GGA TCC TTG AGC AAT
    SFLIE-NdeA1CAG GGG AAT CAT ATG GCA GCA ATA
    SFLIE-BamB1GTG TTG TCA GGA TCC GCT TAA ACC
    SFLGB-NdeA1TGC GGA GGA CAT ATG CTC GAC AGG
    SFLGB-BamB2TAA CAG CGC GGA TCC TTA GTT TCC
    SFLGC-NdeA1GGA AAC TAA CAT ATG GCG CTG TTA
    SFLGC-BamB1GAC ATA CGC GGA TCC TTT ACT GGC
    SFLGF-NdeA1CGG GAT AGC CAT ATG GAT CGC GCA
    SFLGF-BamB1AAA ATG TGG ATC CTT AAC TCA TCG
    SFLGD-NdeAAAG GAG GCG CAT ATG TCT ATT GCC
    SFLGD-BamBCTG ACT CCT GGA TCC TGT AAG GGC
    SFLGL-NdeAGGA GAA GGA TCA TAT GCG TAT CAG
    SFLGL-BamBTTC GTG ATA GGA TCC AAA AAG AGG
    • ↵a Restriction sites are indicated by underlining; start and stop codons (where present on the primer) are indicated by italics.

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Substrate Specificity Classes and the Recognition Signal for Salmonella Type III Flagellar Export
Takanori Hirano, Tohru Minamino, Keiichi Namba, Robert M. Macnab
Journal of Bacteriology Apr 2003, 185 (8) 2485-2492; DOI: 10.1128/JB.185.8.2485-2492.2003

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Substrate Specificity Classes and the Recognition Signal for Salmonella Type III Flagellar Export
Takanori Hirano, Tohru Minamino, Keiichi Namba, Robert M. Macnab
Journal of Bacteriology Apr 2003, 185 (8) 2485-2492; DOI: 10.1128/JB.185.8.2485-2492.2003
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