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SIGNAL TRANSDUCTION

Phosphorylation of the Pseudomonas aeruginosa Response Regulator AlgR Is Essential for Type IV Fimbria-Mediated Twitching Motility

Cynthia B. Whitchurch, Tatiana E. Erova, Jacqui A. Emery, Jennifer L. Sargent, Jonathan M. Harris, Annalese B. T. Semmler, Michael D. Young, John S. Mattick, Daniel J. Wozniak
Cynthia B. Whitchurch
1ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience
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Tatiana E. Erova
2Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina
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Jacqui A. Emery
1ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience
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Jennifer L. Sargent
1ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience
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Jonathan M. Harris
1ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience
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Annalese B. T. Semmler
1ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience
3Department of Biochemistry, University of Queensland, Brisbane, Queensland 4072, Australia
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Michael D. Young
1ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience
3Department of Biochemistry, University of Queensland, Brisbane, Queensland 4072, Australia
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John S. Mattick
1ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience
3Department of Biochemistry, University of Queensland, Brisbane, Queensland 4072, Australia
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Daniel J. Wozniak
2Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, North Carolina
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  • For correspondence: dwozniak@wfubmc.edu
DOI: 10.1128/JB.184.16.4544-4554.2002
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  • FIG. 1.
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    FIG. 1.

    (A) Alignment of the amino-terminal phosphorylation domains of AlgR, YehT, CheY, and NtrC. X, variable sequences between conserved domains of each protein. Overlined sequences are highly conserved among all response regulators, and conserved aspartates and lysines are depicted in red. In CheY and NtrC, the aspartate which aligns with AlgR D54 is the site of phosphorylation by CheA or NtrB, respectively (33, 34). (B) A ribbon diagram for AlgR model. Homology-based model structure for the AlgR receiver domain was constructed using the structure of the NarL nitrate response regulator protein as a template. The α-helices are blue, β-sheets are red, and loops are gray. Asp54 and Asp85 are depicted in stick form and colored according to standard CPK. Two views are shown: the first is down the axis of a barrel formed by the peripheral helices and the second is at right angles to this and shows the two aspartates lying in the same plane as the mouth of the barrel. (C) Comparison of wild-type AlgR and AlgR D54N receiver domain structures. The panel on the left is an overlay of wild-type AlgR receiver domain (yellow) with AlgR D54N (blue) showing very little structural disturbance of the domain structure. The position of residue 54 in these structures is indicated in red. The right panel is a ribbon diagram of the region surrounding residue 54 (shown in stick form) with the wild-type aspartate depicted in blue and the substituted asparagine overlaid in red.

  • FIG. 2.
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    FIG. 2.

    (A) AlgR phosphorylation studies. Phosphorylation assays were conducted as described in Materials and Methods. Lanes contain 100 ng of CheA and/or 1 μg of AlgR or AlgR derivatives (+, addition of protein). The positions of the phosphorylated forms of CheA and AlgR are indicated on the side. Note that CheA can phosphorylate AlgR and AlgR D85N but not AlgR D54N or AlgR D54N D85N. (B) AlgR D54N competes with wild-type AlgR for access to CheA. A competition experiment was performed using 1 μg of wild-type AlgR and different amounts of AlgR D54N (0.5, 1, and 5 μg). The conditions used in the autophosphorylation of CheA, phosphotransfer from CheA to AlgR, and detection are identical to those described for panel A. Lane 1, 100 ng of CheA alone; lanes 2, 3, and 4, 100 ng of CheA and 0.5, 1, or 5 μg of AlgR D54N, respectively; lane 5, 100 ng of CheA and 1 μg of wild-type AlgR; lanes 6, 7, and 8, 100 ng of CheA, 1 μg of wild-type AlgR, and either 0.5, 1, or 5 μg of AlgR D54N, respectively. (C) EMSA of AlgR or AlgR derivatives binding to algD sequences. The fragment used in this assay contains two AlgR-binding sites and is located from −324 to −424 relative to the algD transcription start site. Duplicate samples of AlgR or the various AlgR derivatives (100 ng; protein source depicted below the gel) were tested for binding to algD sequences. The positions of unbound algD as well as the AlgR-algD complexes are indicated.

  • FIG. 3.
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    FIG. 3.

    Twitching motility phenotypes of PAKalgR mutants. (A) Subsurface twitching motility assay of P. aeruginosa PAK (wild type) and mutants PAKpilA::Tcr, S39 (algR), PAKalgR7, PAKalgR10, PAKalgR11, and PAKalgD::Tcr. Bars, 1 cm. (B) Light microscopy of zones of twitching motility showing colony expansion zones obtained at the interstitial surface between the glass coverslip and Gelgro media for PAK (wild type), PAKpilA::Tcr, and S39. Micrographs were taken after 2 to 4 h of incubation (PAK) or 20 h (PAKpilA::Tcr and S39) at 37°C. Bar, 50 μm. In all images, the colony is situated to the left of the image.

  • FIG. 4.
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    FIG. 4.

    Type IV fimbrial subunit production in algR mutants. (A) ELISA against whole-cell samples of the following P. aeruginosa strains: PAK (▪), PAKpilA::Tcr (▴), S39 (×), PAKalgR7 (|), PAKalgR10 (•), and PAKalgR11 (♦). Type IV fimbriae were detected using antipilin antiserum and are indicative of levels of surface fimbriae in these strains. Shown are immunoblots of the PilA subunit of sheared surface fimbriae (B) and PilA subunit (C) remaining in whole-cell samples after surface fimbriae had been sheared off indicated strains. The pilV mutant (R306), which is defective in assembly of the fimbrial structure, was included in these assays to control for fimbrial subunit contribution to surface samples as a result of cell lysis.

  • FIG. 5.
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    FIG. 5.

    AlgR is required for biofilm formation. (A) Subsurface twitching motility assay of P. aeruginosa PAO1 (wild type) and the indicated algR mutant strains. (B) Biofilm formation was assayed every 2 h during initiation using the microtiter plate assay (29). Surface-attached cells were stained with crystal violet, the stain was solubilized in ethanol, and the absorbance was analyzed at 600 nm (A600). The wild-type (WT) strain and strains expressing the various AlgR mutant proteins are indicated.

  • FIG. 6.
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    FIG. 6.

    Electrostatic potential diagrams for the wild-type and D85N mutant AlgR receiver domains. Homology-based model structure for the AlgR receiver domain was constructed using the structure of the NarL nitrate response regulator protein as a template. Electrostatic potential is represented as a mesh contoured to yield isosurfaces with charges of ±3.0. Negative potential is red and positive potential is blue. There is very little positive potential visible in this image. Electrostatic potentials have also been mapped to a solvent-accessible molecular surface constructed with a 1.4-Å probe to show AlgR surface topology. Accessible surface is colored as for electrostatic potential except that uncharged areas are white. The positions of Asp/Asn85 are indicated. Viewpoint is along the helical barrel.

Tables

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  • TABLE 1.

    Strains, plasmids, and primers used in this study

    Strain, plasmid, or oligonucleotideDescription or sequenceaSource or reference
    E. coli
        DH5α 80lacZΔM15Promega
        JM109e14−mcrA recA1 endA1 gyrA96 thi-1 hsdR17(rK− mK+) supE44 relA1 Δ(lac-proAB) [F′ traD36 proAB lacIqZΔM15]Promega
        MV1184 41
    P. aeruginosa
        PAKNonmucoid P. aeruginosa strain KD. Bradley
        PAKpilA::TcrPreviously referred to as AWK 44
        R306PAKpilV::Tn5-B21 2
        S39PAKalgR::Tn5-B21 45
        PAKalgD::TcrTcr cassette insertion in EcoRI site of algDThis study
        PAKalgR7PAK encoding AlgR D54NThis study
        PAKalgR10PAK encoding AlgR D85NThis study
        PAKalgR11PAK encoding AlgR D54N D85NThis study
        PAO1Nonmucoid P. aeruginosa strain O1
        WFPA1 algD::tet in PAO1This study
        WFPA12 algR::aac1 in PAO1This study
        WFPA8 algR7 in PAO1 (encoding AlgR D54N)This study
        WFPA13 algR10 in PAO1 (encoding AlgR D85N)This study
        WFPA16 algR11 in PAO1 (encoding AlgR D54N D85N)This study
        WFPA6 algR gene replacement of WFPA12This study
    Plasmids
        pSM-TETSource of Tcr cassette 27
        pRIC380 P. aeruginosa suicide vector 1
        pUK21Kmr cloning vector 42
        pUCPKS P. aeruginosa-E. coli shuttle vector 43
        pGEMT-easy E. coli cloning vectorPromega
        pUCPAlgR algR cloned into pUCPKS 45
        pGemAlgRD54N algR7 cloned into pGEMT-easyThis study
        pUCPAlgRD54N algR7 cloned into pUCPKSThis study
        pDJW106 algR cloned in pALTER-1 21
        pJK223R1Source of AlgR 19
        pUS152 algR7 in pALTER-1 21
        pUS164 algR10 in pALTER-1 21
        pUS165 algR11 in pALTER-1 21
        pUS162 algR7 in pJK223R1 (source of AlgR D54N)This study
        pUS170 algR10 in pJK223R1 (source of AlgR D85N)This study
        pUS172 algR11 in pJK223R1 (source of AlgR D54N D85N)This study
        pUS150Wild-type algR in gene replacement vector pEX100T 21
        pUS157 algR7 in gene replacement vector pEX100T 21
        pUS166 algR10 in gene replacement vector pDJW525 21
        pUS168 algR11 in gene replacement vector pDJW525 21
        pRS1Source of His-CheAR. Bourret
        pDJW220 algD cloned in pALTER-1 5
    Primers
        algR-Forward5′-TAAAGCGAGTCTCAGCGTCG-3′This study
        algR-Reverse5′-GGGACGACATGGGATATTCC-3′This study
        algD-Forward5′-CGCGGATCCGAGGTGAATGCGATGCG-3′This study
        algD-Reverse5′-TGCTCTAGACTAGGAGCAGATGCCCTC-3′This study
        algD335′-AGCCCTTGTGGCGAATAGGC-3′This study
        algD365′-GAATTGGGGAAAAGTCTGTG-3′This study
    • ↵ a Tcr, tetracycline resistance; aac1, gentamicin resistance; Kmr, kanamycin resistance.

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Phosphorylation of the Pseudomonas aeruginosa Response Regulator AlgR Is Essential for Type IV Fimbria-Mediated Twitching Motility
Cynthia B. Whitchurch, Tatiana E. Erova, Jacqui A. Emery, Jennifer L. Sargent, Jonathan M. Harris, Annalese B. T. Semmler, Michael D. Young, John S. Mattick, Daniel J. Wozniak
Journal of Bacteriology Aug 2002, 184 (16) 4544-4554; DOI: 10.1128/JB.184.16.4544-4554.2002

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Phosphorylation of the Pseudomonas aeruginosa Response Regulator AlgR Is Essential for Type IV Fimbria-Mediated Twitching Motility
Cynthia B. Whitchurch, Tatiana E. Erova, Jacqui A. Emery, Jennifer L. Sargent, Jonathan M. Harris, Annalese B. T. Semmler, Michael D. Young, John S. Mattick, Daniel J. Wozniak
Journal of Bacteriology Aug 2002, 184 (16) 4544-4554; DOI: 10.1128/JB.184.16.4544-4554.2002
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KEYWORDS

Bacterial Proteins
Fimbriae, Bacterial
Pseudomonas aeruginosa
Trans-Activators

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