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GENETICS AND MOLECULAR BIOLOGY

Effects of bfp Mutations on Biogenesis of Functional Enteropathogenic Escherichia coli Type IV Pili

Ravi P. Anantha, Kelly D. Stone, Michael S. Donnenberg
Ravi P. Anantha
Division of Infectious Diseases, Department of Medicine and Graduate Program in Molecular and Cell Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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Kelly D. Stone
Division of Infectious Diseases, Department of Medicine and Graduate Program in Molecular and Cell Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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Michael S. Donnenberg
Division of Infectious Diseases, Department of Medicine and Graduate Program in Molecular and Cell Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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DOI: 10.1128/JB.182.9.2498-2506.2000
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  • Fig. 1.
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    Fig. 1.

    Location of bfp mutants and complementing fragments. Lines beneath the genes show the fragments of the cluster used to construct complementing and control plasmids. For each mutant except bfpA and bfpP mutants, a plasmid containing the fragment ending within the corresponding mutated gene served as the control plasmid and a plasmid containing the fragment ending within the gene immediately downstream of the mutated gene served as the complementing plasmid. For the bfpA andbfpP mutants, the vector served as the control plasmid and the vector containing the corresponding gene served as the complementing plasmid. The upper row of restriction sites shows the beginning and endpoint of each fragment. The lower row of restriction sites indicates where the kanamycin cassette was inserted into each gene. Restriction enzyme abbreviations: B, BamHI; BB,BstBI, H, HindIII; Hp, HpaI; K,KpnI; L, BalI; M, MfeI; N,NruI; P, PstI; S, SpeI; SB,SnaBI; X, XbaI.

  • Fig. 2.
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    Fig. 2.

    Expression and processing of bundlin in wild-type EPEC and mutant strains. Whole-cell lysates of cells grown in DMEM were prepared, separated by SDS-PAGE electrophoresis on a 15% polyacrylamide gel, and analyzed by immunoblotting with an antibundlin monoclonal antibody. Lanes 2 to 9 contain wild-type EPEC and mutant strains, lanes 10 to 15 contain complemented mutant strains, and lanes 16 to 21 contain mutants with control plasmids. Lane 1, pMSD205, unprocessed bundlin control; lane 2, wild-type (WT) EPEC strain E2348/69; lanes 3, 10, and 16, bfpA mutant strain UMD901; lanes 4, 11, and 17, bfpG mutant strain UMD928; lanes 5, 12, and 18, bfpB mutant strain UMD923; lanes 6, 13, and 19,bfpC mutant strain UMD924; lanes 7, 14, and 20,bfpD mutant strain UMD926; lanes 8, 15, and 21,bfpP mutant strain UMD932; lane 9, bfpH mutant strain UMD918.

  • Fig. 3.
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    Fig. 3.

    Localized adherence of wild-type EPEC and mutant strains to epithelial cells. HEp-2 cells were incubated with bacteria for 3 h, washed, fixed, and stained with Giemsa stain. Slides were examined by light microscopy with a 63× objective lens. (A) Wild-type EPEC strain E2348/69. (B) bfpH mutant strain UMD918. (C)bfpC mutant strain UMD924. (D) bfpC mutant strain UMD924 containing plasmid pRPA103. Mutant and complemented strains not shown were indistinguishable from strains UMD924 and UMD924(pRPA103).

  • Fig. 4.
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    Fig. 4.

    Expression of BFP by wild-type EPEC and mutant strains. Strains were grown in DMEM, spotted on Formvar-copper-coated grids, negatively stained with phosphotungstic acid, and examined by electron microscopy. (A) Wild-type EPEC strain E2348-69. (B) bfpHmutant strain UMD918. (C) bfpG mutant strain UMD928. (D)bfpG mutant strain UMD928 containing plasmid pRPA104. Mutant and complemented strains not pictured were indistinguishable from strains UMD928 and UMD928(pRPA104), respectively. Bars, 500 (A to C) and 200 (D) nm.

  • Fig. 5.
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    Fig. 5.

    Distribution of bundlin in membrane fractions from recombinant E. coli strain DH5α containing the entirebfp gene cluster on plasmid pKDS302 (A), containingbfpA on plasmid pMSD230 and bfpP on plasmid pDN19PB (B), and containing bfpA on plasmid pMSD230 and abfpP deletion on plasmid pDN19PBΔ (C). Fractions collected from the sucrose flotation density gradients were analyzed by immunoblotting with antibodies against bundlin. NADH oxidase activity was measured and displayed as a percentage of the total NADH oxidase activity per fraction. Fractions were loaded on SDS–15% PAGE gels in order from the top to the bottom of the gradient (left to right, respectively). The density of each fraction is indicated above each blot. Variations from the linear trend of increasing density can be ascribed to pipetting error. Data are representative of two separate experiments with similar results. Numbers to the left of each panel indicate molecular mass(es) in kilodaltons.

  • Fig. 6.
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    Fig. 6.

    Redistribution of previously fractionated prebundlin from low-density (A) and high-density (B) fractions after a second round of sucrose flotation density gradient fractionation. Fractions collected from the sucrose flotation gradient were analyzed by immunoblotting with antibodies against bundlin. NADH oxidase activity was measured and displayed as a percentage of the total NADH oxidase activity per fraction. Fractions were loaded on SDS–15% PAGE gels in order from the top to the bottom of the gradient (left to right, respectively). The density of each fraction is indicated above each blot. Variations from the linear trend of increasing density can be ascribed to pipetting error. Numbers to the left of the panels indicate molecular mass in kilodaltons.

Tables

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  • Table 1.

    Strains and plasmids used in this study

    Strain or plasmidGenotype or featuresSource or reference
    Strains
     E2348/69Prototype O127:H6 EPEC strain 25
     UMD901E2348/69 bfpA C129S 50
     UMD928E2348/69bfpG::aphA3 This study
     UMD923E2348/69bfpB::aphA3 This study
     UMD924E2348/69bfpC::aphA3 This study
     UMD926E2348/69bfpD::aphA3 This study
     UMD932E2348/69bfpP::aphA3 This study
     UMD918E2348/69bfpH::aphA3 This study
     DH5α supE44 ΔlacU169(φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 36
     DH5αλpir DH5α(λpir) 27
    Plasmids
     pBluescriptHigh-copy-number cloning vectorStratagene
     pMSD205 bfpA gene cloned into PCR1000 under control of an isopropyl-β-d-thiogalactopyranoside-inducible promoter 13
     pRPA1001-kbBamHI-HindIII fragment of the bfpcluster containing bfpA cloned into pWKS30This study
     pRPA1013-kb BamHI-BalI fragment of thebfp cluster containing bfpA-bfpB cloned into pWKS30This study
     pRPA1034.1-kbBamHI-XbaI fragment of the bfp cluster containing bfpA-bfpC cloned into pWKS30This study
     pRPA1042.3-kb BamHI-HindIII fragment of the bfp cluster containing bfpA-bfpG cloned into pWKS30This study
     pRPA1066.5-kbBamHI-KpnI fragment of the bfp cluster containing bfpA-bfpD cloned into pWKS30This study
     pRPA1071.3-kb PstI-BamHI fragment of the bfp cluster containing bfpP cloned into pWKS30This study
     pCVD442Positive-selection suicide vector 14
     pRK2073pRK2 derivative suitable for mobilization of plasmids 10
     pKDS302Entire bfp cluster cloned into pTRC99A under control of trc promoter 40
     pMSD230 bfpA cloned into pTRC99A under control oftrc promoterThis study
     pMSD2335.3-kbBamHI fragment of the bfp cluster containingbfpA-bfpU cloned into pWKS30This study
     pDN19PB bfpP cloned into pDN19 51
     pDN19PBΔ bfpP with a 93-bp deletion cloned into pDN19 51
     pUC18K aphA3 gene cloned into pUC18, creating a nonpolar kanamycin cassette 27
     pUC18K2pUC18K with 1 bp added after the ATG start codonK. Jarvis and J. Kaper
     pUC18K3pUC18K with 2 bp added after the ATG start codonK. Jarvis and J. Kaper
  • Table 2.

    Autoaggregation of wild-type and bfp mutant EPEC strains

    StrainGenotypeAggregation index ± SDa% of wild-type valuePvalue
    E2348/69Wild type80.2 ± 19.3100
    UMD901 bfpA 2.0 ± 0.72.50.004b
    UMD901(pRPA100) bfpA(pbfpA+ )43.8 ± 11.154.60.005c
    UMD928 bfpG 0.8 ± 0.51.00.004b
    UMD928(pRPA104) bfpG(pbfpA+G+ )17.0 ± 3.521.20.002c
    UMD923 bfpB 0.8 ± 0.30.90.004b
    UMD923(pRPA101) bfpB(pbfpA+G+B+ )42.3 ± 11.852.70.006c
    UMD924 bfpC 2.0 ± 0.52.40.004b
    UMD924(pRPA103) bfpC(pbfpA+G+B+C+ )6.4 ± 1.77.90.008c
    UMD926 bfpD 1.0 ± 0.91.20.003b
    UMD926(pRPA106) bfpD((pbfpA+G+B+C+D+ )19.8 ± 11.124.70.051c
    UMD932 bfpP 2.0 ± 0.92.50.004b
    UMD932(pRPA107) bfpP(pbfpP+ )11.5 ± 1.414.30.001c
    UMD918 bfpH 50.2 ± 5.862.60.03b
    • ↵a Values shown are mean values from four separate experiments ± standard deviations (SDs).

    • ↵b Versus wild type.

    • ↵c Versus corresponding mutant.

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Effects of bfp Mutations on Biogenesis of Functional Enteropathogenic Escherichia coli Type IV Pili
Ravi P. Anantha, Kelly D. Stone, Michael S. Donnenberg
Journal of Bacteriology May 2000, 182 (9) 2498-2506; DOI: 10.1128/JB.182.9.2498-2506.2000

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Effects of bfp Mutations on Biogenesis of Functional Enteropathogenic Escherichia coli Type IV Pili
Ravi P. Anantha, Kelly D. Stone, Michael S. Donnenberg
Journal of Bacteriology May 2000, 182 (9) 2498-2506; DOI: 10.1128/JB.182.9.2498-2506.2000
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