Article Figures & Data
Figures
- 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.
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.
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.
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.
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.
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
- Table 1.
Strains and plasmids used in this study
Strain or plasmid Genotype or features Source or reference Strains E2348/69 Prototype O127:H6 EPEC strain 25 UMD901 E2348/69 bfpA C129S 50 UMD928 E2348/69bfpG::aphA3 This study UMD923 E2348/69bfpB::aphA3 This study UMD924 E2348/69bfpC::aphA3 This study UMD926 E2348/69bfpD::aphA3 This study UMD932 E2348/69bfpP::aphA3 This study UMD918 E2348/69bfpH::aphA3 This study DH5α supE44 ΔlacU169(φ80 lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 36 DH5αλpir DH5α(λpir) 27 Plasmids pBluescript High-copy-number cloning vector Stratagene pMSD205 bfpA gene cloned into PCR1000 under control of an isopropyl-β-d-thiogalactopyranoside-inducible promoter 13 pRPA100 1-kbBamHI-HindIII fragment of the bfpcluster containing bfpA cloned into pWKS30 This study pRPA101 3-kb BamHI-BalI fragment of thebfp cluster containing bfpA-bfpB cloned into pWKS30 This study pRPA103 4.1-kbBamHI-XbaI fragment of the bfp cluster containing bfpA-bfpC cloned into pWKS30 This study pRPA104 2.3-kb BamHI-HindIII fragment of the bfp cluster containing bfpA-bfpG cloned into pWKS30 This study pRPA106 6.5-kbBamHI-KpnI fragment of the bfp cluster containing bfpA-bfpD cloned into pWKS30 This study pRPA107 1.3-kb PstI-BamHI fragment of the bfp cluster containing bfpP cloned into pWKS30 This study pCVD442 Positive-selection suicide vector 14 pRK2073 pRK2 derivative suitable for mobilization of plasmids 10 pKDS302 Entire bfp cluster cloned into pTRC99A under control of trc promoter 40 pMSD230 bfpA cloned into pTRC99A under control oftrc promoter This study pMSD233 5.3-kbBamHI fragment of the bfp cluster containingbfpA-bfpU cloned into pWKS30 This 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 pUC18K2 pUC18K with 1 bp added after the ATG start codon K. Jarvis and J. Kaper pUC18K3 pUC18K with 2 bp added after the ATG start codon K. Jarvis and J. Kaper - Table 2.
Autoaggregation of wild-type and bfp mutant EPEC strains
Strain Genotype Aggregation index ± SDa % of wild-type value Pvalue E2348/69 Wild type 80.2 ± 19.3 100 UMD901 bfpA 2.0 ± 0.7 2.5 0.004b UMD901(pRPA100) bfpA(pbfpA+ ) 43.8 ± 11.1 54.6 0.005c UMD928 bfpG 0.8 ± 0.5 1.0 0.004b UMD928(pRPA104) bfpG(pbfpA+G+ ) 17.0 ± 3.5 21.2 0.002c UMD923 bfpB 0.8 ± 0.3 0.9 0.004b UMD923(pRPA101) bfpB(pbfpA+G+B+ ) 42.3 ± 11.8 52.7 0.006c UMD924 bfpC 2.0 ± 0.5 2.4 0.004b UMD924(pRPA103) bfpC(pbfpA+G+B+C+ ) 6.4 ± 1.7 7.9 0.008c UMD926 bfpD 1.0 ± 0.9 1.2 0.003b UMD926(pRPA106) bfpD((pbfpA+G+B+C+D+ ) 19.8 ± 11.1 24.7 0.051c UMD932 bfpP 2.0 ± 0.9 2.5 0.004b UMD932(pRPA107) bfpP(pbfpP+ ) 11.5 ± 1.4 14.3 0.001c UMD918 bfpH 50.2 ± 5.8 62.6 0.03b
