Article Figures & Data
Figures
- FIG. 1.
Synthesis and in vivo activity of EGFP-Im2 hybrid protein. (A) Immunoblot analysis of EGFP-Im2 hybrid protein. Strain MC4100 cells containing plasmids encoding EGFP-Im2 (lanes 1 and 5), Im2 (lanes 2 and 4), and His6-EGFP (lanes 3 and 6) were induced with 0.5 mg of arabinose/ml (lanes 1 and 5) or 100 μM IPTG (isopropyl-β-d-thiogalactopyranoside) (lanes 2 to 4 and lane 6) for 30 min at 37°C. Cell extracts (A 600 = 0.3) were immunoblotted, and the presence of various proteins was detected with antibodies directed against colicin E2 (lanes 1 to 3) and GFP (lanes 4 to 6). *, Contaminant protein recognized by antibodies directed against colicin E2. (B) Intracellular location of EGFP-Im2. Cells (T) producing EGFP-Im2 were induced with 0.5 mg of arabinose/ml for 30 min at 37°C and separated into cytoplasmic (C), periplasmic (P), and membrane (M) fractions as described in Materials and Methods. Fractions of 0.5 unit (A 600) were tested for the presence of EGFP-Im2 by immunoblotting with anti-GFP. The same fractions were tested for the presence of periplasmic maltose-binding protein, membrane-associated TolA, and cytoplasmic EF-Tu markers. (C) Expression and distribution of EGFP-Im2 fusion protein. MC4100 cells expressing (panels 3 and 4) or not (panels 1 and 2) EGFP-Im2 protein were visualized by fluorescence microscopy with an integration time of 3 s (panels 2 and 4) or by phase-contrast of the same field (panels 1 and 3). (D) Biological phenotype of EGFP-Im2 protein. Agar plates were overlaid with E. coli MC4100 cells expressing (middle and right panels, with [+] or without [−] 0.5 mg of arabinose/ml, respectively) or not (left panel) the EGFP-Im2 protein. Then, 1 μl of a serial dilution of either colicin A or colicin E2 was dropped onto each plate. The absence of zones of killing indicates biological resistance to ColE2 by the expressed EGFP-Im2 protein.
- FIG. 2.
Association of EGFP-Im2 with colicin E2 observed by gel filtration chromatography. The physical association of the two proteins was monitored by gel filtration chromatography as described in Materials and Methods. (A) The elution volumes of individual standard proteins are indicated: ferritin (440,000 Da), alcohol dehydrogenase (200,000 Da), β-amylase (150,000), bovine serum albumin (67,000 Da), and ovalbumin (43,000 Da). Elution of protein from a Superdex 200 column was recorded by tracing the A 280 profile. (B) Elution profiles of EGFP-Im2 (150 μg), ColE2 (250 μg) and stoichiometric amount of ColE2 (250 μg) and EGFP-Im2 (150 μg) incubated together before they were applied to the column. The three profiles correspond to three independent experiments, and each individual peaks are annotated. (C) Elution profile of ColE2 (250 μg) preincubated with a twofold molar excess of EGFP-Im2 (300 μg).
- FIG. 3.
Purified EGFP-Im2 inhibits the DNase activity of colicin E2. (A) Nuclease activity of ColE2 and its inhibition by EGFP-Im2. Ethidium bromide-stained agarose gel of electrophoresed target DNA (corresponding to 150 ng of linearized pUC18) after a 1-h incubation at 37°C with digestion buffer (lanes 1 and 14), purified EGFP-Im2 (0.43 μg, lane 2), increasing molar ratios of EGFP-Im2/ColE2 (0.3:1, 0.6:1, 1:1, and 1.3:1, respectively, lanes 3 to 6), refolded ColE2 (0.73 μg, lane 7), increasing molar ratios of Im2/ColE2 (0.3:1, 0.6:1, 1:1, and 1.3:1, respectively, lanes 8 to 11), native ColE2 complex (0.86 μg, lane 12), or refolded Im2 (0.12 μg, lane 13). (B) In vivo activity of ColE2/EGFP-Im2. Plate was overlaid with E. coli MC4100 cells. Portions (1 μl) of serial dilutions of free ColE2 (lane 1), reconstituted ColE2/EGFP-Im2 complex (lanes 2 to 3), reconstituted ColE2/Im2 complex (lanes 4 to 5), or native ColE2/Im2 complex (lane 6) were dropped onto plates. Large clear zones indicate bacterial cell death. Lane 1, unbound ColE2 (0.5 mg/ml); lanes 2 and 3, increasing molar ratios of EGFP-Im2/ColE2 (0.3:1 and 1.3:1, respectively); lanes 4 to 5, increasing molar ratios of Im2/ColE2 (0.3:1 and 1.3:1, respectively); lane 6, native ColE2/Im2 complex (0.5 mg/ml).
- FIG. 4.
Release of immunity protein from ColE2-treated cells. (A) Wild-type and btuB mutant strains incubated with ColE2/EGFP-Im2. The wild-type MC4100 strain and btuB mutant derivative were grown in LB medium at 37°C to an OD600 of 0.75, followed by incubation for 30 min with ColE2/EGFP-Im2 at a multiplicity of 400. Pellet (P) and supernatant (S) were separated by centrifugation, and the content of each fraction was analyzed by Western blotting with monoclonal antibodies directed against GFP (panel EGFP-Im2) or by the overlay method with purified ColA-Im2 (32), which specifically recognizes ColE2 (panel ColE2) (see Materials and Methods). Anti-mouse secondary antibodies were labeled with Alexa Fluor 488 dye (Molecular Probes). (B) Wild-type MC4100 strain incubated with free ColE2 or EGFP-Im2. Cells were prepared as described above but incubated with free ColE2 or EGFP-Im2 as indicated.
- FIG. 5.
Kinetics of immunity protein release. Wild-type strain MC4100 was grown in LB medium at 37°C to an OD600 of 0.75 and incubated with ColE2/EGFP-Im2 at a multiplicity of 400 at 37°C (A) or 4°C (B). At the indicated times, pellet (P) and supernatant (S) were separated by centrifugation, and the content of each fraction was analyzed by Western blotting with antibody directed against GFP (panel 2) or by the overlay method with purified ColA-Im2, which specifically recognizes ColE2 (panel 1).
- FIG. 6.
Release of EGFP-Im2 required functional import machinery. (A) Wild-type MC4100 strain, the ompF mutant derivative, and the ompF ompC mutant strain SM1005 were incubated with ColE2/EGFP-Im2. Cells and samples (P, pellet; S, supernatant) were prepared as described in the legend to Fig. 4. (B) Wild-type C600 strain and tolA, tolB, and tolR mutant derivatives were incubated with ColE2/EGFP-Im2. Cells and samples (P, pellet; S, supernatant) were prepared as described in the legend to Fig. 4.
- FIG. 7.
Protease accessibility of EGFP-Im2. (A) Wild-type MC4100 strain was grown in LB medium at 37°C to an OD600 of 0.75 and incubated with ColE2/EGFP-Im2 at a multiplicity of 400 at 37°C. At the indicated times, the cells (T) were separated into cytoplasmic (C), periplasmic (P), and membrane (M) fractions as described in Materials and Methods. Fractions of 0.3 unit (A 600, sample at 5 min) or of 1 unit (A 600, sample at 20 min) were tested for the presence of EGFP-Im2 by immunoblotting with anti-GFP. (B) Wild-type MC4100 cells were prepared as described above and incubated with ColE2/EGFP-Im2 at a multiplicity of 400 at 37°C. At the indicated times trypsin (8.3 μM) was (+) or was not (−) added for 5 min at 37°C, and digestion was stopped by a molar excess of trypsin inhibitor. Pellet (P) and supernatant (S) were separated by centrifugation, and the content of each fraction was analyzed by Western blotting with antibody directed against GFP. (C) Purified EGFP-Im2 (100 nM, panel 1) and ColE2/EGFP-Im2 (100 nM, panel 2) were incubated with (+) or without (−) trypsin (8.3 μM) at 37°C for 5 min, and digestion was stopped by a molar excess of trypsin inhibitor. The presence of EGFP-Im2 was analyzed by Western blotting with antibody directed against GFP.
