Article Figures & Data
Figures
- FIG. 1.
Purification of HdeA and HdeB. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gels (16%) and stained with Coomassie brilliant blue. Lane 1, periplasmic extract from uninduced strain BL21(DE3) pET-21a-hdeB; lane 2, periplasmic extract from BL21(DE3) pET-21a-hdeB induced for 3 h with IPTG; lane 3, 5 μg of purified HdeB; lane 4, 5 μg of purified HdeA. The positions of molecular mass markers are indicated on the left.
- FIG. 2.
Acid-induced aggregation of periplasmic extracts. SDS-PAGE analyses of the pellet (A) and supernatant (B) fractions of periplasmic extracts (20 μg each) from the hdeA mutant, the hdeB mutant, and their parent after an acid treatment at pH 2 or at pH 3 for 60 min (a control was done at pH 7).
- FIG. 3.
Prevention of periplasmic-protein aggregation at pH 2. Shown are SDS-PAGE analyses of the pellet (A) and supernatant (B) fractions of periplasmic extracts (20 μg) from the hdeA mutant and its parental strain after incubation for 60 min at pH 2, either alone or in the presence of purified (15 μg each) HdeA, HdeB, or both (7.5 μg each). (C) Densitometric scan of the protein distributions of periplasmic extracts from the wild-type (wt) strain and the hdeB mutant (not shown in panel A) supplemented with HdeA or HdeB, as indicated. We checked that the intensities of the protein bands (excluding the HdeA/B bands) in pellets and supernatants added up to similar amounts in all cases.
- FIG. 4.
Prevention of periplasmic-protein aggregation at pH 3. (A and B) SDS-PAGE analyses of pellet (A) and supernatant (B) fractions of periplasmic extracts (20 μg) from the hdeA mutant and its parental strain after incubation for 60 min at pH 3, either alone or in the presence of purified (15 μg each) HdeA, HdeB, or both (7.5 μg each). (B) Densitometric scan of protein distributions of periplasmic extracts from the wild-type (wt) strain and the hdeA mutant supplemented with HdeA or HdeB, as indicated. We checked that the intensities of the protein bands (excluding the HdeA/B bands) in pellets and supernatants added up to similar amounts in all cases.
- FIG. 5.
Solubilization of GAPDH, ADH, and OppA at acidic pH. (A) SDS-PAGE analyses of the supernatant and pellet fractions of GAPDH (10 μg) that were subjected to treatment for 60 min at pH 3 or 2, either alone or in the presence of (5 μg each) HdeA, HdeB, or both. (B) SDS-PAGE analyses of the supernatant fractions of ADH (10 μg) that were subjected to treatment for 60 min at pH 3 or 2, either alone or in the presence of (5 μg each) HdeA, HdeB, or both. (C) SDS-PAGE analyses of the supernatant fractions of OppA (10 μg) that were subjected to treatment for 60 min at pH 3 or 2, either alone or in the presence of (5 μg each) HdeA, HdeB, or both. The molecular masses (monomeric forms) of HdeA, HdeB, GAPDH, ADH, and OppA were 10 kDa, 9 kDa, 36 kDa, 36 kDa, and 61 kDa, respectively.
- FIG. 6.
ANS fluorescence of HdeB and HdeA at neutral and acidic pHs. The intensities of ANS fluorescence (100 μM in H2SO4 solution at pH 2 or pH 3 or in 10 mM Tris, pH 8) in the presence of 7 μM HdeA or 7 μM HdeB were measured after excitation at 395 nm.
- FIG. 7.
Oligomeric forms of HdeB at neutral and acidic pHs. For experiments at pH 7.5, the column was equilibrated in 20 mM Tris, pH 7.5, 100 mM NaCl at 20°C; loaded with 20 μl of HdeB (2.4 mg/ml); and eluted at a flow rate of 0.5 ml/min. For experiments at pH 3 and pH 2, the column was equilibrated with 150 mM Na2SO4 adjusted to these pHs with sulfuric acid and loaded with 20 μl of HdeB and/or HdeA (2.4 mg/ml each) equilibrated at the pH of the column. Proteins were detected by their absorbance at 280 nm. Blue dextran (2 MDa), yellow dextran (20,000 Da), cytochrome c (12,500 Da), and vitamin B12 (1,382 Da) were used as molecular mass standards.
- FIG. 8.
Acid stress sensitivities of the hdeA and hdeB mutants and of those mutants complemented with plasmids pBAD33-hdeA, pBAD33-hdeB, and pBAD33-hdeAB. The mutants, either uncomplemented (0) or complemented with pBAD33-hdeA (A), pBAD33-hdeB (B), or pBAD33-hdeAB (AB), and their parent were subjected to acid stress in LB medium adjusted to pH 3 (stippled bars) or pH 2 (shaded bars) for 90 min at 37°C, as described in Materials and Methods, and survival patterns were determined by plating the bacteria overnight on LB agar plates at pH 7. The results are the averages ± standard errors of the mean of three experiments. The colony counts of the mutants were normalized to those of the parental strain. All of the strains displayed similar viabilities at pH 7.
