Protein folding in the periplasm in the absence of primary oxidant DsbA: modulation of redox potential in periplasmic space via OmpL porin

Abstract

Disulfide bond formation in Escherichia coli is a catalyzed reaction accomplished by DsbA. We found that null mutations in a new porin gene, ompL, allowed a total bypass of the DsbA requirement for protein oxidation. These mutations acted as extragenic null suppressors for dsbA, and restored normal folding of alkaline phosphatase and relieved sensitivity to dithiothreitol. ompL dsbA double mutants were completely like wild-type mutants in terms of motility and lack of mucoidy. This suppression was not dependent on DsbC and DsbG, since the oxidation status of these proteins was unaltered in ompL dsbA strains. Purified OmpL allowed diffusion of small solutes, including sugars, but the suppression was not dependent on the carbon sources used. Suppression by ompL null mutations required DsbB, leading us to propose a hypothesis that DsbB oxidizes yet unidentified, low-molecular-weight redox agents in the periplasm. These oxidized agents accumulate and substitute for DsbA if their leakage into the medium is prevented by the absence of OmpL, presumed to form a specific channel for their diffusion.

Fig. 1. Restriction map of the ompL gene and surrounding DNA sequences. Columns on the right hand indicate the ability of the different clones to complement for M13 resistance and to complement for dsbA mutant phenotypes (sensitivity to DTT and AP^–). The strains SR2701, SR2772 and SR1791 correspond to single Tn10 insertions at positions 495, 537 and 553, respectively.

Fig. 2. Subcellular fractionation of OmpL and its purification. Cultures of BL21(DE3) containing ompL gene (pCD695), cloned in pET-22b under transcription of the T7 polymerase promoter, were grown in M9 minimal medium at 37°C. Expression of T7 RNA polymerase was induced by the addition of 1 mM IPTG for 4 h. Lane 1 (‘Induction’) is the total extract from induced bacteria carrying pCD695 (ompL gene), lane 2 (‘OMPs’) represents the outer-membrane fractions after LDS extraction and lane 3 (‘OmpL’) contains purified OmpL protein after LDS extraction made from BL21(DE3) ompA^– ompC^– ompF^– lamB^– followed by gel filtration. Proteins were resolved by SDS–PAGE (12.5%). The gel was stained with Coomassie Brilliant Blue. The arrows on the left represent the position of OmpF, OmpC and OmpL. On the right are molecular weight size standards (Bio-Rad).

Fig. 3. Predicted topology of OmpL. Using the Chou and Fasman (1974) program, the secondary structure prediction was carried out. The amino acid residues in bold indicate potential β-sheets. The signal sequence cleavage site is marked with a downward arrow. E, predicted extended sheets; T, turns. The highly conserved G and F at the C-terminus, which are present in β-barrel OMPs, are indicated in big bold letters and underlined.

Fig. 4. Linear uptake of sugars by the OmpL porin. Here we show the dependence of diffusion rates of two sugars on the concentration of OmpL porin in proteoliposomes. The permeation rates of arabinose and glucose were measured using the reconstituted proteoliposome assay with different amounts of OmpL. Proteoliposomes were prepared as described earlier (Nikaido et al., 1991).

Fig. 5. Restoration of folding status of AP in vivo by introduction of ompL or null mutation in dsbA^– background (A). Isogenic cultures of wild type, dsbA::Tn10, ompL::Tn10 and their various double mutants were grown in LB medium up to an OD of 0.2 at 595 nm. Cultures were spun, washed and resuspended in low phosphate M63 medium. Bacterial cells were harvested at 0.5 OD, TCA precipitated and resuspended in buffer containing 1% SDS and 10 mM AMS. Samples were run on 12% SDS–PAGE and proteins visualized using antibodies against AP. To check the folding status of DsbG at the same time, antibodies against DsbG were used in the immunoblot assay. The positions of reduced (red) and oxidized (ox) forms are indicated. Note that there is no change in reduced status of DsbG. (B) The dependence on functional copy of dsbB for the suppression noted with the lack of ompL. As can be seen in lane 2 (‘dsbB^–’) and lane 3 (‘dsbB^–ompL^–’), there is no restoration of AP into the oxidized state as compared with dsbA^–ompL^– (A).

Fig. 6. The proposed model for restoration of oxidative environment in the absence of OmpL porin. We postulate the presence of one or more species of reduced molecules in the periplasm that is/are a substrate for DsbB. Under normal conditions it is pumped out by the OmpL channel and with the presence of DsbA periplasm stays oxidizing, AP⁺ (A). (B) In the absence of DsbA oxidant, the periplasm is relatively reduced and hence AP^–. (C) Here the DsbB is free to oxidize the reducing compounds and these compounds are not pumped out via the OmpL channel, hence periplasm stays oxidizing, being AP⁺. In the absence of OmpL and DsbB (D), such a reducing molecule as well as DsbA is not oxidized and thus the periplasm stays reducing, leading to an AP^– phenotype.