The Oligomeric Form of the Escherichia coli Dps Protein Depends on the Availability of Iron Ions.

The Dps protein of Escherichia coli, which combines ferroxidase activity and the ability to bind DNA, is effectively used by bacteria to protect their genomes from damage. Both activities depend on the integrity of this multi-subunit protein, which has an inner cavity for iron oxides; however, the diversity of its oligomeric forms has only been studied fragmentarily. Here, we show that iron ions stabilize the dodecameric form of Dps. This was found by electrophoretic fractionation and size exclusion chromatography, which revealed several oligomers in highly purified protein samples and demonstrated their conversion to dodecamers in the presence of 1 mM Mohr's salt. The transmission electron microscopy data contradicted the assumption that the stabilizing effect is given by the optimal core size formed in the inner cavity of Dps. The charge state of iron ions was evaluated using Mössbauer spectroscopy, which showed the presence of Fe3O4, rather than the expected Fe2O3, in the sample. Assuming that Fe2+ can form additional inter-subunit contacts, we modeled the interaction of FeO and Fe2O3 with Dps, but the binding sites with putative functionality were predicted only for Fe2O3. The question of how the dodecameric form can be stabilized by ferric oxides is discussed.

Interaction of phospholipase A of the E. coli outer membrane with the inhibitors of eucaryotic phospholipases A2 and their effect on the Ca²âº-induced permeabilization of the bacterial membrane.

Phospholipase A of the bacterial outer membrane (OMPLA) is a ß-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A2--palmitoyl trifluoromethyl ketone (PACOCF3) and aristolochic acid (AA)--with OMPLA and considers a possible involvement of the enzyme in the Ca²âº-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF3 and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF3, AA, and bromophenacyl bromide inhibit the Ca²âº-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca²âº and heat shock. The cell viability was not affected by the eucaryotic phospholipases A2 inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.