Supramolecular organizations in the aerobic respiratory chain of Escherichia coli

Abstract

a b s t r a c tThe organization of respiratory chain complexes in supercomplexes has been shown in the mitochondria of several eukaryotes and in the cell membranes of some bacteria. These supercomplexes are suggested to be important for oxidative phosphorylation efficiency and to prevent the formation of reactive oxygen species.Here we describe, for the first time, the identification of supramolecular organizations in the aerobic respiratory chain of Escherichia coli, including a trimer of succinate dehydrogenase. Furthermore, two heterooligomerizations have been shown: one resulting from the association of the NADH:quinone oxidoreductases NDH-1 and NDH-2, and another composed by the cytochrome bo 3 quinol:oxygen reductase, cytochrome bd quinol:oxygen reductase and formate dehydrogenase (fdo). These results are supported by blue native-electrophoresis, mass spectrometry and kinetic data of wild type and mutant E . coli strains.

The aerobic respiratory chain of Escherichia coli: from genes to supercomplexes

“…Protein concentration was determined using the bicinchoninic acid (BCA) protein assay from Pierce (Smith et al, 1985). Membrane solubilization was performed with 6 g digitonin per gram of protein, in a buffer containing 50 mM NaCl, 5 mM aminocaproic acid, 1 mM EDTA, 2 mM PMSF and 50 mM imidazole/HCl, pH 6, as previously described (Sousa et al, 2011). Solubilized membranes (150 mg per lane) were resolved in a 3-10 % acrylamide gradient blue native-PAGE (BN-PAGE) gel (Krause & Seelert, 2008;Schägger & von Jagow, 1991;Wittig et al, 2006), and NADH : NBT (nitro blue tetrazolium) and succinate : NBT oxidoreductase activities were detected in the gel (Zerbetto et al, 1997).…”

“…Solubilized membranes (12.5 mg) were also applied to the top of a continuous sucrose gradient (0.3-1.5 M) in a buffer containing 15 mM Tris/HCl, pH 7, 20 mM KCl and 0.2 % digitonin, resolved by ultracentrifugation at 4 uC (20 h, 150 000 g) (Dudkina et al, 2005;Sousa et al, 2011) and collected in 1 ml fractions. Membrane samples (50 or 100 mg) were resolved by SDS-PAGE (Laemmli, 1970), transferred onto a PVDF membrane essentially according to (Towbin et al, 1979), and detected with polyclonal antibodies against subunits of respiratory chain complexes.…”

“…The presence of the supercomplexes of the E. coli respiratory chain described previously (Sousa et al, 2011) was studied during bacterial growth to establish the growth phase in which these supramolecular structures are assembled. With this purpose, BN-PAGE-resolved membranes obtained from cells collected at ML, ES, MS and LS were probed for NADH : NBT and succinate : NBT oxidoreductase activities (Fig.…”

“…The apparent redundancy of the NADH dehydrogenases and oxygen reductases, which is also observed in other prokaryotes, may provide this bacterium with the required robustness to adjust to a wide range of environmental conditions (Pereira et al, 2004), like temperature, carbon source or oxygen availability. The supramolecular organization of the aerobic respiratory chain in supercomplexes has already been studied (see Dudkina et al, 2010 for a review), namely in bacteria such as Paracoccus denitrificans (Stroh et al, 2004) and Aquifex aeolicus (Prunetti et al, 2010), and more recently in the cytoplasmic membrane of E. coli (Sousa et al, 2011). In the last, supercomplexes composed by NDH-1 and NDH-2, and by the aerobic formate dehydrogenase (FdoGHI) (Benoit et al, 1998) and cytochrome bo 3 have been described.…”

SdhE Is a Conserved Protein Required for Flavinylation of Succinate Dehydrogenase in Bacteria

“…2B). Crystal structure and BN-PAGE data have shown that functional SDH is trimeric with a molecular mass of 360 kDa (33,34). Finally, native WT and ⌬sdhE membranes were separated by BN-PAGE (Fig.…”