External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency in Yarrowia lipolytica.

Alternative NADH:ubiquinone oxidoreductases are single subunit enzymes capable of transferring electrons from NADH to ubiquinone without contributing to the proton gradient across the respiratory membrane. The obligately aerobic yeast Yarrowia lipolytica has only one such enzyme, encoded by the NDH2 gene and located on the external face of the mitochondrial inner membrane. In sharp contrast to ndh2 deletions, deficiencies in nuclear genes for central subunits of proton pumping NADH:ubiquinone oxidoreductases (complex I) are lethal. We have redirected NDH2 to the internal face of the mitochondrial inner membrane by N-terminally attaching the mitochondrial targeting sequence of NUAM, the largest subunit of complex I. Lethality of complex I mutations was rescued by the internal, but not the external version of alternative NADH:ubiquinone oxidoreductase. Internal NDH2 also permitted growth in the presence of complex I inhibitors such as 2-decyl-4-quinazolinyl amine (DQA). Functional expression of NDH2 on both sides of the mitochondrial inner membrane indicates that alternative NADH:ubiquinone oxidoreductase requires no additional components for catalytic activity. Our findings also demonstrate that shuttle mechanisms for the transfer of redox equivalents from the matrix to the cytosolic side of the mitochondrial inner membrane are insufficient in Y. lipolytica.

Aerotaxis in Desulfovibrio.

Aerotaxis of two sulphate-reducing bacteria, the freshwater strain Desulfovibrio desulfuricans CSN (DSM 9104) and the marine strain Desulfovibrio oxyclinae N13 (DSM 11498), was studied using capillary microslides, microscopy and oxygen microsensors. The bacteria formed ring-shaped bands in oxygen diffusion gradients surrounding O2 bubbles, which were placed into anoxic sulphate-free cell suspensions in capillary microslides. The radial expansion of the oxic volume by diffusion was stopped by aerobic respiration. Bands were formed by cells avoiding high O2 levels near the O2 bubble, as well as by cells entering from the surrounding anoxic zone. At the inner edge of the bands, O2 levels of up to 20% air saturation (50 microM O2) were found, while the outer edge always coincided with the oxic-anoxic interface. Ring diameters and O2 concentrations at the inner edge of the band depended on the cell density and the strain used in the suspension. Band formation did not occur in the absence of an electron donor (5mM lactate) or when N2 gas bubbles were used. Both strains were highly motile with velocities of approximately equals 32 microm s(-1) during forward runs, and 7 microm s(-1) during backward runs respectively. Within the bands, cells moved in circles of about 20 microm diameter, while cells outside the band exhibited straighter or only slightly bent traces. It is concluded that the capacity of respiration at high rates and the positive and negative aerotactical responses of Desulfovibrio provide an efficient strategy for removing O2 from the habitat in situations where sufficient electron donors and high cell densities are present.