Boosted Membrane Potential as Bioenergetic Response to Anoxia in Dinoroseobacter shibae

Dinoroseobacter shibae DFL 12(T) is a metabolically versatile member of the world-wide abundant Roseobacter clade. As an epibiont of dinoflagellates D. shibae is subjected to rigorous changes in oxygen availability. It has been shown that it loses up to 90% of its intracellular ATP when exposed to anoxic conditions. Yet, D. shibae regenerates its ATP level quickly when oxygen becomes available again. In the present study we focused on the bioenergetic aspects of the quick recovery and hypothesized that the proton-motive force decreases during anoxia and gets restored upon re-aeration. Therefore, we analyzed Delta pH and the membrane potential (Delta psi) during the oxic-anoxic transitions. To visualize changes of Delta psi we used fluorescence microscopy and the carbocyanine dyes DiOC(2) (3; 3,3'-Diethyloxacarbocyanine Iodide) and JC-10. In control experiments the Delta psi-decreasing effects of the chemiosmotic inhibitors CCCP (carbonyl cyanide m-chlorophenyl hydrazone),TCS(3,3',4',5-tetrachlorosalicylanilide) and gramicidin were tested on D. shibae and Gram-negative and -positive control bacteria (Escherichia coli and Micrococcus luteus). We found that Delta pH is not affected by short-term anoxia and does not contribute to the quick ATP regeneration in D. shibae. By contrast, Delta psi was increased during anoxia, which was astonishing since none of the control organisms behaved that way. Our study shows physiological and bioenergetical aspects comparing to previous studies on transcriptomic responses to the transition from aerobic to nitrate respiration in D. shibae. For the lifestyle as an epibiont of a dinoflagellate, the ability to stand phases of temporary oxygen depletion is beneficial. With a boosted Delta psi, the cells are able to give their ATP regeneration a flying start, once oxygen is available again.