Boosted Membrane Potential as Bioenergetic Response to Anoxia in Dinoroseobacter shibae.

Christian Kirchhoff,Heribert Cypionka

doi:10.3389/fmicb.2017.00695

Abstract

Dinoroseobacter shibae DFL 12T 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 ΔpH and the membrane potential (ΔΨ) during the oxic-anoxic transitions. To visualize changes of ΔΨ we used fluorescence microscopy and the carbocyanine dyes DiOC2 (3; 3,3′-Diethyloxacarbocyanine Iodide) and JC-10. In control experiments the ΔΨ-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 ΔpH is not affected by short-term anoxia and does not contribute to the quick ATP regeneration in D. shibae. By contrast, ΔΨ 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 ΔΨ, the cells are able to give their ATP regeneration a flying start, once oxygen is available again.

Highlights

Dinoroseobacter shibae is a member of the metabolically versatile Roseobacter clade and can be found in marine and fresh water habitats world-wide (Kolber et al, 2001; Béjà et al, 2002)
We show that the membrane potential of D. shibae is strongly influenced by short-term anoxia – in an unexpected way – while pH is not affected
The used carbocyanine dyes for -visualization are usually applied in combination with flow cytometry (Novo et al, 1999; Jiao et al, 2004; Zhen et al, 2014)

Summary

Introduction

Dinoroseobacter shibae is a member of the metabolically versatile Roseobacter clade and can be found in marine and fresh water habitats world-wide (Kolber et al, 2001; Béjà et al, 2002). It belongs to the aerobic anoxygenically phototrophic bacteria. The cells are able to switch from an aerobic to anaerobic lifestyle (Piekarski et al, 2009). The oxic-anoxic shift causes dramatic changes of the cell physiology. Laass et al (2014) reported that D. shibae undergoes a metabolic crisis during the transition from aerobic to anaerobic growth As epibionts of dinoflagellates (Biebl et al, 2005) that can shuttle between oxic and anoxic conditions (Wagner-Döbler et al, 2010), this versatility may be helpful.

Methods

Results

Conclusion