Vacuolar respiration of nitrate coupled to energy conservation in filamentous Beggiatoaceae Martin Beutler, 1,2 * Jana Milucka, 1 Susanne Hinck, 1 Frank Schreiber, 1† Jörg Brock, 1 Marc Mußmann, 1 Heide N. Schulz-Vogt 1 and Dirk de Beer 1 1 Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany. 2 bionsys GmbH, Fahrenheitstraße 1, 28359 Bremen, Germany. Summary We show that the nitrate storing vacuole of the sulfide-oxidizing bacterium Candidatus Allobeggiatoa halophila has an electron transport chain (ETC), which generates a proton motive force (PMF) used for cellu- lar energy conservation. Immunostaining by antibod- ies showed that cytochrome c oxidase, an ETC protein and a vacuolar ATPase are present in the vacuolar membrane and cytochrome c in the vacuolar lumen. The effect of different inhibitors on the vacuolar pH was studied by pH imaging. Inhibition of vacuolar ATPases and pyrophosphatases resulted in a pH decrease in the vacuole, showing that the proton gra- dient over the vacuolar membrane is used for ATP and pyrophosphate generation. Blockage of the ETC decreased the vacuolar PMF, indicating that the proton gradient is build up by an ETC. Furthermore, addition of nitrate resulted in an increase of the vacuolar PMF. Inhibition of nitrate reduction, led to a decreased PMF. Nitric oxide was detected in vacuoles of cells exposed to nitrate showing that nitrite, the product of nitrate reduction, is reduced inside the vacuole. These find- ings show consistently that nitrate respiration contrib- utes to the high proton concentration within the vacuole and the PMF over the vacuolar membrane is actively used for energy conservation. Introduction Filamentous sulfur bacteria of the family Beggiatoaceae are sulfide-oxidizing bacteria that can form extensive white mats covering sulfidic sediments. These bacteria are found world-wide in coastal marine sediments, deep- sea cold and hot seeps, as well as in hypersaline and freshwater sediments (Teske and Nelson, 2006). They belong to the largest known prokaryotes and are closely related to the filamentous Candidatus Marithioploca, and the non-filamentous genus Thiomargarita (Salman et al., 2011). The different species of Beggiatoaceae typically have filament diameters of 1–30 mm but some species can have diameters of more than 100 mm. In the larger filaments most of the cell volume is taken up by a vacuole, whereas the cytoplasm is restricted to a thin outer layer of 1–2 mm (Jannasch et al., 1989). Vacuoles are a rare feature among smaller bacteria, but occur frequently in larger (> 5 mm diameter) sulfur bacteria and cyanobacte- ria. The large vacuoles could allow the cells to grow to large size without being limited by diffusion (Larkin and Henk, 1989), and give the organisms structural rigidity necessary to exploit a larger ambient space than smaller filaments do (Jannasch et al., 1989). An extensive electron-microscopic study on large Candidatus Marithio- ploca spp. concluded that the vacuole is most likely an invagination of the cytoplasm (Maier et al., 1990). It has been shown that the vacuoles of sulfur oxidizers can contain extremely high nitrate concentrations (> 500 mM) (Fossing et al., 1995; McHatton et al., 1996; Schulz et al., 1999) and are used for the storage of this electron acceptor. The stored nitrate enables the filaments to survive long periods without oxygen (Hinck et al., 2007; Preisler et al., 2007). The use of nitrate in Beggiatoaceae as an alternative electron acceptor in addition to oxygen has been shown in several studies (Sweerts et al., 1990; McHatton et al., 1996; Kamp et al., 2006). With the exception of the nitrate-depleted vacuoles of specific hydrothermal vent Beggiatoaceae (Kalanetra et al., 2004), vacuolation and nitrate storage coincide in published 16S rRNA phylogenies (Ahmad et al., 2006; Peixoto de Albuquerque et al., 2010); most likely, these Beggiatoaceae share a similar genomic mechanism for nitrate storage. So far none of the vacuolated, nitrate-accumulating sulfur bacteria has been grown in pure culture. Therefore, working hypotheses for a presumed metabolism of these abundant bacteria were derived from studies of cleaned filaments, enrichment cultures or from genomic analyses. Mußmann and colleagues (2007) obtained partial genome Received 27 July, 2010; revised 4 July, 2012; accepted 23 July, 2012. *For correspondence. E-mail mbeutler@bionsys.de; Tel. (+49) 421 2028 834; Fax (+49) 421 2028 580. † Present address: Eawag – Swiss Federal Institute of Aquatic Science and Technology, Department of Environmental Microbiology, Überlandstrasse 133, 8600 Dübendorf, Switzerland. Environmental Microbiology (2012) doi:10.1111/j.1462-2920.2012.02851.x © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd