Systematic and Applied Microbiology 34 (2011) 293–302 Contents lists available at ScienceDirect Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm Sulfur-metabolizing bacterial populations in microbial mats of the Nakabusa hot spring, Japan Kyoko Kubo a,b,∗ , Katrin Knittel a,∗ , Rudolf Amann a , Manabu Fukui c , Katsumi Matsuura b a Max Planck Institute for Marine Microbiology, Celsiusstr. 1, D-28359 Bremen, Germany b Department of Biological Sciences, Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan c The Institute of Low Temperature Science, Hokkaido University, Kita-19, Nishi-8, Kita-ku, Sapporo 060-0819, Japan article info Article history: Received 28 September 2010 Keywords: Sulfur cycle Anoxygenic photosynthetic bacteria Sulfate-reducing bacteria Sulfur-oxidizing bacteria Alkaline hot spring Microbial mat Sulfurihydrogenibium Aquificae abstract At the Nakabusa hot spring, Japan, dense olive-green microbial mats develop in regions where the slightly alkaline, sulfidic effluent has cooled to 65 ◦ C. The microbial community of such mats was ana- lyzed by focusing on the diversity, as well as the in situ distribution and function of bacteria involved in sulfur cycling. Analyses of 16S rRNA and functional genes (aprA, pufM) suggested the importance of three thermophilic bacterial groups: aerobic chemolithotrophic sulfide-oxidizing species of the genus Sulfurihydrogenibium (Aquificae), anaerobic sulfate-reducing species of the genera Thermodesulfobac- terium/Thermodesulfatator, and filamentous anoxygenic photosynthetic species of the genus Chloroflexus. A new oligonucleotide probe specific for Sulfurihydrogenibium was designed and optimized for catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). In situ hybridizations of thin mat sections showed a heterogeneous vertical distribution of Sulfurihydrogenibium and Chloroflexus. Sulfu- rihydrogenibium dominated near the mat surface (50% of the total mat biovolume), while Chloroflexus dominated in deeper layers (up to 64% of the total mat biovolume). Physiological experiments monitoring in vitro changes of sulfide concentration indicated slight sulfide production by sulfate-reducing bacteria under anoxic-dark conditions, sulfide consumption by photosynthetic bacteria under anoxic-light condi- tions and strong sulfide oxidation by chemolithotrophic members of Aquificae under oxic-dark condition. We therefore propose that Sulfurihydrogenibium spp. act as highly efficient scavengers of oxygen from the spring water, thus creating a favorable, anoxic environment for Chloroflexus and Thermodesulfobac- terium/Thermodesulfatator in deeper layers. © 2011 Elsevier GmbH. All rights reserved. Introduction Microbial mats develop in a wide range of aquatic habitats, such as geothermal hot springs, hypersaline ponds, marine cold seeps or hydrothermal vents. On the deep sea floor, light is absent and fil- amentous mat-forming chemoautotrophic sulfur bacteria develop, while microbial mats from terrestrial hot springs are also often composed of phototrophic bacteria [9]. In this environment, two types of phototrophs contribute to the formation of mats: (i) oxy- genic phototrophs (cyanobacteria) growing autotrophically with water and carbon dioxide as the sole electron donor and carbon source, respectively, and (ii) anoxygenic phototrophs growing by photosynthesis without producing oxygen. ∗ Corresponding authors at: Max Planck Institute for Marine Microbiology, Celsiusstr. 1, D-28359 Bremen, Germany. Tel.: +49 421 2028 936; fax: +49 421 2028 580. E-mail addresses: kkubo@mpi-bremen.de (K. Kubo), kknittel@mpi-bremen.de (K. Knittel). One of the best investigated hot springs is the slightly alka- line, sulfidic hot spring at Nakabusa, Nagano Prefecture, Japan. This site is well known for the formation of dense, colorful microbial mats (Fig. 1). Due to high temperatures of up to 70 ◦ C, the ther- mophilic microorganisms in the mats are protected from grazing by higher organisms like insects (Matsuura, personal communica- tion) [4]. The spring water is of volcanic origin and contains various reduced sulfur compounds in high concentrations, which can be used as electron donors for microbial growth [27]. The tempera- ture and sulfide concentration in Nakabusa spring water are the key factors structuring the microbial community [27]. Close to the source, the temperature is approximately 75 ◦ C, which is beyond the tolerance of any cyanobacteria. However, at this point, stream- ers extend from gray-colored mats, and 16S rRNA gene sequences from sulfide-oxidizing (Aquifex spp., Sulfurihydrogenibium spp.) and sulfate-reducing bacteria (Thermodesulfobacterium-affiliated species) have been retrieved from the streamers [27]. At low sul- fide concentrations (<0.1 mM), filamentous Aquifex-like bacteria dominated, while at high sulfide concentrations (>0.1 mM) large sausage-shaped Sulfurihydrogenibium-like bacteria dominated the 0723-2020/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.syapm.2010.12.002