Abstract
So-called “sulfur-turf” microbial mats in sulfide containing hot springs (55–70°C, pH 7.3–8.3) in Japan were dominated by a large sausage-shaped bacterium (LSSB) that is closely related to the genus Sulfurihydrogenibium. Several previous reports proposed that the LSSB would be involved in sulfide oxidation in hot spring. However, the LSSB has not been isolated yet, thus there has been no clear evidence showing whether it possesses any genes and enzymes responsible for sulfide oxidation. To verify this, we investigated sulfide oxidation potential in the LSSB using a metagenomic approach and subsequent biochemical analysis. Genome fragments of the LSSB (a total of 3.7 Mb sequence including overlapping fragments) were obtained from the metagenomic fosmid library constructed from genomic DNA of the sulfur-turf mats. The sequence annotation clearly revealed that the LSSB possesses sulfur oxidation-related genes coding sulfide dehydrogenase (SD), sulfide-quinone reductase and sulfite dehydrogenase. The gene encoding SD, the key enzyme for sulfide oxidation, was successfully cloned and heterologously expressed in Escherichia coli. The purified recombinant enzyme clearly showed SD activity with optimum temperature and pH of 60°C and 8.0, respectively, which were consistent with the environmental conditions in the hot spring where the sulfur-turf thrives. Furthermore, the affinity of SD to sulfide was relatively high, which also reflected the environment where the sulfide could be continuously supplied. This is the first report showing that the LSSB harbors sulfide oxidizing metabolism adapted to the hot spring environment and can be involved in sulfide oxidation in the sulfur-turf microbial mats.
Highlights
A wide variety of microbial mats develop in geothermal hot springs, and form the unique microbial ecosystems composed of physiologically and phylogenetically diverse microbes
Microscopic observation indicated the microbial community in the sulfur-turf was dominated by a large sausage-shaped bacterium (LSSB) (5–40 mm in cell length) with a large amount of elemental sulfur particles around its cells (Fig. 1)
In the 16S rRNA gene clone library analysis, a total of 99 clonal sequences were grouped into nine operational taxonomic units (OTUs) (ST-01 to ST-09) that were affiliated with the five bacterial phyla, Aquificae (92% of total clones), Deinococcus/Thermus (3%), Firmicutes (3%), Thermodesulfobacteria (1%) and Armatimonadetes (1%) (Table S1, Fig. S1) [30]
Summary
A wide variety of microbial mats develop in geothermal hot springs, and form the unique microbial ecosystems composed of physiologically and phylogenetically diverse microbes. The sulfur-turf mats are dominated by an uncultured large sausage-shaped bacterium (LSSB) with a large amount of elemental sulfur particles around its cells. All of the isolates show rod-shaped cells with less than 2.8 mm long and are thermophilic, microaerophilic, elemental sulfur- and thiosulfate-utilizing chemolithoautotrophic organisms. The LSSB is, as the name implies, sausage shaped and gigantic bacterium with length of 5–20 mm This conspicuous organism is widespread in sulfide-containing hot springs. Some previous studies proposed that the uncultured LSSB could be associated with sulfide oxidation in the sulfur-turf microbial mats in the hot springs. No report shows that any known Sulfurihydrogenibium isolates closely related to the LSSB can utilize sulfide, they are known to oxidize elemental sulfur and thiosulfate. There is no direct evidence in genetic and biochemical respects showing that LSSB possesses sulfide oxidation metabolism so far due to the lack of its genomic information as well as the axenic culture
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