Molecular Characterization of Community Structures and Sulfur Metabolism within Microbial Streamers in Japanese Hot Springs

Abstract

Community structures of submerged microbial slime streamers (SMSS) in sulfide-containing hot springs at 72 to 80°C at Nakabusa and Yumata, Japan, were investigated by molecular analysis based on the 16S rRNA gene. The SMSS were classified into two consortia; consortium I occurred at lower levels of sulfide in the hot springs (less than 0.1 mM), and consortium II dominated when the sulfide levels were higher (more than 0.1 mM). The dominant cell morphotypes in consortium I were filamentous and small rod-shaped cells. The filamentous cells hybridized with fluorescent oligonucleotide probes for the domain Bacteria, the domain Archaea, and the family Aquificaceae. Our analysis of the denaturing gradient gel electrophoresis (DGGE) bands by using reverse transcription (RT)-PCR amplification with two primer sets (Eub341-F with the GC clamp and Univ907R for the Bacteria and Eub341-F with the GC clamp and Arch915R) indicated that dominant bands were phylogenetically related to microbes in the genus Aquifex. On the other hand, consortium II was dominated by long, small, rod-shaped cells, which hybridized with the oligonucleotide probe S-‫-ء‬Tdes-0830-a-A-20 developed in this study for the majority of as-yet-uncultivated microbes in the class Thermodesulfobacteria. The dominant DGGE band obtained by PCR and RT-PCR was affiliated with the genus Sulfurihydrogenibium. Moreover, our analysis of dissimilatory sulfite reductase (DSR) gene sequences retrieved from both consortia revealed a high frequency of DSR genes corresponding to the DSR of Thermodesulfobacteria-like microorganisms. Using both sulfide monitoring and 35 SO 4 2؊ tracer experiments, we observed microbial sulfide production and consumption by SMSS, suggesting that there is in situ sulfide production by as-yet-uncultivated Thermodesulfobacteria-like microbes and there is in situ sulfide consumption by Sulfurihydrogenibium-like microbes within the SMSS in the Nakabusa and Yumata hot springs.Over the past decade, studies of the microbiology of hightemperature terrestrial hot springs by both molecular-ecological and culture-based approaches have revealed phylogenetic and physiological diversity (20-23, 32, 37, 38, 40, 55). While culture-dependent approaches are effective for physiological characterization of isolated thermophiles, they are not useful for providing comprehensive ecophysiological information about the microbial populations in thermophilic environments. One reason for this is that cultivated microbes from hightemperature habitats may frequently represent minor members of a microbial community whose representation is similar to the representation found in the native setting (52).Sulfate-reducing bacteria (SRB), which obtain energy from dissimilatory sulfate reduction, are widespread in anoxic environments and play an important role in the sulfur cycle (53). Several strains belonging to the genus Thermodesulfobacterium, which includes thermophilic and gram-negative SRB, have been isolated from sediments and filamentous microbial communities in terrestrial...

Sulfurihydrogenibium yellowstonense sp. nov., an extremely thermophilic, facultatively heterotrophic, sulfur-oxidizing bacterium from Yellowstone National Park, and emended descriptions of the genus Sulfurihydrogenibium, Sulfurihydrogenibium subterraneum and Sulfurihydrogenibium azorense

“…Recently, two species of the genus Sulfurihydrogenibium were isolated and characterized as strict chemolithoautotrophs capable of using numerous electron donors and acceptors [H 2 , thiosulfate, elemental sulfur, sulfite, Fe(II), arsenite and selenite as electron donors and molecular oxygen, nitrate, Fe(III), arsenate and selenate as electron acceptors] Aguiar et al, 2004). Members of the genus Sulfurihydrogenibium play a significant role in sulfur-cycling (Skirnisdottir et al, 2000;Takai et al, 2002;Nakagawa & Fukui, 2003) and iron mineralization (Reysenbach et al, 1999) in terrestrial hot spring environments. Based on the phylogenetic analysis of 16S The GenBank/EMBL/DDBJ accession number for the partial 16S rRNA gene sequence of strain SS-5 T is AY686713.…”

Caldimicrobium thiodismutans sp. nov., a sulfur-disproportionating bacterium isolated from a hot spring, and emended description of the genus Caldimicrobium

“…The 16S rRNA gene sequence of strain TF1 T was identical or very closely related to partial sequences of the 16S rRNA genes that had been detected in the Nakabusa hot spring (Nakagawa & Fukui, 2003;Kubo et al, 2011;Everroad et al, 2012). In the maximum-likelihood phylogenetic tree, the novel isolate was shown to be a member of the family Thermodesulfobacteriaceae (Fig.…”

“…Strain TF1 T was isolated from a neutral to slightly alkaline hot spring in central Japan, Nakabusa hot spring (368 239 N 1378 459 E), where the microbial community structure has been investigated by culture-independent methods (Nakagawa & Fukui, 2003;Kubo et al, 2011;Everroad et al, 2012). A sample of submerged microbial streamer was obtained from a pool of spring water (around 75 8C, pH 8.6-8.7).…”

Thermodesulfatator atlanticus sp. nov., a thermophilic, chemolithoautotrophic, sulfate-reducing bacterium isolated from a Mid-Atlantic Ridge hydrothermal vent

“…The type strain is AT1325 Over the past decades, microbiological investigations of a range of high-temperature marine and terrestrial environments have revealed the presence of a phylogenetically, metabolically and physiologically diverse community of thermophilic prokaryotes endemic to these particular habitats. Among the ubiquitous thermophilic taxa, members of the class Thermodesulfobacteria are commonly retrieved in hot biotopes, regardless of the geographical provenance of the samples (Skirnisdottir et al, 2000;Nakagawa & Fukui, 2003;Nakagawa et al, 2005).…”