Filamentous "Epsilonproteobacteria" dominate microbial mats from sulfidic cave springs.
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Journal: 2004/January - Applied and Environmental Microbiology
ISSN: 0099-2240
PUBMED: 12957939
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Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where microbial mats dominated by filamentous morphotypes are found. The full-cycle rRNA approach, including 16S rRNA gene retrieval and fluorescence in situ hybridization (FISH), was used to identify these filaments. The majority of the obtained 16S rRNA gene clones from the mats were affiliated with the "Epsilonproteobacteria" and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was closely related to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic springs, and sulfidic caves (97 to 99% sequence similarity), while group II formed a novel clade moderately related to deep-sea hydrothermal vent symbionts (90 to 94% sequence similarity). FISH with newly designed probes for both groups specifically stained filamentous bacteria within the mats. FISH-based quantification of the two filament groups in six different microbial mat samples from Lower Kane Cave showed that LKC group II dominated five of the six mat communities. This study further expands our perceptions of the diversity and geographic distribution of "Epsilonproteobacteria" in extreme environments and demonstrates their biogeochemical importance in subterranean ecosystems.
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Appl Environ Microbiol 69(9): 5503-5511
PMID: 12957939

Filamentous “<em>Epsilonproteobacteria</em>” Dominate Microbial Mats from Sulfidic Cave Springs

Research Group for Microbial Geochemistry, Department of Geological Sciences, University of Texas at Austin, Austin, Texas 78712, Lehrstuhl für Mikrobiologie, Technische Universität München, D-85350 Freising, Germany, Department of Integrative Biology, Brigham Young University, Provo, Utah 84602, Department of Microbial Ecology, Institute of Ecology and Conservation Biology, Vienna University, A-1090 Vienna, Austria4
Corresponding author. Mailing address: Department of Geological Sciences, University of Texas at Austin, 1 University Station C1100, Austin, TX 78712-0254. Phone: (512) 471-5413. Fax: (512) 471-5766. E-mail: ude.saxetu.liam@legnea.
Received 2003 Feb 6; Accepted 2003 May 2.
Copyright © 2003, American Society for Microbiology

Abstract

Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where microbial mats dominated by filamentous morphotypes are found. The full-cycle rRNA approach, including 16S rRNA gene retrieval and fluorescence in situ hybridization (FISH), was used to identify these filaments. The majority of the obtained 16S rRNA gene clones from the mats were affiliated with the “Epsilonproteobacteria” and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was closely related to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic springs, and sulfidic caves (97 to 99% sequence similarity), while group II formed a novel clade moderately related to deep-sea hydrothermal vent symbionts (90 to 94% sequence similarity). FISH with newly designed probes for both groups specifically stained filamentous bacteria within the mats. FISH-based quantification of the two filament groups in six different microbial mat samples from Lower Kane Cave showed that LKC group II dominated five of the six mat communities. This study further expands our perceptions of the diversity and geographic distribution of “Epsilonproteobacteria” in extreme environments and demonstrates their biogeochemical importance in subterranean ecosystems.

Abstract

Caves containing hydrogen sulfide-rich springs represent less than 10% of all known caves globally (42). However, these caves serve as access points into sulfidic groundwater aquifers, typically associated with geothermal regions and oil-field basins, which play an important role in global sulfur cycling. The microbial communities colonizing sulfidic cave habitats have recently received attention due to their chemolithoautotrophic metabolism that can sustain complex ecosystems in the subsurface (48) and their geomicrobiological impact due to acid production (14, 60). Filamentous bacteria dominate subaqueous cave microbial mats, and from phylogenetic analyses, stable isotope evidence, and aqueous geochemistry surveys, populations are considered to be chemolithoautotrophic, aerobic to microaerophilic sulfur-oxidizing bacteria (5, 19, 48, 59). Recently, two cultivation-independent studies based on the phylogeny of bacterial community 16S rRNA genes characterized filamentous microbial mats from the Sulphur River of Parker Cave, Kentucky, and Cesspool Cave, Virginia (5, 14). In both 16S rRNA gene libraries, most clones were affiliated with uncharacterized environmental groups within the “Epsilonproteobacteria,” but the actual abundance and the morphotype(s) of the respective organisms were not established. Additionally, cultivation of any of these filamentous bacteria from sulfidic cave mats has been unsuccessful to date. The only sulfur bacteria isolated from these caves are gram-negative, rod-shaped thiobacilli (14, 60).

The most commonly studied genera of the “Epsilonproteobacteria,” Helicobacter and Campylobacter, are often associated with the gastrointestinal tract of animals as pathogens (13, 40). Other major phylogenetic groups within the “Epsilonproteobacteria” include Arcobacter, Wolinella, Sulfurospirillum, and Thiovulum, commonly found in natural settings as living cells or in symbiotic association with animals (13, 40, 51). Recently, members of the Arcobacter were also detected in activated sludge from wastewater treatment plants (53). Members of the Thiovulum phylogenetic group have been described from many natural habitats, some of which are considered extreme environments, including caves (5, 14), springs (47), groundwater associated with oil (17, 22, 64), marine water and muds (29, 58), deep-sea hydrothermal vent sites (9, 26, 36, 45), and vent-associated metazoans (8, 27). However, relatively little is known about the ecology or physiology of most of these “Epsilonproteobacteria,” as cultured representatives for most of the detected environmental clone groups are missing. Moreover, there are few detailed studies describing the occurrence of “Epsilonproteobacteria” from terrestrial environments compared to the relatively large number of such investigations from marine habitats.

In this study we investigated bacterial communities from filamentous microbial mats associated with aphotic sulfidic springs in Lower Kane Cave, a system located in north-central Wyoming. Using the full-cycle rRNA approach, including the construction of 16S rRNA gene clone libraries and quantitative fluorescence in situ hybridization (FISH), we report on the occurrence of two distinct epsilonproteobacterial filament groups within the microbial mats in the cave. Quantitative FISH with the newly designed 16S rRNA-targeted oligonucleotide probes for both groups revealed that members of one of the groups dominated the mat communities analyzed.

Acknowledgments

Special thanks to the Bureau of Land Management, Cody office, for cooperation in permitting this research. We thank S. Engel, T. Dogwiler, and R. Payn for field assistance and K. Crandall for laboratory support and critical insights.

This research was supported by a National Science Foundation LExEn grant (EAR-0085576) and in part by Brigham Young University and the Geology Foundation of the University of Texas at Austin.

Acknowledgments

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