Order-specific 16S rRNA-targeted oligonucleotide probes for (hyper)thermophilic archaea and bacteria

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Abstract
New oligonucleotide probes were designed and evaluated for application in fluorescence in situ hybridization (FISH) studies on (hyper)thermophilic microbial communities—Arglo32, Tcoc164, and Aqui1197 target the 16S rRNA of Archaeoglobales, Thermococcales, and Aquificales, respectively. Both sequence information and experimental evaluation showed high coverage and specificity of all three probes. The signal intensity of Aqui1197 was improved by addition of a newly designed, unlabeled “helper” oligonucleotide, hAqui1045. It was shown that in addition to its function as a probe for Aquificales, Aqui1197 is suitable as a supplementary probe to extend the coverage of the domain-specific bacterial probe EUB338. In sediments from two hydrothermal seeps on Vulcano Island, Italy, the microbial community structure was analyzed by FISH with both established and the new oligonucleotide probes, showing the applicability of Arglo32, Tcoc164, and Aqui1197/hAqui1045 to natural samples. At both sites, all major groups of (hyper)thermophiles, except for methanogens, were detected: Crenarchaeota (19%, 16%), Thermococcales (14%, 22%), Archaeoglobales (14%, 12%), Aquificales (5%, 8%), Thermotoga/Thermosipho spp. (12%, 9%), Thermus sp. (12%, none), and thermophilic Bacillus sp. (12%, 8%).

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