Archaeal diversity in a terrestrial acidic spring field revealed by a novel PCR primer targeting archaeal 16S rRNA genes

Abstract

The phylogenetic diversity of archaeal 16S rRNA genes in a thermoacidic spring field of Ohwakudani, Hakone, Japan, was investigated by PCR-based analysis using a novel Archaea-specific primer designed in the present study. Clone libraries of archaeal 16S rRNA genes were constructed from hot water (78 °C) and mud (28 °C) samples by PCR using a newly designed forward primer and a previously reported forward primer with reverse primers. Most phylotypes found in the libraries from the hot water sample were related to cultured (hyper)thermophiles. The phylotypes and their detection frequencies from the hot water sample were similar for the libraries amplified with the two different primer sets. In contrast, phylotypes having a low similarity (<95%) to cultured Archaea were found in the libraries from the mud sample. Some of the phylotypes were relatively close to members of Thermoplasmata (80-93% similarity) and the others were not clearly affiliated with Crenarchaeota and Euryarchaeota, but related to Thaumarchaeota and Korarchaeota. The phylotypes and their detection frequencies were significantly different between the two libraries of the mud sample. Our results from the PCR-based analysis using the redesigned primer suggest that more diverse, uncultured Archaea are present in acidic environments at a low temperature than previously recognized.

Predominant Acidilobus-Like Populations from Geothermal Environments in Yellowstone National Park Exhibit Similar Metabolic Potential in Different Hypoxic Microbial Communities

“…Nearly identical full-length 16S rRNA gene sequences have been observed in these same sites upon repetitive sampling (4), which indicates a consistent presence of this specific phylotype in acidic (pH 3 to 6), thermal (70°C to 80°C) habitats of YNP. Related Acidilobus populations are also observed in sulfidic thermal habitats in Kamchatka and Japan (28,53); however, the 16S rRNA gene sequences of the YNP populations are only 97% identical to either Acidilobus sulfurireducens or Ac. saccharovorans, which were isolated from Yellowstone National Park (54) and Kamchatka, Russia (17), respectively.…”

The Archaea Community Associated with Lava-Formed Gotjawal Forest Soil in Jeju, Korea

“…A total of five euryarchaeal clones belonged to Thermoplasma-related group III (TRG-III) [19]. The phylotypes in TRG-III were related to environmental clones recovered from acidic environments [20]- [22]. One euryarchaeal clone was related to methanogens of the Methanosaetaceae family (Figure 2(b)).…”

Functional Gene Analysis of Freshwater Iron-Rich Flocs at Circumneutral pH and Isolation of a Stalk-Forming Microaerophilic Iron-Oxidizing Bacterium

“…In the end, the culture showing growth at the highest dilution was designated strain OYT1 (deposited in JCM as JCM 18545 and in DSMZ as DSM 26810). Purity was confirmed by microscopic observation, streaking cell suspensions on R2A agar plates to check for the presence of contaminating heterotrophs, and repeated partial sequencing of the 16S rRNA genes amplified by PCR using the bacterial and archaeal universal primer sets Bac27F (26) or Arc9F (27) and Uni1406R (18), as described below. Cells were stained with SYBR green I for visualization by fluorescence microscopy.…”

“…DNA was extracted from the samples (approximately 0.5 g) using the FastDNA spin kit for soil and the FastPrep instrument (MP Biomedicals, Santa Ana, CA, USA). PCR was performed with the following oligonucleotide primers: Bac27F (26) and Uni1406R (18) for the bacterial 16S rRNA gene, Arc9F (27) and Uni1406R for the archaeal 16S rRNA gene, A189f and A682r (28) for pmoA, and cbbM343F and cbbM1226R (29) for cbbM. In addition, PCRs for pmoA and cbbM were also performed for the DNA extracts from the iron-rich microbial mat collected previously in Budo Pond, Hiroshima University, Japan (20).…”