In this work we used Crenarchaeota
-specific primers to detect Crenarchaeota
in thermal habitats of Kamchatka, the Lake Baikal region, and Iceland, which had different pH and temperature values. Furthermore, we adopted these primers for carrying out DGGE analysis in order to reveal the phylogenetic position of the detected Crenarchaeota.
All sequences obtained in the course of this work belonged to Crenarchaeota
. These results, as well as our previous data (38
), demonstrate the specificity of the Cren7F-Cren518R primer pair to Crenarchaeota
So far, the majority of the cultured Crenarchaeota
are hyperthermophilic neutrophiles which have an optimal growth temperature of 80 to 90°C. Scarce extreme thermophiles with growth temperature optima of 65 to 70°C are acidophiles and predominantly aerobes or facultative anaerobes belonging to Sulfolobales
, with Thermocladium modestius
and the two species of Caldisphaera
as the only obligate anaerobes in this group (7
). Among the crenarchaeotal sequences detected by us, there were sequences related to cultivated organisms of the orders Thermoproteales
and to the Acidilobus
group. The temperature and pH characteristics of the sampling sites where these sequences were detected were in good agreement with those of known cultivated representatives of the genera Thermoproteus
), and Caldisphaera
In our search for new thermophilic Crenarchaeota
, we studied many neutral hot springs that had water temperatures ranging from 55 to 70°C, i.e., exhibiting conditions not optimal for known cultured Crenarchaeota.
Five crenarchaeotal sequences, four from different sites of Uzon Caldera, Kamchatka, and one from Hverakjalki, Iceland, belonged to the deep-branching Crenarchaeota
lineage of clone pSL12, which has been proposed to form a clade with the marine nonthermophilic group of the Crenarchaeota
). Sequences belonging to this lineage were also found in hot springs of Yellowstone National Park, as well as of Iceland, Italy, and Thailand (26
). Another deep-branching lineage of Crenarchaeota
(clone pJP89 lineage), previously found only in Obsidian Pool, Yellowstone National Park (3
), was detected by us in Icelandic hot springs (phylotypes Is9, Is5, and V5).
In one of the samples from Lake Baikal region (BL1017), we found a phylotype distantly related to a deep-branching Crenarchaeota
lineage (clone pJP41 lineage), observed previously in Obsidian Pool, Yellowstone National Park (3
), and the Hveragerði area, Iceland (31
). Our attempts to cultivate this organism under various cultivation conditions led us to the conclusion that the pJP41-related Crenarchaeota
could be moderately thermophilic, neutrophilic, or moderately alkaliphilic anaerobic organotrophs not dependent on the presence of elemental sulfur.
Given the trees constructed by Pace et al. (2
) and our tree (Fig. ), at least two thermophilic lineages occur within the radiation of nonthermophilic Crenarchaeota
—the clone pSL12 lineage and the clone pJP89 lineage. In contrast, the clone pJP41 phylogenetic lineage appears to be an early offspring of the (hyper)thermophilic Crenarchaeota
. These phylogenetic positions are supported by the occurrence of signatures 289C:311G, 501G, and 504A in clones pSL12 and pJP89 and our phylotypes related to them and by the presence of only 289C:311G in pJP41 and our phylotypes related to it (Table ).
Sequence signatures of 16S rRNAs of uncultured moderately thermophilic Crenarchaeota
The distribution of the four groups of uncultured thermophilic crenarchaeotes (the pSL12, pJP89, pJP41, and “Fervidococcus
” groups) in natural environments is shown in Fig. in temperature-pH coordinates. It appears from the diagram that pSL12-related sequences were previously found in a rather narrow range of temperatures (74 to 81°C) and pH values (5.7 to 7.6). Geochemical data for the hot springs of Iceland were not reported, whereas Obsidian Pool is known to contain H2
S and high concentrations of H2
, and NH4+
). Our data on Uzon Caldera, Kamchatka, showed that this group of organisms is also present in hot springs having temperatures from 57°C to 75°C. The habitats of these organisms in Uzon Caldera are characterized by the presence of sulfide and low redox conditions. These observations confirm that the deep-branching phylogenetic lineage of nonthermophilic crenarchaeotes is not limited to low-temperature environments but actually includes thermophilic subbranches. These organisms are most probably moderate thermophiles and neutrophiles. Furthermore, as we mentioned above, the clone pSL12 lineage is adjacent to the group of nonthermophilic marine and soil Crenarchaeota
, in which the presence of amoA
) or nitrification capacity (13
) has repeatedly been demonstrated by metagenomics or cultural methods, respectively. It cannot be excluded that the microorganisms related to the clone pSL12 lineage function as nitrifiers in the hot springs of Uzon Caldera, where ammonia ions are present.
FIG. 4. Temperature and pH of hot springs where phylotypes related to the pSL12 (squares), pJP89 (asterisks), pJP41 (triangles), and “Fervidococcus” (circles) lineages were found. Filled symbols represent data obtained in this work; empty symbols (more ...)
The information on the habitats of pJP41-related and pJP89-related organisms is rather scarce and consists only of Obsidian Pool parameters (74 to 81°C and pH 5.7 to 7.0). This information is extended by our data for the Icelandic hot spring (69°C; pH 6.5), where a pJP89-related phylotype was detected, and for the Baikal region hot spring (59°C; pH 8.8), where the pJP41-related organism was found. The growth characteristics of the pJP41-related crenarchaeote present in enrichment culture BL1017 (60 to 65°C and pH 7.0 to 7.5) were somewhere in between the parameters of these two natural habitats. Baikal habitat differs from Obsidian Pool by the absence of sulfide although the redox potential of water there was also low, corresponding to the anaerobic nature of the pJP41-related crenarchaeote BL1017.
Monitoring with Crenarchaeota
-specific primers also allowed us to isolate the first representative of “uncultured Desulfurococcales
,” which we have named “F. fontis
.” Environmental clones of the “Fervidococcus
” group were found in Yellowstone National Park in the Sylvan and Bison springs (33
), which have temperatures of 81 to 82.6°C and pH values of 5.5 to 8.1, and in the Hveragerði area (Iceland) (31
), but geochemical data on the hot springs of Iceland were not reported. According to our data, the temperature and pH range of the hot springs where the “Fervidococcus
”-related phylotypes were detected is very broad. These data are in good agreement with the “F. fontis
” growth parameters.
It follows from the diagram in Fig. that there could be a large and diverse group of Crenarchaeota
that inhabit terrestrial hot springs with temperatures that are moderate (55 to 70°C) compared with the parameters of the usual habitats of hyperthermophilic cultured Crenarchaeota
. The adaptation of the former Crenarchaeota
group to moderate-temperature habitats is evidenced by the data on the G+C contents of their 16S rRNAs. The G+C contents of the 16S rRNAs are known to correlate with the temperature growth optima of the organisms (18
). Our analysis showed that the G+C content of the 16S rRNA fragment flanked by Cren7F and Cren518R primers also shows good correlation with the temperature optimum. For example, the G+C content of this fragment in the Desulfurococcales
in the GreenGenes database is 62 to 72 mol%, whereas that of marine nonthermophilic Crenarchaeota
is 45 to 51 mol%. The G+C content of this fragment in our pSL12-related phylotypes was 56 to 57 mol%, which correlates with the temperature characteristics of their habitats. In our “Fervidococcus
”-related phylotypes, this value was 62 to 63 mol%, which correlates with the temperature characteristics of habitats and with those of “F. fontis
Thus, the combination of culture-independent and cultivation-based methods applied to microbial communities of hot springs of Russia and Iceland allowed us to extend the knowledge of the biodiversity of Crenarchaeota representatives. This work also sheds light on the ecological function of some Crenarchaeota groups commonly referred to as uncultivated. “F. fontis” is a strict anaerobe, organotroph, and neutrophile, growing at temperatures lower than other anaerobic neutrophilic Crenarchaeota. Thus, the organisms of this group share an ecological niche with many thermophilic bacteria. This seems to explain how these organisms have so far escaped cultivation under laboratory conditions as they are outcompeted by bacteria at lower temperatures and by hyperthermophilic archaea at higher temperatures.