The results of all four original articles demonstrate that a large number of previously “unknown” Archaea exist in "non-extreme" habitats, i.e. boreal forest soils, pelagic water of a boreal forest lake and temperate estuary sediment.
In all four articles the same PCR primers and similar PCR conditions were used. However, the rRNA sequences obtained were phylogenetically very diverse. For example, no euryarchaeotal sequences were found in any of the soil samples, whereas half of the sequences retrieved from the water samples were euryarchaeotal. This would indicate that the results obtained were not biased towards particular groups, but reflected the actual diversity and distribution of Archaea in these environments.
All 57 sequenced clones were aligned using ARB editor which found only some non-complementary secondary structure helix positions. This observation supports the results of the CHECK_CHIMERA program and suggests that the obtained sequences are not chimeras.
Paper I was among the very first reports describing the existence of Crenarchaeota in soil. The results of papers I and II provide evidence that boreal forest soil contains a diverse Crenarchaeota community. They also show that Crenarchaeota are found in soils, which have been subjected to strong ecological and physico-chemical treatments such as clear-cutting and burning. The results indicate that diverse representatives of the Crenarchaeota form a stable part of the highly variable microbial community in boreal forest soil, which provides a nutritionally rich, but physically labile environment, with periods of fast freezing and thawing. Very little is known of the role or metabolic activity of Archaea in soil, except for methanogenic genera belonging to the Euryarchaeota. The novel Archaea found in soil are phylogenetically distant from the Euryarchaeota, and form a cluster distinct from any other cultured Crenarchaeota. It is therefore not possible to infer their ecological role from other known Archaea.
In paper III, new non-thermophilic Crenarchaeota 16S rRNA gene sequences were recovered from estuarine sediment samples. Phylogenetic analysis revealed that these sequences were closely related to marine and freshwater-lake Crenarchaeota 16S rRNA gene sequences retrieved from a diverse range of geographical sites, such as North America (MacGregor et al., 1997), the Antarctic Ocean (Delong et al., 1994) and Japan (Li et al., 1999). Most of the sequences were isolated from the top layer of the sediment cores sampled and may suggest an aerobic metabolism. However, the phylogenetic distance between these novel sequences and the cultivated members of the Crenarchaeota prevents any inference of their physiological properties.
Two sequences were isolated from a layer of active iron oxidation, suggesting that these microorganisms might be involved in biogeochemical processes that occur in this zone of the sediments. Two other sequences from this study were affiliated with euryarchaeotal sequences retrieved from deep-sea sediment (Vetriani et al., 1999) indicating that these microorganisms are present in a wide range of environments. Although the molecular and phylogenetic data collected in this study cannot help in inferring an ecological role for these microorganisms in the environment, these findings are of fundamental value for understanding the complexity of estuarine ecosystems.
Sequences detected in the pelagic water of a boreal forest lake (paper IV) using a rRNA approach belonged to members of the Crenarchaeota and Euryarchaeota. Using fluorescent in situ hybridization, it was possible to detect euryarchaeotal microorganisms and quantify their abundance in the water samples. An important step forward in paper IV (comparing to papers I, II and III) was the visualization of euryarchaeal organisms by FISH. These results indicated that euryarchaeal sequences obtained by PCR did not originate from DNA extracted from dead or dormant cells, but from active cells present in the water samples.
Positive hybridization signals were not obtained using Crenarchaeota-specific probes CREN499, CREN512 and CREN569 probes. Hybridization with the Euryarchaeota specific probes, on the other hand, detected as many cells as the general Archaea probe. The available probes appeared to detect only Euryarchaea species and the total number of Archaea in the samples is therefore probably underestimated. In theory, PCR can identify a single copy of a gene with appropriate condition and primers (Saiki et al., 1988) and FISH can detect a single cell (Poulsen et al., 1993). However, both methods have limitations when analyzing environmental samples. For example, humic compounds present in sediment and soil samples can decrease the efficiency of the PCR by inhibiting DNA polymerase activity (Fritze et al., 1999). Several factors can affect successful fluorescence in situ hybridization as well. The contrasting FISH and PCR results could be due to a low numbers of Crenarchaeota cells in the samples, and/or low ribosome contents in the cells (Roszak and Colwell, 1987; Kotler et al., 1993). Other possible reasons include physical-chemical factors, e.g. reduced penetration of oligonucleotide probes through the cell walls of Crenarchaeota; or probes targeting regions of 16S rRNA which are inaccessible due to secondary structure conformation. The permeation process during hybridization is difficult to adjust so that organisms with different cell envelope structures are simultaneously detected. Improvement of this process might reveal more Crenarchaeota in lake water samples.
The existence of Archaea in the freshwater environment was expected as methanogens have been found previously in freshwater ecosystems. However, the large diversity of retrieved archaeal 16S rRNA sequences, and the low sequence similarity to previously recovered sequences from pelagic interfaces, was surprising.
Original papers I, II and III have shown that representatives of the Crenarchaeota inhabit boreal forest soils and estuarine sediment. Paper IV have shown that they also occupy pelagic habitats in a boreal forest lake. It was found that in all three biotopes, the crenarchaeotal 16S rRNA gene sequences were not only diverse, but formed novel phylogenetic clusters distinct from sequences recovered from other environments. The Crenarchaeota found in the boreal forest lake water were significantly different from Crenarchaeota found in the boreal forest soil and estuarine sediment samples. This may be due to the different geographical location of each sampling site, or inherent differences in the properties of each biotope examined.
The Euryarchaeota detected in water samples occupy a very specific niche. As many of the sequences were only distantly related to any of the main metabolic subgroups of Euryarchaeota (namely the methanogens or halophiles), their metabolic properties and role in the environment could not be inferred.