Over 200 years ago Alexander von Humboldt (1808) observed that plant and animal diversity peaks at tropical latitudes and decreases toward the poles, a trend he attributed to more favorable temperatures in the tropics. Studies to date suggest that this temperature-diversity gradient is weak or nonexistent for Bacteria and Archaea. To test the impacts of temperature as well as pH on bacterial and archaeal diversity, we performed pyrotag sequencing of 16S rRNA genes retrieved from 165 soil, sediment and biomat samples of 36 geothermal areas in Canada and New Zealand, covering a temperature range of 7.5-99 1C and a pH range of 1.8-9.0. This represents the widest ranges of temperature and pH yet examined in a single microbial diversity study. Species richness and diversity indices were strongly correlated to temperature, with R 2 values up to 0.62 for neutralalkaline springs. The distributions were unimodal, with peak diversity at 24 1C and decreasing diversity at higher and lower temperature extremes. There was also a significant pH effect on diversity; however, in contrast to previous studies of soil microbial diversity, pH explained less of the variability (13-20%) than temperature in the geothermal samples. No correlation was observed between diversity values and latitude from the equator, and we therefore infer a direct temperature effect in our data set. These results demonstrate that temperature exerts a strong control on microbial diversity when considered over most of the temperature range within which life is possible.

The exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced by Gluconacetobacter xylinus or the EPS produced by Beijerinckia indica. The latter is a heteropolysaccharide comprised primarily of L-guluronic acid, D-glucose, and D-glycero-D-mannoheptose.13 C-labeled EPS and 13 C-labeled cellulose were purified from bacterial cultures grown on [ 13 C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from 13 C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However, B. indica EPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylum Planctomycetes. In one incubation, members of the Planctomycetes made up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo-or monosaccharides, the predominance of Planctomycetes suggested that they were primary degraders of EPS. Other bacteria assimilating B. indica EPS included members of the Verrucomicrobia, candidate division OD1, and the Armatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species. Some major phyla of the domain Bacteria, like the Proteobacteria and Actinobacteria, are well-known from cultivation studies. Others, like Acidobacteria, Verrucomicrobia, and Planctomycetes, have few cultured species, although they make up large proportions of the bacterial 16S rRNA genes detected in environments such as soil (1). One theory proposed to explain the difficulty in culturing bacteria from these phyla is that most are K selected as opposed to r selected. Typical r-selected bacteria grow rapidly on simple monomeric substrates in nutrient-rich environments, conditions presented by most microbiological media (2, 3). On the other hand, K-selected bacteria have a more efficient cell metabolism and strong competitive ability and grow slowly on recalcitrant, complex substances (3-5). Several studies have demonstrated that members of difficult-to-culture phyla like Acidobacteria, Verrucomicrobia, and Planctomycetes can be cultivated using polysaccharides, such as xylan, xanthan, and pectin, as energy sources (e.g., see references 6 to 8). The heteropolysaccharide gellan, which is often used as a gelling agent as an alternative to agar, can also act as an energy source for some soil bacteria (9), in part explaining the observation that it improves cultu...

and LAY possessed only a soluble form of methane monooxygenase (sMMO) and lacked intracytoplasmic membranes. Growth occurred only on methane and methanol; the latter was the preferred growth substrate. mRNA transcripts of sMMO were detectable in cells when either methane or both methane and methanol were available. Carbon was assimilated via the serine and ribulose-bisphosphate (RuBP) pathways; nitrogen was fixed via an oxygen-sensitive nitrogenase. Strains AR4 T , SOP9 and LAY were moderately acidophilic, mesophilic organisms capable of growth between pH 3.5 and 7.2 (optimum pH 4.8-5.2) and at 4-33 6C (optimum 20-23 6C).The major cellular fatty acid was 18 : 1v7c and the quinone was Q-10. The DNA G+C content was 55.6-57.5 mol%. The isolates belonged to the family Beijerinckiaceae of the class Alphaproteobacteria and were most closely related to the sMMO-possessing methanotrophs of the genus Methylocella (96.4-97.0 % 16S rRNA gene sequence similarity), particulate MMO (pMMO)-possessing methanotrophs of the genus Methylocapsa (96.1-97.0 %), facultative methylotrophs of the genus Methylovirgula (96.1-96.3 %) and non-methanotrophic organotrophs of the genus Beijerinckia (96.5-97.0 %). Phenotypically, strains AR4 T , SOP9 and LAY were most similar to Methylocella species, but differed from members of this genus by cell morphology, greater tolerance of low pH, detectable activities of RuBP pathway enzymes and inability to grow on multicarbon compounds. Therefore, we propose a novel genus and species, Methyloferula stellata gen. nov., sp. nov., to accommodate strains AR4 The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences and partial mmoX gene sequences of strains AR4 T , SOP9 and LAY are FR686343-FR686345 (16S rRNA gene) and FR686346-FR686348 (mmoX), respectively, and those for the partial mxaF, nifH and rbcL sequences of strain AR4T are FR686349, FR686351 and FR686352, respectively.

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Analysis of the increasing wealth of metagenomic data collected from diverse environments can lead to the discovery of novel branches on the tree of life. Here we analyse 5.2 Tb of metagenomic data collected globally to discover a novel bacterial phylum (‘Candidatus Kryptonia') found exclusively in high-temperature pH-neutral geothermal springs. This lineage had remained hidden as a taxonomic ‘blind spot' because of mismatches in the primers commonly used for ribosomal gene surveys. Genome reconstruction from metagenomic data combined with single-cell genomics results in several high-quality genomes representing four genera from the new phylum. Metabolic reconstruction indicates a heterotrophic lifestyle with conspicuous nutritional deficiencies, suggesting the need for metabolic complementarity with other microbes. Co-occurrence patterns identifies a number of putative partners, including an uncultured Armatimonadetes lineage. The discovery of Kryptonia within previously studied geothermal springs underscores the importance of globally sampled metagenomic data in detection of microbial novelty, and highlights the extraordinary diversity of microbial life still awaiting discovery.