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Hot Spring Metagenomic Sequences Reveal New Bacterial Phylum

NEW YORK (GenomeWeb) – An international team reported online today in Nature Communications that it has detected signs of a previously unappreciated bacterial phylum in metagenomic sequences stemming from hot springs in the US, Canada, and China.

"It's not every day that you find a completely new phylum," first author Emiley Eloe-Fadrosh, a researcher with the Department of Energy's Joint Genome Institute, said in a statement. "With all the studies that have been conducted in hot springs, there's an assumption that all novelty has been found. But we found these unknown lineages in high abundance."

Eloe-Fadrosh and colleagues tracked down sequences from the phylum, dubbed Candidatus Kryptonia, when they sifted through terabytes of metagenomic sequence data from the Integrated Microbial Genomes with Microbiome Samples database, focusing in on contigs with previously undescribed ribosomal RNA gene sequences.

The search first led to four bacterial genera from geothermal spring sites that fell in a previously undiscovered lineage, though additional examples of these genera turned up during the team's single-cell analyses of samples from the sites.

Based on genome sequences for the original four bacteria, the group began reconstructing information about their lifestyle, metabolic capabilities, and exposures to bacteria-infecting phage viruses, which were estimated using CRISPR-Cas system sequences.

"While a lot of research and media attention is gathering around the biotechnological applications of the CRISPR-Cas system, we are very excited about using it as a powerful tool in reconstructing the infection history of the organisms, as well as a fingerprint to uncover and trace the correlated viruses," co-author Nikos Kyrpides, also at JGI, said in a statement.

Starting from 5.2 terabytes of metagenomic sequence data, the team put together hundreds of contigs containing bacterial small subunit ribosomal RNA gene sequences.

When they attempted to match these up with rRNA gene profiles in the existing phylogenetic tree, the researchers found that one microbe didn't fit into any known bacterial lineage.

Instead it fell into a new lineage in the Fibrobacteres-Chlorobi-Bacteroidetes superfamily, alongside three more genera detected only from sequences in the metagenomic collection.

The microbes — which shared more than 97 percent small subunit rRNA sequence identity, on average — came from neutral pH samples collected in Nevada's Great Boiling Spring, the Dewar Creek Spring in Canada, and two sites in China: the Gongxiaoshe and Jinze geothermal springs.

To confirm the existence of bacteria from this new lineage, the researchers used single-cell amplification and sequencing to put together 18 Ca. Kryptonia genomes from cells obtained at three of the hot springs. They also visualized the microbes with fluorescence in situ hybridization experiment targeting the newly identified rRNA sequences.

Still other representatives from the phylum turned up — at three more very hot, pH-neutral locales — when the group did a broader search for Ca. Kryptonia sequences in thousands of metagenomic datasets.

In each of the sequenced Ca. Kryptonia genomes, the researchers uncovered a fusion between the CRISPR-Cas subtype I-B and subtype III-A that seemed capable of functioning in bacterial defense processes based on genes present.

From the types of spacer sequences carried in the CRISPR-Cas system, the team speculated that a Ca. Kryptobacter tengchongensis species from the Jinze hot spring has faced off against many viruses, while a species from the Dewar Creek spring showed signs of more limited CRISPR-Cas activity.

Finally, genome sequence data offered a peek at potential metabolic promise and pitfalls in Ca. Kryptonia. For example, the absence of some amino acid and vitamin-producing pathways hinted at reliance on other members of the microbial community.

"Our study suggests that dependency on other organisms within the geothermal spring community might be a more common occurrence than previously appreciated," the authors wrote, "perhaps contributing to challenges in obtaining many of these lineages as isolated monocultures."