NEW YORK (GenomeWeb News) – Japanese researchers have sequenced the first complete genome of a bacterial symbiont found within a termite gut.
Using an Applied Biosystems 3730 Sanger sequencer and a GS 20 pyrosequencing system from Roche’s 454 Life Sciences subsidiary, the researchers sequenced the genome of a yet uncultured bacterium — dubbed Rs-D17 — directly from its host cell. The results, which appeared online this week in the Proceedings of the National Academy of Sciences, provide clues about how the bacterium flourishes inside the termite’s belly as well as ways in which it functions interdependently with its host species.
In recent years, there has been much interest in understanding how termites process dead plant material, particularly because they may use enzymes that could be useful for biofuel production. And, some predict, the microorganisms living within termites may hold some answers to termite biology.
For instance, last year, researchers from the Department of Energy’s Joint Genome Institute and the California Institute of Technology published a metagenomic analysis of the organisms found in the midgut of a higher termite species — work that they suggested could ultimately contribute to generating new cellulosic biofuels.
For the current study, though, researchers focused on just one bacterial species, called Rs-D17, which lives inside another single-celled organism found in the gut of the termite Reticulitermes speratus. To sequence Rs-D17, the researchers collected the bacteria from one host protist cell at a time by micromanipulation. Then they directly sequenced the Rs-D17 bacterial cells by isothermal whole-genome amplification.
“Our strategy, acquiring a complete genome from a small number of almost pure genomovars, enabled the precise identification of pseudogenes and duplicated genes without ambiguity,” lead author Yuichi Hongoh, an environmental and molecular biologist at Japan’s RIKEN research institute, and colleagues wrote.
The researchers applied a hybrid sequencing strategy using the ABI and 454 platforms to generate data about Rs-D17’s 1,125,857 base-pair genome. The team reported complete coverage by Sanger sequencing and 97.7 percent coverage by 454 pyrosequencing at 42-fold redundancy.
Hongoh and colleagues then reassembled a circular Rs-D17 chromosome and assessed salient features of the genome. It contained 761 putative protein-coding genes and as many as 121 pseudogenes. Six regions of the genome appear to have been recently duplicated. The researchers also obtained sequence data for three circular plasmids ranging in size from 5,362 to 11,650 base pairs.
By doing sequence analysis, the team was able to identify genes related to the Rs-D17’s energy metabolism, defense mechanisms, and biosynthesis pathways. They also analyzed 59 genes that are unique to Rs-D17 and don’t share homology with sequences in public databases.
“This complete genome from a termite gut symbiont revealed the importance of provision of amino acids and cofactors by gut bacteria,” the authors noted. “[T]he Rs-D17 genome retains abundant genes for biosynthesis of those compounds despite its reduced genome size.”
The team speculated that such genes may help the symbiont fix atmospheric nitrogen and supply nitrogen-based compounds to termites — an important feature, since a termite’s wood-based diet is nitrogen poor. Similarly, other genes seemed to reflect Rs-D17’s adaptation to its environment and symbiotic relationship with termites and protists. But, they added, the adaptations have probably left the bacterial species unable to survive outside its host.
“The Rs-D17 genome shares the known characteristics of obligately intracellular symbionts, such as small genome size, small number of RNA genes, streamlining adaptation, few repertories of available carbon and energy sources, a weak cell wall and loss of regulators, defense mechanism, and transporters,” the authors explained.
Since the 16S rRNA sequence was already available for Rs-D17, they compared these sequences to check the purity of the clones analyzed. The majority of the clones sequenced — 84 of 89 — belonged to one Rs-D17 phylotype.
They also looked for genomic variation within this group by analyzing a region between the 16S and 23S rRNA genes in 48 clones. Overall, the results suggest that Rs-D17 genomes remain fairly consistent within each protist cell but vary widely between bacteria found in different protist cells.
The latest study adds to the body of knowledge related to the gut bacteria that help termites get the nutrients they need and break down plant material. And, the authors emphasized, the analysis provides a level of understanding about symbiotic relationships between specific microorganisms that isn’t possible by metagenomics alone.