Localizing transcripts to single cells suggests an important role of uncultured deltaproteobacteria in the termite gut hydrogen economy

Abstract

Identifying microbes responsible for particular environmental functions is challenging, given that most environments contain an uncultivated microbial diversity. Here we combined approaches to identify bacteria expressing genes relevant to catabolite flow and to locate these genes within their environment, in this case the gut of a "lower," wood-feeding termite. First, environmental transcriptomics revealed that 2 of the 23 formate dehydrogenase (FDH) genes known in the system accounted for slightly more than one-half of environmental transcripts. FDH is an essential enzyme of H 2 metabolism that is ultimately important for the assimilation of lignocellulose-derived energy by the insect. Second, single-cell PCR analysis revealed that two different bacterial types expressed these two transcripts. The most commonly transcribed FDH in situ is encoded by a previously unappreciated deltaproteobacterium, whereas the other FDH is spirochetal. Third, PCR analysis of fractionated gut contents demonstrated that these bacteria reside in different spatial niches; the spirochete is free-swimming, whereas the deltaproteobacterium associates with particulates. Fourth, the deltaproteobacteria expressing FDH were localized to protozoa via hybridization chain reaction-FISH, an approach for multiplexed, spatial mapping of mRNA and rRNA targets. These results underscore the importance of making direct vs. inference-based genespecies associations, and have implications in higher termites, the most successful termite lineage, in which protozoa have been lost from the gut community. Contrary to expectations, in higher termites, FDH genes related to those from the protozoan symbiont dominate, whereas most others were absent, suggesting that a successful gene variant can persist and flourish after a gut perturbation alters a major environmental niche.acetogenesis | RNA-Seq | microfluidic digital PCR

Mapping a multiplexed zoo of mRNA expression

“…The fact that amplification polymers carry up to hundreds of fluorophores (Choi et al, 2014) makes it possible to achieve high signal-to-background even when autofluorescence is high [e.g. in whole-mount vertebrate embryos (Choi et al, 2014;McLennan et al, 2015;Huss et al, 2015), in thick mouse brain sections (Sylwestrak et al, 2016) or in bacteria contained within environmental samples or other organisms (Rosenthal et al, 2013;Yamaguchi et al, 2015;Nikolakakis et al, 2015)]. The resulting HCR signal is stable for at least 1 week in zebrafish embryos stored in solution (Fig.…”

Acetogenesis from H2plus CO2and nitrogen fixation by an endosymbiotic spirochete of a termite-gut cellulolytic protist

“…In the case of the endosymbiont of Trichonympha, the sequenced genome carries neither acetyl-CoA pathway genes nor nitrogen fixation genes. However, associations of the second bacterial symbionts, which are different species depending on the Trichonympha lineages, have been reported, and they likely involve H 2 metabolism (19,41,42). One of them belongs to the genus Desulfovibrio, and genes for sulfate reduction and hydrogenase are identified (41).…”

“…One of them belongs to the genus Desulfovibrio, and genes for sulfate reduction and hydrogenase are identified (41). Another is an unclassified bacterial species in Deltaproteobacteria, and investigations of its formate dehydrogenase gene imply its importance for gut H 2 economy, possibly as a reductive acetogen (19).…”

“…One species, Treponema primitia, is known to carry out reductive acetogenesis and another, Treponema azotonutricium, is a nitrogen fixer (12)(13)(14)(15). Indeed, gene-based analyses suggest that treponemes are often major contributors for reductive acetogenesis and, to a lesser extent, nitrogen fixation in the gut microbial community (16)(17)(18)(19).…”

“…In one of these endosymbionts, an ability of nitrogen fixation is also predicted (20). Furthermore, gene inventory studies suggest the importance of protist-associated bacteria for H 2 metabolism or nitrogen fixation in termite guts (19,22). Therefore, the protist-associated bacteria are very likely to play important roles in the gut metabolism, but their actual activities and contributions are rarely evaluated and our knowledge of the symbiotic relationships is still fragmentary.…”