Diversity of Formyltetrahydrofolate Synthetases in the Guts of the Wood-Feeding Cockroach Cryptocercus punctulatus and the Omnivorous Cockroach Periplaneta americana

We examined the diversity of a marker gene for homoacetogens in two cockroach gut microbial communities. Formyltetrahydrofolate synthetase (FTHFS or fhs) libraries prepared from a wood-feeding cockroach, Cryptocercus punctulatus, were dominated by sequences that affiliated with termite gut treponemes. No spirochete-like sequences were recovered from the omnivorous roach Periplaneta americana, which was dominated by Firmicutes-like sequences

To DGC or not to DGC: oxygen guarding in the termite Zootermopsis nevadensis (Isoptera: Termopsidae)

The ability of some insects to engage in complex orchestrations of tracheal gas exchange has been well demonstrated, but its evolutionary origin remains obscure. According to a recently proposed hypothesis, insects may employ spiracular control of gas exchange to guard tissues against long-term oxidative damage by using the discontinuous gas-exchange cycle (DGC) to limit internal oxygen partial pressure (P_(O_2)). This manuscript describes a different approach to oxygen guarding in the lower termite Zootermopsis nevadensis. These insects do not display a DGC but respond to elevated oxygen concentrations by restricting spiracular area, resulting in a transient decline in CO_2 emission. High internal CO_2 concentrations are then maintained; restoring normoxia results in a transient reciprocal increase in CO_2 emission caused by release of excess endotracheal CO_2. These changes in spiracular area reflect active guarding of low internal O_2 concentrations and demonstrate that regulation of endotracheal hypoxia takes physiological priority over prevention of CO_2 build-up. This adaptation may reflect the need to protect oxygen-sensitive symbionts (or, gut bug guarding). Termites may eschew the DGC because periodic flushing of the tracheal system with air may harm the obligate anaerobes upon which the lower termites depend for survival on their native diet of chewed wood

Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite

From the standpoints of both basic research and biotechnology, there is considerable interest in reaching a clearer understanding of the diversity of biological mechanisms employed during lignocellulose degradation. Globally, termites are an extremely successful group of wood-degrading organisms and are therefore important both for their roles in carbon turnover in the environment and as potential sources of biochemical catalysts for efforts aimed at converting wood into biofuels. Only recently have data supported any direct role for the symbiotic bacteria in the gut of the termite in cellulose and xylan hydrolysis. Here we use a metagenomic analysis of the bacterial community resident in the hindgut paunch of a wood-feeding ‘higher’ Nasutitermes species (which do not contain cellulose-fermenting protozoa) to show the presence of a large, diverse set of bacterial genes for cellulose and xylan hydrolysis. Many of these genes were expressed in vivo or had cellulase activity in vitro, and further analyses implicate spirochete and fibrobacter species in gut lignocellulose degradation. New insights into other important symbiotic functions including H_2 metabolism, CO_2-reductive acetogenesis and N_2 fixation are also provided by this first system-wide gene analysis of a microbial community specialized towards plant lignocellulose degradation. Our results underscore how complex even a 1-μl environment can be

Probing Individual Environmental Bacteria for Viruses by Using Microfluidic Digital PCR

Viruses may very well be the most abundant biological entities on the planet. Yet neither metagenomic studies nor classical phage isolation techniques have shed much light on the identity of the hosts of most viruses. We used a microfluidic digital polymerase chain reaction (PCR) approach to physically link single bacterial cells harvested from a natural environment with a viral marker gene. When we implemented this technique on the microbial community residing in the termite hindgut, we found genus-wide infection patterns displaying remarkable intragenus selectivity. Viral marker allelic diversity revealed restricted mixing of alleles between hosts, indicating limited lateral gene transfer of these alleles despite host proximity. Our approach does not require culturing hosts or viruses and provides a method for examining virus-bacterium interactions in many environments

Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria

Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA–based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment

Disentangling hindgut metabolism in the American cockroach through single-cell genomics and metatranscriptomics

Omnivorous cockroaches host a complex hindgut microbiota comprised of insect-specific lineages related to those found in mammalian omnivores. Many of these organisms have few cultured representatives, thereby limiting our ability to infer the functional capabilities of these microbes. Here we present a unique reference set of 96 high-quality single cell-amplified genomes (SAGs) from bacterial and archaeal cockroach gut symbionts. We additionally generated cockroach hindgut metagenomic and metatranscriptomic sequence libraries and mapped them to our SAGs. By combining these datasets, we are able to perform an in-depth phylogenetic and functional analysis to evaluate the abundance and activities of the taxa in vivo. Recovered lineages include key genera within Bacteroidota, including polysaccharide-degrading taxa from the genera Bacteroides, Dysgonomonas, and Parabacteroides, as well as a group of unclassified insect-associated Bacteroidales. We also recovered a phylogenetically diverse set of Firmicutes exhibiting a wide range of metabolic capabilities, including—but not limited to—polysaccharide and polypeptide degradation. Other functional groups exhibiting high relative activity in the metatranscriptomic dataset include multiple putative sulfate reducers belonging to families in the Desulfobacterota phylum and two groups of methanogenic archaea. Together, this work provides a valuable reference set with new insights into the functional specializations of insect gut symbionts and frames future studies of cockroach hindgut metabolism

Multispecies diel transcriptional oscillations in open ocean heterotrophic bacterial assemblages

Oscillating diurnal rhythms of gene transcription, metabolic activity, and behavior are found in all three domains of life. However, diel cycles in naturally occurring heterotrophic bacteria and archaea have rarely been observed. Here, we report time-resolved whole-genome transcriptome profiles of multiple, naturally occurring oceanic bacterial populations sampled in situ over 3 days. As anticipated, the cyanobacterial transcriptome exhibited pronounced diel periodicity. Unexpectedly, several different heterotrophic bacterioplankton groups also displayed diel cycling in many of their gene transcripts. Furthermore, diel oscillations in different heterotrophic bacterial groups suggested population-specific timing of peak transcript expression in a variety of metabolic gene suites. These staggered multispecies waves of diel gene transcription may influence both the tempo and the mode of matter and energy transformation in the sea.Gordon and Betty Moore Foundation (GBMF 492.01)Gordon and Betty Moore Foundation (GBMF 3777)National Science Foundation (U.S.) (Grant EF0424599)David & Lucile Packard Foundatio

Distinct dissolved organic matter sources induce rapid transcriptional responses in coexisting populations of Prochlorococcus, Pelagibacter and the OM60 clade

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in Environmental Microbiology 16 (2014): 2815-2830, doi:10.1111/1462-2920.12254.A considerable fraction of the Earth's organic carbon exists in dissolved form in seawater. To investigate the roles of planktonic marine microbes in the biogeochemical cycling of this dissolved organic matter (DOM), we performed controlled seawater incubation experiments and followed the responses of an oligotrophic surface water microbial assemblage to perturbations with DOM derived from an axenic culture of Prochlorococcus, or high-molecular weight DOM concentrated from nearby surface waters. The rapid transcriptional responses of both Prochlorococcus and Pelagibacter populations suggested the utilization of organic nitrogen compounds common to both DOM treatments. Along with these responses, both populations demonstrated decreases in gene transcripts associated with nitrogen stress, including those involved in ammonium acquisition. In contrast, responses from low abundance organisms of the NOR5/OM60 gammaproteobacteria were observed later in the experiment, and included elevated levels of gene transcripts associated with polysaccharide uptake and oxidation. In total, these results suggest that numerically dominant oligotrophic microbes rapidly acquire nitrogen from commonly available organic sources, and also point to an important role for carbohydrates found within the DOM pool for sustaining the less abundant microorganisms in these oligotrophic systems.This work was supported by a National Science Foundation Science and Technology Center Award EF0424599 (E.F.D and D.M.K.), grants to D.M.K., D.J.R and E.F.D from the Gordon and Betty Moore Foundation, a gift from the Agouron Institute (to E.F.D.) and a fellowship (202180) to A.K.S. from the Canadian Institutes of Health Research (CIHR)

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

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 gene– species 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

Development and quantitative analyses of a universal rRNA-subtraction protocol for microbial metatranscriptomics

Metatranscriptomes generated by pyrosequencing hold significant potential for describing functional processes in complex microbial communities. Meeting this potential requires protocols that maximize mRNA recovery by reducing the relative abundance of ribosomal RNA, as well as systematic comparisons to identify methodological artifacts and test for reproducibility across data sets. Here, we implement a protocol for subtractive hybridization of bacterial rRNA (16S and 23S) that uses sample-specific probes and is applicable across diverse environmental samples. To test this method, rRNA-subtracted and unsubtracted transcriptomes were sequenced (454 FLX technology) from bacterioplankton communities at two depths in the oligotrophic open ocean, yielding 10 data sets representing ~350 Mbp. Subtractive hybridization reduced bacterial rRNA transcript abundance by 40–58%, increasing recovery of non-rRNA sequences up to fourfold (from 12% to 20% of total sequences to 40–49%). In testing this method, we established criteria for detecting sequences replicated artificially via pyrosequencing errors and identified such replicates as a significant component (6–39%) of total pyrosequencing reads. Following replicate removal, statistical comparisons of reference genes (identified via BLASTX to NCBI-nr) between technical replicates and between rRNA-subtracted and unsubtracted samples showed low levels of differential transcript abundance (<0.2% of reference genes). However, gene overlap between data sets was remarkably low, with no two data sets (including duplicate runs from the same pyrosequencing library template) sharing greater than 17% of unique reference genes. These results indicate that pyrosequencing captures a small subset of total mRNA diversity and underscores the importance of reliable rRNA subtraction procedures to enhance sequencing coverage across the functional transcript pool.Agouron InstituteGordon and Betty Moore FoundationUnited States. Dept. of Energy. Office of ScienceNational Science Foundation (U.S.) (NSF Science and Technology Center Award EF0424599