AnnoView enables large-scale analysis, comparison, and visualization of microbial gene neighborhoods.

The analysis and comparison of gene neighborhoods is a powerful approach for exploring microbial genome structure, function, and evolution. Although numerous tools exist for genome visualization and comparison, genome exploration across large genomic databases or user-generated datasets remains a challenge. Here, we introduce AnnoView, a web server designed for interactive exploration of gene neighborhoods across the bacterial and archaeal tree of life. Our server offers users the ability to identify, compare, and visualize gene neighborhoods of interest from 30 238 bacterial genomes and 1672 archaeal genomes, through integration with the comprehensive Genome Taxonomy Database and AnnoTree databases. Identified gene neighborhoods can be visualized using pre-computed functional annotations from different sources such as KEGG, Pfam and TIGRFAM, or clustered based on similarity. Alternatively, users can upload and explore their own custom genomic datasets in GBK, GFF or CSV format, or use AnnoView as a genome browser for relatively small genomes (e.g. viruses and plasmids). Ultimately, we anticipate that AnnoView will catalyze biological discovery by enabling user-friendly search, comparison, and visualization of genomic data. AnnoView is available at http://annoview.uwaterloo.ca.

Comammox Nitrospira among dominant ammonia oxidizers within aquarium biofilter microbial communities.

Nitrification by aquarium biofilters transforms ammonia waste (NH3/NH4+) to less toxic nitrate (NO3-) via nitrite (NO2-). Prior to the discovery of complete ammonia-oxidizing ("comammox" or CMX) Nitrospira, previous research revealed that ammonia-oxidizing archaea (AOA) dominated over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Here, we profiled aquarium biofilter microbial communities and quantified the abundance of all three known ammonia oxidizers using 16S rRNA gene sequencing and quantitative PCR (qPCR), respectively. Biofilter and water samples were each collected from representative residential and commercial freshwater and saltwater aquaria. Distinct biofilter microbial communities were associated with freshwater and saltwater biofilters. Comammox Nitrospira amoA genes were detected in all 38 freshwater biofilter samples (average CMX amoA genes: 2.2 × 103 ± 1.5 × 103 copies/ng) and dominant in 30, whereas AOA were present in 35 freshwater biofilter samples (average AOA amoA genes: 1.1 × 103 ± 2.7 × 103 copies/ng) and only dominant in 7 of them. The AOB were at relatively low abundance within biofilters (average of 3.2 × 101 ± 1.1 × 102 copies of AOB amoA genes/ng of DNA), except for the aquarium with the highest ammonia concentration. For saltwater biofilters, AOA or AOB were differentially abundant, with no comammox Nitrospira detected. Additional sequencing of Nitrospira amoA genes revealed differential distributions, suggesting niche adaptation based on water chemistry (e.g., ammonia, carbonate hardness, and alkalinity). Network analysis of freshwater microbial communities demonstrated positive correlations between nitrifiers and heterotrophs, suggesting metabolic and ecological interactions within biofilters. These results demonstrate that comammox Nitrospira plays a previously overlooked, but important role in home aquarium biofilter nitrification. IMPORTANCE: Nitrification is a crucial process that converts toxic ammonia waste into less harmful nitrate that occurs in aquarium biofilters. Prior research found that ammonia-oxidizing archaea (AOA) were dominant over ammonia-oxidizing bacteria (AOB) in freshwater aquarium biofilters. Our study profiled microbial communities of aquarium biofilters and quantified the abundance of all currently known groups of aerobic ammonia oxidizers. The findings reveal that complete ammonia-oxidizing (comammox) Nitrospira were present in all freshwater aquarium biofilter samples in high abundance, challenging our previous understanding of aquarium nitrification. We also highlight niche adaptation of ammonia oxidizers based on salinity. The network analysis of freshwater biofilter microbial communities revealed significant positive correlations among nitrifiers and other community members, suggesting intricate interactions within biofilter communities. Overall, this study expands our understanding of nitrification in aquarium biofilters, emphasizes the role of comammox Nitrospira, and highlights the value of aquaria as microcosms for studying nitrifier ecology.

Impact of dry density and incomplete saturation on microbial growth in bentonite clay for nuclear waste storage.

AIMS: Many countries are in the process of designing a deep geological repository (DGR) for long-term storage of used nuclear fuel. For several designs, used fuel containers will be placed belowground, with emplacement tunnels being backfilled using a combination of highly compacted powdered bentonite clay buffer boxes surrounded by a granulated "gapfill" bentonite. To limit the potential for microbiologically influenced corrosion of used fuel containers, identifying conditions that suppress microbial growth is critical for sustainable DGR design. This study investigated microbial communities in powdered and gapfill bentonite clay incubated in oxic pressure vessels at dry densities between 1.1 g cm-3 (i.e. below repository target) and 1.6 g cm-3 (i.e. at or above repository target) as a 1-year time series. RESULTS: Our results showed an initial (i.e. 1 month) increase in the abundance of culturable heterotrophs associated with all dry densities <1.6 g cm-3, which reveals growth during transient low-pressure conditions associated with the bentonite saturation process. Following saturation, culturable heterotroph abundances decreased to those of starting material by the 6-month time point for all 1.4 and 1.6 g cm-3 pressure vessels, and the most probable numbers of culturable sulfate-reducing bacteria (SRB) remained constant for all vessels and time points. The 16S rRNA gene sequencing results showed a change in microbial community composition from the starting material to the 1-month time point, after which time most samples were dominated by sequences associated with Pseudomonas, Bacillus, Cupriavidus, and Streptomyces. Similar taxa were identified as dominant members of the culture-based community composition, demonstrating that the dominant members of the clay microbial communities are viable. Members of the spore-forming Desulfosporosinus genus were the dominant SRB for both clay and culture profiles. CONCLUSIONS: After initial microbial growth while bentonite was below target pressure in the early phases of saturation, microbial growth in pressure vessels with dry densities of at least 1.4 g cm-3 was eventually suppressed as bentonite neared saturation.

Microbial communities change along the 300 km length of the Grand River for extreme high- and low-flow regimes.

The Grand River watershed is the largest catchment in southern Ontario. The river's northern and southern sections are influenced by agriculture, whereas central regions receive wastewater effluent and urban runoff. To characterize in-river microbial communities, as they relate to spatial and environmental factors, we conducted two same-day sampling events along the entire 300 km length of the river, representing contrasting flow seasons (high flow spring melt and low flow end of summer). Through high-throughput sequencing of 16S rRNA genes, we assessed the relationship between river microbiota and spatial and physicochemical variables. Flow season had a greater impact on communities than spatial or diel effects and profiles diverged with distance between sites under both flow conditions, but low-flow profiles exhibited higher beta diversity. High-flow profiles showed greater species richness and increased presence of soil and sediment taxa, which may relate to increased input from terrestrial sources. Total suspended solids, dissolved inorganic carbon, and distance from headwaters significantly explained microbial community variation during the low-flow event, whereas conductivity, sulfate, and nitrite were significant explanatory factors for spring melt. This study establishes a baseline for the Grand River's microbial community, serving as a foundation for modeling the microbiology of anthropogenically impacted freshwater systems affected by lotic processes.

Large Fractionation in Iron Isotopes Implicates Metabolic Pathways for Iron Cycling in Boreal Shield Lakes.

Stable Fe isotopes have only recently been measured in freshwater systems, mainly in meromictic lakes. Here we report the δ56Fe of dissolved, particulate, and sediment Fe in two small dimictic boreal shield headwater lakes: manipulated eutrophic Lake 227, with annual cyanobacterial blooms, and unmanipulated oligotrophic Lake 442. Within the lakes, the range in δ56Fe is large (ca. -0.9 to +1.8‰), spanning more than half the entire range of natural Earth surface samples. Two layers in the water column with distinctive δ56Fe of dissolved (dis) and particulate (spm) Fe were observed, despite differences in trophic states. In the epilimnia of both lakes, a large Δ56Fedis-spm fractionation of 0.4-1‰ between dissolved and particulate Fe was only observed during cyanobacterial blooms in Lake 227, possibly regulated by selective biological uptake of isotopically light Fe by cyanobacteria. In the anoxic layers in both lakes, upward flux from sediments dominates the dissolved Fe pool with an apparent Δ56Fedis-spm fractionation of -2.2 to -0.6‰. Large Δ56Fedis-spm and previously published metagenome sequence data suggest active Fe cycling processes in anoxic layers, such as microaerophilic Fe(II) oxidation or photoferrotrophy, could regulate biogeochemical cycling. Large fractionation of stable Fe isotopes in these lakes provides a potential tool to probe Fe cycling and the acquisition of Fe by cyanobacteria, with relevance for understanding biogeochemical cycling of Earth's early ferruginous oceans.

Microbiology of barrier component analogues of a deep geological repository.

Canada is currently implementing a site selection process to identify a location for a deep geological repository (DGR) for the long-term storage of Canada's used nuclear fuel, wherein used nuclear fuel bundles will be sealed inside copper-coated carbon steel containers, encased in highly compacted bentonite clay buffer boxes, and sealed deep underground in a stable geosphere. Because a DGR must remain functional for a million years, it is important to examine ancient natural systems that serve as analogues for planned DGR components. Specifically, studying the microbiology of natural analogue components of a DGR is important for developing an understanding of the types of microorganisms that may be able to grow and influence the long-term stability of a DGR. This study explored the abundance, viability, and composition of microorganisms in several ancient natural analogues using a combination of cultivation and cultivation-independent approaches. Samples were obtained from the Tsukinuno bentonite deposit (Japan) that formed ∼10 mya, the Opalinus Clay formation (Switzerland) that formed ∼174 mya, and Canadian shield crystalline rock from Northern Ontario that formed ∼2.7 bya. Analysis of 16S rRNA gene amplicons revealed that three of the ten Tsukinuno bentonite samples analyzed were dominated by putative aerobic heterotrophs and fermenting bacteria from the phylum Actinobacteria, whereas five of the Tsukinuno bentonite samples were dominated by sequences associated with putative acidophilic chemolithoautotrophs capable of sulfur reduction. The remaining Tsukinuno bentonite samples, the Northern Ontario rock samples, and the Opalinus Clay samples generated inconsistent replicate 16S rRNA gene profiles and were associated primarily with contaminant sequences, suggesting that the microbial profiles detected were not sample-specific but spurious. Culturable aerobic heterotroph abundances were relatively low for all Tsukinuno bentonite samples, culturable anaerobic heterotrophs were only detected in half of the Tsukinuno samples, and sulfate-reducing bacteria (SRB) were only detected in one Tsukinuno sample by cultivation. Culture-specific 16S rRNA gene profiles from Tsukinuno clay samples demonstrated the presence of phyla Bacteroidetes, Proteobacteria, Actinobacteria, and Firmicutes among aerobic heterotroph cultures and additional bacteria from the phyla Actinobacteria and Firmicutes from anaerobic heterotroph plate incubations. Only one nucleic acid sequence detected from a culture was also associated with its corresponding clay sample profile, suggesting that nucleic acids from culturable bacteria were relatively rare within the clay samples. Sequencing of DNA extracted from the SRB culture revealed that the taxon present in the culture was affiliated with the genus Desulfosporosinus, which has been found in related bentonite clay analyses. Although the crystalline rock and Opalinus Clay samples were associated with inconsistent, likely spurious 16S rRNA gene profiles, we show evidence for viable and detectable microorganisms within several Tsukinuno natural analogue bentonite samples.

Monitoring Microbial Populations and Antibiotic Resistance Gene Enrichment Associated with Arctic Waste Stabilization Ponds.

Wastewater management in the Canadian Arctic is challenging due to climate extremes, small population sizes, and lack of conventional infrastructure for wastewater treatment. Although many northern communities use waste stabilization ponds (WSPs) as their primary form of wastewater treatment, few studies have explored WSP microbial communities and assessed effluent impacts on receiving waters from a microbiological perspective. Here, we used 16S rRNA gene and metagenome sequencing to characterize WSP and receiving water microbial communities for two time points bracketing the spring WSP thaw in Baker Lake (Nunavut) and compared these results to other Nunavut WSPs in Cambridge Bay and Kugluktuk. Most amplicon sequence variants (ASVs) recovered from these WSP samples belonged to the phylum Proteobacteria, with considerable variation between the three locations and only six ASVs shared among the WSPs at >0.2% relative abundance. Wastewater indicator ASVs for the Baker Lake WSP were identified, and few indicator ASVs were detected in samples originating from other upstream or downstream sites. The metagenomic data revealed a strong enrichment of antibiotic resistance genes for WSP samples relative to downstream and reference samples, especially for genes associated with macrolide resistance. Together, our results provide a baseline characterization for WSP microbial communities, demonstrate how indicator ASVs can be used to monitor attenuation and dilution of effluent microorganisms, and reveal that WSPs can serve as hot spots for antibiotic resistance genes.IMPORTANCE Given that the microbial communities of Arctic waste stabilization ponds (WSPs) are poorly studied to date, our characterization of multiple WSP systems and time points provides important baseline data that will assist with ongoing monitoring of effluent impacts on downstream aquatic ecosystems in the Arctic. This research also identifies indicator amplicon sequence variants (ASVs) of WSPs that will be helpful for future monitoring for WSP effluent attenuation and demonstrates that WSP microbial communities are enriched in antibiotic resistance genes. Given operational and infrastructure changes anticipated for wastewater treatment systems in the Arctic, baseline data such as these are essential for further development of safe and effective wastewater treatment systems.

Comprehensive skin microbiome analysis reveals the uniqueness of human skin and evidence for phylosymbiosis within the class Mammalia.

Skin is the largest organ of the body and represents the primary physical barrier between mammals and their external environment, yet the factors that govern skin microbial community composition among mammals are poorly understood. The objective of this research was to generate a skin microbiota baseline for members of the class Mammalia, testing the effects of host species, geographic location, body region, and biological sex. Skin from the back, torso, and inner thighs of 177 nonhuman mammals was sampled, representing individuals from 38 species and 10 mammalian orders. Animals were sampled from farms, zoos, households, and the wild. The DNA extracts from all skin swabs were amplified by PCR and sequenced, targeting the V3-V4 regions of bacterial and archaeal 16S rRNA genes. Previously published skin microbiome data from 20 human participants, sampled and sequenced using an identical protocol to the nonhuman mammals, were included to make this a comprehensive analysis. Human skin microbial communities were distinct and significantly less diverse than all other sampled mammalian orders. The factor most strongly associated with microbial community data for all samples was whether the host was a human. Within nonhuman samples, host taxonomic order was the most significant factor influencing skin microbiota, followed by the geographic location of the habitat. By comparing the congruence between host phylogeny and microbial community dendrograms, we observed that Artiodactyla (even-toed ungulates) and Perissodactyla (odd-toed ungulates) had significant congruence, providing evidence of phylosymbiosis between skin microbial communities and their hosts.

Seasonal habitat drives intestinal microbiome composition in anadromous Arctic char (Salvelinus alpinus).

Intestinal microbial communities from 362 anadromous Arctic char (Salvelinus alpinus) from the high Arctic Kitikmeot region, Nunavut, Canada, were characterized using high-throughput 16S rRNA gene sequencing. The resulting bacterial communities were compared across four seasonal habitats that correspond to different stages of annual migration. Arctic char intestinal communities differed by sampling site, salinity and stages of freshwater residence. Although microbiota from fish sampled in brackish water were broadly consistent with taxa seen in other anadromous salmonids, they were enriched with putative psychrophiles, including the nonluminous gut symbiont Photobacterium iliopiscarium that was detected in >90% of intestinal samples from these waters. Microbiota from freshwater-associated fish were less consistent with results reported for other salmonids, and highly variable, possibly reflecting winter fasting behaviour of these char. We identified microbiota links to age for those fish sampled during the autumn upriver migration, but little impact of the intestinal content and water microbiota on the intestinal community. The strongest driver of intestinal community composition was seasonal habitat, and this finding combined with identification of psychrophiles suggested that water temperature and migratory behaviour are key to understanding the relationship between Arctic char and their symbionts.

Pygenprop: a Python library for programmatic exploration and comparison of organism genome properties.

SUMMARY: A critical step in comparative genomics is the identification of differences in the presence/absence of encoded biochemical pathways among organisms. Our library, Pygenprop, facilitates these comparisons using data from the Genome Properties database. Pygenprop is written in Python and, unlike existing libraries, it is compatible with a variety of tools in the Python data science ecosystem, such as Jupyter Notebooks for interactive analyses and scikit-learn for machine learning. Pygenprop assigns YES, NO, or PARTIAL support for each property based on InterProScan annotations of open reading frames from an organism's genome. The library contains classes for representing the Genome Properties database as a whole and methods for detecting differences in property assignments between organisms. As the Genome Properties database grows, we anticipate widespread adoption of Pygenprop for routine genome analyses and integration within third-party bioinformatics software. AVAILABILITY AND IMPLEMENTATION: Pygenprop is written in Python and is compatible with versions 3.6 or higher. Source code is available under Apache Licence Version 2 at https://github.com/Micromeda/pygenprop. The package can be installed from both PyPi (https://pypi.org/project/pygenprop) and Anaconda (https://anaconda.org/lbergstrand/pygenprop). Documentation is available on Read the Docs (http://pygenprop.rtfd.io/).

"Candidatus Nitrosotenuis aquarius," an Ammonia-Oxidizing Archaeon from a Freshwater Aquarium Biofilter.

Ammonia is a metabolic waste product excreted by aquatic organisms that causes toxicity when it accumulates. Aquaria and aquaculture systems therefore use biological filters that promote the growth of nitrifiers to convert ammonia to nitrate. Ammonia-oxidizing bacteria (AOB) have been isolated from aquarium biofilters and are available as commercial supplements, but recent evidence suggests that ammonia-oxidizing archaea (AOA) are abundant in aquarium biofilters. In this study, we report the cultivation and closed genome sequence of the novel AOA representative "Candidatus Nitrosotenuis aquarius," which was enriched from a freshwater aquarium biofilter. "Ca Nitrosotenuis aquarius" oxidizes ammonia stoichiometrically to nitrite with a concomitant increase in thaumarchaeotal cells and a generation time of 34.9 h. "Ca Nitrosotenuis aquarius" has an optimal growth temperature of 33°C, tolerates up to 3 mM NH4Cl, and grows optimally at 0.05% salinity. Transmission electron microscopy revealed that "Ca Nitrosotenuis aquarius" cells are rod shaped, with a diameter of ∼0.4 µm and length ranging from 0.6 to 3.6 µm. In addition, these cells possess surface layers (S-layers) and multiple proteinaceous appendages. Phylogenetically, "Ca Nitrosotenuis aquarius" belongs to the group I.1a Thaumarchaeota, clustering with environmental sequences from freshwater aquarium biofilters, aquaculture systems, and wastewater treatment plants. The complete 1.70-Mbp genome contains genes involved in ammonia oxidation, bicarbonate assimilation, flagellum synthesis, chemotaxis, S-layer production, defense, and protein glycosylation. Incubations with differential inhibitors indicate that "Ca Nitrosotenuis aquarius"-like AOA contribute to ammonia oxidation within the aquarium biofilter from which it originated.IMPORTANCE Nitrification is a critical process for preventing ammonia toxicity in engineered biofilter environments. This work describes the cultivation and complete genome sequence of a novel AOA representative enriched from a freshwater aquarium biofilter. In addition, despite the common belief in the aquarium industry that AOB mediate ammonia oxidation, the present study suggests an in situ role for "Ca Nitrosotenuis aquarius"-like AOA in freshwater aquarium biofilters.

Agricultural soil denitrifiers possess extensive nitrite reductase gene diversity.

Denitrification transforms nitrogen applied as fertilizer and emits N2 O, which is a potent greenhouse gas. Very little is known about the identities of abundant and active denitrifiers in agricultural soils. We coupled DNA stable-isotope probing (DNA-SIP) with flow-through reactors (FTRs) to detect active agricultural soil denitrifiers. The FTRs were incubated with nitrate and 13 C6 -glucose under anoxic conditions and sampled at multiple time points. Labelled DNA from active microorganisms was analyzed by 16S rRNA gene fingerprinting, amplicon and shotgun metagenomic sequencing. Taxonomic representation of heavy fractions was consistent across sites and time points, including Betaproteobacteria (71%; Janthinobacterium, Acidovorax, Azoarcus and Dechloromonas), Alphaproteobacteria (8%; Rhizobium), Gammaproteobacteria (4%; Pseudomonas) and Actinobacteria (4%; Streptomycetaceae). Most nitrite-reductase reads from heavy DNA annotated to the copper-containing form (nirK). Assigned taxonomies of active denitrifiers based on reads matching the nirK gene were comparable to those obtained through nitric oxide (norB) and RNA polymerase (rpoB) annotations but not the nitrous oxide reductase gene (nosZ). Analysis of recovered metagenomes from heavy DNA demonstrated extensive nirK sequence family diversity, including novel taxonomic groups that are not captured by existing primers.

Temporal Variations of Microbiota Associated with the Immature Stages of Two Florida Culex Mosquito Vectors.

Microbiota associated with mosquito vector populations impact several traits of mosquitoes, including survival, reproduction, control, and immunity against pathogens. The influence of seasonal variations and mosquito species on mosquito gut microbiota is poorly understood. We sought to determine whether the mosquito microbiota associated with immature stages of two congeners (Culex coronator and Culex nigripalpus) differ temporally and between the two species. Using high throughput 16S rRNA gene sequence analysis, we characterized bacterial and archaeal communities found in the immature stages of the two Culex mosquito species sampled over three seasons to compare the diversity of bacteria between the two species. Beta diversity analyses of the larval microbiota sequences revealed that the two Culex species differed significantly, both temporally within each species and between the two species. Bacteria in Cx. coronator larvae were dominated by Alphaproteobacteria, mainly associated with Roseoccocus and unidentified species of Rhizobiales, and two unidentified species of Cyanobacteria. In contrast, Cx. nigripalpus was dominated by Thorsellia anophelis (Gammaproteobacteria), Clostridium, an unidentified species of Ruminococcacae (Clostridiales), and additional unidentified species associated with Erysipelotrichaceae (Erysipelotrichales), Bacteroidales, and Mollicutes. Results of our study revealed both seasonal and interspecies differences in bacterial community composition associated with the immature stages of Cx. coronator and Cx. nigripalpus vector populations in Florida. These results have important implications for our understanding of the underlying factors of variations in disease transmission among seasons, susceptibility to various pesticides, and other biotic factors, including the role of the microbiota on the spread of invasive species. In addition, our results suggest close associations of certain bacteria species with each of the two Culex species that will be further targeted for their potential in the development of microbial-based control strategies.

MetAnnotate: function-specific taxonomic profiling and comparison of metagenomes.

BACKGROUND: Metagenomes provide access to the taxonomic composition and functional capabilities of microbial communities. Although metagenomic analysis methods exist for estimating overall community composition or metabolic potential, identifying specific taxa that encode specific functions or pathways of interest can be more challenging. Here we present MetAnnotate, which addresses the common question: "which organisms perform my function of interest within my metagenome(s) of interest?" MetAnnotate uses profile hidden Markov models to analyze shotgun metagenomes for genes and pathways of interest, classifies retrieved sequences either through a phylogenetic placement or best hit approach, and enables comparison of these profiles between metagenomes. RESULTS: Based on a simulated metagenome dataset, the tool achieves high taxonomic classification accuracy for a broad range of genes, including both markers of community abundance and specific biological pathways. Lastly, we demonstrate MetAnnotate by analyzing for cobalamin (vitamin B12) synthesis genes across hundreds of aquatic metagenomes in a fraction of the time required by the commonly used Basic Local Alignment Search Tool top hit approach. CONCLUSIONS: MetAnnotate is multi-threaded and installable as a local web application or command-line tool on Linux systems. Metannotate is a useful framework for general and/or function-specific taxonomic profiling and comparison of metagenomes.

Norwegian deep-water coral reefs: cultivation and molecular analysis of planktonic microbial communities.

Deep-sea coral reefs do not receive sunlight and depend on plankton. Little is known about the plankton composition at such reefs, even though they constitute habitats for many invertebrates and fish. We investigated plankton communities from three reefs at 260-350 m depth at hydrocarbon fields off the mid-Norwegian coast using a combination of cultivation and small subunit (SSU) rRNA gene and transcript sequencing. Eight months incubations of a reef water sample with minimal medium, supplemented with carbon dioxide and gaseous alkanes at in situ-like conditions, enabled isolation of mostly Alphaproteobacteria (Sulfitobacter, Loktanella), Gammaproteobacteria (Colwellia) and Flavobacteria (Polaribacter). The relative abundance of isolates in the original sample ranged from ∼ 0.01% to 0.80%. Comparisons of bacterial SSU sequences from filtered plankton of reef and non-reef control samples indicated high abundance and metabolic activity of primarily Alphaproteobacteria (SAR11 Ia), Gammaproteobacteria (ARCTIC96BD-19), but also of Deltaproteobacteria (Nitrospina, SAR324). Eukaryote SSU sequences indicated metabolically active microalgae and animals, including codfish, at the reef sites. The plankton community composition varied between reefs and differed between DNA and RNA assessments. Over 5000 operational taxonomic units were detected, some indicators of reef sites (e.g. Flavobacteria, Cercozoa, Demospongiae) and some more active at reef sites (e.g. Gammaproteobacteria, Ciliophora, Copepoda).

Vegetation-associated impacts on arctic tundra bacterial and microeukaryotic communities.

The Arctic is experiencing rapid vegetation changes, such as shrub and tree line expansion, due to climate warming, as well as increased wetland variability due to hydrological changes associated with permafrost thawing. These changes are of global concern because changes in vegetation may increase tundra soil biogeochemical processes that would significantly enhance atmospheric CO2 concentrations. Predicting the latter will at least partly depend on knowing the structure, functional activities, and distributions of soil microbes among the vegetation types across Arctic landscapes. Here we investigated the bacterial and microeukaryotic community structures in soils from the four principal low Arctic tundra vegetation types: wet sedge, birch hummock, tall birch, and dry heath. Sequencing of rRNA gene fragments indicated that the wet sedge and tall birch communities differed significantly from each other and from those associated with the other two dominant vegetation types. Distinct microbial communities were associated with soil pH, ammonium concentration, carbon/nitrogen (C/N) ratio, and moisture content. In soils with similar moisture contents and pHs (excluding wet sedge), bacterial, fungal, and total eukaryotic communities were correlated with the ammonium concentration, dissolved organic nitrogen (DON) content, and C/N ratio. Operational taxonomic unit (OTU) richness, Faith's phylogenetic diversity, and the Shannon species-level index (H') were generally lower in the tall birch soil than in soil from the other vegetation types, with pH being strongly correlated with bacterial richness and Faith's phylogenetic diversity. Together, these results suggest that Arctic soil feedback responses to climate change will be vegetation specific not just because of distinctive substrates and environmental characteristics but also, potentially, because of inherent differences in microbial community structure.

Developmental succession of the microbiome of Culex mosquitoes.

BACKGROUND: The native microflora associated with mosquitoes have important roles in mosquito development and vector competence. Sequencing of bacterial V3 region from 16S rRNA genes across the developmental stages of Culex mosquitoes (early and late larval instars, pupae and adults) was used to test the hypothesis that bacteria found in the larval stage of Culex are transstadially transmitted to the adult stage, and to compare the microbiomes of field-collected versus laboratory-reared mosquitoes. RESULTS: Beta diversity analysis revealed that bacterial community structure differed among three life stages (larvae, pupae and adults) of Culex tarsalis. Although only ~2% of the total number of bacterial OTUs were found in all stages, sequences from these OTUs accounted for nearly 82% of the total bacterial sequences recovered from all stages. Thorsellia (Gammaproteobacteria) was the most abundant bacterial taxon found across all developmental stages of field-collected Culex mosquitoes, but was rare in mosquitoes from laboratory-reared colonies. The proportion of Thorsellia sequences in the microbiomes of mosquito life stages varied ontogenetically with the greatest proportions recovered from the pupae of C. tarsalis and the lowest from newly emerged adults. The microbiome of field-collected late instar larvae was not influenced significantly by differences in the microbiota of the habitat due to habitat age or biopesticide treatments. The microbiome diversity was the greatest in the early instar larvae and the lowest in laboratory-reared mosquitoes. CONCLUSIONS: Bacterial communities in early instar C. tarsalis larvae were significantly more diverse when compared to late instar larvae, pupae and newly emerged adults. Some of the bacterial OTUs found in the early instar larvae were also found across developmental stages. Thorsellia dominated the bacterial communities in field-collected immature stages but occurred at much lower relative abundance in adults. Differences in microbiota observed in larval habitats did not influence bacterial community profiles of late instar larvae or adults. However, bacterial communities in laboratory-reared C. tarsalis larvae differed significantly from the field. Determining the role of Thorsellia in mosquitoes and its distribution across different species of mosquitoes warrants further investigation.

Evaluating bias of illumina-based bacterial 16S rRNA gene profiles.

Massively parallel sequencing of 16S rRNA genes enables the comparison of terrestrial, aquatic, and host-associated microbial communities with sufficient sequencing depth for robust assessments of both alpha and beta diversity. Establishing standardized protocols for the analysis of microbial communities is dependent on increasing the reproducibility of PCR-based molecular surveys by minimizing sources of methodological bias. In this study, we tested the effects of template concentration, pooling of PCR amplicons, and sample preparation/interlane sequencing on the reproducibility associated with paired-end Illumina sequencing of bacterial 16S rRNA genes. Using DNA extracts from soil and fecal samples as templates, we sequenced pooled amplicons and individual reactions for both high (5- to 10-ng) and low (0.1-ng) template concentrations. In addition, all experimental manipulations were repeated on two separate days and sequenced on two different Illumina MiSeq lanes. Although within-sample sequence profiles were highly consistent, template concentration had a significant impact on sample profile variability for most samples. Pooling of multiple PCR amplicons, sample preparation, and interlane variability did not influence sample sequence data significantly. This systematic analysis underlines the importance of optimizing template concentration in order to minimize variability in microbial-community surveys and indicates that the practice of pooling multiple PCR amplicons prior to sequencing contributes proportionally less to reducing bias in 16S rRNA gene surveys with next-generation sequencing.

Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.

Bacteria responsible for cellulose hydrolysis in situ are poorly understood, largely because of the relatively recent development of cultivation-independent methods for their detection and characterization. This study combined DNA stable-isotope probing (DNA-SIP) and metagenomics for identifying active bacterial communities that assimilated carbon from glucose and cellulose in Arctic tundra microcosms. Following DNA-SIP, bacterial fingerprint analysis of gradient fractions confirmed isotopic enrichment. Sequenced fingerprint bands and clone library analysis of 16S rRNA genes identified active bacterial taxa associated with cellulose-associated labelled DNA, including Bacteroidetes (Sphingobacteriales), Betaproteobacteria (Burkholderiales), Alphaproteobacteria (Caulobacteraceae), and Chloroflexi (Anaerolineaceae). We also compared glycoside hydrolase metagenomic profiles from bulk soil and heavy DNA recovered from DNA-SIP incubations. Active populations consuming [(13)C]glucose and [(13)C]cellulose were distinct, based on ordinations of light and heavy DNA. Metagenomic analysis demonstrated a ∼3-fold increase in the relative abundance of glycoside hydrolases in DNA-SIP libraries over bulk-soil libraries. The data also indicate that multiple displacement amplification introduced bias into the resulting metagenomic analysis. This research identified DNA-SIP incubation conditions for glucose and cellulose that were suitable for Arctic tundra soil and confirmed that DNA-SIP enrichment can increase target gene frequencies in metagenomic libraries.