A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities.

We present a programmable droplet-based microfluidic device that combines the reconfigurable flow-routing capabilities of integrated microvalve technology with the sample compartmentalization and dispersion-free transport that is inherent to droplets. The device allows for the execution of user-defined multistep reaction protocols in 95 individually addressable nanoliter-volume storage chambers by consecutively merging programmable sequences of picoliter-volume droplets containing reagents or cells. This functionality is enabled by "flow-controlled wetting," a droplet docking and merging mechanism that exploits the physics of droplet flow through a channel to control the precise location of droplet wetting. The device also allows for automated cross-contamination-free recovery of reaction products from individual chambers into standard microfuge tubes for downstream analysis. The combined features of programmability, addressability, and selective recovery provide a general hardware platform that can be reprogrammed for multiple applications. We demonstrate this versatility by implementing multiple single-cell experiment types with this device: bacterial cell sorting and cultivation, taxonomic gene identification, and high-throughput single-cell whole genome amplification and sequencing using common laboratory strains. Finally, we apply the device to genome analysis of single cells and microbial consortia from diverse environmental samples including a marine enrichment culture, deep-sea sediments, and the human oral cavity. The resulting datasets capture genotypic properties of individual cells and illuminate known and potentially unique partnerships between microbial community members.

MetaPathways: a modular pipeline for constructing pathway/genome databases from environmental sequence information.

BACKGROUND: A central challenge to understanding the ecological and biogeochemical roles of microorganisms in natural and human engineered ecosystems is the reconstruction of metabolic interaction networks from environmental sequence information. The dominant paradigm in metabolic reconstruction is to assign functional annotations using BLAST. Functional annotations are then projected onto symbolic representations of metabolism in the form of KEGG pathways or SEED subsystems. RESULTS: Here we present MetaPathways, an open source pipeline for pathway inference that uses the PathoLogic algorithm to map functional annotations onto the MetaCyc collection of reactions and pathways, and construct environmental Pathway/Genome Databases (ePGDBs) compatible with the editing and navigation features of Pathway Tools. The pipeline accepts assembled or unassembled nucleotide sequences, performs quality assessment and control, predicts and annotates noncoding genes and open reading frames, and produces inputs to PathoLogic. In addition to constructing ePGDBs, MetaPathways uses MLTreeMap to build phylogenetic trees for selected taxonomic anchor and functional gene markers, converts General Feature Format (GFF) files into concatenated GenBank files for ePGDB construction based on third-party annotations, and generates useful file formats including Sequin files for direct GenBank submission and gene feature tables summarizing annotations, MLTreeMap trees, and ePGDB pathway coverage summaries for statistical comparisons. CONCLUSIONS: MetaPathways provides users with a modular annotation and analysis pipeline for predicting metabolic interaction networks from environmental sequence information using an alternative to KEGG pathways and SEED subsystems mapping. It is extensible to genomic and transcriptomic datasets from a wide range of sequencing platforms, and generates useful data products for microbial community structure and function analysis. The MetaPathways software package, installation instructions, and example data can be obtained from http://hallam.microbiology.ubc.ca/MetaPathways.

Metagenomics of hydrocarbon resource environments indicates aerobic taxa and genes to be unexpectedly common.

Oil in subsurface reservoirs is biodegraded by resident microbial communities. Water-mediated, anaerobic conversion of hydrocarbons to methane and CO2, catalyzed by syntrophic bacteria and methanogenic archaea, is thought to be one of the dominant processes. We compared 160 microbial community compositions in ten hydrocarbon resource environments (HREs) and sequenced twelve metagenomes to characterize their metabolic potential. Although anaerobic communities were common, cores from oil sands and coal beds had unexpectedly high proportions of aerobic hydrocarbon-degrading bacteria. Likewise, most metagenomes had high proportions of genes for enzymes involved in aerobic hydrocarbon metabolism. Hence, although HREs may have been strictly anaerobic and typically methanogenic for much of their history, this may not hold today for coal beds and for the Alberta oil sands, one of the largest remaining oil reservoirs in the world. This finding may influence strategies to recover energy or chemicals from these HREs by in situ microbial processes.

Inoculum dose-disease response relationships for the pea root rot pathogen, Aphanomyces euteiches, are dependent on soil type and other pathogens.

The oomycete pathogen, Aphanomyces euteiches, was implicated for the first time in pea and lentil root rot in Saskatchewan and Alberta in 2012 and 2013. Subsequent surveys from 2014 to 2017 revealed that Aphanomyces root rot (ARR) was widespread across the Canadian prairies. The absence of effective chemical, biological, and cultural controls and lack of genetic resistance leave only one management option: avoidance. The objectives of this study were to relate oospore levels in autoclaved and non-autoclaved soils to ARR severity across soil types from the vast prairie landscape and to determine the relationship of measured DNA quantity of A. euteiches using droplet digital PCR or quantitative PCR to the initial oospore inoculum dose in soils. These objectives support a future end goal of creating a rapid assessment method capable of categorizing root rot risk in field soil samples to aid producers with pulse crop field selection decisions. The ARR severity to oospore dose relationship was statistically significantly affected by the soil type and location from which soils were collected and did not show a linear relationship. For most soil types, ARR did not develop at oospore levels below 100/g soil, but severity rose above this level, confirming a threshold level of 100 oospores/g soil for disease development. For most soil types, ARR severity was significantly higher in non-autoclaved compared to autoclaved treatments, demonstrating the role that other pathogens play in increasing disease severity. There was a significant linear relationship between DNA concentrations measured in soil and oospore inoculum concentration, although the strength of the relationship was better for some soil types, and in some soil types, DNA measurement results underestimated the number of oospores. This research is important for developing a root rot risk assessment system for the Canadian prairies based on soil inoculum quantification, following field validation of soil quantification and relationship to root rot disease severity.

The effect of wheat genotype on the microbiome is more evident in roots and varies through time.

Crop breeding has traditionally ignored the plant-associated microbial communities. Considering the interactions between plant genotype and associated microbiota is of value since different genotypes of the same crop often harbor distinct microbial communities which can influence the plant phenotype. However, recent studies have reported contrasting results, which led us to hypothesize that the effect of genotype is constrained by growth stages, sampling year and plant compartment. To test this hypothesis, we sampled bulk soil, rhizosphere soil and roots of 10 field-grown wheat genotypes, twice per year, for 4 years. DNA was extracted and regions of the bacterial 16 S rRNA and CPN60 genes and the fungal ITS region were amplified and sequenced. The effect of genotype was highly contingent on the time of sampling and on the plant compartment sampled. Only for a few sampling dates, were the microbial communities significantly different across genotypes. The effect of genotype was most often significant for root microbial communities. The three marker genes used provided a highly coherent picture of the effect of genotype. Taken together, our results confirm that microbial communities in the plant environment strongly vary across compartments, growth stages, and years, and that this can mask the effect of genotype.

Plant Holobiont Theory: The Phytomicrobiome Plays a Central Role in Evolution and Success.

Under natural conditions, plants are always associated with a well-orchestrated community of microbes-the phytomicrobiome. The nature and degree of microbial effect on the plant host can be positive, neutral, or negative, and depends largely on the environment. The phytomicrobiome is integral for plant growth and function; microbes play a key role in plant nutrient acquisition, biotic and abiotic stress management, physiology regulation through microbe-to-plant signals, and growth regulation via the production of phytohormones. Relationships between the plant and phytomicrobiome members vary in intimacy, ranging from casual associations between roots and the rhizosphere microbial community, to endophytes that live between plant cells, to the endosymbiosis of microbes by the plant cell resulting in mitochondria and chloroplasts. If we consider these key organelles to also be members of the phytomicrobiome, how do we distinguish between the two? If we accept the mitochondria and chloroplasts as both members of the phytomicrobiome and the plant (entrained microbes), the influence of microbes on the evolution of plants becomes so profound that without microbes, the concept of the "plant" is not viable. This paper argues that the holobiont concept should take greater precedence in the plant sciences when referring to a host and its associated microbial community. The inclusivity of this concept accounts for the ambiguous nature of the entrained microbes and the wide range of functions played by the phytomicrobiome in plant holobiont homeostasis.

The Coevolution of Plants and Microbes Underpins Sustainable Agriculture.

Terrestrial plants evolution occurred in the presence of microbes, the phytomicrobiome. The rhizosphere microbial community is the most abundant and diverse subset of the phytomicrobiome and can include both beneficial and parasitic/pathogenic microbes. Prokaryotes of the phytomicrobiome have evolved relationships with plants that range from non-dependent interactions to dependent endosymbionts. The most extreme endosymbiotic examples are the chloroplasts and mitochondria, which have become organelles and integral parts of the plant, leading to some similarity in DNA sequence between plant tissues and cyanobacteria, the prokaryotic symbiont of ancestral plants. Microbes were associated with the precursors of land plants, green algae, and helped algae transition from aquatic to terrestrial environments. In the terrestrial setting the phytomicrobiome contributes to plant growth and development by (1) establishing symbiotic relationships between plant growth-promoting microbes, including rhizobacteria and mycorrhizal fungi, (2) conferring biotic stress resistance by producing antibiotic compounds, and (3) secreting microbe-to-plant signal compounds, such as phytohormones or their analogues, that regulate aspects of plant physiology, including stress resistance. As plants have evolved, they recruited microbes to assist in the adaptation to available growing environments. Microbes serve themselves by promoting plant growth, which in turn provides microbes with nutrition (root exudates, a source of reduced carbon) and a desirable habitat (the rhizosphere or within plant tissues). The outcome of this coevolution is the diverse and metabolically rich microbial community that now exists in the rhizosphere of terrestrial plants. The holobiont, the unit made up of the phytomicrobiome and the plant host, results from this wide range of coevolved relationships. We are just beginning to appreciate the many ways in which this complex and subtle coevolution acts in agricultural systems.

Nitrogen- and phosphorus-starved Triticum aestivum show distinct belowground microbiome profiles.

Many plants have natural partnerships with microbes that can boost their nitrogen (N) and/or phosphorus (P) acquisition. To assess whether wheat may have undiscovered associations of these types, we tested if N/P-starved Triticum aestivum show microbiome profiles that are simultaneously different from those of N/P-amended plants and those of their own bulk soils. The bacterial and fungal communities of root, rhizosphere, and bulk soil samples from the Historical Dryland Plots (Lethbridge, Canada), which hold T. aestivum that is grown both under N/P fertilization and in conditions of extreme N/P-starvation, were taxonomically described and compared (bacterial 16S rRNA genes and fungal Internal Transcribed Spacers-ITS). As the list may include novel N- and/or P-providing wheat partners, we then identified all the operational taxonomic units (OTUs) that were proportionally enriched in one or more of the nutrient starvation- and plant-specific communities. These analyses revealed: a) distinct N-starvation root and rhizosphere bacterial communities that were proportionally enriched, among others, in OTUs belonging to families Enterobacteriaceae, Chitinophagaceae, Comamonadaceae, Caulobacteraceae, Cytophagaceae, Streptomycetaceae, b) distinct N-starvation root fungal communities that were proportionally enriched in OTUs belonging to taxa Lulworthia, Sordariomycetes, Apodus, Conocybe, Ascomycota, Crocicreas, c) a distinct P-starvation rhizosphere bacterial community that was proportionally enriched in an OTU belonging to genus Agrobacterium, and d) a distinct P-starvation root fungal community that was proportionally enriched in OTUs belonging to genera Parastagonospora and Phaeosphaeriopsis. Our study might have exposed wheat-microbe connections that can form the basis of novel complementary yield-boosting tools.

Temporal and spatial archaeal colonization of hydrothermal vent deposits.

Thermocouple arrays were deployed on two deep-sea hydrothermal vents at Guaymas Basin (27 degrees 0.5'N, 111 degrees 24.5'W) in order to measure in situ temperatures at which microorganisms colonize the associated mineral deposits. Intact sections of three structures that formed around the arrays were collected after 4 and 72 day deployments (named BM4, BM72 and TS72). Archaeal diversity associated with discreet subsamples collected across each deposit was determined by polymerase chain reaction amplification of 16S rRNA genes. Spatial differences in archaeal diversity were observed in all deposits and appeared related to in situ temperature. In BM4, no 16S rRNA genes were detected beyond about 1.5 cm within the sample (> 200 degrees C). Phylotypes detected on the outside of this deposit belong to taxonomic groups containing mesophiles and (hyper)thermophiles, whereas only putative hyperthermophiles were detected 1.5 cm inside the structure (approximately 110 degrees C). In contrast, the more moderate thermal gradient recorded across TS72 was associated with a deeper colonization (2-3 cm inside the deposit) of putative hyperthermophilic phylotypes. Although our study does not provide a precise assessment of the highest temperature for the existence of microbial habitats inside the deposits, archaeal 16S rRNA genes were detected directly next to thermocouples that measured 110 degrees C (Methanocaldococcus spp. in BM4) and 116 degrees C (Desulfurococcaceae in TS72). The successive array deployments conducted at the Broken Mushroom (BM) site also revealed compositional differences in archaeal communities associated with immature (BM4) and mature chimneys (BM72) formed by the same fluids. These differences suggest a temporal transition in the primary carbon sources used by the archaeal communities, with potential CO(2)/H(2) methanogens prevalent in BM4 being replaced by possible methylotroph or acetoclastic methanogens and heterotrophs in BM72. This study is the first direct assessment of in situ conditions experienced by microorganisms inhabiting actively forming hydrothermal deposits at different stages of structure development.

Cyanotoxin degradation activity and mlr gene expression profiles of a Sphingopyxis sp. isolated from Lake Champlain, Canada.

A bacterium capable of degrading five microcystin (MC) variants, microcystin-LR, YR, LY, LW and LF at an initial total concentration of 50 µg l-1 in less than 16 hours was isolated from Missisquoi Bay, in the south of Quebec, Canada. Phylogenetic analysis of the 16S rRNA gene sequence identified the bacterium as Sphingopyxis sp., designated strain MB-E. It was shown that microcystin biodegradation activity was reduced at acidic and basic pH values. Even though no biodegradation occurred at pH values of 5.05 and 10.23, strain MB-E was able to degrade MCLR and MCYR at pH 9.12 and all five MCs variants tested at pH 6.1. Genomic sequencing revealed that strain MB-E contained the microcystin degrading gene cluster, including the mlrA, mlrB, mlrC and mlrD genes, and transcriptomic analysis demonstrated that all of these genes were induced during the degradation of MCLR alone or in the mixture of all five MCs. This novel transcriptomic analysis showed that the expression of the mlr gene cluster was similar for MCLR alone, or the mixture of MCs, and appeared to be related to the total concentration of substrate. The results suggested that the bacterium used the same pathway for the degradation of all MC variants.

Salix purpurea Stimulates the Expression of Specific Bacterial Xenobiotic Degradation Genes in a Soil Contaminated with Hydrocarbons.

The objectives of this study were to uncover Salix purpurea-microbe xenobiotic degradation systems that could be harnessed in rhizoremediation, and to identify microorganisms that are likely involved in these partnerships. To do so, we tested S. purpurea's ability to stimulate the expression of 10 marker microbial oxygenase genes in a soil contaminated with hydrocarbons. In what appeared to be a detoxification rhizosphere effect, transcripts encoding for alkane 1-monooxygenases, cytochrome P450 monooxygenases, laccase/polyphenol oxidases, and biphenyl 2,3-dioxygenase small subunits were significantly more abundant in the vicinity of the plant's roots than in bulk soil. This gene expression induction is consistent with willows' known rhizoremediation capabilities, and suggests the existence of S. purpurea-microbe systems that target many organic contaminants of interest (i.e. C4-C16 alkanes, fluoranthene, anthracene, benzo(a)pyrene, biphenyl, polychlorinated biphenyls). An enhanced expression of the 4 genes was also observed within the bacterial orders Actinomycetales, Rhodospirillales, Burkholderiales, Alteromonadales, Solirubrobacterales, Caulobacterales, and Rhizobiales, which suggest that members of these taxa are active participants in the exposed partnerships. Although the expression of the other 6 marker genes did not appear to be stimulated by the plant at the community level, signs of additional systems that rest on their expression by members of the orders Solirubrobacterales, Sphingomonadales, Actinomycetales, and Sphingobacteriales were observed. Our study presents the first transcriptomics-based identification of microbes whose xenobiotic degradation activity in soil appears stimulated by a plant. It paints a portrait that contrasts with the current views on these consortia's composition, and opens the door for the development of laboratory test models geared towards the identification of root exudate characteristics that limit the efficiency of current willow-based rhizoremediation applications.

Molecular tools for investigating ANME community structure and function.

Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H(4)MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabolism. These include reactions catalyzed by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase and methyl-coenzyme M reductase (MCR). The latter enzyme is central to C-H bond formation and cleavage underlying methanogenic and reverse methanogenic phenotypes. Here, we describe a set of novel tools for the detection and quantification of H4MPT-linked C1 transfer reactions mediated by uncultivated anaerobic methane-oxidizing archaea (ANME). These tools include polymerase chain reaction primers targeting ANME MCR subunit A subgroups and protein extraction methods from marine sediments compatible with high-resolution mass spectrometry for profiling community structure and functional dynamics.

Phylogenetic characterization of the bacterial assemblage associated with mucous secretions of the hydrothermal vent polychaete Paralvinella palmiformis.

As part of an ongoing examination of microbial diversity associated with hydrothermal vent polychaetes of the family Alvinellidae, we undertook a culture-independent molecular analysis of the bacterial assemblage associated with mucous secretions of the Northeastern Pacific vent polychaete Paralvinella palmiformis. Using a molecular 16S rDNA-based phylogenetic approach, clone libraries were constructed from two samples collected from active sulfide edifices in two hydrothermal vent fields. In both cases, clone libraries were largely dominated by epsilon-Proteobacteria. Phylotypes belonging to the Cytophaga-Flavobacteria and to the Verrucomicrobia were also largely represented within the libraries. The remaining sequences were related to the taxonomic groups Fusobacteria, Green non-sulfur bacteria, Firmicutes, gamma- and delta-Proteobacteria. To our knowledge, this is the first report of the presence of Verrucomicrobia, Fusobacteria and green non-sulfur bacteria on hydrothermal edifices. The potential functions of the detected bacteria are discussed in terms of productivity, recycling of organic matter and detoxification within the P. palmiformis microhabitat.

Desulfurobacterium crinifex sp. nov., a novel thermophilic, pinkish-streamer forming, chemolithoautotrophic bacterium isolated from a Juan de Fuca Ridge hydrothermal vent and amendment of the genus Desulfurobacterium.

A novel thermophilic, chemolithoautotrophic bacterium, designated as NE1206(T), was isolated from a Juan de Fuca Ridge hydrothermal vent sample (tubes of the annelid polychaete Paralvinella sulfincola attached to small pieces of hydrothermal chimney). The cells were rod-shaped (1.2-3.5 x 0.4-0.7 microm), occurring as single motile rods or forming macroscopic aggregates visible as pinkish to brownish streamers. The new isolate was anaerobic. It grew between 50 and 70 degrees C (optimum 60-65 degrees C; doubling time approximately 1 h 15 min at 60 degrees C), between pH 5.0 and 7.5 (optimum pH around 6.0-6.5) and at sea salts concentrations between 20 and 40 g l(-1 )(optimum 30 g l(-1)). Cells grew chemolithoautotrophically in an H(2)/CO(2) atmosphere (80/20, v/v; 200 kPa). Molecular hydrogen was the sole electron donor used by the strain. Nitrate and elemental sulfur served as electron acceptors, yielding ammonia and hydrogen sulfide, respectively (nitrate reduction supported higher growth rates than sulfur reduction). The G+C content of the genomic DNA was 36.7+/-0.8 mol%. Phylogenetic analyses of the 16S rRNA gene located the strain within the genus Desulfurobacterium. However, the novel isolate possesses physiological and biochemical characteristics that differ from the previously described species of this genus. We propose that the isolate represents a novel species, Desulfurobacterium crinifex sp. nov. The type strain is NE1206(T) (DSM 15218(T), CIP 107649(T)). An amendment of the genus Desulfurobacterium description is proposed, based on the phenotypic characteristics of the novel species.