Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activity in vitro for 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.

Microbial functional diversity across biogeochemical provinces in the central Pacific Ocean.

Enzymes catalyze key reactions within Earth's life-sustaining biogeochemical cycles. Here, we use metaproteomics to examine the enzymatic capabilities of the microbial community (0.2 to 3 µm) along a 5,000-km-long, 1-km-deep transect in the central Pacific Ocean. Eighty-five percent of total protein abundance was of bacterial origin, with Archaea contributing 1.6%. Over 2,000 functional KEGG Ontology (KO) groups were identified, yet only 25 KO groups contributed over half of the protein abundance, simultaneously indicating abundant key functions and a long tail of diverse functions. Vertical attenuation of individual proteins displayed stratification of nutrient transport, carbon utilization, and environmental stress. The microbial community also varied along horizontal scales, shaped by environmental features specific to the oligotrophic North Pacific Subtropical Gyre, the oxygen-depleted Eastern Tropical North Pacific, and nutrient-rich equatorial upwelling. Some of the most abundant proteins were associated with nitrification and C1 metabolisms, with observed interactions between these pathways. The oxidoreductases nitrite oxidoreductase (NxrAB), nitrite reductase (NirK), ammonia monooxygenase (AmoABC), manganese oxidase (MnxG), formate dehydrogenase (FdoGH and FDH), and carbon monoxide dehydrogenase (CoxLM) displayed distributions indicative of biogeochemical status such as oxidative or nutritional stress, with the potential to be more sensitive than chemical sensors. Enzymes that mediate transformations of atmospheric gases like CO, CO2, NO, methanethiol, and methylamines were most abundant in the upwelling region. We identified hot spots of biochemical transformation in the central Pacific Ocean, highlighted previously understudied metabolic pathways in the environment, and provided rich empirical data for biogeochemical models critical for forecasting ecosystem response to climate change.

Normalizing Metagenomic Hi-C Data and Detecting Spurious Contacts Using Zero-Inflated Negative Binomial Regression.

High-throughput chromosome conformation capture (Hi-C) has recently been applied to natural microbial communities and revealed great potential to study multiple genomes simultaneously. Several extraneous factors may influence chromosomal contacts rendering the normalization of Hi-C contact maps essential for downstream analyses. However, the current paucity of metagenomic Hi-C normalization methods and the ignorance for spurious interspecies contacts weaken the interpretability of the data. Here, we report on two types of biases in metagenomic Hi-C experiments: explicit biases and implicit biases, and introduce HiCzin, a parametric model to correct both types of biases and remove spurious interspecies contacts. We demonstrate that the normalized metagenomic Hi-C contact maps by HiCzin result in lower biases, higher capability to detect spurious contacts, and better performance in metagenomic contig clustering.

Microbial communities can predict the ecological condition of headwater streams.

Humanity's reliance on clean water and the ecosystem services provided makes identifying efficient and effective ways to assess the ecological condition of streams ever more important. We used high throughput sequencing of the 16S rRNA region to explore relationships between stream microbial communities, environmental attributes, and assessments of stream ecological condition. Bacteria and archaea in microbial community samples collected from the water column and from stream sediments during spring and summer were used to replicate standard assessments of ecological condition performed with benthic macroinvertebrate collections via the Benthic Index of Biotic Integrity (BIBI). Microbe-based condition assessments were generated at different levels of taxonomic resolution from phylum to OTU (Operational Taxonomic Units) in order to understand appropriate levels of taxonomic aggregation. Stream sediment microbial communities from both spring and summer were much better than the water column at replicating BIBI condition assessment results. Accuracies were as high as 100% on training data used to build the models and up to 80% on validation data used to assess predictions. Assessments using all OTUs usually had the highest accuracy on training data, but were lower on validation data due to overfitting. In contrast, assessments at the order-level had similar performance accuracy for validation data, and a reduced subset of orders also performed well, suggesting the method could be generalized to other watersheds. Subsets of the important orders responded similarly to environmental gradients compared to the entire community, where strong shifts in community structure occurred for known aquatic stressors such as pH, dissolved organic carbon, and nitrate nitrogen. The results suggest the stream microbes may be useful for assessing the ecological condition of streams and especially useful for stream restorations where many eukaryotic taxa have been eliminated due to prior degradation and are unable to recolonize.

Headwater Stream Microbial Diversity and Function across Agricultural and Urban Land Use Gradients.

Anthropogenic activity impacts stream ecosystems, resulting in a loss of diversity and ecosystem function; however, little is known about the response of aquatic microbial communities to changes in land use. Here, microbial communities were characterized in 82 headwater streams across a gradient of urban and agricultural land uses using 16S rRNA gene amplicon sequencing and compared to a rich data set of physicochemical variables and traditional benthic invertebrate indicators. Microbial diversity and community structures differed among watersheds with high agricultural, urban, and forested land uses, and community structure differed in streams classified as being in good, fair, poor, and very poor condition using benthic invertebrate indicators. Microbial community similarity decayed with geodesic distance across the study region but not with environmental distance. Stream community respiration rates ranged from 21.7 to 1,570 mg O2 m-2 day-1 and 31.9 to 3,670 mg O2 m-2 day-1 for water column and sediments, respectively, and correlated with nutrients associated with anthropogenic influence and microbial community structure. Nitrous oxide (N2O) concentrations ranged from 0.22 to 4.41 µg N2O liter-1; N2O concentration was negatively correlated with forested land use and was positively correlated with dissolved inorganic nitrogen concentrations. Our findings suggest that stream microbial communities are impacted by watershed land use and can potentially be used to assess ecosystem health.IMPORTANCE Stream ecosystems are frequently impacted by changes in watershed land use, resulting in altered hydrology, increased pollutant and nutrient loads, and habitat degradation. Macroinvertebrates and fish are strongly affected by changes in stream conditions and are commonly used in biotic indices to assess ecosystem health. Similarly, microbes respond to environmental stressors, and changes in community composition alter key ecosystem processes. The response of microbes to habitat degradation and their role in global biogeochemical cycles provide an opportunity to use microbes as a monitoring tool. Here, we identify stream microbes that respond to watershed urbanization and agricultural development and demonstrate that microbial diversity and community structure can be used to assess stream conditions and ecosystem functioning.