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Identifying differentially abundant microbes is a common goal of microbiome studies. Multiple methods are used interchangeably for this purpose in the literature. Yet, there are few large-scale studies systematically exploring the appropriateness of using these tools interchangeably, and the scale and significance of the differences between them. Here, we compare the performance of 14 differential abundance testing methods on 38 16S rRNA gene datasets with two sample groups. We test for differences in amplicon sequence variants and operational taxonomic units (ASVs) between these groups. Our findings confirm that these tools identified drastically different numbers and sets of significant ASVs, and that results depend on data pre-processing. For many tools the number of features identified correlate with aspects of the data, such as sample size, sequencing depth, and effect size of community differences. ALDEx2 and ANCOM-II produce the most consistent results across studies and agree best with the intersect of results from different approaches. Nevertheless, we recommend that researchers should use a consensus approach based on multiple differential abundance methods to help ensure robust biological interpretations. Many microbiome differential abundance methods are available, but it lacks systematic comparison among them. Here, the authors compare the performance of 14 differential abundance testing methods on 38 16S rRNA gene datasets with two sample groups, and show ALDEx2 and ANCOM-II produce the most consistent results.

Unraveling the drivers controlling community assembly is a central issue in ecology. Although it is generally accepted that selection, dispersal, diversification and drift are major community assembly processes, defining their relative importance is very challenging. Here, we present a framework to quantitatively infer community assembly mechanisms by phylogenetic bin-based null model analysis (iCAMP). iCAMP shows high accuracy (0.93–0.99), precision (0.80–0.94), sensitivity (0.82–0.94), and specificity (0.95–0.98) on simulated communities, which are 10–160% higher than those from the entire community-based approach. Application of iCAMP to grassland microbial communities in response to experimental warming reveals dominant roles of homogeneous selection (38%) and ‘drift’ (59%). Interestingly, warming decreases ‘drift’ over time, and enhances homogeneous selection which is primarily imposed on Bacillales. In addition, homogeneous selection has higher correlations with drought and plant productivity under warming than control. iCAMP provides an effective and robust tool to quantify microbial assembly processes, and should also be useful for plant and animal ecology. Studies of microbial community assembly mechanisms typically use metrics for turnover within the whole community. Here, the authors develop an alternative approach based on turnover within lineages and dissect mechanistic change in grassland bacterial assembly under experimental warming.

While footprinting analysis of ATAC-seq data can theoretically enable investigation of transcription factor (TF) binding, the lack of a computational tool able to conduct different levels of footprinting analysis has so-far hindered the widespread application of this method. Here we present TOBIAS, a comprehensive, accurate, and fast footprinting framework enabling genome-wide investigation of TF binding dynamics for hundreds of TFs simultaneously. We validate TOBIAS using paired ATAC-seq and ChIP-seq data, and find that TOBIAS outperforms existing methods for bias correction and footprinting. As a proof-of-concept, we illustrate how TOBIAS can unveil complex TF dynamics during zygotic genome activation in both humans and mice, and propose how zygotic Dux activates cascades of TFs, binds to repeat elements and induces expression of novel genetic elements. Footprinting analysis allows genome-wide investigation of transcription factor (TF) binding on chromatin. Here the authors developed a framework termed TOBIAS aimed at identifying footprints of chromatin-associated proteins from ATAC-seq accessibility profiles and apply it to zygotic development datasets.

Pseudomonas syringae is a Gram-negative and model pathogenic bacterium that causes plant diseases worldwide. Here, we set out to identify binding motifs for all 301 annotated transcription factors (TFs) of P. syringae using HT-SELEX. We successfully identify binding motifs for 100 TFs. We map functional interactions between the TFs and their targets in virulence-associated pathways, and validate many of these interactions and functions using additional methods such as ChIP-seq, electrophoretic mobility shift assay (EMSA), RT-qPCR, and reporter assays. Our work identifies 25 virulence-associated master regulators, 14 of which had not been characterized as TFs before. The authors set out to identify binding motifs for all 301 transcription factors of a plant pathogenic bacterium, Pseudomonas syringae , using HT-SELEX. They successfully identify binding motifs for 100 transcription factors, infer their binding sites on the genome, and validate the predicted interactions and functions.

Plant response to drought stress involves fungi and bacteria that live on and in plants and in the rhizosphere, yet the stability of these myco- and micro-biomes remains poorly understood. We investigate the resistance and resilience of fungi and bacteria to drought in an agricultural system using both community composition and microbial associations. Here we show that tests of the fundamental hypotheses that fungi, as compared to bacteria, are (i) more resistant to drought stress but (ii) less resilient when rewetting relieves the stress, found robust support at the level of community composition. Results were more complex using all-correlations and co-occurrence networks. In general, drought disrupts microbial networks based on significant positive correlations among bacteria, among fungi, and between bacteria and fungi. Surprisingly, co-occurrence networks among functional guilds of rhizosphere fungi and leaf bacteria were strengthened by drought, and the same was seen for networks involving arbuscular mycorrhizal fungi in the rhizosphere. We also found support for the stress gradient hypothesis because drought increased the relative frequency of positive correlations. Fungi are expected to be more resistant and less resilient than bacteria to environmental disturbances. Here, the authors report complex responses by microbial co-occurrence networks to drought in an agricultural system, challenging simple predictions of fungal and bacterial drought responses.

Microbial composition and functions in the rhizosphere—an important microbial hotspot—are among the most fascinating yet elusive topics in microbial ecology. We used 557 pairs of published 16S rDNA amplicon sequences from the bulk soils and rhizosphere in different ecosystems around the world to generalize bacterial characteristics with respect to community diversity, composition, and functions. The rhizosphere selects microorganisms from bulk soil to function as a seed bank, reducing microbial diversity. The rhizosphere is enriched in Bacteroidetes, Proteobacteria, and other copiotrophs. Highly modular but unstable bacterial networks in the rhizosphere (common for r -strategists) reflect the interactions and adaptations of microorganisms to dynamic conditions. Dormancy strategies in the rhizosphere are dominated by toxin–antitoxin systems, while sporulation is common in bulk soils. Functional predictions showed that genes involved in organic compound conversion, nitrogen fixation, and denitrification were strongly enriched in the rhizosphere (11–182%), while genes involved in nitrification were strongly depleted. Understanding soil microbiota dynamics is key the development of soil-based sustainable agriculture and conservation strategies. This meta-analysis shows that bulk soil functions as a seed bank for the rhizosphere, which encompasses a rich microbiota adapted to dynamic conditions in hotpots.

“Plastisphere”, microbial communities colonizing plastic debris, has sparked global concern for marine ecosystems. Microbiome inhabiting this novel human-made niche has been increasingly characterized; however, whether the plastisphere holds crucial roles in biogeochemical cycling remains largely unknown. Here we evaluate the potential of plastisphere in biotic and abiotic denitrification and nitrous oxide (N 2 O) production in estuaries. Biofilm formation provides anoxic conditions favoring denitrifiers. Comparing with surrounding bulk water, plastisphere exhibits a higher denitrifying activity and N 2 O production, suggesting an overlooked N 2 O source. Regardless of plastisphere and bulk water, bacterial and fungal denitrifications are the main regulators for N 2 O production instead of chemodenitrification. However, the contributions of bacteria and fungi in the plastisphere are different from those in bulk water, indicating a distinct N 2 O production pattern in the plastisphere. These findings pinpoint plastisphere as a N 2 O source, and provide insights into roles of the new biotope in biogeochemical cycling in the Anthropocene. The roles of marine plastisphere in global nitrogen cycling are largely unknown. Here, the authors indicate that the plastisphere could act as a potential source of N2O production, which is mainly regulated by the biotic denitrification

The perturbations of the gut microbiota and metabolites are closely associated with the progression of inflammatory bowel disease (IBD). However, inconsistent findings across studies impede a comprehensive understanding of their roles in IBD and their potential as reliable diagnostic biomarkers. To address this challenge, here we comprehensively analyze 9 metagenomic and 4 metabolomics cohorts of IBD from different populations. Through cross-cohort integrative analysis (CCIA), we identify a consistent characteristic of commensal gut microbiota. Especially, three bacteria, namely Asaccharobacter celatus , Gemmiger formicilis , and Erysipelatoclostridium ramosum , which are rarely reported in IBD. Metagenomic functional analysis reveals that essential gene of Two-component system pathway, linked to fecal calprotectin, are implicated in IBD. Metabolomics analysis shows 36 identified metabolites with significant differences, while the roles of these metabolites in IBD are still unknown. To further elucidate the relationship between gut microbiota and metabolites, we construct multi-omics biological correlation (MOBC) maps, which highlights gut microbial biotransformation deficiencies and significant alterations in aminoacyl-tRNA synthetases. Finally, we identify multi-omics biomarkers for IBD diagnosis, validated across multiple global cohorts (AUROC values ranging from 0.92 to 0.98). Our results offer valuable insights and a significant resource for developing mechanistic hypotheses on host-microbiome interactions in IBD. Gut microbiota play pivotal roles in IBD. Here, Ning et al . use a multi-omics approach to characterize gut microbiota and metabolites alterations, and potential pathogenic bacteria associated with IBD, with the aim to help develop more precise biomarkers for IBD diagnosis and drug targets

Factors driving microbial community composition and diversity are well established but the relationship with microbial functioning is poorly understood, especially at large scales. We analysed microbial biodiversity metrics and distribution of potential functional groups along a gradient of increasing land-use perturbation, detecting over 79,000 bacterial and 25,000 fungal OTUs in 715 sites across 24 European countries. We found the lowest bacterial and fungal diversity in less-disturbed environments (woodlands) compared to grasslands and highly-disturbed environments (croplands). Highly-disturbed environments contain significantly more bacterial chemoheterotrophs, harbour a higher proportion of fungal plant pathogens and saprotrophs, and have less beneficial fungal plant symbionts compared to woodlands and extensively-managed grasslands. Spatial patterns of microbial communities and predicted functions are best explained when interactions among the major determinants (vegetation cover, climate, soil properties) are considered. We propose guidelines for environmental policy actions and argue that taxonomical and functional diversity should be considered simultaneously for monitoring purposes. “Factors influencing soil microbiota functioning remain understudied. Here, the authors describe bacterial and fungal diversity across Europe and along a gradient of land-use perturbation, observing that the occurrence of pathogens, symbionts and saprotrophs varied among cropland, woodland and grassland.”

CRISPR is becoming an indispensable tool in biological research. Once known as the bacterial immune system against invading viruses, the programmable capacity of the Cas9 enzyme is now revolutionizing diverse fields of medical research, biotechnology, and agriculture. CRISPR-Cas9 is no longer just a gene-editing tool; the application areas of catalytically impaired inactive Cas9, including gene regulation, epigenetic editing, chromatin engineering, and imaging, now exceed the gene-editing functionality of WT Cas9. Here, we will present a brief history of gene-editing tools and describe the wide range of CRISPR-based genome-targeting tools. We will conclude with future directions and the broader impact of CRISPR technologies. CRISPR has rapidly become an indispensable tool for biological research. Here Mazhar Adli reviews the current toolbox for editing and manipulating the genome and looks toward future developments in this fast moving field.