AnnoTree: visualization and exploration of a functionally annotated microbial tree of life

Mendler, Kerrin; Chen, Han; Parks, Donovan H.; Lobb, Briallen; Hug, Laura; Doxey, Andrew C.

doi:10.1093/nar/gkz246

Cited by 249 publications

(279 citation statements)

References 42 publications

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“…Many contigs from these libraries are phage-associated (30%; table S4, 141 figure 2A) and many encode genes of unknown function (figure S4); these are both hallmarks of known 142 acrs (Borges et al, 2017;Pawluk et al, 2017;Sontheimer and Davidson, 2017). Additionally, we 143 observed that Cas9 homologs are found in 15 of the 18 bacterial genera identified in these selections 144 (83%, table S4), a significant enrichment over the 12% of bacterial genera that harbor Cas9 (567/4822) 145 from a representative set of ~24,000 bacterial genomes (Mendler et al, 2018) (p=4x10 -21 , χ² test). This 146 enrichment strongly supports our hypothesis that many of the recovered contigs encode true acrs rather 147 than artifacts of functional selection, since phages and MGEs must encounter Cas9 to benefit from the 148 protective effect of acrs.…”

Section: Introduction 14mentioning

confidence: 70%

Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

Forsberg

Bhatt

Schmidtke

et al. 2019

Preprint

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1 CRISPR-Cas systems protect bacteria and archaea from phages and other mobile genetic elements, 2 which use small anti-CRISPR (Acr) proteins to overcome CRISPR-Cas immunity. Because they are 3 difficult to identify, the natural diversity and impact of Acrs on microbial ecosystems is underappreciated. 4To overcome this discovery bottleneck, we developed a high-throughput functional selection that isolates 5 acr genes based on their ability to inhibit CRISPR-Cas function. Using this selection, we discovered ten 6 DNA fragments from human oral and fecal metagenomes that antagonize Streptococcus pyogenes Cas9 7 (SpyCas9). The most potent acr discovered, acrIIA11, was recovered from a Lachnospiraceae phage 8 and is among the strongest known SpyCas9 inhibitors. AcrIIA11 homologs are distributed across multiple 9 bacterial phyla and many divergent homologs inhibit SpyCas9. We show that AcrIIA11 antagonizes 10 SpyCas9 using a different mechanism than that of previously characterized inhibitors. Our study 11 highlights the power of functional selections to uncover widespread Cas9 inhibitors within diverse 12 microbiomes. 13 4 an Acr protein Watters et al., 2018). These clever strategies have revealed many 49 candidate acrs. But they have also highlighted the difficulty of finding new acr genes based on homology, 50 since acrs share little sequence conservation (Sontheimer and Davidson, 2017). As a result, most acrs 51 almost certainly lie unrecognized among the many genes of unknown function in phages, plasmids, and 52 other MGEs (Hatfull, 2015). 53 54To overcome the challenges associated with anti-CRISPR discovery, we devised a functional 55 metagenomic selection that identifies acr genes from any cloned DNA, based on their ability to protect a 56 plasmid from CRISPR-Cas-mediated destruction. Because functional metagenomics selects for a 57 function of interest from large clone libraries (Handelsman, 2004), it is well-suited to identify individual 58 genes like acrs that have strong fitness impacts (Iqbal et al., 2014;Forsberg et al., 2015; Forsberg et al., 59 2016; Genee et al., 2016). This approach may be particularly useful for Acr discovery because Acrs are 60 expressed from single genes and function readily in many genetic backgrounds (Pawluk et al., 2016a; 61 Rauch et al., 2017). 62 63Using this functional selection, we find that many unrelated metagenomic clones from human oral 64 and gut microbiomes protect against Streptococcus pyogenes Cas9 (SpyCas9), the variant used most 65 commonly for gene editing applications (Knott and Doudna, 2018). We identify a broadly distributed but 66 previously undescribed Acr from the most potent Cas9-antagonizing clone in our libraries. This Acr, 67 named AcrIIA11, binds both SpyCas9 and double-stranded DNA (dsDNA) and exhibits a novel mode of 68 SpyCas9 antagonism, protecting both plasmids and phages from immune restriction. Thus, our functional 69 approach reveals not only new classes of Acrs, but also new mechanisms of CRISPR-Cas antagonism. 70 71Results 72 73 A functio...

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Section: Introduction 14mentioning

confidence: 70%

Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

Forsberg

Bhatt

Schmidtke

et al. 2019

Preprint

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“…The observation that distributions of traits are variable, sometimes patchy, and potentially decoupled from phylogenetic relatedness raises the possibility that more complex, enzyme-specific patterns might underlie processes contributing to metabolic diversity in the CPR. To address this, we drew upon trait distributions to compute two metrics -one to quantify the average branch length of clades in which a trait is conserved (phylogenetic depth) and a second, patchiness, related to the number of gains/losses of a binary trait over a tree (see Materials and Methods) (Mendler et al, 2019) . These metrics were then integrated to create an 'evolutionary profile' for each trait.…”

Section: Functionally Linked Metabolic Genes Display Different 'Evolumentioning

confidence: 99%

The rise of diversity in metabolic platforms across the Candidate Phyla Radiation

Jaffe

Castelle

Carnevali

et al. 2019

Preprint

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A unifying feature of the bacterial Candidate Phyla Radiation (CPR) is a limited and highly variable repertoire of biosynthetic capabilities. However, the distribution of metabolic traits across the CPR and the evolutionary processes underlying them are incompletely resolved. Here, we selected~1,000 genomes of CPR bacteria from diverse environments to construct a robust internal phylogeny that was consistent across two unlinked marker sets. Mapping of glycolysis, the pentose phosphate pathway, and pyruvate metabolism onto the tree showed that some components of these pathways are sparsely distributed and that similarity between metabolic platforms is only partially predicted by phylogenetic relationships. To evaluate the extent to which gene loss and lateral gene transfer have shaped trait distribution , we analyzed the patchiness of gene presence in a phylogenetic context, examined the phylogenetic depth of clades with shared traits, and compared the reference tree topology with those of specific metabolic proteins. While the central glycolytic pathway in CPR is widely conserved and has likely been shaped primarily by vertical transmission, there is evidence for both gene loss and transfer especially in steps that convert glucose into fructose 1,6-bisphosphate and glycerate 3P into pyruvate. Additionally, the distribution of Group 3 and Group 4-related NiFe hydrogenases is patchy and suggests multiple events of ancient gene transfer. Overall, patterns of gene gain and loss, including acquisition of accessory traits in independent transfer events, may have been driven by shifts in host-derived resources and led to sparse but varied genetic inventories. INTRODUCTION :

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“…The list of transformable species keeps growing, now including species that had long been considered incapable of transformation 12 . In addition, from the recent database AnnoTree grouping nearly 24,000 bacterial genomes 13 , we uncover that transformation-specific genes required for the uptake of eDNA (comEC, dprA) are extremely conserved (Fig. 1).…”

Section: Introductionmentioning

confidence: 92%

Bacterial transformation buffers environmental fluctuations through the reversible integration of mobile genetic elements

Carvalho

Fouchet

Danesh

et al. 2019

Preprint

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Horizontal gene transfer (HGT) is known to promote the spread of genes in bacterial communities, which is of primary importance to human health when these genes provide resistance to antibiotics. Among the main HGT mechanisms, natural transformation stands out as being widespread and encoded by the bacterial core genome. From an evolutionary perspective, transformation is often viewed as a mean to generate genetic diversity and mixing within bacterial populations. However, another recent paradigm proposes that its main evolutionary function would be to cure bacterial genomes from their parasitic mobile genetic elements (MGEs). Here, we propose to combine these two seemingly opposing points of view because MGEs, although costly for bacterial cells, can carry functions that are point-in-time beneficial to bacteria under stressful conditions (e.g. antibiotic resistance genes under antibiotic exposure). Using computational modeling, we show that, in stochastic environments (unpredictable stress exposure), an intermediate transformation rate maximizes bacterial fitness by allowing the reversible integration of MGEs carrying resistance genes but costly for the replication of host cells. By ensuring such reversible genetic diversification (acquisition then removal of MGEs), transformation would be a key mechanism for stabilizing the bacterial genome in the long term, which would explain its striking conservation.

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AnnoTree: visualization and exploration of a functionally annotated microbial tree of life

Cited by 249 publications

References 42 publications

Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

Functional metagenomics-guided discovery of potent Cas9 inhibitors in the human microbiome

The rise of diversity in metabolic platforms across the Candidate Phyla Radiation

Bacterial transformation buffers environmental fluctuations through the reversible integration of mobile genetic elements

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