Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems.

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA1 (msDNA). Despite decades of research on the biosynthesis of msDNA2, the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports3,4. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities.

The bacterial genetic determinants of Escherichia coli capacity to cause bloodstream infections in humans.

Escherichia coli is both a highly prevalent commensal and a major opportunistic pathogen causing bloodstream infections (BSI). A systematic analysis characterizing the genomic determinants of extra-intestinal pathogenic vs. commensal isolates in human populations, which could inform mechanisms of pathogenesis, diagnostic, prevention and treatment is still lacking. We used a collection of 912 BSI and 370 commensal E. coli isolates collected in France over a 17-year period (2000-2017). We compared their pangenomes, genetic backgrounds (phylogroups, STs, O groups), presence of virulence-associated genes (VAGs) and antimicrobial resistance genes, finding significant differences in all comparisons between commensal and BSI isolates. A machine learning linear model trained on all the genetic variants derived from the pangenome and controlling for population structure reveals similar differences in VAGs, discovers new variants associated with pathogenicity (capacity to cause BSI), and accurately classifies BSI vs. commensal strains. Pathogenicity is a highly heritable trait, with up to 69% of the variance explained by bacterial genetic variants. Lastly, complementing our commensal collection with an older collection from 1980, we predict that pathogenicity continuously increased through 1980, 2000, to 2010. Together our findings imply that E. coli exhibit substantial genetic variation contributing to the transition between commensalism and pathogenicity and that this species evolved towards higher pathogenicity.

Genome wide association study of Escherichia coli bloodstream infection isolates identifies genetic determinants for the portal of entry but not fatal outcome.

Escherichia coli is an important cause of bloodstream infections (BSI), which is of concern given its high mortality and increasing worldwide prevalence. Finding bacterial genetic variants that might contribute to patient death is of interest to better understand infection progression and implement diagnostic methods that specifically look for those factors. E. coli samples isolated from patients with BSI are an ideal dataset to systematically search for those variants, as long as the influence of host factors such as comorbidities are taken into account. Here we performed a genome-wide association study (GWAS) using data from 912 patients with E. coli BSI from hospitals in Paris, France. We looked for associations between bacterial genetic variants and three patient outcomes (death at 28 days, septic shock and admission to intensive care unit), as well as two portals of entry (urinary and digestive tract), using various clinical variables from each patient to account for host factors. We did not find any association between genetic variants and patient outcomes, potentially confirming the strong influence of host factors in influencing the course of BSI; we however found a strong association between the papGII operon and entrance of E. coli through the urinary tract, which demonstrates the power of bacterial GWAS when applied to actual clinical data. Despite the lack of associations between E. coli genetic variants and patient outcomes, we estimate that increasing the sample size by one order of magnitude could lead to the discovery of some putative causal variants. Given the wide adoption of bacterial genome sequencing of clinical isolates, such sample sizes may be soon available.

ChemGAPP: a tool for chemical genomics analysis and phenotypic profiling.

MOTIVATION: High-throughput chemical genomic screens produce informative datasets, providing valuable insights into unknown gene function on a genome-wide level. However, there is currently no comprehensive analytic package publicly available. We developed ChemGAPP to bridge this gap. ChemGAPP integrates various steps in a streamlined and user-friendly format, including rigorous quality control measures to curate screening data. RESULTS: ChemGAPP provides three sub-packages for different chemical-genomic screens: ChemGAPP Big for large-scale screens; ChemGAPP Small for small-scale screens; and ChemGAPP GI for genetic interaction screens. ChemGAPP Big, tested against the Escherichiacoli KEIO collection, revealed reliable fitness scores which displayed biologically relevant phenotypes. ChemGAPP Small demonstrated significant changes in phenotype in a small-scale screen. ChemGAPP GI was benchmarked against three sets of genes with known epistasis types and successfully reproduced each interaction type. AVAILABILITY AND IMPLEMENTATION: ChemGAPP is available at https://github.com/HannahMDoherty/ChemGAPP, as a standalone Python package as well as Streamlit applications.

Major role of iron uptake systems in the intrinsic extra-intestinal virulence of the genus Escherichia revealed by a genome-wide association study.

The genus Escherichia is composed of several species and cryptic clades, including E. coli, which behaves as a vertebrate gut commensal, but also as an opportunistic pathogen involved in both diarrheic and extra-intestinal diseases. To characterize the genetic determinants of extra-intestinal virulence within the genus, we carried out an unbiased genome-wide association study (GWAS) on 370 commensal, pathogenic and environmental strains representative of the Escherichia genus phylogenetic diversity and including E. albertii (n = 7), E. fergusonii (n = 5), Escherichia clades (n = 32) and E. coli (n = 326), tested in a mouse model of sepsis. We found that the presence of the high-pathogenicity island (HPI), a ~35 kbp gene island encoding the yersiniabactin siderophore, is highly associated with death in mice, surpassing other associated genetic factors also related to iron uptake, such as the aerobactin and the sitABCD operons. We confirmed the association in vivo by deleting key genes of the HPI in E. coli strains in two phylogenetic backgrounds. We then searched for correlations between virulence, iron capture systems and in vitro growth in a subset of E. coli strains (N = 186) previously phenotyped across growth conditions, including antibiotics and other chemical and physical stressors. We found that virulence and iron capture systems are positively correlated with growth in the presence of numerous antibiotics, probably due to co-selection of virulence and resistance. We also found negative correlations between virulence, iron uptake systems and growth in the presence of specific antibiotics (i.e. cefsulodin and tobramycin), which hints at potential "collateral sensitivities" associated with intrinsic virulence. This study points to the major role of iron capture systems in the extra-intestinal virulence of the genus Escherichia.

The impact of the genetic background on gene deletion phenotypes in Saccharomyces cerevisiae.

Loss-of-function (LoF) mutations associated with disease do not manifest equally in different individuals. The impact of the genetic background on the consequences of LoF mutations remains poorly characterized. Here, we systematically assessed the changes in gene deletion phenotypes for 3,786 gene knockouts in four Saccharomyces cerevisiae strains and 38 conditions. We observed 18.5% of deletion phenotypes changing between pairs of strains on average with a small fraction conserved in all four strains. Conditions causing higher wild-type growth differences and the deletion of pleiotropic genes showed above-average changes in phenotypes. In addition, we performed a genome-wide association study (GWAS) for growth under the same conditions for a panel of 925 yeast isolates. Gene-condition associations derived from GWAS were not enriched for genes with deletion phenotypes under the same conditions. However, cases where the results were congruent indicate the most likely mechanism underlying the GWAS signal. Overall, these results show a high degree of genetic background dependencies for LoF phenotypes.

pyseer: a comprehensive tool for microbial pangenome-wide association studies.

Summary: Genome-wide association studies (GWAS) in microbes have different challenges to GWAS in eukaryotes. These have been addressed by a number of different methods. pyseer brings these techniques together in one package tailored to microbial GWAS, allows greater flexibility of the input data used, and adds new methods to interpret the association results. Availability and implementation: pyseer is written in python and is freely available at https://github.com/mgalardini/pyseer, or can be installed through pip. Documentation and a tutorial are available at http://pyseer.readthedocs.io. Supplementary information: Supplementary data are available at Bioinformatics online.

A resource of variant effect predictions of single nucleotide variants in model organisms.

The effect of single nucleotide variants (SNVs) in coding and noncoding regions is of great interest in genetics. Although many computational methods aim to elucidate the effects of SNVs on cellular mechanisms, it is not straightforward to comprehensively cover different molecular effects. To address this, we compiled and benchmarked sequence and structure-based variant effect predictors and we computed the impact of nearly all possible amino acid and nucleotide variants in the reference genomes of Homo sapiens, Saccharomyces cerevisiae and Escherichia coli Studied mechanisms include protein stability, interaction interfaces, post-translational modifications and transcription factor binding sites. We apply this resource to the study of natural and disease coding variants. We also show how variant effects can be aggregated to generate protein complex burden scores that uncover protein complex to phenotype associations based on a set of newly generated growth profiles of 93 sequenced S. cerevisiae strains in 43 conditions. This resource is available through mutfunc (www.mutfunc.com), a tool by which users can query precomputed predictions by providing amino acid or nucleotide-level variants.

COLOMBOS v3.0: leveraging gene expression compendia for cross-species analyses.

COLOMBOS is a database that integrates publicly available transcriptomics data for several prokaryotic model organisms. Compared to the previous version it has more than doubled in size, both in terms of species and data available. The manually curated condition annotation has been overhauled as well, giving more complete information about samples' experimental conditions and their differences. Functionality-wise cross-species analyses now enable users to analyse expression data for all species simultaneously, and identify candidate genes with evolutionary conserved expression behaviour. All the expression-based query tools have undergone a substantial improvement, overcoming the limit of enforced co-expression data retrieval and instead enabling the return of more complex patterns of expression behaviour. COLOMBOS is freely available through a web application at http://colombos.net/. The complete database is also accessible via REST API or downloadable as tab-delimited text files.

MeDuSa: a multi-draft based scaffolder.

MOTIVATION: Completing the genome sequence of an organism is an important task in comparative, functional and structural genomics. However, this remains a challenging issue from both a computational and an experimental viewpoint. Genome scaffolding (i.e. the process of ordering and orientating contigs) of de novo assemblies usually represents the first step in most genome finishing pipelines. RESULTS: In this article we present MeDuSa (Multi-Draft based Scaffolder), an algorithm for genome scaffolding. MeDuSa exploits information obtained from a set of (draft or closed) genomes from related organisms to determine the correct order and orientation of the contigs. MeDuSa formalizes the scaffolding problem by means of a combinatorial optimization formulation on graphs and implements an efficient constant factor approximation algorithm to solve it. In contrast to currently used scaffolders, it does not require either prior knowledge on the microrganisms dataset under analysis (e.g. their phylogenetic relationships) or the availability of paired end read libraries. This makes usability and running time two additional important features of our method. Moreover, benchmarks and tests on real bacterial datasets showed that MeDuSa is highly accurate and, in most cases, outperforms traditional scaffolders. The possibility to use MeDuSa on eukaryotic datasets has also been evaluated, leading to interesting results.

Evolution of Intra-specific Regulatory Networks in a Multipartite Bacterial Genome.

Reconstruction of the regulatory network is an important step in understanding how organisms control the expression of gene products and therefore phenotypes. Recent studies have pointed out the importance of regulatory network plasticity in bacterial adaptation and evolution. The evolution of such networks within and outside the species boundary is however still obscure. Sinorhizobium meliloti is an ideal species for such study, having three large replicons, many genomes available and a significant knowledge of its transcription factors (TF). Each replicon has a specific functional and evolutionary mark; which might also emerge from the analysis of their regulatory signatures. Here we have studied the plasticity of the regulatory network within and outside the S. meliloti species, looking for the presence of 41 TFs binding motifs in 51 strains and 5 related rhizobial species. We have detected a preference of several TFs for one of the three replicons, and the function of regulated genes was found to be in accordance with the overall replicon functional signature: house-keeping functions for the chromosome, metabolism for the chromid, symbiosis for the megaplasmid. This therefore suggests a replicon-specific wiring of the regulatory network in the S. meliloti species. At the same time a significant part of the predicted regulatory network is shared between the chromosome and the chromid, thus adding an additional layer by which the chromid integrates itself in the core genome. Furthermore, the regulatory network distance was found to be correlated with both promoter regions and accessory genome evolution inside the species, indicating that both pangenome compartments are involved in the regulatory network evolution. We also observed that genes which are not included in the species regulatory network are more likely to belong to the accessory genome, indicating that regulatory interactions should also be considered to predict gene conservation in bacterial pangenomes.

Exploring the dynamics of bacterial community composition in soil: the pan-bacteriome approach.

We performed a longitudinal study (repeated observations of the same sample over time) to investigate both the composition and structure of temporal changes of bacterial community composition in soil mesocosms, subjected to three different treatments (water and 5 or 25 mg kg(-1) of dried soil Cd(2+)). By analogy with the pan genome concept, we identified a core bacteriome and an accessory bacteriome. Resident taxa were assigned to the core bacteriome, while occasional taxa were assigned to the accessory bacteriome. Core and accessory bacteriome represented roughly 35 and 50 % of the taxa detected, respectively, and were characterized by different taxonomic signatures from phylum to genus level while 15 % of the taxa were found to be unique to a particular sample. In particular, the core bacteriome was characterized by higher abundance of members of Planctomycetes, Actinobacteria, Verrucomicrobia and Acidobacteria, while the accessory bacteriome included more members of Firmicutes, Clamydiae and Proteobacteria, suggesting potentially different responses to environmental changes of members from these phyla. We conclude that the pan-bacteriome model may be a useful approach to gain insight for modeling bacterial community structure and inferring different abilities of bacteria taxa.

DuctApe: a suite for the analysis and correlation of genomic and OmniLog™ Phenotype Microarray data.

Addressing the functionality of genomes is one of the most important and challenging tasks of today's biology. In particular the ability to link genotypes to corresponding phenotypes is of interest in the reconstruction and biotechnological manipulation of metabolic pathways. Over the last years, the OmniLog™ Phenotype Microarray (PM) technology has been used to address many specific issues related to the metabolic functionality of microorganisms. However, computational tools that could directly link PM data with the gene(s) of interest followed by the extraction of information on gene-phenotype correlation are still missing. Here we present DuctApe, a suite that allows the analysis of both genomic sequences and PM data, to find metabolic differences among PM experiments and to correlate them with KEGG pathways and gene presence/absence patterns. As example, an application of the program to four bacterial datasets is presented. The source code and tutorials are available at http://combogenomics.github.io/DuctApe/.

The DivJ, CbrA and PleC system controls DivK phosphorylation and symbiosis in Sinorhizobium meliloti.

Sinorhizobium meliloti is a soil bacterium that invades the root nodules it induces on Medicago sativa, whereupon it undergoes an alteration of its cell cycle and differentiates into nitrogen-fixing, elongated and polyploid bacteroid with higher membrane permeability. In Caulobacter crescentus, a related alphaproteobacterium, the principal cell cycle regulator, CtrA, is inhibited by the phosphorylated response regulator DivK. The phosphorylation of DivK depends on the histidine kinase DivJ, while PleC is the principal phosphatase for DivK. Despite the importance of the DivJ in C. crescentus, the mechanistic role of this kinase has never been elucidated in other Alphaproteobacteria. We show here that the histidine kinases DivJ together with CbrA and PleC participate in a complex phosphorylation system of the essential response regulator DivK in S. meliloti. In particular, DivJ and CbrA are involved in DivK phosphorylation and in turn CtrA inactivation, thereby controlling correct cell cycle progression and the integrity of the cell envelope. In contrast, the essential PleC presumably acts as a phosphatase of DivK. Interestingly, we found that a DivJ mutant is able to elicit nodules and enter plant cells, but fails to establish an effective symbiosis suggesting that proper envelope and/or low CtrA levels are required for symbiosis.

Anti-CRISPR Anopheles mosquitoes inhibit gene drive spread under challenging behavioural conditions in large cages.

CRISPR-based gene drives have the potential to spread within populations and are considered as promising vector control tools. A doublesex-targeting gene drive was able to suppress laboratory Anopheles mosquito populations in small and large cages, and it is considered for field application. Challenges related to the field-use of gene drives and the evolving regulatory framework suggest that systems able to modulate or revert the action of gene drives, could be part of post-release risk-mitigation plans. In this study, we challenge an AcrIIA4-based anti-drive to inhibit gene drive spread in age-structured Anopheles gambiae population under complex feeding and behavioural conditions. A stochastic model predicts the experimentally-observed genotype dynamics in age-structured populations in medium-sized cages and highlights the necessity of large-sized cage trials. These experiments and experimental-modelling framework demonstrate the effectiveness of the anti-drive in different scenarios, providing further corroboration for its use in controlling the spread of gene drive in Anopheles.

Reduced ambiguity and improved interpretability of bacterial genome-wide associations using gene-cluster-centric k-mers.

The wide adoption of bacterial genome sequencing and encoding both core and accessory genome variation using k-mers has allowed bacterial genome-wide association studies (GWAS) to identify genetic variants associated with relevant phenotypes such as those linked to infection. Significant limitations still remain because of k-mers being duplicated across gene clusters and as far as the interpretation of association results is concerned, which affects the wider adoption of GWAS methods on microbial data sets. We have developed a simple computational method (panfeed) that explicitly links each k-mer to their gene cluster at base-resolution level, which allows us to avoid biases introduced by a global de Bruijn graph as well as more easily map and annotate associated variants. We tested panfeed on two independent data sets, correctly identifying previously characterized causal variants, which demonstrates the precision of the method, as well as its scalable performance. panfeed is a command line tool written in the python programming language and is available at https://github.com/microbial-pangenomes-lab/panfeed.

Epistatic interactions between the high pathogenicity island and other iron uptake systems shape Escherichia coli extra-intestinal virulence.

The intrinsic virulence of extra-intestinal pathogenic Escherichia coli is associated with numerous chromosomal and/or plasmid-borne genes, encoding diverse functions such as adhesins, toxins, and iron capture systems. However, the respective contribution to virulence of those genes seems to depend on the genetic background and is poorly understood. Here, we analyze genomes of 232 strains of sequence type complex STc58 and show that virulence (quantified in a mouse model of sepsis) emerged in a sub-group of STc58 due to the presence of the siderophore-encoding high-pathogenicity island (HPI). When extending our genome-wide association study to 370 Escherichia strains, we show that full virulence is associated with the presence of the aer or sit operons, in addition to the HPI. The prevalence of these operons, their co-occurrence and their genomic location depend on strain phylogeny. Thus, selection of lineage-dependent specific associations of virulence-associated genes argues for strong epistatic interactions shaping the emergence of virulence in E. coli.

The DNA cytosine methylome revealed two methylation motifs in the upstream regions of genes related to morphological and physiological differentiation in Streptomyces coelicolor A(3)2 M145.

DNA methylation is an epigenetic modification detected in both prokaryotic and eukaryotic genomic DNAs. In bacteria, the importance of 5-methylcytosine (m5C) in gene expression has been less investigated than in eukaryotic systems. Through dot-blot analysis employing m5C antibodies against chromosomal DNA, we have previously demonstrated that m5C influences the differentiation of Streptomyces coelicolor A(3)2 M145 in solid sporulating and liquid non-sporulating complex media. Here, we mapped the methylated cytosines of the M145 strain growing in the defined Maltose Glutamate (MG) liquid medium. Sequencing of the M145 genome after bisulfite treatment (BS-sequencing) evidenced 3360 methylated cytosines and the two methylation motifs, GGCmCGG and GCCmCG, in the upstream regions of 321 genes. Besides, the role of cytosine methylation was investigated using the hypo-methylating agent 5'-aza-2'-deoxycytidine (5-aza-dC) in S. coelicolor cultures, demonstrating that m5C affects both growth and antibiotic biosynthesis. Finally, quantitative reverse-transcription polymerase-chain-reaction (RT-qPCR) analysis of genes containing the methylation motifs in the upstream regions showed that 5-aza-dC treatment influenced their transcriptional levels and those of the regulatory genes for two antibiotics. To the best of our knowledge, this is the first study that reports the cytosine methylome of S. coelicolor M145, supporting the crucial role ascribed to cytosine methylation in controlling bacterial gene expression.

High-throughput functional characterization of protein phosphorylation sites in yeast.

Phosphorylation is a critical post-translational modification involved in the regulation of almost all cellular processes. However, fewer than 5% of thousands of recently discovered phosphosites have been functionally annotated. In this study, we devised a chemical genetic approach to study the functional relevance of phosphosites in Saccharomyces cerevisiae. We generated 474 yeast strains with mutations in specific phosphosites that were screened for fitness in 102 conditions, along with a gene deletion library. Of these phosphosites, 42% exhibited growth phenotypes, suggesting that these are more likely functional. We inferred their function based on the similarity of their growth profiles with that of gene deletions and validated a subset by thermal proteome profiling and lipidomics. A high fraction exhibited phenotypes not seen in the corresponding gene deletion, suggestive of a gain-of-function effect. For phosphosites conserved in humans, the severity of the yeast phenotypes is indicative of their human functional relevance. This high-throughput approach allows for functionally characterizing individual phosphosites at scale.

Exploring the symbiotic pangenome of the nitrogen-fixing bacterium Sinorhizobium meliloti.

BACKGROUND: Sinorhizobium meliloti is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the Medicago sativa plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of S. meliloti strains AK83 and BL225C, we sequenced the complete genomes for these two strains. RESULTS: With sizes of 7.14 Mbp and 6.97 Mbp, respectively, the genomes of AK83 and BL225C are larger than the laboratory strain Rm1021. The core genome of Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of genes that were present only in the accessory genome, consequently responsible for phenotypic diversity and putatively involved in plant-bacterium interaction. Notably, the symbiosis inefficient AK83 lacked several genes required for microaerophilic growth inside nodules, while several genes for accessory functions related to competition, plant invasion and bacteroid tropism were identified only in AK83 and BL225C strains. Presence and extent of polymorphism in regulons of transcription factors involved in symbiotic interaction were also analyzed. Our results indicate that regulons are flexible, with a large number of accessory genes, suggesting that regulons polymorphism could also be a key determinant in the variability of symbiotic performances among the analyzed strains. CONCLUSIONS: In conclusions, the extended comparative genomics approach revealed a variable subset of genes and regulons that may contribute to the symbiotic diversity.