Chance Favors the Prepared Genomes: Horizontal Transfer Shapes the Emergence of Antibiotic Resistance Mutations in Core Genes.

Bacterial lineages acquire novel traits at diverse rates in part because the genetic background impacts the successful acquisition of novel genes by horizontal transfer. Yet, how horizontal transfer affects the subsequent evolution of core genes remains poorly understood. Here, we studied the evolution of resistance to quinolones in Escherichia coli accounting for population structure. We found 60 groups of genes whose gain or loss induced an increase in the probability of subsequently becoming resistant to quinolones by point mutations in the gyrase and topoisomerase genes. These groups include functions known to be associated with direct mitigation of the effect of quinolones, with metal uptake, cell growth inhibition, biofilm formation, and sugar metabolism. Many of them are encoded in phages or plasmids. Although some of the chronologies may reflect epidemiological trends, many of these groups encoded functions providing latent phenotypes of antibiotic low-level resistance, tolerance, or persistence under quinolone treatment. The mutations providing resistance were frequent and accumulated very quickly. Their emergence was found to increase the rate of acquisition of other antibiotic resistances setting the path for multidrug resistance. Hence, our findings show that horizontal gene transfer shapes the subsequent emergence of adaptive mutations in core genes. In turn, these mutations further affect the subsequent evolution of resistance by horizontal gene transfer. Given the substantial gene flow within bacterial genomes, interactions between horizontal transfer and point mutations in core genes may be a key to the success of adaptation processes.

Bacteria have numerous distinctive groups of phage-plasmids with conserved phage and variable plasmid gene repertoires.

Plasmids and temperate phages are key contributors to bacterial evolution. They are usually regarded as very distinct. However, some elements, termed phage-plasmids, are known to be both plasmids and phages, e.g. P1, N15 or SSU5. The number, distribution, relatedness and characteristics of these phage-plasmids are poorly known. Here, we screened for these elements among ca. 2500 phages and 12000 plasmids and identified 780 phage-plasmids across very diverse bacterial phyla. We grouped 92% of them by similarity of gene repertoires to eight defined groups and 18 other broader communities of elements. The existence of these large groups suggests that phage-plasmids are ancient. Their gene repertoires are large, the average element is larger than an average phage or plasmid, and they include slightly more homologs to phages than to plasmids. We analyzed the pangenomes and the genetic organization of each group of phage-plasmids and found the key phage genes to be conserved and co-localized within distinct groups, whereas genes with homologs in plasmids are much more variable and include most accessory genes. Phage-plasmids are a sizeable fraction of the sequenced plasmids (∼7%) and phages (∼5%), and could have key roles in bridging the genetic divide between phages and other mobile genetic elements.

Phylogenetic background and habitat drive the genetic diversification of Escherichia coli.

Escherichia coli is mostly a commensal of birds and mammals, including humans, where it can act as an opportunistic pathogen. It is also found in water and sediments. We investigated the phylogeny, genetic diversification, and habitat-association of 1,294 isolates representative of the phylogenetic diversity of more than 5,000 isolates from the Australian continent. Since many previous studies focused on clinical isolates, we investigated mostly other isolates originating from humans, poultry, wild animals and water. These strains represent the species genetic diversity and reveal widespread associations between phylogroups and isolation sources. The analysis of strains from the same sequence types revealed very rapid change of gene repertoires in the very early stages of divergence, driven by the acquisition of many different types of mobile genetic elements. These elements also lead to rapid variations in genome size, even if few of their genes rise to high frequency in the species. Variations in genome size are associated with phylogroup and isolation sources, but the latter determine the number of MGEs, a marker of recent transfer, suggesting that gene flow reinforces the association of certain genetic backgrounds with specific habitats. After a while, the divergence of gene repertoires becomes linear with phylogenetic distance, presumably reflecting the continuous turnover of mobile element and the occasional acquisition of adaptive genes. Surprisingly, the phylogroups with smallest genomes have the highest rates of gene repertoire diversification and fewer but more diverse mobile genetic elements. This suggests that smaller genomes are associated with higher, not lower, turnover of genetic information. Many of these genomes are from freshwater isolates and have peculiar traits, including a specific capsule, suggesting adaptation to this environment. Altogether, these data contribute to explain why epidemiological clones tend to emerge from specific phylogenetic groups in the presence of pervasive horizontal gene transfer across the species.

Causes and Consequences of Bacteriophage Diversification via Genetic Exchanges across Lifestyles and Bacterial Taxa.

Bacteriophages (phages) evolve rapidly by acquiring genes from other phages. This results in mosaic genomes. Here, we identify numerous genetic transfers between distantly related phages and aim at understanding their frequency, consequences, and the conditions favoring them. Gene flow tends to occur between phages that are enriched for recombinases, transposases, and nonhomologous end joining, suggesting that both homologous and illegitimate recombination contribute to gene flow. Phage family and host phyla are strong barriers to gene exchange, but phage lifestyle is not. Even if we observe four times more recent transfers between temperate phages than between other pairs, there is extensive gene flow between temperate and virulent phages, and between the latter. These predominantly involve virulent phages with large genomes previously classed as low gene flux, and lead to the preferential transfer of genes encoding functions involved in cell energetics, nucleotide metabolism, DNA packaging and injection, and virion assembly. Such exchanges may contribute to the observed twice larger genomes of virulent phages. We used genetic transfers, which occur upon coinfection of a host, to compare phage host range. We found that virulent phages have broader host ranges and can mediate genetic exchanges between narrow host range temperate phages infecting distant bacterial hosts, thus contributing to gene flow between virulent phages, as well as between temperate phages. This gene flow drastically expands the gene repertoires available for phage and bacterial evolution, including the transfer of functional innovations across taxa.

Atypical organizations and epistatic interactions of CRISPRs and cas clusters in genomes and their mobile genetic elements.

Prokaryotes use CRISPR-Cas systems for adaptive immunity, but the reasons for the frequent existence of multiple CRISPRs and cas clusters remain poorly understood. Here, we analysed the joint distribution of CRISPR and cas genes in a large set of fully sequenced bacterial genomes and their mobile genetic elements. Our analysis suggests few negative and many positive epistatic interactions between Cas subtypes. The latter often result in complex genetic organizations, where a locus has a single adaptation module and diverse interference mechanisms that might provide more effective immunity. We typed CRISPRs that could not be unambiguously associated with a cas cluster and found that such complex loci tend to have unique type I repeats in multiple CRISPRs. Many chromosomal CRISPRs lack a neighboring Cas system and they often have repeats compatible with the Cas systems encoded in trans. Phages and 25 000 prophages were almost devoid of CRISPR-Cas systems, whereas 3% of plasmids had CRISPR-Cas systems or isolated CRISPRs. The latter were often compatible with the chromosomal cas clusters, suggesting that plasmids can co-opt the latter. These results highlight the importance of interactions between CRISPRs and cas present in multiple copies and in distinct genomic locations in the function and evolution of bacterial immunity.

CRISPRCasdb a successor of CRISPRdb containing CRISPR arrays and cas genes from complete genome sequences, and tools to download and query lists of repeats and spacers.

In Archaea and Bacteria, the arrays called CRISPRs for 'clustered regularly interspaced short palindromic repeats' and the CRISPR associated genes or cas provide adaptive immunity against viruses, plasmids and transposable elements. Short sequences called spacers, corresponding to fragments of invading DNA, are stored in-between repeated sequences. The CRISPR-Cas systems target sequences homologous to spacers leading to their degradation. To facilitate investigations of CRISPRs, we developed 12 years ago a website holding the CRISPRdb. We now propose CRISPRCasdb, a completely new version giving access to both CRISPRs and cas genes. We used CRISPRCasFinder, a program that identifies CRISPR arrays and cas genes and determine the system's type and subtype, to process public whole genome assemblies. Strains are displayed either in an alphabetic list or in taxonomic order. The database is part of the CRISPR-Cas++ website which also offers the possibility to analyse submitted sequences and to download programs. A BLAST search against lists of repeats and spacers extracted from the database is proposed. To date, 16 990 complete prokaryote genomes (16 650 bacteria from 2973 species and 340 archaea from 300 species) are included. CRISPR-Cas systems were found in 36% of Bacteria and 75% of Archaea strains. CRISPRCasdb is freely accessible at https://crisprcas.i2bc.paris-saclay.fr/.

Genetic exchanges are more frequent in bacteria encoding capsules.

Capsules allow bacteria to colonize novel environments, to withstand numerous stresses, and to resist antibiotics. Yet, even though genetic exchanges with other cells should be adaptive under such circumstances, it has been suggested that capsules lower the rates of homologous recombination and horizontal gene transfer. We analysed over one hundred pan-genomes and thousands of bacterial genomes for the evidence of an association between genetic exchanges (or lack thereof) and the presence of a capsule system. We found that bacteria encoding capsules have larger pan-genomes, higher rates of horizontal gene transfer, and higher rates of homologous recombination in their core genomes. Accordingly, genomes encoding capsules have more plasmids, conjugative elements, transposases, prophages, and integrons. Furthermore, capsular loci are frequent in plasmids, and can be found in prophages. These results are valid for Bacteria, independently of their ability to be naturally transformable. Since we have shown previously that capsules are commonly present in nosocomial pathogens, we analysed their co-occurrence with antibiotic resistance genes. Genomes encoding capsules have more antibiotic resistance genes, especially those encoding efflux pumps, and they constitute the majority of the most worrisome nosocomial bacteria. We conclude that bacteria with capsule systems are more genetically diverse and have fast-evolving gene repertoires, which may further contribute to their success in colonizing novel niches such as humans under antibiotic therapy.

CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins.

CRISPR (clustered regularly interspaced short palindromic repeats) arrays and their associated (Cas) proteins confer bacteria and archaea adaptive immunity against exogenous mobile genetic elements, such as phages or plasmids. CRISPRCasFinder allows the identification of both CRISPR arrays and Cas proteins. The program includes: (i) an improved CRISPR array detection tool facilitating expert validation based on a rating system, (ii) prediction of CRISPR orientation and (iii) a Cas protein detection and typing tool updated to match the latest classification scheme of these systems. CRISPRCasFinder can either be used online or as a standalone tool compatible with Linux operating system. All third-party software packages employed by the program are freely available. CRISPRCasFinder is available at https://crisprcas.i2bc.paris-saclay.fr.

Abundance and co-occurrence of extracellular capsules increase environmental breadth: Implications for the emergence of pathogens.

Extracellular capsules constitute the outermost layer of many bacteria, are major virulence factors, and affect antimicrobial therapies. They have been used as epidemiological markers and recently became vaccination targets. Despite the efforts to biochemically serotype capsules in a few model pathogens, little is known of their taxonomic and environmental distribution. We developed, validated, and made available a computational tool, CapsuleFinder, to identify capsules in genomes. The analysis of over 2500 prokaryotic genomes, accessible in a database, revealed that ca. 50% of them-including Archaea-encode a capsule. The Wzx/Wzy-dependent capsular group was by far the most abundant. Surprisingly, a fifth of the genomes encode more than one capsule system-often from different groups-and their non-random co-occurrence suggests the existence of negative and positive epistatic interactions. To understand the role of multiple capsules, we queried more than 6700 metagenomes for the presence of species encoding capsules and showed that their distribution varied between environmental categories and, within the human microbiome, between body locations. Species encoding capsules, and especially those encoding multiple capsules, had larger environmental breadths than the other species. Accordingly, capsules were more frequent in environmental bacteria than in pathogens and, within the latter, they were more frequent among facultative pathogens. Nevertheless, capsules were frequent in clinical samples, and were usually associated with fast-growing bacteria with high infectious doses. Our results suggest that capsules increase the environmental range of bacteria and make them more resilient to environmental perturbations. Capsules might allow opportunistic pathogens to profit from empty ecological niches or environmental perturbations, such as those resulting from antibiotic therapy, to colonize the host. Capsule-associated virulence might thus be a by-product of environmental adaptation. Understanding the role of capsules in natural environments might enlighten their function in pathogenesis.

Integrative and conjugative elements and their hosts: composition, distribution and organization.

Conjugation of single-stranded DNA drives horizontal gene transfer between bacteria and was widely studied in conjugative plasmids. The organization and function of integrative and conjugative elements (ICE), even if they are more abundant, was only studied in a few model systems. Comparative genomics of ICE has been precluded by the difficulty in finding and delimiting these elements. Here, we present the results of a method that circumvents these problems by requiring only the identification of the conjugation genes and the species' pan-genome. We delimited 200 ICEs and this allowed the first large-scale characterization of these elements. We quantified the presence in ICEs of a wide set of functions associated with the biology of mobile genetic elements, including some that are typically associated with plasmids, such as partition and replication. Protein sequence similarity networks and phylogenetic analyses revealed that ICEs are structured in functional modules. Integrases and conjugation systems have different evolutionary histories, even if the gene repertoires of ICEs can be grouped in function of conjugation types. Our characterization of the composition and organization of ICEs paves the way for future functional and evolutionary analyses of their cargo genes, composed of a majority of unknown function genes.

Regulation of genetic flux between bacteria by restriction-modification systems.

Restriction-modification (R-M) systems are often regarded as bacteria's innate immune systems, protecting cells from infection by mobile genetic elements (MGEs). Their diversification has been recently associated with the emergence of particularly virulent lineages. However, we have previously found more R-M systems in genomes carrying more MGEs. Furthermore, it has been suggested that R-M systems might favor genetic transfer by producing recombinogenic double-stranded DNA ends. To test whether R-M systems favor or disfavor genetic exchanges, we analyzed their frequency with respect to the inferred events of homologous recombination and horizontal gene transfer within 79 bacterial species. Genetic exchanges were more frequent in bacteria with larger genomes and in those encoding more R-M systems. We created a recognition target motif predictor for Type II R-M systems that identifies genomes encoding systems with similar restriction sites. We found more genetic exchanges between these genomes, independently of their evolutionary distance. Our results reconcile previous studies by showing that R-M systems are more abundant in promiscuous species, wherein they establish preferential paths of genetic exchange within and between lineages with cognate R-M systems. Because the repertoire and/or specificity of R-M systems in bacterial lineages vary quickly, the preferential fluxes of genetic transfer within species are expected to constantly change, producing time-dependent networks of gene transfer.

Identification and analysis of integrons and cassette arrays in bacterial genomes.

Integrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program - IntegronFinder - to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covariance models, for integron-integrases using HMM profiles, and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as α-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within integrons and their stabilization in the new genome.

A widespread family of polymorphic toxins encoded by temperate phages.

BACKGROUND: Polymorphic toxins (PTs) are multi-domain bacterial exotoxins belonging to distinct families that share common features in terms of domain organization. PTs are found in all major bacterial clades, including many toxic effectors of type V and type VI secretion systems. PTs modulate the dynamics of microbial communities by killing or inhibiting the growth of bacterial competitors lacking protective immunity proteins. RESULTS: In this work, we identified a novel widespread family of PTs, named MuF toxins, which were exclusively encoded within temperate phages and their prophages. By analyzing the predicted proteomes of 1845 bacteriophages and 2464 bacterial genomes, we found that MuF-containing proteins were frequently part of the DNA packaging module of tailed phages. Interestingly, MuF toxins were abundant in the human gut microbiome. CONCLUSIONS: Our results uncovered the presence of the MuF toxin family in the temperate phages of Firmicutes. The MuF toxin family is likely to play an important role in the ecology of the human microbiota where pathogens and commensal species belonging to the Firmicutes are abundant. We propose that MuF toxins could be delivered by phages into host bacteria and either influence the lysogeny decision or serve as bacterial weapons by inhibiting the growth of competing bacteria.

Comparative genomic analysis of Acinetobacter strains isolated from murine colonic crypts.

BACKGROUND: A restricted set of aerobic bacteria dominated by the Acinetobacter genus was identified in murine intestinal colonic crypts. The vicinity of such bacteria with intestinal stem cells could indicate that they protect the crypt against cytotoxic and genotoxic signals. Genome analyses of these bacteria were performed to better appreciate their biodegradative capacities. RESULTS: Two taxonomically different clusters of Acinetobacter were isolated from murine proximal colonic crypts, one was identified as A. modestus and the other as A. radioresistens. Their identification was performed through biochemical parameters and housekeeping gene sequencing. After selection of one strain of each cluster (A. modestus CM11G and A. radioresistens CM38.2), comparative genomic analysis was performed on whole-genome sequencing data. The antibiotic resistance pattern of these two strains is different, in line with the many genes involved in resistance to heavy metals identified in both genomes. Moreover whereas the operon benABCDE involved in benzoate metabolism is encoded by the two genomes, the operon antABC encoding the anthranilate dioxygenase, and the phenol hydroxylase gene cluster are absent in the A. modestus genomic sequence, indicating that the two strains have different capacities to metabolize xenobiotics. A common feature of the two strains is the presence of a type IV pili system, and the presence of genes encoding proteins pertaining to secretion systems such as Type I and Type II secretion systems. CONCLUSIONS: Our comparative genomic analysis revealed that different Acinetobacter isolated from the same biological niche, even if they share a large majority of genes, possess unique features that could play a specific role in the protection of the intestinal crypt.

Metagenomic assessment of the interplay between the environment and the genetic diversification of Acinetobacter.

Most bacteria have poorly characterized environmental reservoirs and unknown closely related species. This hampers the study of bacterial evolutionary ecology because both the environment and the genetic background of ancestral lineages are unknown. We combined metagenomics, comparative genomics and phylogenomics to overcome this limitation, to identify novel taxa and to propose environments where they can be isolated. We applied this method to characterize the ecological distribution of known and novel lineages of Acinetobacter spp. We observed two major environmental transitions at deep phylogenetic levels, splitting the genus into three ecologically differentiated clades. One of these has rapidly shifted towards host-association by acquiring genes involved in bacteria-eukaryote interactions. We show that environmental perturbations affect species distribution in predictable ways: bovines have very diverse communities of Acinetobacter, unless they were administered antibiotics, in which case they show highly uniform communities of Acinetobacter spp. that resemble those of humans. Our results uncover the diversity of bacterial lineages, overpassing the limitations of classical cultivation methods and highlight the role of the environment in shaping their evolution.

Metabolic diversity of the emerging pathogenic lineages of Klebsiella pneumoniae.

Multidrug resistant and hypervirulent clones of Klebsiella pneumoniae are emerging pathogens. To understand the association between genotypic and phenotypic diversity in this process, we combined genomic, phylogenomic and phenotypic analysis of a diverse set of K. pneumoniae and closely related species. These species were able to use an unusually large panel of metabolic substrates for growth, many of which were shared between all strains. We analysed the substrates used by only a fraction of the strains, identified some of their genetic basis, and found that many could not be explained by the phylogeny of the strains. Puzzlingly, few traits were associated with the ecological origin of the strains. One noticeable exception was the ability to use D-arabinose, which was much more frequent in hypervirulent strains. The broad carbon and nitrogen core metabolism of K. pneumoniae might contribute to its ability to thrive in diverse environments. Accordingly, even the hypervirulent and multidrug resistant clones have the metabolic signature of ubiquitous bacteria. The apparent few metabolic differences between hypervirulent, multi-resistant and environmental strains may favour the emergence of dual-risk strains that combine resistance and hypervirulence.

Pervasive domestication of defective prophages by bacteria.

Integrated phages (prophages) are major contributors to the diversity of bacterial gene repertoires. Domestication of their components is thought to have endowed bacteria with molecular systems involved in secretion, defense, warfare, and gene transfer. However, the rates and mechanisms of domestication remain unknown. We used comparative genomics to study the evolution of prophages within the bacterial genome. We identified over 300 vertically inherited prophages within enterobacterial genomes. Some of these elements are very old and might predate the split between Escherichia coli and Salmonella enterica. The size distribution of prophage elements is bimodal, suggestive of rapid prophage inactivation followed by much slower genetic degradation. Accordingly, we observed a pervasive pattern of systematic counterselection of nonsynonymous mutations in prophage genes. Importantly, such patterns of purifying selection are observed not only on accessory regions but also in core phage genes, such as those encoding structural and lysis components. This suggests that bacterial hosts select for phage-associated functions. Several of these conserved prophages have gene repertoires compatible with described functions of adaptive prophage-derived elements such as bacteriocins, killer particles, gene transfer agents, or satellite prophages. We suggest that bacteria frequently domesticate their prophages. Most such domesticated elements end up deleted from the bacterial genome because they are replaced by analogous functions carried by new prophages. This puts the bacterial genome in a state of continuous flux of acquisition and loss of phage-derived adaptive genes.

A PNPase dependent CRISPR System in Listeria.

The human bacterial pathogen Listeria monocytogenes is emerging as a model organism to study RNA-mediated regulation in pathogenic bacteria. A class of non-coding RNAs called CRISPRs (clustered regularly interspaced short palindromic repeats) has been described to confer bacterial resistance against invading bacteriophages and conjugative plasmids. CRISPR function relies on the activity of CRISPR associated (cas) genes that encode a large family of proteins with nuclease or helicase activities and DNA and RNA binding domains. Here, we characterized a CRISPR element (RliB) that is expressed and processed in the L. monocytogenes strain EGD-e, which is completely devoid of cas genes. Structural probing revealed that RliB has an unexpected secondary structure comprising basepair interactions between the repeats and the adjacent spacers in place of canonical hairpins formed by the palindromic repeats. Moreover, in contrast to other CRISPR-Cas systems identified in Listeria, RliB-CRISPR is ubiquitously present among Listeria genomes at the same genomic locus and is never associated with the cas genes. We showed that RliB-CRISPR is a substrate for the endogenously encoded polynucleotide phosphorylase (PNPase) enzyme. The spacers of the different Listeria RliB-CRISPRs share many sequences with temperate and virulent phages. Furthermore, we show that a cas-less RliB-CRISPR lowers the acquisition frequency of a plasmid carrying the matching protospacer, provided that trans encoded cas genes of a second CRISPR-Cas system are present in the genome. Importantly, we show that PNPase is required for RliB-CRISPR mediated DNA interference. Altogether, our data reveal a yet undescribed CRISPR system whose both processing and activity depend on PNPase, highlighting a new and unexpected function for PNPase in "CRISPRology".

The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts.

The roles of restriction-modification (R-M) systems in providing immunity against horizontal gene transfer (HGT) and in stabilizing mobile genetic elements (MGEs) have been much debated. However, few studies have precisely addressed the distribution of these systems in light of HGT, its mechanisms and its vectors. We analyzed the distribution of R-M systems in 2261 prokaryote genomes and found their frequency to be strongly dependent on the presence of MGEs, CRISPR-Cas systems, integrons and natural transformation. Yet R-M systems are rare in plasmids, in prophages and nearly absent from other phages. Their abundance depends on genome size for small genomes where it relates with HGT but saturates at two occurrences per genome. Chromosomal R-M systems might evolve under cycles of purifying and relaxed selection, where sequence conservation depends on the biochemical activity and complexity of the system and total gene loss is frequent. Surprisingly, analysis of 43 pan-genomes suggests that solitary R-M genes rarely arise from the degradation of R-M systems. Solitary genes are transferred by large MGEs, whereas complete systems are more frequently transferred autonomously or in small MGEs. Our results suggest means of testing the roles for R-M systems and their associations with MGEs.