Evolution of homologous recombination rates across bacteria.

Bacteria are nonsexual organisms but are capable of exchanging DNA at diverse degrees through homologous recombination. Intriguingly, the rates of recombination vary immensely across lineages where some species have been described as purely clonal and others as "quasi-sexual." However, estimating recombination rates has proven a difficult endeavor and estimates often vary substantially across studies. It is unclear whether these variations reflect natural variations across populations or are due to differences in methodologies. Consequently, the impact of recombination on bacterial evolution has not been extensively evaluated and the evolution of recombination rate-as a trait-remains to be accurately described. Here, we developed an approach based on Approximate Bayesian Computation that integrates multiple signals of recombination to estimate recombination rates. We inferred the rate of recombination of 162 bacterial species and one archaeon and tested the robustness of our approach. Our results confirm that recombination rates vary drastically across bacteria; however, we found that recombination rate-as a trait-is conserved in several lineages but evolves rapidly in others. Although some traits are thought to be associated with recombination rate (e.g., GC-content), we found no clear association between genomic or phenotypic traits and recombination rate. Overall, our results provide an overview of recombination rate, its evolution, and its impact on bacterial evolution.

Tractidigestivibacter montrealensis sp. nov., a new member of human gut microbiota isolated from a healthy volunteer.

Strain KD21T, isolated from the fecal sample of a healthy female volunteer, is a strictly anaerobic, non-motile, Gram-staining-positive, saccharolytic small rod that does not produce spores. Strain KD21T was able to grow in the range of temperature 28°C-37°C (optimum, 37 °C), pH 6.0-8.0 (optimum, pH 7.0), and with 0-5.0 g/l NaCl (optimum, 0 g/l NaCl). Bacteria cells reduced nitrates to nitrites. Its major fatty acids were C18:1ω9c, C16:0, C18:0, and summed in feature 8 (C18:1ω7c and/or C18:1ω6c). 16S rRNA gene phylogenetic analysis revealed that KD21T is a member of the genus Tractidigestivibacter and is distinct from any species with validly published names. The sequence showed 98.48% similarity with T. scatoligenes SK9K4T. The DNA G + C content of strain KD21T was 62.6 mol%. The DNA-DNA hybridization and OrthoANI values between strain KD21T and T. scatoligenes SK9K4T were 40.2% and 90.2%, respectively. Differences in phenotypic, phylogenetic, chemotaxonomic, and genomic characteristics indicated that strain KD21T represents a novel species within the genus Tractidigestivibacter. The name T. montrealensis sp. nov. is proposed and the type strain is KD21T (= CSUR Q8103T =  DSM 115111T).

Genome-wide transcriptome analysis of the orphan crop tef (Eragrostis tef (Zucc.) Trotter) under long-term low calcium stress.

Calcium (Ca2+) is one of the essential mineral nutrients for plant growth and development. However, the effects of long-term Ca2+ deficiency in orphan crops such as Tef [(Eragrostis tef) (Zucc.) Trotter], which accumulate high levels of Ca in the grains, remained unknown. Tef is a staple crop for nearly 70 million people in East Africa, particularly in Ethiopia and Eritrea. It is one of the most nutrient-dense grains, and is also more resistant to marginal soils and climatic conditions than main cereals like corn, wheat, and rice. In this study, tef plants were grown in a hydroponic solution containing optimum (1 mM) or low (0.01 mM) Ca2+, and plant growth parameters and whole-genome transcriptome were analyzed. Ca+2-deficient plants exhibited leaf necrosis, leaf curling, and growth stunting symptoms. Ca2+ deficiency significantly decreased root and shoot Ca, potassium (K), and copper content in both root and shoots. At the same time, it greatly increased root iron (Fe) content, suggesting the role of Ca2+ in the uptake and/or translocation of these minerals. Transcriptomic analysis using RNA-seq revealed that members of Ca2+ channels, including the cyclic nucleotide-gated channels and glutamate receptor-like channels, Ca2+-transporters, Ca2+-binding proteins and Ca2+-dependent protein kinases were differentially regulated by Ca+2 treatment. Moreover, several Fe/metal transporters, including members of vacuolar Fe transporters, yellow stripe-like, natural resistance-associated macrophage protein, and oligo-peptide transporters, were differentially regulated between shoot and root in response to Ca2+ treatment. Taken together, our findings suggest that Ca2+ deficiency affects plant growth and mineral accumulation by regulating the transcriptomes of several transporters and signaling genes.

Gene flow and introgression are pervasive forces shaping the evolution of bacterial species.

BACKGROUND: Although originally thought to evolve clonally, studies have revealed that most bacteria exchange DNA. However, it remains unclear to what extent gene flow shapes the evolution of bacterial genomes and maintains the cohesion of species. RESULTS: Here, we analyze the patterns of gene flow within and between >2600 bacterial species. Our results show that fewer than 10% of bacterial species are truly clonal, indicating that purely asexual species are rare in nature. We further demonstrate that the taxonomic criterion of ~95% genome sequence identity routinely used to define bacterial species does not accurately represent a level of divergence that imposes an effective barrier to gene flow across bacterial species. Interruption of gene flow can occur at various sequence identities across lineages, generally from 90 to 98% genome identity. This likely explains why a ~95% genome sequence identity threshold has empirically been judged as a good approximation to define bacterial species. Our results support a universal mechanism where the availability of identical genomic DNA segments required to initiate homologous recombination is the primary determinant of gene flow and species boundaries in bacteria. We show that these barriers of gene flow remain porous since many distinct species maintain some level of gene flow, similar to introgression in sexual organisms. CONCLUSIONS: Overall, bacterial evolution and speciation are likely shaped by similar forces driving the evolution of sexual organisms. Our findings support a model where the interruption of gene flow-although not necessarily the initial cause of speciation-leads to the establishment of permanent and irreversible species borders.

Unexpected myriad of co-occurring viral strains and species in one of the most abundant and microdiverse viruses on Earth.

Viral genetic microdiversity drives adaptation, pathogenicity, and speciation and has critical consequences for the viral-host arms race occurring at the strain and species levels, which ultimately impact microbial community structure and biogeochemical cycles. Despite the fact that most efforts have focused on viral macrodiversity, little is known about the microdiversity of ecologically important viruses on Earth. Recently, single-virus genomics discovered the putatively most abundant ocean virus in temperate and tropical waters: the uncultured dsDNA virus vSAG 37-F6 infecting Pelagibacter, the most abundant marine bacteria. In this study, we report the cooccurrence of up to ≈1,500 different viral strains (>95% nucleotide identity) and ≈30 related species (80-95% nucleotide identity) in a single oceanic sample. Viral microdiversity was maintained over space and time, and most alleles were the result of synonymous mutations without any apparent adaptive benefits to cope with host translation codon bias and efficiency. Gene flow analysis used to delimitate species according to the biological species concept (BSC) revealed the impact of recombination in shaping vSAG 37-F6 virus and Pelagibacter speciation. Data demonstrated that this large viral microdiversity somehow mirrors the host species diversity since ≈50% of the 926 analyzed Pelagibacter genomes were found to belong to independent BSC species that do not significantly engage in gene flow with one another. The host range of this evolutionarily successful virus revealed that a single viral species can infect multiple Pelagibacter BSC species, indicating that this virus crosses not only formal BSC barriers but also biomes since viral ancestors are found in freshwater.

Evolution of Chi motifs in Proteobacteria.

Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex not only allows bacteria to repair DNA double-strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis, and Staphylococcus aureus. In this study, we detected putative Chi motifs in a large dataset of Proteobacteria and identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

CoreCruncher: Fast and Robust Construction of Core Genomes in Large Prokaryotic Data Sets.

The core genome represents the set of genes shared by all, or nearly all, strains of a given population or species of prokaryotes. Inferring the core genome is integral to many genomic analyses, however, most methods rely on the comparison of all the pairs of genomes; a step that is becoming increasingly difficult given the massive accumulation of genomic data. Here, we present CoreCruncher; a program that robustly and rapidly constructs core genomes across hundreds or thousands of genomes. CoreCruncher does not compute all pairwise genome comparisons and uses a heuristic based on the distributions of identity scores to classify sequences as orthologs or paralogs/xenologs. Although it is much faster than current methods, our results indicate that our approach is more conservative than other tools and less sensitive to the presence of paralogs and xenologs. CoreCruncher is freely available from: https://github.com/lbobay/CoreCruncher. CoreCruncher is written in Python 3.7 and can also run on Python 2.7 without modification. It requires the python library Numpy and either Usearch or Blast. Certain options require the programs muscle or mafft.

Recombination events are concentrated in the spike protein region of Betacoronaviruses.

The Betacoronaviruses comprise multiple subgenera whose members have been implicated in human disease. As with SARS, MERS and now SARS-CoV-2, the origin and emergence of new variants are often attributed to events of recombination that alter host tropism or disease severity. In most cases, recombination has been detected by searches for excessively similar genomic regions in divergent strains; however, such analyses are complicated by the high mutation rates of RNA viruses, which can produce sequence similarities in distant strains by convergent mutations. By applying a genome-wide approach that examines the source of individual polymorphisms and that can be tested against null models in which recombination is absent and homoplasies can arise only by convergent mutations, we examine the extent and limits of recombination in Betacoronaviruses. We find that recombination accounts for nearly 40% of the polymorphisms circulating in populations and that gene exchange occurs almost exclusively among strains belonging to the same subgenus. Although experimental studies have shown that recombinational exchanges occur at random along the coronaviral genome, in nature, they are vastly overrepresented in regions controlling viral interaction with host cells.

Impact of homologous recombination on core genome phylogenies.

BACKGROUND: Core genome phylogenies are widely used to build the evolutionary history of individual prokaryote species. By using hundreds or thousands of shared genes, these approaches are the gold standard to reconstruct the relationships of large sets of strains. However, there is growing evidence that bacterial strains exchange DNA through homologous recombination at rates that vary widely across prokaryote species, indicating that core genome phylogenies might not be able to reconstruct true phylogenies when recombination rate is high. Few attempts have been made to evaluate the robustness of core genome phylogenies to recombination, but some analyses suggest that reconstructed trees are not always accurate. RESULTS: In this study, we tested the robustness of core genome phylogenies to various levels of recombination rates. By analyzing simulated and empirical data, we observed that core genome phylogenies are relatively robust to recombination rates; nevertheless, our results suggest that many reconstructed trees are not completely accurate even when bootstrap supports are high. We found that some core genome phylogenies are highly robust to recombination whereas others are strongly impacted by it, and we identified that the robustness of core genome phylogenies to recombination is highly linked to the levels of selective pressures acting on a species. Stronger selective pressures lead to less accurate tree reconstructions, presumably because selective pressures more strongly bias the routes of DNA transfers, thereby causing phylogenetic artifacts. CONCLUSIONS: Overall, these results have important implications for the application of core genome phylogenies in prokaryotes.

Strain Structure and Dynamics Revealed by Targeted Deep Sequencing of the Honey Bee Gut Microbiome.

Host-associated microbiomes can be critical for the health and proper development of animals and plants. The answers to many fundamental questions regarding the modes of acquisition and microevolution of microbiome communities remain to be established. Deciphering strain-level dynamics is essential to fully understand how microbial communities evolve, but the forces shaping the strain-level dynamics of microbial communities remain largely unexplored, mostly because of methodological issues and cost. Here, we used targeted strain-level deep sequencing to uncover the strain dynamics within a host-associated microbial community using the honey bee gut microbiome as a model system. Our results revealed that amplicon sequencing of conserved protein-coding gene regions using species-specific primers is a cost-effective and accurate method for exploring strain-level diversity. In fact, using this method we were able to confirm strain-level results that have been obtained from whole-genome shotgun sequencing of the honey bee gut microbiome but with a much higher resolution. Importantly, our deep sequencing approach allowed us to explore the impact of low-frequency strains (i.e., cryptic strains) on microbiome dynamics. Results show that cryptic strain diversity is not responsible for the observed variations in microbiome composition across bees. Altogether, the findings revealed new fundamental insights regarding strain dynamics of host-associated microbiomes.IMPORTANCE The factors driving fine-scale composition and dynamics of gut microbial communities are poorly understood. In this study, we used metagenomic amplicon deep sequencing to decipher the strain dynamics of two key members of the honey bee gut microbiome. Using this high-throughput and cost-effective approach, we were able to confirm results from previous large-scale whole-genome shotgun (WGS) metagenomic sequencing studies while also gaining additional insights into the community dynamics of two core members of the honey bee gut microbiome. Moreover, we were able to show that cryptic strains are not responsible for the observed variations in microbiome composition across bees.

CoreSimul: a forward-in-time simulator of genome evolution for prokaryotes modeling homologous recombination.

BACKGROUND: Prokaryotes are asexual, but these organisms frequently engage in homologous recombination, a process that differs from meiotic recombination in sexual organisms. Most tools developed to simulate genome evolution either assume sexual reproduction or the complete absence of DNA flux in the population. As a result, very few simulators are adapted to model prokaryotic genome evolution while accounting for recombination. Moreover, many simulators are based on the coalescent, which assumes a neutral model of genomic evolution, and those are best suited for organisms evolving under weak selective pressures, such as animals and plants. In contrast, prokaryotes are thought to be evolving under much stronger selective pressures, suggesting that forward-in-time simulators are better suited for these organisms. RESULTS: Here, I present CoreSimul, a forward-in-time simulator of core genome evolution for prokaryotes modeling homologous recombination. Simulations are guided by a phylogenetic tree and incorporate different substitution models, including models of codon selection. CONCLUSIONS: CoreSimul is a flexible forward-in-time simulator that constitutes a significant addition to the limited list of available simulators applicable to prokaryote genome evolution.

Factors driving effective population size and pan-genome evolution in bacteria.

BACKGROUND: Knowledge of population-level processes is essential to understanding the efficacy of selection operating within a species. However, attempts at estimating effective population sizes (Ne) are particularly challenging in bacteria due to their extremely large census populations sizes, varying rates of recombination and arbitrary species boundaries. RESULTS: In this study, we estimated Ne for 153 species (152 bacteria and one archaeon) defined under a common framework and found that ecological lifestyle and growth rate were major predictors of Ne; and that contrary to theoretical expectations, Ne was unaffected by recombination rate. Additionally, we found that Ne shapes the evolution and diversity of total gene repertoires of prokaryotic species. CONCLUSION: Together, these results point to a new model of genome architecture evolution in prokaryotes, in which pan-genome sizes, not individual genome sizes, are governed by drift-barrier evolution.

Biological species in the viral world.

Due to their dependence on cellular organisms for metabolism and replication, viruses are typically named and assigned to species according to their genome structure and the original host that they infect. But because viruses often infect multiple hosts and the numbers of distinct lineages within a host can be vast, their delineation into species is often dictated by arbitrary sequence thresholds, which are highly inconsistent across lineages. Here we apply an approach to determine the boundaries of viral species based on the detection of gene flow within populations, thereby defining viral species according to the biological species concept (BSC). Despite the potential for gene transfer between highly divergent genomes, viruses, like the cellular organisms they infect, assort into reproductively isolated groups and can be organized into biological species. This approach revealed that BSC-defined viral species are often congruent with the taxonomic partitioning based on shared gene contents and host tropism, and that bacteriophages can similarly be classified in biological species. These results open the possibility to use a single, universal definition of species that is applicable across cellular and acellular lifeforms.

ConSpeciFix: classifying prokaryotic species based on gene flow.

Summary: Classification of prokaryotic species is usually based on sequence similarity thresholds, which are easy to apply but lack a biologically-relevant foundation. Here, we present ConSpeciFix, a program that classifies prokaryotes into species using criteria set forth by the Biological Species Concept, thereby unifying species definition in all domains of life. Availability and implementation: ConSpeciFix's webserver is freely available at www.conspecifix.com. The local version of the program can be freely downloaded from https://github.com/Bobay-Ochman/ConSpeciFix. ConSpeciFix is written in Python 2.7 and requires the following dependencies: Usearch, MCL, MAFFT and RAxML.

Antibiotics reduce genetic diversity of core species in the honeybee gut microbiome.

The gut microbiome plays a key role in animal health, and perturbing it can have detrimental effects. One major source of perturbation to microbiomes, in humans and human-associated animals, is exposure to antibiotics. Most studies of how antibiotics affect the microbiome have used amplicon sequencing of highly conserved 16S rRNA sequences, as in a recent study showing that antibiotic treatment severely alters the species-level composition of the honeybee gut microbiome. But because the standard 16S rRNA-based methods cannot resolve closely related strains, strain-level changes could not be evaluated. To address this gap, we used amplicon sequencing of protein-coding genes to assess effects of antibiotics on fine-scale genetic diversity of the honeybee gut microbiota. We followed the population dynamics of alleles within two dominant core species of the bee gut community, Gilliamella apicola and Snodgrassella alvi, following antibiotic perturbation. Whereas we observed a large reduction in genetic diversity in G. apicola, S. alvi diversity was mostly unaffected. The reduction in G. apicola diversity accompanied an increase in the frequency of several alleles, suggesting resistance to antibiotic treatment. We find that antibiotic perturbation can cause major shifts in diversity and that the extent of these shifts can vary substantially across species. Thus, antibiotics impact not only species composition, but also allelic diversity within species, potentially affecting hosts if variants with particular functions are reduced or eliminated. Overall, we show that amplicon sequencing of protein-coding genes, without clustering into operational taxonomic units, provides an accurate picture of the fine-scale dynamics of microbial communities over time.

Impact of Recombination on the Base Composition of Bacteria and Archaea.

The mutational process in bacteria is biased toward A and T, and most species are GC-rich relative to the mutational input to their genome. It has been proposed that the shift in base composition is an adaptive process-that natural selection operates to increase GC-contents-and there is experimental evidence that bacterial strains with GC-rich versions of genes have higher growth rates than those strains with AT-rich versions expressing identical proteins. Alternatively, a nonadaptive process, GC-biased gene conversion (gBGC), could also increase the GC-content of DNA due to the mechanistic bias of gene conversion events during recombination. To determine what role recombination plays in the base composition of bacterial genomes, we compared the spectrum of nucleotide polymorphisms introduced by recombination in all microbial species represented by large numbers of sequenced strains. We found that recombinant alleles are consistently biased toward A and T, and that the magnitude of AT-bias introduced by recombination is similar to that of mutations. These results indicate that recombination alone, without the intervention of selection, is unlikely to counteract the AT-enrichment of bacterial genomes.

The Evolution of Bacterial Genome Architecture.

The genome architecture of bacteria and eukaryotes evolves in opposite directions when subject to genetic drift, a difference that can be ascribed to the fact that bacteria exhibit a mutational bias that deletes superfluous sequences, whereas eukaryotes are biased toward large insertions. Expansion of eukaryotic genomes occurs through the addition of non-functional sequences, such as repetitive sequences and transposable elements, whereas variation in bacterial genome size is largely due to the acquisition and loss of functional accessory genes. These properties create the situation in which eukaryotes with very similar numbers of genes can have vastly different genome sizes, while in bacteria, gene number scales linearly with genome size. Some bacterial genomes, however, particularly those of species that undergo bottlenecks due to recent association with hosts, accumulate pseudogenes and mobile elements, conferring them a low gene content relative to their genome size. These non-functional sequences are gradually eroded and eliminated after long-term association with hosts, with the result that obligate symbionts have the smallest genomes of any cellular organism. The architecture of bacterial genomes is shaped by complex and diverse processes, but for most bacterial species, genome size is governed by a non-adaptive process, i.e., genetic drift coupled with a mutational bias toward deletions. Thus, bacteria with small effective population sizes typically have the smallest genomes. Some marine bacteria counter this near-universal trend: despite having immense population sizes, selection, not drift, acts to reduce genome size in response to metabolic constraints in their nutrient-limited environment.

Human symbionts inject and neutralize antibacterial toxins to persist in the gut.

The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes--one of two major phyla in the gut--also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.