Reconciling Ecological and Genomic Divergence among Lineages of Listeria under an "Extended Mosaic Genome Concept"

Homologous recombination is a major force mechanism driving bacterial evolution, host adaptability, and acquisition of novel virulence traits. Pectobacterium parmentieri is a plant bacterial pathogen distributed worldwide, primarily affecting potatoes, by causing soft rot and blackleg diseases. The goal of this investigation was to understand the impact of homologous recombination on the genomic evolution of P. parmentieri. Analysis of P. parmentieri genomes using Roary revealed a dynamic pan-genome with 3,742 core genes and over 55% accessory genome variability. Bayesian population structure analysis identified 7 lineages, indicating species heterogeneity. ClonalFrameML analysis displayed 5,125 recombination events, with the lineage 4 exhibiting the highest events. fastGEAR analysis identified 486 ancestral and 941 recent recombination events ranging from 43 bp to 119 kb and 36 bp to 13.96 kb, respectively, suggesting ongoing adaptation. Notably, 11% (412 genes) of the core genome underwent recent recombination, with lineage 1 as the main donor. The prevalence of recent recombination (double compared to ancient) events implies continuous adaptation, possibly driven by global potato trade. Recombination events were found in genes involved in vital cellular processes (DNA replication, DNA repair, RNA processing, homeostasis, and metabolism), pathogenicity determinants (type secretion systems, cell-wall degrading enzymes, iron scavengers, lipopolysaccharides (LPS), flagellum, etc.), antimicrobial compounds (phenazine and colicin) and even CRISPR-Cas genes. Overall, these results emphasize the potential role of homologous recombination in P. parmentieri's evolutionary dynamics, influencing host colonization, pathogenicity, adaptive immunity, and ecological fitness.

Recombination is essential to microbial evolution, and is involved in the spread of antibiotic resistance, antigenic variation, and adaptation to the host niche. However, assessing the impact of homologous recombination on accessory genes which are only present in a subset of strains of a given species remains challenging due to their complex phylogenetic relationships. Quantifying homologous recombination for accessory genes (which are important for niche-specific adaptations) in comparison to core genes (which are present in all strains and have essential functions) is critical to understanding how selection acts on variation to shape species diversity and genome structures of bacteria. Here, we apply a computationally efficient, non-phylogenetic approach to measure homologous recombination rates in the core and accessory genome using >100,000 whole genome sequences from Streptococcus pneumoniae and several additional species. By analyzing diverse sets of sequence clusters, we show that core genes often have higher recombination rates than accessory genes, and for some bacterial species the associated effect sizes for these differences are pronounced. In a subset of species, we find that gene frequency and homologous recombination rate are positively correlated. For S. pneumoniae and several additional species, we find that while the recombination rate is higher for the core genome, the mutational divergence is lower, indicating that divergence-based homologous recombination barriers could contribute to differences in recombination rates between the core and accessory genome. Homologous recombination may therefore play a key role in increasing the efficiency of selection in the most conserved parts of the genome.

Recombination is essential to microbial evolution, and is involved in the spread of antibiotic resistance, antigenic variation, and adaptation to the host niche. However, assessing the impact of homologous recombination on accessory genes which are only present in a subset of strains of a given species remains challenging due to their complex phylogenetic relationships. Quantifying homologous recombination for accessory genes (which are important for niche-specific adaptations) in comparison to core genes (which are present in all strains and have essential functions) is critical to understanding how selection acts on variation to shape species diversity and genome structures of bacteria. Here, we apply a computationally efficient, non-phylogenetic approach to measure homologous recombination rates in the core and accessory genome using >100,000 whole genome sequences from Streptococcus pneumoniae and several additional species. By analyzing diverse sets of sequence clusters, we show that core genes often have higher recombination rates than accessory genes, and for some bacterial species the associated effect sizes for these differences are pronounced. In a subset of species, we find that gene frequency and homologous recombination rate are positively correlated. For S. pneumoniae and several additional species, we find that while the recombination rate is higher for the core genome, the mutational divergence is lower, indicating that divergence-based homologous recombination barriers could contribute to differences in recombination rates between the core and accessory genome. Homologous recombination may therefore play a key role in increasing the efficiency of selection in the most conserved parts of the genome.

The global movement of bees for agricultural pollination services can affect local pollinator populations via hybridization. When commercial bumblebees are of the same species but of different geographic origin, intraspecific hybridization may result in beneficial integration of new genetic variation, or alternatively may disrupt locally adapted gene complexes. However, neither the existence nor the extent of genomic introgression and evolutionary divergence between wild and commercial bumblebees is fully understood. We obtained whole‐genome sequencing data from wild and commercial Bombus terrestris collected from sites in Southern Sweden with and without long‐term use of commercially imported B. terrestris. We search for evidence of introgression, dispersal and genome‐wide differentiation in a comparative genomic analysis of wild and commercial bumblebees. Commercial B. terrestris were found in natural environments near sites where commercial bumblebees were used, as well as drifting wild B. terrestris in commercial bumblebee colonies. However, we found no evidence for widespread, recent genomic introgression of commercial B. terrestris into local wild conspecific populations. We found that wild B. terrestris had significantly higher nucleotide diversity (Nei's pi, π), while the number of segregating sites (Watterson's theta, θw) was higher in commercial B. terrestris. A highly divergent region on chromosome 11 was identified in commercial B. terrestris and found to be enriched with structural variants. The genes present in this region are involved in flight muscle contraction and structure and pathogen immune response, providing evidence for differing evolutionary processes operating in wild and commercial B. terrestris. We did not find evidence for recent introgression, suggesting that co‐occurring commercial B. terrestris have not disrupted evolutionary processes in wild B. terrestris populations.

BackgroundComparative genomics provides insights into the diversification of bacterial species. Bacterial speciation usually takes place with lasting homologous recombination, which not only acts as a cohering force between diverging lineages but brings advantageous alleles favored by natural selection, and results in ecologically distinct species, e.g., frequent host shift in Xanthomonas pathogenic to various plants.ResultsUsing whole-genome sequences, we examined the genetic divergence in Xanthomonas campestris that infected Brassicaceae, and X. citri, pathogenic to a wider host range. Genetic differentiation between two incipient races of X. citri pv. mangiferaeindicae was attributable to a DNA fragment introduced by phages. In contrast to most portions of the genome that had nearly equivalent levels of genetic divergence between subspecies as a result of the accumulation of point mutations, 10% of the core genome involving with homologous recombination contributed to the diversification in Xanthomonas, as revealed by the correlation between homologous recombination and genomic divergence. Interestingly, 179 genes were under positive selection; 98 (54.7%) of these genes were involved in homologous recombination, indicating that foreign genetic fragments may have caused the adaptive diversification, especially in lineages with nutritional transitions. Homologous recombination may have provided genetic materials for the natural selection, and host shifts likely triggered ecological adaptation in Xanthomonas. To a certain extent, we observed positive selection nevertheless contributed to ecological divergence beyond host shifting.ConclusionAltogether, mediated with lasting gene flow, species formation in Xanthomonas was likely governed by natural selection that played a key role in helping the deviating populations to explore novel niches (hosts) or respond to environmental cues, subsequently triggering species diversification.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1369-8) contains supplementary material, which is available to authorized users.

BackgroundBecause biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen.ResultsPhage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical.ConclusionsSignificant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.

2.AbstractHaemophilus influenzaeis part of the human nasopharyngeal microbiota and a pathogen causing invasive disease. The extensive genetic diversity observed inH. influenzaenecessitates discriminatory analytical approaches to evaluate its population structure. This study developed a core genome MLST (cgMLST) scheme forH. influenzaeusing pangenome analysis tools and validated the cgMLST scheme using datasets consisting of complete reference genomes (N=14) and high-quality draftH. influenzaegenomes (N=2,297). The draft genome dataset was divided into a development (N=921) and a validation dataset (N=1,376). The development dataset was used to identify potential core genes with the validation dataset used to refine the final core gene list to ensure the reliability of the proposed cgMLST scheme. Functional classifications were made for all resulting core genes. Phylogenetic analyses were performed using both allelic profiles and nucleotide sequence alignments of the core genome to test congruence, as assessed by Spearman’s correlation and Ordinary Least Square linear regression tests. Preliminary analyses using the development dataset identified 1,067 core genes, which were refined to 1,037 with the validation dataset. More than 70% of core genes were predicted to encode proteins essential for metabolism or genetic information processing. Phylogenetic and statistical analyses indicated that the core genome allelic profile accurately represented phylogenetic relatedness among the isolates (R2= 0.945). We used this cgMLST scheme to define a high-resolution population structure forH. influenzae, which enhances the genomic analysis of this clinically relevant human pathogen.3.Impact statementDiscriminatingH. influenzaevariants and evaluating population structure has been challenging and largely unstandardised. To address this, we have developed a cgMLST scheme forH. influenzae.Since an accurate typing approach relies on precise reflection of the underlying population structure, we explored various methods to define the scheme. The core genes included in this scheme were predicted to encode functions in essential biological pathways, such as metabolism and genetic information processing, and could be reliably assembled from short-read sequence data. Single-linkage clustering, based on core genome allelic profiles, showed high congruence to genealogy reconstructed by Maximum-Likelihood (ML) methods from the core genome nucleotide alignment. The cgMLST scheme v1 enables rapid and accurate depiction of high-resolutionH. influenzaepopulation structure, and making this scheme accessible via the PubMLST database, ensures that microbiology reference laboratories and public health authorities worldwide can use it for genomic surveillance.4.Data summaryTheH. influenzaecgMLST scheme is accessible viahttps://pubmlst.org/organisms/haemophilus-influenzae. The list of isolate IDs available publicly frompubmlst.orgis provided in Supplementary File 1. The pipeline for cgMLST scheme development and validation is published athttps://www.protocols.io/private/EF6DB7FE429311EEB8630A58A9FEAC02. All in-house R and Python scripts for data processing and analysis are available fromhttps://gitfront.io/r/user-4399403/ZHt8DArALHcY/cgmlst-hinf/.

Intraspecific trait variability has important consequences for the function and stability of marine ecosystems. Here we examine variation in the ability to use nitrate across hundreds of Prochlorococcus genomes to better understand the modes of evolution influencing intraspecific allocation of ecologically important functions. Nitrate assimilation genes are absent in basal lineages but occur at an intermediate frequency that is randomly distributed within recently emerged clades. The distribution of nitrate assimilation genes within clades appears largely governed by vertical inheritance, gene loss, and homologous recombination. By mapping this process onto a model of Prochlorococcus’ macroevolution, we propose that niche-constructing adaptive radiations and subsequent niche partitioning set the stage for loss of nitrate assimilation genes from basal lineages as they specialized to lower light levels. Retention of these genes in recently emerged lineages has likely been facilitated by selection as they sequentially partitioned into niches where nitrate assimilation conferred a fitness benefit.

Nucleotide sequence data are being harnessed to identify species, even in cases in which organisms themselves are neither in hand nor witnessed. But how genome-wide sequence divergence maps to species status is far from clear. While gene sequence divergence is commonly used to delineate bacterial species, its correspondence to established species boundaries has yet to be explored across eukaryotic taxa. Because the processes underlying gene flow differ fundamentally between prokaryotes and eukaryotes, these domains are likely to differ in the relationship between reproductive isolation and genome-wide sequence divergence. In prokaryotes, homologous recombination, the basis of gene flow, depends directly on the degree of genomic sequence divergence, whereas in sexually reproducing eukaryotes, reproductive incompatibility can stem from changes in very few genes. Guided by measures of genome-wide sequence divergence in bacteria, we gauge how genomic criteria correspond to species boundaries in eukaryotes. In recognized species of eukaryotes, levels of gene sequence divergence within species are typically very small, averaging <1% across protein-coding regions in most animals, plants, and fungi. There are even instances in which divergence between sister species is the same or less than that among conspecifics. In contrast, bacterial species, defined as populations exchanging homologous genes, show levels of divergence both within and between species that are considerably higher. Although no single threshold delineates species, eukaryotic populations with >1% genome-wide sequence divergence are likely separate species, whereas prokaryotic populations with 1% divergence are still able to recombine and thus can be considered the same species.

Ideonella and Thermobifida were the most promising bacterial candidates for degrading plastic polymers. A comparative pan- and phylogenomic analysis of 33 Ideonella and Thermobifida strains was done to determine their plastic degradation potential, niche adaptation and speciation. Our study disclosed that more accessory genes in the strains showed phenotypic plasticity, according to the BPGA data. Pan and core genes were employed for the phylogenetic reconstruction. Pathway enrichment analyses scrutinized the functional roles of the core and adaptive-associated genes. KEGG annotation revealed that most genes were associated with the metabolism of amino acids and carbohydrates. The detailed COG analysis disclosed that approximately 40% of the pan genes performed metabolic functions. The unique gene pool consisted of genes chiefly involved in "general function prediction" and "amino acid transport and metabolism". Our in silico study revealed that these strains could assist in agronomic applications in the future since they devour nitrogen compounds and their central metabolic pathways are involved in amino acid metabolism. The rational selection of strains of Ideonella is far more effective at depolymerising plastics than Thermobifida. A greater number of unique genes, 1701 and 692, were identified for Ideonella sakaiensis 201-F6 and Thermobifida alba DSM-43795, respectively. Furthermore, we examined the singletons involved in xenobiotic catabolism. The unique singleton data were used to construct a supertree. To characterize the conserved patterns, we used SMART and MEME to identify domain and transmembrane regions in the unique protein sequences. Therefore, our study unraveled the genomic insights into the ecology-driven speciation of Ideonella and Thermobifida.

The genomic architecture underlying ecological divergence and ecological speciation with gene flow is still largely unknown for most organisms. One central question is whether divergence is genome-wide or localized in 'genomic mosaics' during early stages when gene flow is still pronounced. Empirical work has so far been limited, and the relative impacts of gene flow and natural selection on genomic patterns have not been fully explored. Here, we use ecotypes of Atlantic cod to investigate genomic patterns of diversity and population differentiation in a natural system characterized by high gene flow and large effective population sizes, properties which theoretically could restrict divergence in local genomic regions. We identify a genomic region of strong population differentiation, extending over approximately 20 cM, between pairs of migratory and stationary ecotypes examined at two different localities. Furthermore, the region is characterized by markedly reduced levels of genetic diversity in migratory ecotype samples. The results highlight the genomic region, or 'genomic island', as potentially associated with ecological divergence and suggest the involvement of a selective sweep. Finally, we also confirm earlier findings of localized genomic differentiation in three other linkage groups associated with divergence among eastern Atlantic populations. Thus, although the underlying mechanisms are still unknown, the results suggest that 'genomic mosaics' of differentiation may even be found under high levels of gene flow and that marine fishes may provide insightful model systems for studying and identifying initial targets of selection during ecological divergence.

ContextProcesses that shape genomic and ecological divergence can reveal important evolutionary dynamics to inform the conservation of threatened species. Fontainea is a genus of rainforest shrubs and small trees including critically endangered and threatened species restricted to narrow, but complex geographic and ecological regions. Several species of Fontainea are subject to spatially explicit conditions and experience limited intra-specific gene flow, likely generating genetic differentiation and local adaptation.ObjectivesHere, we explored the genetic and ecological mechanisms underlying patterns of diversification in two, closely related threatened Fontainea species. Our aim was to compare spatial patterns of genetic variation between the vulnerable Fontainea australis (Southern Fontainea) and critically endangered F. oraria (Coastal Fontainea), endemic to the heterogeneous subtropical region of central, eastern Australia, where large-scale clearing has severely reduced rainforest habitat to a fraction (< 1%) of its pre-European settlement extent.MethodsWe used a set of 10,000 reduced-representation markers to infer genetic relationships and the drivers of spatial genetic variation across the two species. In addition, we employed a combination of univariate and multivariate genome-environment association analysis using a set of topo-climatic variables to explore potential patterns of local adaptation as a factor impacting genomic divergence.ResultsOur study revealed that Coastal Fontainea have a close genetic relationship with Southern Fontainea. We showed that isolation by distance has played a key role in their genetic variation, indicating that vicariance can explain the spatial genetic distribution of the two species. Genotype-environment analyses showed a strong association with temperature and topographic features, suggesting adaptation to localised thermal environments. We used a multivariate redundancy analysis to identify a range of putatively adapted loci associated with local environmental conditions.ConclusionsDivergent selection at the local-habitat scale as a result of dispersal limitations and environmental heterogeneity (including physical barriers) are likely contributors to adaptive divergence between the two Fontainea species. Our findings have presented evidence to indicate that Southern and Coastal Fontainea were comprised of distinct genetic groups and ecotypes, that together may form a single species continuum, with further phenotype research suggested to confirm the current species boundaries. Proactive conservation actions, including assisted migration to enhance the resilience of populations lacking stress-tolerant single nucleotide polymorphisms (SNPs) may be required to secure the long-term future of both taxa. This is especially vital for the critically endangered Coastal Fontainea given projections of habitat decline for the species under future climate scenarios.

Multiscale comparative pathogenomic analysis of Vibrio anguillarum linking serotype diversity, genomic plasticity and pathogenicity

Streptococcus suis is a major porcine and zoonotic pathogen responsible for significant economic losses in the pig industry and an increasing number of human cases. Multiple isolates of S. suis show marked genomic diversity. Here, we report the analysis of whole genome sequences of nine pig isolates that caused disease typical of S. suis and had phenotypic characteristics of S. suis, but their genomes were divergent from those of many other S. suis isolates. Comparison of protein sequences predicted from divergent genomes with those from normal S. suis reduced the size of core genome from 793 to only 397 genes. Divergence was clear if phylogenetic analysis was performed on reduced core genes and MLST alleles. Phylogenies based on certain other genes (16S rRNA, sodA, recN, and cpn60) did not show divergence for all isolates, suggesting recombination between some divergent isolates with normal S. suis for these genes. Indeed, there is evidence of recent recombination between the divergent and normal S. suis genomes for 249 of 397 core genes. In addition, phylogenetic analysis based on the 16S rRNA gene and 132 genes that were conserved between the divergent isolates and representatives of the broader Streptococcus genus showed that divergent isolates were more closely related to S. suis. Six out of nine divergent isolates possessed a S. suis-like capsule region with variation in capsular gene sequences but the remaining three did not have a discrete capsule locus. The majority (40/70), of virulence-associated genes in normal S. suis were present in the divergent genomes. Overall, the divergent isolates extend the current diversity of S. suis species but the phenotypic similarities and the large amount of gene exchange with normal S. suis gives insufficient evidence to assign these isolates to a new species or subspecies. Further, sampling and whole genome analysis of more isolates is warranted to understand the diversity of the species.

Mismatch repair: praying for genome stability