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Pseudocercospora (previously Mycosphaerella) fijiensis is a hemibiotroph fungus and the causal agent of black Sigatoka disease, one of the most significant threats to banana production worldwide. Only a few genomics reports have paid any attention to effector proteins, which are key players in pathogenicity. These reports focus on canonical effectors: small secreted proteins, rich in cysteines, containing a signal peptide and no transmembrane domain. Thus, bias in previous reports has resulted in the non-canonical effectors being, in effect, excluded from the discussion of effectors in P. fijiensis pathogenicity. Here, using WideEffHunter and EffHunter, bioinformatic tools which identify non-canonical and canonical effectors, respectively, we predict, for the first time, the full effectorome of P. fijiensis. This complete effectorome comprises 5179 proteins: 240 canonical and 4939 non-canonical effectors. Protein families related to key functions of the hemibiotrophic lifestyle, such as Salicylate hydroxylase and Isochorismatase, are widely represented families of effectors in the P. fijiensis genome. An analysis of the gene distribution in core and dispensable scaffolds of both classes of effectors revealed a novel genomic structure of the effectorome. The majority of the effectors (canonical and non-canonical) were found to be harbored in the core scaffolds, while dispensable scaffolds harbored less than 10% of the effectors, all of which were non-canonical. Additionally, we found the motifs RXLR, YFWxC, LysM, EAR, [Li]xAR, PDI, CRN, and ToxA in the effectors of P. fijiensis. This novel genomic structure of effectors (more enriched in the core than in the dispensable genome), as well as the occurrence of effector motifs which were also observed in four other fungi, evidences that these phenomena are not unique to P. fijiensis; rather, they are widely occurring characteristics of effectors in other fungi.

Pseudocercospora (previously Mycosphaerella) fijiensis is a hemibiotroph fungus and the causal agent of black Sigatoka disease, one of the most significant threats to banana production worldwide. Only a few genomics reports have paid any attention to effector proteins, which are key players in pathogenicity. These reports focus on canonical effectors: small secreted proteins, rich in cysteines, containing a signal peptide and no transmembrane domain. Thus, bias in previous reports has resulted in the non-canonical effectors being, in effect, excluded from the discussion of effectors in P. fijiensis pathogenicity. Here, using WideEffHunter and EffHunter, bioinformatic tools which identify non-canonical and canonical effectors, respectively, we predict, for the first time, the full effectorome of P. fijiensis. This complete effectorome comprises 5179 proteins: 240 canonical and 4939 non-canonical effectors. Protein families related to key functions of the hemibiotrophic lifestyle, such as Salicylate hydroxylase and Isochorismatase, are widely represented families of effectors in the P. fijiensis genome. An analysis of the gene distribution in core and dispensable scaffolds of both classes of effectors revealed a novel genomic structure of the effectorome. The majority of the effectors (canonical and non-canonical) were found to be harbored in the core scaffolds, while dispensable scaffolds harbored less than 10% of the effectors, all of which were non-canonical. Additionally, we found the motifs RXLR, YFWxC, LysM, EAR, [Li]xAR, PDI, CRN, and ToxA in the effectors of P. fijiensis. This novel genomic structure of effectors (more enriched in the core than in the dispensable genome), as well as the occurrence of effector motifs which were also observed in four other fungi, evidences that these phenomena are not unique to P. fijiensis; rather, they are widely occurring characteristics of effectors in other fungi. [ABSTRACT FROM AUTHOR]

In the present work, we carried out a comparative genomic analysis to trace the evolutionary trajectory of the bacterial species that make up the Liquorilactobacillus genus, from the identification of genes and speciation/adaptation mechanisms in their unique characteristics to the identification of the pattern grouping these species. We present phylogenetic relationships between Liquorilactobacillus and related taxa such as Bacillus, basal lactobacilli and Ligilactobacillus, highlighting evolutionary divergences and lifestyle transitions across different taxa. The species of this genus share a core genome of 1023 genes, distributed in all COGs, which made it possible to characterize it as Liquorilactobacillus sensu lato: few amino acid auxotrophy, low genes number for resistance to antibiotics and general and specific cellular reprogramming mechanisms for environmental responses. These species were divided into four clades, with diversity being enhanced mainly by the diversity of genes involved in sugar metabolism. Clade 1 presented lower (< 70%) average amino acid identity with the other clades, with exclusive or absent genes, and greater distance in the genome compared to clades 2, 3 and 4. The data pointed to an ancestor of clades 2, 3 and 4 as being the origin of the genus Ligilactobacillus, while the species of clade 1 being closer to the ancestral Bacillus. All these traits indicated that the species of clade 1 could be soon separated in a distinct genus. [ABSTRACT FROM AUTHOR]

BACKGROUND: Cumulative sequencing efforts have yielded enough genomes to construct pangenomes for dozens of bacterial species and elucidate intraspecies gene conservation. Given the diversity of organisms for which this is achievable, similar analyses for ancestral species are feasible through the integration of pangenomics and phylogenetics, promising deeper insights into the nature of ancient life. RESULTS: We construct pangenomes for 183 bacterial species from 54,085 genomes and identify their core genomes using a novel statistical model to estimate genome-specific error rates and underlying gene frequencies. The core genomes are then integrated into a phylogenetic tree to reconstruct the core genome of the last bacterial common ancestor (LBCA), yielding three main results: First, the gene content of modern and ancestral core genomes are diverse at the level of individual genes but are similarly distributed by functional category and share several poorly characterized genes. Second, the LBCA core genome is distinct from any individual modern core genome but has many fundamental biological systems intact, especially those involving translation machinery and biosynthetic pathways to all major nucleotides and amino acids. Third, despite this metabolic versatility, the LBCA core genome likely requires additional non-core genes for viability, based on comparisons with the minimal organism, JCVI-Syn3A. CONCLUSIONS: These results suggest that many cellular systems commonly conserved in modern bacteria were not just present in ancient bacteria but were nearly immutable with respect to short-term intraspecies variation. Extending this analysis to other domains of life will likely provide similar insights into more distant ancestral species.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Abstract Background Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. Results Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. Conclusion In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Background: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. Results: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. Conclusion: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research. [ABSTRACT FROM AUTHOR]

BackgroundWith the exponential growth of publicly available genome sequences, pangenome analyses have provided increasingly complete pictures of genetic diversity for many microbial species. However, relatively few studies have scaled beyond single pangenomes to compare global genetic diversity both within and across different species. We present here several methods for "comparative pangenomics" that can be used to contextualize multi-pangenome scale genetic diversity with gene function for multiple species at multiple resolutions: pangenome shape, genes, sequence variants, and positions within variants.ResultsApplied to 12,676 genomes across 12 microbial pathogenic species, we observed several shared resolution-specific patterns of genetic diversity: First, pangenome openness is associated with species' phylogenetic placement. Second, relationships between gene function and frequency are conserved across species, with core genomes enriched for metabolic and ribosomal genes and accessory genomes for trafficking, secretion, and defense-associated genes. Third, genes in core genomes with the highest sequence diversity are functionally diverse. Finally, certain protein domains are consistently mutation enriched across multiple species, especially among aminoacyl-tRNA synthetases where the extent of a domain's mutation enrichment is strongly function-dependent.ConclusionsThese results illustrate the value of each resolution at uncovering distinct aspects in the relationship between genetic and functional diversity across multiple species. With the continued growth of the number of sequenced genomes, these methods will reveal additional universal patterns of genetic diversity at the pangenome scale.

A strictly aerobic, Gram-stain-negative, rod-shaped, and motile bacterium, designated strain KMM 296, isolated from the coelomic fluid of the mussel Crenomytilus grayanus, was investigated in detail due to its ability to produce a highly active alkaline phosphatase CmAP of the structural family PhoA. A previous taxonomic study allocated the strain to the species Cobetia marina, a member of the family Halomonadaceae of the class Gammaproteobacteria. However, 16S rRNA gene sequencing showed KMM 296's relatedness to Cobetia amphilecti NRIC 0815T. The isolate grew with 0.5–19% NaCl at 4–42 °C and hydrolyzed Tweens 20 and 40 and L-tyrosine. The DNA G+C content was 62.5 mol%. The prevalent fatty acids were C18:1 ω7c, C12:0 3-OH, C18:1 ω7c, C12:0, and C17:0 cyclo. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, and also an unidentified aminolipid, phospholipid, and a few unidentified lipids. The major respiratory quinone was Q-8. According to phylogenomic and chemotaxonomic evidence, and the nearest neighbors, the strain KMM 296 represents a member of the species C. amphilecti. The genome-based analysis of C. amphilecti NRIC 0815T and C. litoralis NRIC 0814T showed their belonging to a single species. In addition, the high similarity between the C. pacifica NRIC 0813T and C. marina LMG 2217T genomes suggests their affiliation to one species. Based on the rules of priority, C. litoralis should be reclassified as a later heterotypic synonym of C. amphilecti, and C. pacifica is a later heterotypic synonym of C. marina. The emended descriptions of the species C. amphilecti and C. marina are also proposed. [ABSTRACT FROM AUTHOR]

ABSTRACTCampylobacter species are typically helical shaped, Gram-negative, and non-spore-forming bacteria. Species in this genus include established foodborne and animal pathogens as well as emerging pathogens. The accumulation of genomic data from the Campylobacter genus has increased exponentially in recent years, accompanied by the discovery of putative new species. At present, the lack of a standardized species boundary complicates distinguishing established and novel species. We defined the Campylobacter genus core genome (500 loci) using publicly available Campylobacter complete genomes (n = 498) and constructed a core genome phylogeny using 2,193 publicly available Campylobacter genomes to examine inter-species diversity and species boundaries. Utilizing 8,440 Campylobacter genomes representing 33 species and 8 subspecies, we found species delineation based on an average nucleotide identity (ANI) cutoff of 94.2% is consistent with the core genome phylogeny. We identified 60 ANI genomic species that delineated Campylobacter species in concordance with previous comparative genetic studies. All pairwise ANI genomic species pairs had in silico DNA-DNA hybridization scores of less than 70%, supporting their delineation as separate species. We provide the tool Campylobacter Genomic Species typer (CampyGStyper) that assigns ANI genomic species to query genomes based on ANI similarities to medoid genomes from each ANI genomic species with an accuracy of 99.96%. The ANI genomic species definitions proposed here allow consistent species definition in the Campylobacter genus and will facilitate the detection of novel species in the future.IMPORTANCEIn recent years, Campylobacter has gained recognition as the leading cause of bacterial gastroenteritis worldwide, leading to a substantial rise in the collection of genomic data of the Campylobacter genus in public databases. Currently, a standardized Campylobacter species boundary at the genomic level is absent, leading to challenges in detecting emerging pathogens and defining putative novel species within this genus. We used a comprehensive representation of genomes of the Campylobacter genus to construct a core genome phylogenetic tree. Furthermore, we found an average nucleotide identity (ANI) of 94.2% as the optimal cutoff to define the Campylobacter species. Using this cutoff, we identified 60 ANI genomic species which provided a standardized species definition and nomenclature. Importantly, we have developed Campylobacter Genomic Species typer (CampyGStyper), which can robustly and accurately assign these ANI genomic species to Campylobacter genomes, thereby aiding pathogen surveillance and facilitating evolutionary and epidemiological studies of existing and emerging pathogens in the genus Campylobacter.

Ralstonia solanacearum species complex (RSSC) strains are bacteria that colonize plant xylem tissue and cause vascular wilt diseases. However, individual strains vary in host range, optimal disease temperatures and physiological traits. To increase our understanding of the evolution, diversity and biology of the RSSC, we performed a meta-analysis of 100 representative RSSC genomes. These 100 RSSC genomes contain 4940 genes on average, and a pangenome analysis found that there are 3262 genes in the core genome (~60 % of the mean RSSC genome) with 13 128 genes in the extensive flexible genome. A core genome phylogenetic tree and a whole-genome similarity matrix aligned with the previously named species ( R. solanacearum , R. pseudosolanacearum , R. syzygii ) and phylotypes (I–IV). These analyses also highlighted a third unrecognized sub-clade of phylotype II. Additionally, we identified differences between phylotypes with respect to gene content and recombination rate, and we delineated population clusters based on the extent of horizontal gene transfer. Multiple analyses indicate that phylotype II is the most diverse phylotype, and it may thus represent the ancestral group of the RSSC. We also used our genome-based framework to test whether the RSSC sequence variant (sequevar) taxonomy is a robust method to define within-species relationships of strains. The sequevar taxonomy is based on alignments of a single conserved gene (egl). Although sequevars in phylotype II describe monophyletic groups, the sequevar system breaks down in the highly recombinogenic phylotype I, which highlights the need for an improved, cost-effective method for genotyping strains in phylotype I. Finally, we enabled quick and precise genome-based identification of newly sequenced RSSC strains by assigning Life Identification Numbers (LINs) to the 100 strains and by circumscribing the RSSC and its sub-groups in the LINbase Web service.

Abstract Background Staphylococcus aureus can infect and adapt to multiple host species. However, our understanding of the genetic and evolutionary drivers of its generalist lifestyle remains inadequate. This is particularly important when considering local populations of S. aureus, where close physical proximity between bacterial lineages and between host species may facilitate frequent and repeated interactions between them. Here, we aim to elucidate the genomic differences between human- and animal-derived S. aureus from 437 isolates sampled from disease cases in the northeast region of the United States. Results Multi-locus sequence typing revealed the existence of 75 previously recognized sequence types (ST). Our population genomic analyses revealed heterogeneity in the accessory genome content of three dominant S. aureus lineages (ST5, ST8, ST30). Genes related to antimicrobial resistance, virulence, and plasmid types were differentially distributed among isolates according to host (human versus non-human) and among the three major STs. Across the entire population, we identified a total of 1,912 recombination events that occurred in 765 genes. The frequency and impact of homologous recombination were comparable between human- and animal-derived isolates. Low-frequency STs were major donors of recombined DNA, regardless of the identity of their host. The most frequently recombined genes (clfB, aroA, sraP) function in host infection and virulence, which were also frequently shared between the rare lineages. Conclusions Taken together, these results show that frequent but variable patterns of recombination among co-circulating S. aureus lineages, including the low-frequency lineages, that traverse host barriers shape the structure of local gene pool and the reservoir of host-associated genetic variants. Our study provides important insights to the genetic and evolutionary factors that contribute to the ability of S. aureus to colonize and cause disease in multiple host species. Our study highlights the importance of continuous surveillance of S. aureus circulating in different ecological host niches and the need to systematically sample from them. These findings will inform development of effective measures to control S. aureus colonization, infection, and transmission across the One Health continuum.

Abstract Background Cumulative sequencing efforts have yielded enough genomes to construct pangenomes for dozens of bacterial species and elucidate intraspecies gene conservation. Given the diversity of organisms for which this is achievable, similar analyses for ancestral species are feasible through the integration of pangenomics and phylogenetics, promising deeper insights into the nature of ancient life. Results We construct pangenomes for 183 bacterial species from 54,085 genomes and identify their core genomes using a novel statistical model to estimate genome-specific error rates and underlying gene frequencies. The core genomes are then integrated into a phylogenetic tree to reconstruct the core genome of the last bacterial common ancestor (LBCA), yielding three main results: First, the gene content of modern and ancestral core genomes are diverse at the level of individual genes but are similarly distributed by functional category and share several poorly characterized genes. Second, the LBCA core genome is distinct from any individual modern core genome but has many fundamental biological systems intact, especially those involving translation machinery and biosynthetic pathways to all major nucleotides and amino acids. Third, despite this metabolic versatility, the LBCA core genome likely requires additional non-core genes for viability, based on comparisons with the minimal organism, JCVI-Syn3A. Conclusions These results suggest that many cellular systems commonly conserved in modern bacteria were not just present in ancient bacteria but were nearly immutable with respect to short-term intraspecies variation. Extending this analysis to other domains of life will likely provide similar insights into more distant ancestral species.

Introduction. Invasive listeriosis is a rare disease posing a threat to high-risk groups and often leading to a fatal outcome. Its causative agent is Listeria monocytogenes, a ubiquitous saprophyte that has turned into an important foodborne pathogen with the growing industry of semi-cooked and ready-to-eat products. The aim of the study is the characterization of L. monocytogenes isolates in the Moscow region and identification of possible causes of susceptibility to infection Materials and methods. The multicenter monitoring of L. monocytogenes was conducted in the Moscow metropolitan area, using bacteriological and genomic methods for description of the pathogen, medical history collection and detailed analysis of patient case summaries. Results. In the cohorts of patients with perinatal listeriosis (PL) and meningitis-septicemia (MS), invasive listeriosis had a year-round occurrence with slight upswings in MarchApril and JulyNovember. During the COVID-19 pandemic, in the MS group, the minimum age of patients decreased to 31 years and the proportion of deaths increased 1.57-fold compared to 20182019. During the pandemic, an increase in the diversity of L. monocytogenes genotypes was observed, along with changes in the spectrum of pathogen genotypes throughout the pandemic stages. During the monitoring, a total of 73 L. monocytogenes clinical isolates belonging to 24 genotypes were described. Seven genotypes belonged to the first phylogenetic lineage (PLI); 14 genotypes belonged to PLII. The PL cohort had the highest proportion of PLI genotypes (52%). In the MS cohort, the group of men had the widest diversity of genotypes, 6 of which were identical to genotypes of food isolates. In the analysed set of isolates, 12 new profiles of internalin genes were identified and described. The whole genome sequencing detected the presence of plasmids in 9 of 58 genomes of clinical isolates. The comparison of core genomes revealed an epidemic relationship between isolates of the same genotype for ST4, ST21, and ST425. Conclusion. The performed study presents a detailed description of the diversity and virulence of L. monocytogenes circulating in the Moscow metropolitan area, thus providing information for timely diagnosis and treatment of invasive listeriosis.

Vibrio parahaemolyticus is a significant cause of foodborne illness, and its incidence worldwide is on the rise. It is thus imperative to develop a straightforward and efficient method for typing strains of this pathogen. In this study, we conducted a pangenome analysis of 75 complete genomes of V. parahaemolyticus and identified the core gene mtlA with the highest degree of variation, which distinguished 44 strains and outperformed traditional seven-gene-based MLST when combined with aer, another core gene with high degree of variation. The mtlA gene had higher resolution to type strains with a close relationship compared to the traditional MLST genes in the phylogenetic tree built by core genomes. Strong positive selection was also detected in the gene mtlA (ω > 1), representing adaptive and evolution in response to the environment. Therefore, the panel of gene mtlA and aer may serve as a tool for the typing of V. parahaemolyticus, potentially contributing to the prevention and control of this foodborne disease.

As the name of the genus Pantoea ("of all sorts and sources") suggests, this genus includes bacteria with a wide range of provenances, including plants, animals, soils, components of the water cycle, and humans. Some members of the genus are pathogenic to plants, and some are suspected to be opportunistic human pathogens; while others are used as microbial pesticides or show promise in biotechnological applications. During its taxonomic history, the genus and its species have seen many revisions. However, evolutionary and comparative genomics studies have started to provide a solid foundation for a more stable taxonomy. To move further toward this goal, we have built a 2,509-gene core genome tree of 437 public genome sequences representing the currently known diversity of the genus Pantoea. Clades were evaluated for being evolutionarily and ecologically significant by determining bootstrap support, gene content di [ABSTRACT FROM AUTHOR]

Streptomyces possess a linear, genetically compartmentalized chromosome. Core genes (conserved across the genus) are grouped in the central part, while terminal regions are populated by poorly conserved genes including clusters encoding antibiotic production. The genome is highly plastic: the Streptomyces pangenome is at least 2 logs larger than the core genome (i.e., all core genes). The Streptomyces chromosome is unstable, exhibiting a stable backbone in the central region and an evolutionary gradient towards fast-evolving arms. The genetic compartmentalization correlates with chromosome 3D folding in the exponential phase. The distal rRNA (rrn) operons delimit a highly structured and expressed region termed the 'central compartment', exhibiting structural features distinct from those of the 'terminal compartments', which are almost transcriptionally quiescent. The chromosome spatial conformation changes over the developmental cycle from a rather open to a closed conformation. Streptomyces are prolific producers of specialized metabolites with applications in medicine and agriculture. Remarkably, these bacteria possess a large linear chromosome that is genetically compartmentalized: core genes are grouped in the central part, while the ends are populated by poorly conserved genes including antibiotic biosynthetic gene clusters. The genome is highly unstable and exhibits distinct evolutionary rates along the chromosome. Recent chromosome conformation capture (3C) and comparative genomics studies have shed new light on the interplay between genome dynamics in space and time. Here, we review insights that illustrate how the balance between chance (random genome variations) and necessity (structural and functional constraints) may have led to the emergence of spatial structuring of the Streptomyces chromosome. [ABSTRACT FROM AUTHOR]

At the genome level, microorganisms are highly adaptable both in terms of allele and gene composition. Such heritable traits emerge in response to different environmental niches and can have a profound influence on microbial community dynamics. As a consequence, any individual genome or population will contain merely a fraction of the total genetic diversity of any operationally defined "species", whose ecological potential can thus be only fully understood by studying all of their genomes and the genes therein. This concept, known as the pangenome, is valuable for studying microbial ecology and evolution, as it partitions genomes into core (present in all the genomes from a species, and responsible for housekeeping and species‐level niche adaptation among others) and accessory regions (present only in some, and responsible for intra‐species differentiation). Here we present SuperPang, an algorithm producing pangenome assemblies from a set of input genomes of varying quality, including metagenome‐assembled genomes (MAGs). SuperPang runs in linear time and its results are complete, non‐redundant, preserve gene ordering and contain both coding and non‐coding regions. Our approach provides a modular view of the pangenome, identifying operons and genomic islands, and allowing to track their prevalence in different populations. We illustrate this by analysing intra‐species diversity in Polynucleobacter, a bacterial genus ubiquitous in freshwater ecosystems, characterized by their streamlined genomes and their ecological versatility. We show how SuperPang facilitates the simultaneous analysis of allelic and gene content variation under different environmental pressures, allowing us to study the drivers of microbial diversification at unprecedented resolution. [ABSTRACT FROM AUTHOR]

ABSTRACTCarbapenemase-producing Enterobacterales have emerged in the last decades as one of the main threats in modern medicine. Enterobacter is a genus increasingly recognized as a key player in carbapenemase diffusion besides Escherichia coli and Klebsiella pneumoniae, but information regarding Citrobacter spp. remained scarce. A collection of 803 isolates of Citrobacter spp. recovered from the French National Center for carbapenem resistance over a 2-year period during 2019–2020 was analyzed. A total of 15 different carbapenemases were identified of which OXA-48 followed by NDM-1 and OXA-181 represented the main enzymes. Phylogenetic analysis of this collection revealed that Citrobacter freundii was the main species among which three main clones are circulating in France, being ST8, ST22, and ST91. ST22 and ST8 were distributed in all regions whereas ST91 demonstrated a regional spread. Analysis of the species diversity revealed that among the C. freundii complex, Citrobacter portucalensis and Citrobacter braakii were also disseminated. We revealed the dissemination of a clone carrying both blaDHA-15 (β-lactamase-encoding gene rarely reported in Enterobacterales) and blaOXA-48 in the north of France and allowed to identify an outbreak not previously investigated. This study analyzed the largest collection of carbapenemase-producing Citrobacter spp. and allowed deciphering of molecular epidemiology of Citrobacter spp. in France.IMPORTANCEThe emergence of carbapenemase producers in Enterobacterales mostly involves Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex species. However, in France, we observed the emergence and the rapid dissemination of carbapenemase in Citrobacter spp. In this study, we demonstrated that a wide variety of carbapenemases is produced by many different species of Citrobacter spp. However, we clearly identify three high-risk clones of Citrobacter freundii, ST8, ST22, and ST91 that drive the spread of carbapenemase in France. This epidemiological study paves the way of further analysis that would aim to identify the virulence factors involved in this pellicular ability of these three clones to disseminate at the hospital.

As the name of the genus Pantoea (“of all sorts and sources”) suggests, this genus includes bacteria with a wide range of provenances, including plants, animals, soils, components of the water cycle, and humans. Some members of the genus are pathogenic to plants, and some are suspected to be opportunistic human pathogens; while others are used as microbial pesticides or show promise in biotechnological applications. During its taxonomic history, the genus and its species have seen many revisions. However, evolutionary and comparative genomics studies have started to provide a solid foundation for a more stable taxonomy. To move further toward this goal, we have built a 2,509-gene core genome tree of 437 public genome sequences representing the currently known diversity of the genus Pantoea. Clades were evaluated for being evolutionarily and ecologically significant by determining bootstrap support, gene content differences, and recent recombination events. These results were then integrated with genome metadata, published literature, descriptions of named species with standing in nomenclature, and circumscriptions of yet-unnamed species clusters, 15 of which we assigned names under the nascent SeqCode. Finally, genome-based circumscriptions and descriptions of each species and each significant genetic lineage within species were uploaded to the LINbase Web server so that newly sequenced genomes of isolates belonging to any of these groups could be precisely and accurately identified.

Here we review two connected themes in evolutionary microbiology: (a) the nature of gene repertoire variation within species groups (pangenomes) and (b) the concept of metabolite transporters as accessory proteins capable of providing niche-defining "bolt-on" phenotypes. We discuss the need for improved sampling and understanding of pangenome variation in eukaryotic microbes. We then review the factors that shape the repertoire of accessory genes within pangenomes. As part of this discussion, we outline how gene duplication is a key factor in both eukaryotic pangenome variation and transporter gene family evolution. We go on to outline how, through functional characterization of transporter-encoding genes, in combination with analyses of how transporter genes are gained and lost from accessory genomes, we can reveal much about the niche range, the ecology, and the evolution of virulence of microbes. We advocate for the coordinated systematic study of eukaryotic pangenomes through genome sequencing and the functional analysis of genes found within the accessory gene repertoire. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus can infect and adapt to multiple host species. However, our understanding of the genetic and evolutionary drivers of its generalist lifestyle remains inadequate. This is particularly important when considering local populations of S. aureus, where close physical proximity between bacterial lineages and between host species may facilitate frequent and repeated interactions between them. Here, we aim to elucidate the genomic differences between human- and animal-derived S. aureus from 437 isolates sampled from disease cases in the northeast region of the United States. Results: Multi-locus sequence typing revealed the existence of 75 previously recognized sequence types (ST). Our population genomic analyses revealed heterogeneity in the accessory genome content of three dominant S. aureus lineages (ST5, ST8, ST30). Genes related to antimicrobial resistance, virulence, and plasmid types were differentially distributed among isolates according to host (human versus non-human) and among the three major STs. Across the entire population, we identified a total of 1,912 recombination events that occurred in 765 genes. The frequency and impact of homologous recombination were comparable between human- and animal-derived isolates. Low-frequency STs were major donors of recombined DNA, regardless of the identity of their host. The most frequently recombined genes (clfB, aroA, sraP) function in host infection and virulence, which were also frequently shared between the rare lineages. Conclusions: Taken together, these results show that frequent but variable patterns of recombination among co-circulating S. aureus lineages, including the low-frequency lineages, that traverse host barriers shape the structure of local gene pool and the reservoir of host-associated genetic variants. Our study provides important insights to the genetic and evolutionary factors that contribute to the ability of S. aureus to colonize and cause disease in multiple host species. Our study highlights the importance of continuous surveillance of S. aureus circulating in different ecological host niches and the need to systematically sample from them. These findings will inform development of effective measures to control S. aureus colonization, infection, and transmission across the One Health continuum. [ABSTRACT FROM AUTHOR]

Many Bacillus species are essential antibacterial agents, but their antibiosis potential still needs to be elucidated to its full extent. Here, we isolated a soil bacterium, BP9, which has significant antibiosis activity against fungal and bacterial pathogens. BP9 improved the growth of wheat seedlings via active colonization and demonstrated effective biofilm and swarming activity. BP9 sequenced genome contains 4282 genes with a mean G-C content of 45.94% of the whole genome. A single copy concatenated 802 core genes of 28 genomes, and their calculated average nucleotide identity (ANI) discriminated the strain BP9 from Bacillus licheniformis and classified it as Bacillus paralicheniformis. Furthermore, a comparative pan-genome analysis of 40 B. paralicheniformis strains suggested that the genetic repertoire of BP9 belongs to open-type genome species. A comparative analysis of a pan-genome dataset using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cluster of Orthologous Gene groups (COG) revealed the diversity of secondary metabolic pathways, where BP9 distinguishes itself by exhibiting a greater prevalence of loci associated with the metabolism and transportation of organic and inorganic substances, carbohydrate and amino acid for effective inhabitation in diverse environments. The primary secondary metabolites and their genes involved in synthesizing bacillibactin, fencing, bacitracin, and lantibiotics were identified as acquired through a recent Horizontal gene transfer (HGT) event, which contributes to a significant part of the strain`s antimicrobial potential. Finally, we report some genes essential for plant-host interaction identified in BP9, which reduce spore germination and virulence of multiple fungal and bacterial species. The effective colonization, diverse predicted metabolic pathways and secondary metabolites (antibiotics) suggest testing the suitability of strain BP9 as a potential bio-preparation in agricultural fields.

Abstract 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.

Abstract Background Moraxella bovis and Moraxella bovoculi both associate with infectious bovine keratoconjunctivitis (IBK), an economically significant and painful ocular disease that affects cattle worldwide. There are two genotypes of M. bovoculi (genotypes 1 and 2) that differ in their gene content and potential virulence factors, although neither have been experimentally shown to cause IBK. M. bovis is a causative IBK agent, however, not all strains carry a complete assortment of known virulence factors. The goals of this study were to determine the population structure and depth of M. bovis genomic diversity, and to compare core and accessory genes and predicted outer membrane protein profiles both within and between M. bovis and M. bovoculi. Results Phylogenetic trees and bioinformatic analyses of 36 M. bovis chromosomes sequenced in this study and additional available chromosomes of M. bovis and both genotype 1 and 2 M. bovoculi, showed there are two genotypes (1 and 2) of M. bovis. The two M. bovis genotypes share a core of 2015 genes, with 121 and 186 genes specific to genotype 1 and 2, respectively. The two genotypes differ by their chromosome size and prophage content, encoded protein variants of the virulence factor hemolysin, and by their affiliation with different plasmids. Eight plasmid types were identified in this study, with types 1 and 6 observed in 88 and 56% of genotype 2 strains, respectively, and absent from genotype 1 strains. Only type 1 plasmids contained one or two gene copies encoding filamentous haemagglutinin-like proteins potentially involved with adhesion. A core of 1403 genes was shared between the genotype 1 and 2 strains of both M. bovis and M. bovoculi, which encoded a total of nine predicted outer membrane proteins. Conclusions There are two genotypes of M. bovis that differ in both chromosome content and plasmid profiles and thus may not equally associate with IBK. Immunological reagents specifically targeting select genotypes of M. bovis, or all genotypes of M. bovis and M. bovoculi together could be designed from the outer membrane proteins identified in this study.

Objective: To analyse the pan-genome of three black-pigmented periodontal pathogens: Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens. Methods: Pan-genome analyses of 66, 33 and 5 publicly available whole-genome sequences of P. gingivalis, P. intermedia and P. nigrescens, respectively, were performed using Pan-genome Analysis Pipeline software (version 1.2.1; Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, PR China). Phylogenetic trees were constructed based on the entire pangenome and single nucleotide polymorphisms within the core genome. The distribution and abundance of virulence genes in the core and dispensable genomes were also compared in the three species. Results: All three species possess an open pan-genome. The core genome of P. gingivalis, P. inter)media and P. nigrescens included 1001, 1514 and 1745 orthologous groups, respectively, which were mainly related to basic cellular functions such as metabolism. The dispensable genome of P. gingivalis, P. intermedia and P. nigrescens was composed of 2814, 2689 and 906 orthologous groups, respectively, and it was enriched in genes involved in pathogenicity or with unknown functions. Phylogenetic trees presented a clear separation of P. gingivalis, P. intermedia and P. nigrescens, verifying the reclassification of the black-pigmented species. Furthermore, the three species shared almost the same virulence factors involved in adhesion, proteolysis and evasion of host defences. Some of these virulence genes were conserved across species whereas others belonged to the dispensable genome, which might be acquired through horizontal gene transfer. Conclusion: This study highlighted the usefulness of pan-genome analysis to infer evolutionary cues for black-pigmented species, indicating their homology and phylogenomic di [ABSTRACT FROM AUTHOR]

A strictly aerobic, Gram-stain-negative, rod-shaped, and motile bacterium, designated strain KMM 296, isolated from the coelomic fluid of the mussel Crenomytilus grayanus, was investigated in detail due to its ability to produce a highly active alkaline phosphatase CmAP of the structural family PhoA. A previous taxonomic study allocated the strain to the species Cobetia marina, a member of the family Halomonadaceae of the class Gammaproteobacteria. However, 16S rRNA gene sequencing showed KMM 296’s relatedness to Cobetia amphilecti NRIC 0815T. The isolate grew with 0.5–19% NaCl at 4–42 °C and hydrolyzed Tweens 20 and 40 and L-tyrosine. The DNA G+C content was 62.5 mol%. The prevalent fatty acids were C18:1 ω7c, C12:0 3-OH, C18:1 ω7c, C12:0, and C17:0 cyclo. The polar lipid profile was characterized by the presence of phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, and also an unidentified aminolipid, phospholipid, and a few unidentified lipids. The major respiratory quinone was Q-8. According to phylogenomic and chemotaxonomic evidence, and the nearest neighbors, the strain KMM 296 represents a member of the species C. amphilecti. The genome-based analysis of C. amphilecti NRIC 0815T and C. litoralis NRIC 0814T showed their belonging to a single species. In addition, the high similarity between the C. pacifica NRIC 0813T and C. marina LMG 2217T genomes suggests their affiliation to one species. Based on the rules of priority, C. litoralis should be reclassified as a later heterotypic synonym of C. amphilecti, and C. pacifica is a later heterotypic synonym of C. marina. The emended descriptions of the species C. amphilecti and C. marina are also proposed.

BackgroundThe growing importance of the ubiquitous fungal genus Trichoderma (Hypocreales, Ascomycota) requires understanding of its biology and evolution. Many Trichoderma species are used as biofertilizers and biofungicides and T. reesei is the model organism for industrial production of cellulolytic enzymes. In addition, some highly opportunistic species devastate mushroom farms and can become pathogens of humans. A comparative analysis of the first three whole genomes revealed mycoparasitism as the innate feature of Trichoderma. However, the evolution of these traits is not yet understood.ResultsWe selected 12 most commonly occurring Trichoderma species and studied the evolution of their genome sequences. Trichoderma evolved in the time of the Cretaceous-Palaeogene extinction event 66 (±15) mya, but the formation of extant sections (Longibrachiatum, Trichoderma) or clades (Harzianum/Virens) happened in Oligocene. The evolution of the Harzianum clade and section Trichoderma was accompanied by significant gene gain, but the ancestor of section Longibrachiatum experienced rapid gene loss. The highest number of genes gained encoded ankyrins, HET domain proteins and transcription factors. We also identified the Trichoderma core genome, completely curated its annotation, investigated several gene families in detail and compared the results to those of other fungi. Eighty percent of those genes for which a function could be predicted were also found in other fungi, but only 67% of those without a predictable function.ConclusionsOur study presents a time scaled pattern of genome evolution in 12 Trichoderma species from three phylogenetically distant clades/sections and a comprehensive analysis of their genes. The data offer insights in the evolution of a mycoparasite towards a generalist.

Weissella strains are often detected in spontaneously fermented foods. Because of their abilities to produce lactic acid and functional exopolysaccharides as well as their probiotic traits, Weissella spp. improve not only the sensorial properties but also nutritional values of the fermented food products. However, some Weissella species have been associated with human and animal diseases. In the era of vast genomic sequencing, new genomic/genome data are becoming available to the public on daily pace. Detailed genomic analyses are due to provide a full understanding of individual Weissella species. In this study, the genomes of six Weissella paramesenteroides strains were de novo sequenced. The genomes of 42 W. paramesenteroides strains were compared to discover their metabolic and functional potentials in food fermentation. Comparative genomics and metabolic pathway reconstructions revealed that W. paramesenteroides is a compact group of heterofermentative bacteria with good capacity of producing secondary metabolites and vitamin Bs. Since the strains rarely harbored plasmid DNA, they did not commonly possess the genes associated with bacteriocin production. All 42 strains were shown to bear vanT gene from the glycopeptide resistance gene cluster vanG. Yet none of the strains carried virulence genes. [ABSTRACT FROM AUTHOR]

Introduction: Free-living amoebae of the Naegleria genus belong to the major protist clade Heterolobosea and are ubiquitously distributed in soil and freshwater habitats. Of the 47 Naegleria species described, N. fowleri is the only one being pathogenic to humans, causing a rare but fulminant primary amoebic meningoencephalitis. Some Naegleria genome sequences are publicly available, but the genetic basis for Naegleria diversity and ability to thrive in diverse environments (including human brain) remains unclear. Methods: Herein, we constructed a high-quality Naegleria genus pangenome to obtain a comprehensive catalog of genes encoded by these amoebae. For this, we first sequenced, assembled, and annotated six new Naegleria genomes. Results and Discussion: Genome architecture analyses revealed that Naegleria may use genome plasticity features such as ploidy/aneuploidy to modulate their behavior in different environments. When comparing 14 near-to-complete genome sequences, our results estimated the theoretical Naegleria pangenome as a closed genome, with 13,943 genes, including 3,563 core and 10,380 accessory genes. The functional annotations revealed that a large fraction of Naegleria genes show significant sequence similarity with those already described in other kingdoms, namely Animalia and Plantae. Comparative analyses highlighted a remarkable genomic heterogeneity, even for closely related strains and demonstrate that Naegleria harbors extensive genome variability, reflected in different metabolic repertoires. If Naegleria core genome was enriched in conserved genes essential for metabolic, regulatory and survival processes, the accessory genome revealed the presence of genes involved in stress response, macromolecule modifications, cell signaling and immune response. Commonly reported N. fowleri virulence-associated genes were present in both core and accessory genomes, suggesting that N. fowleri’s ability to infect human brain could be related to its unique species-specific genes (mostly of unknown function) and/or to differential gene expression. The construction of Naegleria first pangenome allowed us to move away from a single reference genome (that does not necessarily represent each species as a whole) and to identify essential and dispensable genes in Naegleria evolution, diversity and biology, paving the way for further genomic and postgenomic studies. [ABSTRACT FROM AUTHOR]

Abstract Background With the exponential growth of publicly available genome sequences, pangenome analyses have provided increasingly complete pictures of genetic diversity for many microbial species. However, relatively few studies have scaled beyond single pangenomes to compare global genetic diversity both within and across different species. We present here several methods for “comparative pangenomics” that can be used to contextualize multi-pangenome scale genetic diversity with gene function for multiple species at multiple resolutions: pangenome shape, genes, sequence variants, and positions within variants. Results Applied to 12,676 genomes across 12 microbial pathogenic species, we observed several shared resolution-specific patterns of genetic diversity: First, pangenome openness is associated with species’ phylogenetic placement. Second, relationships between gene function and frequency are conserved across species, with core genomes enriched for metabolic and ribosomal genes and accessory genomes for trafficking, secretion, and defense-associated genes. Third, genes in core genomes with the highest sequence diversity are functionally diverse. Finally, certain protein domains are consistently mutation enriched across multiple species, especially among aminoacyl-tRNA synthetases where the extent of a domain’s mutation enrichment is strongly function-dependent. Conclusions These results illustrate the value of each resolution at uncovering distinct aspects in the relationship between genetic and functional diversity across multiple species. With the continued growth of the number of sequenced genomes, these methods will reveal additional universal patterns of genetic diversity at the pangenome scale.

IntroductionFree-living amoebae of the Naegleria genus belong to the major protist clade Heterolobosea and are ubiquitously distributed in soil and freshwater habitats. Of the 47 Naegleria species described, N. fowleri is the only one being pathogenic to humans, causing a rare but fulminant primary amoebic meningoencephalitis. Some Naegleria genome sequences are publicly available, but the genetic basis for Naegleria diversity and ability to thrive in diverse environments (including human brain) remains unclear.MethodsHerein, we constructed a high-quality Naegleria genus pangenome to obtain a comprehensive catalog of genes encoded by these amoebae. For this, we first sequenced, assembled, and annotated six new Naegleria genomes.Results and DiscussionGenome architecture analyses revealed that Naegleria may use genome plasticity features such as ploidy/aneuploidy to modulate their behavior in different environments. When comparing 14 near-to-complete genome sequences, our results estimated the theoretical Naegleria pangenome as a closed genome, with 13,943 genes, including 3,563 core and 10,380 accessory genes. The functional annotations revealed that a large fraction of Naegleria genes show significant sequence similarity with those already described in other kingdoms, namely Animalia and Plantae. Comparative analyses highlighted a remarkable genomic heterogeneity, even for closely related strains and demonstrate that Naegleria harbors extensive genome variability, reflected in different metabolic repertoires. If Naegleria core genome was enriched in conserved genes essential for metabolic, regulatory and survival processes, the accessory genome revealed the presence of genes involved in stress response, macromolecule modifications, cell signaling and immune response. Commonly reported N. fowleri virulence-associated genes were present in both core and accessory genomes, suggesting that N. fowleri’s ability to infect human brain could be related to its unique species-specific genes (mostly of unknown function) and/or to differential gene expression. The construction of Naegleria first pangenome allowed us to move away from a single reference genome (that does not necessarily represent each species as a whole) and to identify essential and dispensable genes in Naegleria evolution, diversity and biology, paving the way for further genomic and post-genomic studies.

NorA is one of the main native MDR efflux pumps of Staphylococcus aureus, contributing to reduced susceptibility towards fluoroquinolones and biocides, but little is known about its variability within S. aureus or its distribution and conservation among other staphylococci. We screened for sequences homologous to S. aureus norA and found it in 61 out of the 63 Staphylococcus species described. To the best of our knowledge, this is the first study to report the occurrence of norA across the Staphylococcus genus. The norA phylogenetic tree follows the evolutionary relations of staphylococci and the closely related Mammalliicoccus genus. Comparative analyses suggest a conservation of the NorA function in staphylococci. We also analyzed the variability of norA within S. aureus, for which there are several circulating norA alleles, differing up to 10% at the nucleotide level, which may hamper proper norA detection. We demonstrate the applicability of a PCR-based algorithm to detect and differentiate norA alleles in 52 S. aureus representing a wider collection of 89 isolates from different hosts. Our results highlight the prevalence of norAI and norAII in different settings and the association of norA alleles with specific S. aureus clonal lineages. Ultimately, it confirms the applicability of our PCR-based algorithm to rapidly detect and assign the different norA alleles, a trait that may impact antimicrobial efflux capacity and the search for potential NorA inhibitors. [ABSTRACT FROM AUTHOR]

Previous studies were mainly focused on genomic evolution and diversity of type species of Bifidobacterium genus due to their health-promoting effect on host. However, those studies were mainly based on species-level taxonomic resolution, adaptation, and characterization of carbohydrate metabolic features of the bifidobacterial species. Here, a comprehensive analysis of the type strain genome unveils the association of pan-genome evolution with the divergence of metabolic function of the Bifidobacterium genus. This study has also demonstrated that horizontal gene transfer, as well as genome expansion and reduction events, leads to the divergence of metabolic functions in Bifidobacterium genus. Furthermore, the genome-based search of probiotic traits among all the available bifidobacterial type strains gives hints on type species, that could confer health benefits to nutrient-deficient individuals. Altogether, the present study provides insight into the developments of genomic evolution, functional divergence, and potential probiotic type species of the Bifidobacterium genus. [ABSTRACT FROM AUTHOR]

Background: Moraxella bovis and Moraxella bovoculi both associate with infectious bovine keratoconjunctivitis (IBK), an economically significant and painful ocular disease that affects cattle worldwide. There are two genotypes of M. bovoculi (genotypes 1 and 2) that differ in their gene content and potential virulence factors, although neither have been experimentally shown to cause IBK. M. bovis is a causative IBK agent, however, not all strains carry a complete assortment of known virulence factors. The goals of this study were to determine the population structure and depth of M. bovis genomic diversity, and to compare core and accessory genes and predicted outer membrane protein profiles both within and between M. bovis and M. bovoculi. Results: Phylogenetic trees and bioinformatic analyses of 36 M. bovis chromosomes sequenced in this study and additional available chromosomes of M. bovis and both genotype 1 and 2 M. bovoculi, showed there are two genotypes (1 and 2) of M. bovis. The two M. bovis genotypes share a core of 2015 genes, with 121 and 186 genes specific to genotype 1 and 2, respectively. The two genotypes differ by their chromosome size and prophage content, encoded protein variants of the virulence factor hemolysin, and by their affiliation with different plasmids. Eight plasmid types were identified in this study, with types 1 and 6 observed in 88 and 56% of genotype 2 strains, respectively, and absent from genotype 1 strains. Only type 1 plasmids contained one or two gene copies encoding filamentous haemagglutinin-like proteins potentially involved with adhesion. A core of 1403 genes was shared between the genotype 1 and 2 strains of both M. bovis and M. bovoculi, which encoded a total of nine predicted outer membrane proteins. Conclusions: There are two genotypes of M. bovis that differ in both chromosome content and plasmid profiles and thus may not equally associate with IBK. Immunological reagents specifically targeting select genotypes of M. bovis, or all genotypes of M. bovis and M. bovoculi together could be designed from the outer membrane proteins identified in this study. [ABSTRACT FROM AUTHOR]

ABSTRACT Staphylococcus aureus is a Gram-positive pathogen that causes various infections in humans and domestic animals. In China, S. aureus is the most common Gram-positive pathogen that causes clinical infections. However, there are few comprehensive genome-based molecular epidemiology studies to investigate the genotypic background of the major S. aureus clones that are epidemic in China. Here, four S. aureus isolates that were recovered from hospital personnel were sequenced. In combination with whole-genome sequencing (WGS) data of 328 S. aureus strains as references, we performed a comprehensive molecular epidemiology study to reveal the molecular epidemic characterization of S. aureus that is epidemic in China. It was found that 332 S. aureus isolates were phylogenetically categorized into 4 major epidemic groups with different epidemiology phenotypes. Each group has exclusive features in virulence genotypic profiles, antimicrobial resistance genotypic profiles, core and pangenome features representing the differences involved in genetic features, evolutionary processes, and potential future evolutionary directions. Moreover, a comparative core genome analysis of 332 S. aureus isolates indicated several key genes that contributed to differences in molecular epidemic characterization and promoted the adaptive evolutionary process of each group. This study provides a comprehensive understanding of molecular epidemiological characteristics and adaptive evolutionary directions of major S. aureus clones that are epidemic in China. IMPORTANCE Staphylococcus aureus is an important Gram-positive pathogen that is epidemic worldwide and causes various infections in humans and domestic animals. However, there has been relatively little research on comprehensive molecular epidemiology in China. In this research, we reconstructed the phylogenetic relationship based on whole-genome data of strains almost all over China, screened for resistance and virulence genes, and took core and pan genome analysis to perform a comprehensive molecular epidemiology study of S. aureus that is epidemic in China. Our results highlight that there are 4 major epidemic groups with different epidemiology phenotypes after phylogenetic categorization with exclusive genetic features in virulence genotypic profiles, antimicrobial-resistance genotypic profiles, and core and pangenome features, and we found key gene features involved in epidemic transmission and adaptive evolution. Our findings are critical in describing molecular characteristic profiles of S. aureus infection, which could update existing preventive measures and take appropriate strategies.

Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well studied in terms of physiology but whose evolutionary history and taxonomy are unclear. Recent work has suggested that Methylobacterium is much more diverse than thought previously, questioning its status as an ecologically and phylogenetically coherent taxonomic genus. However, taxonomic and evolutionary studies of Methylobacterium have mostly been restricted to model species, often isolated from habitats other than the phyllosphere and have yet to utilize comprehensive phylogenomic methods to examine gene trees, gene content, or synteny. By analyzing 189 Methylobacterium genomes from a wide range of habitats, including the phyllosphere, we inferred a robust phylogenetic tree while explicitly accounting for the impact of horizontal gene transfer (HGT). We showed that Methylobacterium contains four evolutionarily distinct groups of bacteria (namely A, B, C, D), characterized by different genome size, GC content, gene content, and genome architecture, revealing the dynamic nature of Methylobacterium genomes. In addition to recovering 59 described species, we identified 45 candidate species, mostly phyllosphere-associated, stressing the significance of plants as a reservoir of Methylobacterium diversity. We inferred an ancient transition from a free-living lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups (A, B, D), whose early branching in Methylobacterium history has been heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandonment of Methylorubrum. [ABSTRACT FROM AUTHOR]

Understanding the biochemistry and metabolic pathways of cyanide degradation is necessary to improve the efficacy of cyanide bioremediation processes and industrial requirements. We have isolated and sequenced the genome of a cyanide-degrading Bacillus strain from water in contact with mine tailings from Lima, Peru. This strain was classified as Bacillus safensis based on 16S rRNA gene sequencing and core genome analyses and named B. safensis PER-URP-08. We searched for possible cyanide-degradation enzymes in the genome of this strain and identified a putative cyanide dihydratase (CynD) gene similar to a previously characterized CynD from Bacillus pumilus C1. Sequence analysis of CynD from B. safensis and B. pumilus allow us to identify C-terminal residues that differentiate both CynDs. We then cloned, expressed in Escherichia coli, and purified recombinant CynD from B. safensis PER-URP-08 (CynDPER-URP-08) and showed that in contrast to CynD from B. pumilus C1, this recombinant CynD remains active at up to pH 9. We also showed that oligomerization of CynDPER-URP-08 decreases as a function of increased pH. Finally, we demonstrated that transcripts of CynDPER-URP-08 in B. safensis PER-URP-08 are strongly induced in the presence of cyanide. Our results suggest that the use of B. safensis PER-URP-08 and CynDPER-URP-08 as potential tool for cyanide bioremediation warrants further investigation. IMPORTANCE Despite being of environmental concern around the world due to its toxicity, cyanide continues to be used in many important industrial processes. Thus, searching for cyanide bioremediation methods is a matter of societal concern and must be present on the political agenda of all governments. Here, we report the isolation, genome sequencing and characterization of cyanide degradation capacity of a bacterial strain isolated from an industrial mining site in Peru. We characterize a cyanide dehydratase (CynD) homolog from one of these bacteria, Bacillus safensis PER-URP-08. [ABSTRACT FROM AUTHOR]

Fusarium circinatum is an important global pathogen of pine trees. Genome plasticity has been observed in different isolates of the fungus, but no genome comparisons are available. To address this gap, we sequenced and assembled to chromosome level five isolates of F. circinatum. These genomes were analysed together with previously published genomes of F. circinatum isolates, FSP34 and KS17. Multi-sample variant calling identified a total of 461,683 micro variants (SNPs and small indels) and a total of 1828 macro structural variants of which 1717 were copy number variants and 111 were inversions. The variant density was higher on the sub-telomeric regions of chromosomes. Variant annotation revealed that genes involved in transcription, transport, metabolism and transmembrane proteins were overrepresented in gene sets that were affected by high impact variants. A core genome representing genomic elements that were conserved in all the isolates and a non-redundant pangenome representing all genomic elements is presented. Whole genome alignments showed that an average of 93% of the genomic elements were present in all isolates. The results of this study reveal that some genomic elements are not conserved within the isolates and some variants are high impact. The described genome-scale variations will help to inform novel disease management strategies against the pathogen. [ABSTRACT FROM AUTHOR]

Species belonging to the genus Novosphingobium are found in many different habitats and have been identified as metabolically versatile. Through comparative genomic analysis, we identified habitat-specific genes and regulatory hubs that could determine habitat selection for Novosphingobium spp. Genomes from 27 Novosphingobium strains isolated from diverse habitats such as rhizosphere soil, plant surfaces, heavily contaminated soils, and marine and freshwater environments were analyzed. Genome size and coding potential were widely variable, differing significantly between habitats. Phylogenetic relationships between strains were less likely to describe functional genotype similarity than the habitat from which they were isolated. In this study, strains (19 out of 27) with a recorded habitat of isolation, and at least 3 representative strains per habitat, comprised four ecological groups-rhizosphere, contaminated soil, marine, and freshwater. Sulfur acquisition and metabolism were the only core genomic traits to differ significantly in proportion between these ecological groups; for example, alkane sulfonate (ssuABCD) assimilation was found exclusively in all of the rhizospheric isolates. When we examined osmolytic regulation in Novosphingobium spp. through ectoine biosynthesis, which was assumed to be marine habitat specific, we found that it was also present in isolates from contaminated soil, suggesting its relevance beyond the marine system. Novosphingobium strains were also found to harbor a wide variety of mono- and dioxygenases, responsible for the metabolism of several aromatic compounds, suggesting their potential to act as degraders of a variety of xenobiotic compounds. Protein-protein interaction analysis revealed β-barrel outer membrane proteins as habitat-specific hubs in each of the four habitats-freshwater (Saro_1868), marine water (PP1Y_AT17644), rhizosphere (PMI02_00367), and soil (V474_17210). These outer membrane proteins could play a key role in habitat demarcation and extend our understanding of the metabolic versatility of the Novosphingobium species. IMPORTANCE This study highlights the significant role of a microorganism's genetic repertoire in structuring the similarity between Novosphingobium strains. The results suggest that the phylogenetic relationships were mostly influenced by metabolic trait enrichment, which is possibly governed by the microenvironment of each microbe's respective niche. Using core genome analysis, the enrichment of a certain set of genes specific to a particular habitat was determined, which provided insights on the influence of habitat on the distribution of metabolic traits in Novosphingobium strains. We also identified habitat-specific protein hubs, which suggested delineation of Novosphingobium strains based on their habitat. Examining the available genomes of ecologically diverse bacterial species and analyzing the habitat-specific genes are useful for understanding the distribution and evolution of functional and phylogenetic diversity in the genus Novosphingobium.

The availability of multiple gene sequences, and in particular full genome sequence data, for microbial strains has changed how taxonomists delineate subspecies belonging to the Archaea and Bacteria. Well-defined phylogenetic lineages that share higher genome similarity values compared to the widely used species thresholds are often described as subspecies, despite clear evidence of genetic isolation between them. These well-defined lineages, reflecting notable genetic isolation of the core genome represent more recently evolved, unique and sui generis evolutionary units. Because they bear all of the hallmarks of species, most contemporary subspecies likely represent species in their own right. Although there is considerable value in defining intraspecies variation (e.g., pathovar, serovar and symbiovar), the discriminating properties of such units are mostly encoded on accessory subgenomic compartments. We therefore argue that the taxonomic category of subspecies has become irrelevant and propose that its use should be discontinued. This will minimize inconsistencies related to the subjective nature of species-subspecies distinctions. Formal recognition of biologically relevant variation within species based on the accessory genome information will have practical significance in fields such as clinical, industrial and agricultural microbiology.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and responsible for causing life-threatening infections. The emergence of hypervirulent and multidrug-resistant (MDR) S. aureus strains led to challenging issues in antibiotic therapy. Consequently, the morbidity and mortality rates caused by S. aureus infections have a substantial impact on health concerns. The current worldwide prevalence of MRSA infections highlights the need for long-lasting preventive measures and strategies. Unfortunately, effective measures are limited. In this study, we focus on the identification of vaccine candidates and drug target proteins against the 16 strains of MRSA using reverse vaccinology and subtractive genomics approaches. Using the reverse vaccinology approach, 4 putative antigenic proteins were identified; among these, PrsA and EssA proteins were found to be more promising vaccine candidates. We applied a molecular docking approach of selected 8 drug target proteins with the drug-like molecules, revealing that the ZINC4235426 as potential drug molecule with favorable interactions with the target active site residues of 5 drug target proteins viz., biotin protein ligase, HPr kinase/phosphorylase, thymidylate kinase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-L-lysine ligase, and pantothenate synthetase. Thus, the identified proteins can be used for further rational drug or vaccine design to identify novel therapeutic agents for the treatment of multidrug-resistant staphylococcal infection. [ABSTRACT FROM AUTHOR]

Abstract Background The quantity of genomic data is expanding at an increasing rate. Tools for phylogenetic analysis which scale to the quantity of available data are required. To address this need, we present cognac, a user-friendly software package to rapidly generate concatenated gene alignments for phylogenetic analysis. Results We illustrate that cognac is able to rapidly identify phylogenetic marker genes using a data driven approach and efficiently generate concatenated gene alignments for very large genomic datasets. To benchmark our tool, we generated core gene alignments for eight unique genera of bacteria, including a dataset of over 11,000 genomes from the genus Escherichia producing an alignment with 1353 genes, which was constructed in less than 17 h. Conclusions We demonstrate that cognac presents an efficient method for generating concatenated gene alignments for phylogenetic analysis. We have released cognac as an R package ( https://github.com/rdcrawford/cognac ) with customizable parameters for adaptation to diverse applications.

Introduction. Listeriosis is a foodborne infection, especially dangerous for people in at-risk groups. Susceptibility to listeria infection is determined by a complex of reasons: environmental factors, host immune status, and pathogen virulence. The susceptibility to listeriosis can also be aggravated by previous infections, especially viral infections, which demonstrate a steadily increasing number of identified pathogens.The aim of our study was to present molecular and genetic characterization of pathogens causing sporadic invasive listeriosis in a megalopolis, primarily during the peak of influenza and ARVI incidence.Materials and methods. Listeria monocytogenes isolates were collected from 18 hospitalized patients at hospitals in Moscow, from November 2018 to October 2019. The first comparison group was represented by isolates from food products and fish preserves. The second comparison group included previously examined environmental isolates. The clinical isolates were examined by using multilocus sequence typing techniques, including the standard MLST scheme extended by loci of internalin genes. Isolates of the autochthonous genotype (ST7) were compared through whole-genome sequencing and subsequent analysis of the core genome (cgMLST).Results. In cases of invasive listeriosis, 44% of isolates were isolated from patients with listeriosis; 27% of isolates were obtained from patients with meningitis. L. monocytogenes of phylogenetic lineage II prevailed in these groups of cases that occurred when the epidemic threshold for influenza was crossed during the 2018/2019 season. Listeria pneumonia identified in the senior age group occurred during the season of autumn ARVI and was primarily caused by L. monocytogenes of phylogenetic lineage I. The examination of genomes of ST7 isolates demonstrated identity between the core genomes of bacteria isolated from the mother-infant pair. Out of ST7 food isolates most closely related to the clinical ones was the isolate from meat (23 locus differences, the common deletion in the MFS transporter locus). Analyzing invasive listeriosis, the comparison between the list of the identified genotypes and the data from European countries showed that each country had its own specific range of genotypes, though ST7 was detected in all the examined samples.Conclusions. Along with the monitoring of food manufacturing and storage, timely vaccination against seasonal respiratory infections and use of personal protective equipment in public spaces can reduce the risk of listeriosis incidence in at-risk groups.

Probiotics are health-beneficial microorganisms with mainly immunomodulatory and anti-inflammatory properties. Lactobacillus delbrueckii species is a common bacteria used in the dairy industry, and their benefits to hosting health have been reported. This study analyzed the core genome of nine strains of L. delbrueckii species with documented probiotic properties, focusing on genes related to their host health benefits. For this, a combined methodology including several software and databases (BPGA, SPAAN, BAGEL4, BioCyc, KEEG, and InterSPPI) was used to predict the most important characteristics related to L. delbrueckii strains probiose. Comparative genomics analyses revealed that L. delbrueckii probiotic strains shared essential genes related to acid and bile stress response and antimicrobial activity. Other standard features shared by these strains are surface layer proteins and extracellular proteins-encoding genes, with high adhesion profiles that interacted with human proteins of the inflammatory signaling pathways (TLR2/4-MAPK, TLR2/4-NF-κB, and NOD-like receptors). Among these, the PrtB serine protease appears to be a strong candidate responsible for the anti-inflammatory properties reported for these strains. Furthermore, genes with high proteolytic and metabolic activity able to produce beneficial metabolites, such as acetate, bioactive peptides, and B-complex vitamins were also identified. These findings suggest that these proteins can be essential in biological mechanisms related to probiotics’ beneficial effects of these strains in the host.

ABSTRACT The bacterial accessory genome provides the genetic flexibility needed to facilitate environment and host adaptation. In Neisseria gonorrhoeae, known accessory elements include plasmids which can transfer and mediate antimicrobial resistance (AMR); however, chromosomal accessory genes could also play a role in AMR. Here, the gonococcal accessory genome was characterized using gene-by-gene approaches and its association with the core genome and AMR were assessed. The gonococcal accessory gene pool consisted of 247 genes, which were mainly genes located on large mobile genetic elements, phage associated genes, or genes encoding putative secretion systems. Accessory elements showed similar synteny across genomes, indicating either a predisposition for particular genomic locations or ancestral inheritance that are conserved during strain expansion. Significant associations were found between the prevalence of accessory elements and core genome multi-locus sequence types (cgMLST), consistent with a structured gonococcal population despite frequent horizontal gene transfer (HGT). Increased prevalence of putative DNA exchange regulators was significantly associated with AMR, which included a putative secretion system, methyltransferases and a toxin-antitoxin system. Although frequent HGT results in high genetic diversity in the gonococcus, we found that this is mediated by a small gene pool. In fact, a highly organized genome composition was identified with a strong association between the accessory and core genome. Increased prevalence of DNA exchange regulators in antimicrobial resistant isolates suggests that genetic material exchange plays a role in the development or maintenance of AMR. These findings enhance our understanding of gonococcal genome architecture and have important implications for gonococcal population biology. IMPORTANCE The emergence of antimicrobial resistance (AMR) against third generation cephalosporins in Neisseria gonorrhoeae is a major public health concern, as these are antibiotics of last resort for the effective treatment of gonorrhea. Although the resistance mechanisms against this class of antibiotics have not been entirely resolved, resistance against other classes of antibiotics, such as tetracyclines, is known to be mediated through plasmids, which are known gonococcal extra-chromosomal accessory elements. A complete assessment of the chromosomal accessory genome content and its role in AMR has not yet been undertaken. Here, we comprehensively characterize the gonococcal accessory genome to better understand genome architecture as well as the evolution and mechanisms of AMR in this species.

Vibrio vulnificus is an opportunistic pathogen that naturally inhabits sea water globally and is responsible for most vibriosis-related deaths. The consumption of V. vulnificus contaminated seafood and exposure of wounds to Vibrio can result in systemic infection, with increased risks of amputation and extremely high rates of mortality. However, the pathogenicity and virulence factors of V. vulnificus are not fully understood. The genomic characterization of V. vulnificus will be helpful to extend our understanding on V. vulnificus at a genomic level. In this manuscript, the genome of V. vulnificus strain MCCC 1A08743 isolated from contaminated prawns from Zhanjiang, China, was sequenced using Illumina HiSeq X Ten system and annotated through multiple databases. The strain MCCC 1A08743 genome included 4371 protein-coding genes and 117 RNA genes. Average nucleotide identity analysis and core genome phylogenetic analysis revealed that MCCC 1A08743 was most closely related to strains from clinical samples from the United States. Pathogenicity annotation of the MCCC 1A08743 genome, using Virulence Factor Database and Pathogen-Host Interactions database, predicted the pathogenicity of the strain, and this was confirmed using mice infection experiments, which indicated that V. vulnificus strain MCCC 1A08743 could infect C57BL/6J mice and cause liver lesions. This article has an associated First Person interview with the first author of the paper.