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ABSTRACT Despite the importance of intra-species variants of viruses for causing disease and/or disrupting ecosystem functioning, there is no universally applicable standard to define these. A (natural) gap in whole-genome average nucleotide identity (ANI) values around 95% is commonly used to define species, especially for bacteriophages, but whether a similar gap exists within species that can be used to define intra-species units has not been evaluated yet. Whole-genome comparisons among members of 1,016 bacteriophage (Caudoviricetes) species revealed a region of low frequency of ANI values around 99.2%–99.8%, showing threefold or fewer pairs than expected for an even distribution. This second gap is prevalent in viruses infecting various cultured or uncultured hosts from a variety of environments, although a few exceptions to this pattern were also observed (3.7% of total species) and are likely attributed to cultivation biases or other factors. Similar results were observed for a limited set of eukaryotic viruses that are adequately sampled, including SARS-CoV-2, whose ANI-based clusters matched well with the WHO-defined variants of concern, indicating that our findings from bacteriophages might be more broadly applicable and the ANI-based clusters may represent functionally and/or ecologically distinct units. These units appear to be predominantly driven by (high) ecological cohesiveness coupled to either frequent recombination for bacteriophages or selection and clonal evolution for other viruses such as SARS-CoV-2, indicating that fundamentally different underlying mechanisms could lead to similar diversity patterns. Accordingly, we propose the ANI gap approach outlined above for defining viral intra-species units, for which we propose the term genomovars. IMPORTANCEViral species are composed of an ensemble of intra-species variants whose individual dynamics may have major implications for human and animal health and/or ecosystem functioning. However, the lack of universally accepted standards to define these intra-species variants has led researchers to use different approaches for this task, creating inconsistent intra-species units across different viral families and confusion in communication. By comparing hundreds of mostly bacteriophage genomes, we show that there is a widely distributed natural gap in whole-genome average nucleotide identity values in most, but not all, of these species that can be used to define intra-species units. Therefore, these results advance the molecular toolbox for tracking viral intra-species units and should facilitate future epidemiological and environmental studies. 

ABSTRACT Root nodulating rhizobia are nearly ubiquitous in soils and provide the critical service of nitrogen fixation to thousands of legume species, including staple crops. However, the magnitude of fixed nitrogen provided to hosts varies markedly among rhizobia strains, despite host legumes having mechanisms to selectively reward beneficial strains and to punish ones that do not fix sufficient nitrogen. Variation in the services of microbial mutualists is considered paradoxical given host mechanisms to select beneficial genotypes. Moreover, the recurrent evolution of non-fixing symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. Here, we deconstructed hundreds of genome sequences from genotypically and phenotypically diverse Bradyrhizobium strains and revealed mechanisms that generate variation in symbiotic nitrogen fixation. We show that this trait is conferred by a modular system consisting of many extremely large integrative conjugative elements and few conjugative plasmids. Their transmissibility and propensity to reshuffle genes generate new combinations that lead to uncooperative genotypes and make individual partnerships unstable. We also demonstrate that these same properties extend beneficial associations to diverse host species and transfer symbiotic capacity among diverse strains. Hence, symbiotic nitrogen fixation is underpinned by modularity, which engenders flexibility, a feature that reconciles evolutionary robustness and instability. These results provide new insights into mechanisms driving the evolution of mobile genetic elements. Moreover, they yield a new predictive model on the evolution of rhizobial symbioses, one that informs on the health of organisms and ecosystems that are hosts to symbionts and that helps resolve the long-standing paradox. IMPORTANCE Genetic variation is fundamental to evolution yet is paradoxical in symbiosis. Symbionts exhibit extensive variation in the magnitude of services they provide despite hosts having mechanisms to select and increase the abundance of beneficial genotypes. Additionally, evolution of uncooperative symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. We analyzed genome sequences of Bradyrhizobium, bacteria that in symbioses with legume hosts, fix nitrogen, a nutrient essential for ecosystems. We show that genes for symbiotic nitrogen fixation are within elements that can move between bacteria and reshuffle gene combinations that change host range and quality of symbiosis services. Consequently, nitrogen fixation is evolutionarily unstable for individual partnerships, but is evolutionarily stable for legume- Bradyrhizobium symbioses in general. We developed a holistic model of symbiosis evolution that reconciles robustness and instability of symbiosis and informs on applications of rhizobia in agricultural settings. 

ABSTRACT Enterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar discs, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by six Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA , tig , OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions and identifies multiple new genetic determinants of biofilm formation. IMPORTANCE E. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.