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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 Recent genomic analyses have revealed that microbial communities are predominantly composed of persistent, sequence-discrete species and intraspecies units (genomovars), but the mechanisms that create and maintain these units remain unclear. By analyzing closely-related isolate genomes from the same or related samples and identifying recent recombination events using a novel bioinformatics methodology, we show that high ecological cohesiveness coupled to frequent-enough and unbiased (i.e., not selection-driven) horizontal gene flow, mediated by homologous recombination, often underlie these diversity patterns. Ecological cohesiveness was inferred based on greater similarity in temporal abundance patterns of genomes of the same vs. different units, and recombination was shown to affect all sizable segments of the genome (i.e., be genome-wide) and have two times or greater impact on sequence evolution than point mutations. These results were observed in bothSalinibacter ruber, an environmental halophilic organism, andEscherichia coli, the model gut-associated organism and an opportunistic pathogen, indicating that they may be more broadly applicable to the microbial world. Therefore, our results represent a departure compared to previous models of microbial speciation that invoke either ecology or recombination, but not necessarily their synergistic effect, and answer an important question for microbiology: what a species and a subspecies are. 

Abstract Genome search and/or classification typically involves finding the best-match database (reference) genomes and has become increasingly challenging due to the growing number of available database genomes and the fact that traditional methods do not scale well with large databases. By combining k-mer hashing-based probabilistic data structures (i.e. ProbMinHash, SuperMinHash, Densified MinHash and SetSketch) to estimate genomic distance, with a graph based nearest neighbor search algorithm (Hierarchical Navigable Small World Graphs, or HNSW), we created a new data structure and developed an associated computer program, GSearch, that is orders of magnitude faster than alternative tools while maintaining high accuracy and low memory usage. For example, GSearch can search 8000 query genomes against all available microbial or viral genomes for their best matches (n = ∼318 000 or ∼3 000 000, respectively) within a few minutes on a personal laptop, using ∼6 GB of memory (2.5 GB via SetSketch). Notably, GSearch has an O(log(N)) time complexity and will scale well with billions of genomes based on a database splitting strategy. Further, GSearch implements a three-step search strategy depending on the degree of novelty of the query genomes to maximize specificity and sensitivity. Therefore, GSearch solves a major bottleneck of microbiome studies that require genome search and/or classification. 

Abstract Whether prokaryotes, and other microorganisms, form distinct clusters that can be recognized as species remains an issue of paramount theoretical as well as practical consequence in identifying, regulating, and communicating about these organisms. In the past decade, comparisons of thousands of genomes of isolates and hundreds of metagenomes have shown that prokaryotic diversity may be predominantly organized in such sequence‐discrete clusters, albeit organisms of intermediate relatedness between the identified clusters are also frequently found. Accumulating evidence suggests, however, that the latter “intermediate” organisms show enough ecological and/or functional distinctiveness to be considered different species. Notably, the area of discontinuity between clusters often—but not always—appears to be around 85%–95% genome‐average nucleotide identity, consistently among different taxa. More recent studies have revealed remarkably similar diversity patterns for viruses and microbial eukaryotes as well. This high consistency across taxa implies a specific mechanistic process that underlies the maintenance of the clusters. The underlying mechanism may be a substantial reduction in the efficiency of homologous recombination, which mediates (successful) horizontal gene transfer, around 95% nucleotide identity. Deviations from the 95% threshold (e.g., species showing lower intraspecies diversity) may be caused by ecological differentiation that imposes barriers to otherwise frequent gene transfer. While this hypothesis that clusters are driven by ecological differentiation coupled to recombination frequency (i.e., higher recombination within vs. between groups) is appealing, the supporting evidence remains anecdotal. The data needed to rigorously test the hypothesis toward advancing the species concept are also outlined. 

Abstract Despite increasing efforts across various disciplines, the fate, transport, and impact of synthetic plastics on the environment and public health remain poorly understood. To better elucidate the microbial ecology of plastic waste and its potential for biotransformation, we conducted a large-scale analysis of all publicly available meta-omic studies investigating plastics (n = 27) in the environment. Notably, we observed low prevalence of known plastic degraders throughout most environments, except for substantial enrichment in riverine systems. This indicates rivers may be a highly promising environment for discovery of novel plastic bioremediation products. Ocean samples associated with degrading plastics showed clear differentiation from non-degrading polymers, showing enrichment of novel putative biodegrading taxa in the degraded samples. Regarding plastisphere pathogenicity, we observed significant enrichment of antimicrobial resistance genes on plastics but not of virulence factors. Additionally, we report a co-occurrence network analysis of 10 + million proteins associated with the plastisphere. This analysis revealed a localized sub-region enriched with known and putative plastizymes—these may be useful for deeper investigation of nature’s ability to biodegrade man-made plastics. Finally, the combined data from our meta-analysis was used to construct a publicly available database, the Plastics Meta-omic Database (PMDB)—accessible at plasticmdb.org. These data should aid in the integrated exploration of the microbial plastisphere and facilitate research efforts investigating the fate and bioremediation potential of environmental plastic waste. 

Abstract Nitrous oxide (N₂O) is a climate-active gas with emissions predicted to increase due to agricultural intensification. Microbial reduction of N₂O to dinitrogen (N₂) is the major consumption process but microbial N₂O reduction under acidic conditions is considered negligible, albeit strongly acidic soils harbornosZgenes encoding N₂O reductase. Here, we study a co-culture derived from acidic tropical forest soil that reduces N₂O at pH 4.5. The co-culture exhibits bimodal growth with aSerratiasp. fermenting pyruvate followed by hydrogenotrophic N₂O reduction by aDesulfosporosinussp. Integrated omics and physiological characterization revealed interspecies nutritional interactions, with the pyruvate fermentingSerratiasp. supplying amino acids as essential growth factors to the N₂O-reducingDesulfosporosinussp. Thus, we demonstrate growth-linked N₂O reduction between pH 4.5 and 6, highlighting microbial N₂O reduction potential in acidic soils.