Expanding standards in viromics: in silico evaluation of dsDNA viral genome identification, classification, and auxiliary metabolic gene curation.

BACKGROUND: Viruses influence global patterns of microbial diversity and nutrient cycles. Though viral metagenomics (viromics), specifically targeting dsDNA viruses, has been critical for revealing viral roles across diverse ecosystems, its analyses differ in many ways from those used for microbes. To date, viromics benchmarking has covered read pre-processing, assembly, relative abundance, read mapping thresholds and diversity estimation, but other steps would benefit from benchmarking and standardization. Here we use in silico-generated datasets and an extensive literature survey to evaluate and highlight how dataset composition (i.e., viromes vs bulk metagenomes) and assembly fragmentation impact (i) viral contig identification tool, (ii) virus taxonomic classification, and (iii) identification and curation of auxiliary metabolic genes (AMGs). RESULTS: The in silico benchmarking of five commonly used virus identification tools show that gene-content-based tools consistently performed well for long (≥3 kbp) contigs, while k-mer- and blast-based tools were uniquely able to detect viruses from short (≤3 kbp) contigs. Notably, however, the performance increase of k-mer- and blast-based tools for short contigs was obtained at the cost of increased false positives (sometimes up to ∼5% for virome and ∼75% bulk samples), particularly when eukaryotic or mobile genetic element sequences were included in the test datasets. For viral classification, variously sized genome fragments were assessed using gene-sharing network analytics to quantify drop-offs in taxonomic assignments, which revealed correct assignations ranging from ∼95% (whole genomes) down to ∼80% (3 kbp sized genome fragments). A similar trend was also observed for other viral classification tools such as VPF-class, ViPTree and VIRIDIC, suggesting that caution is warranted when classifying short genome fragments and not full genomes. Finally, we highlight how fragmented assemblies can lead to erroneous identification of AMGs and outline a best-practices workflow to curate candidate AMGs in viral genomes assembled from metagenomes. CONCLUSION: Together, these benchmarking experiments and annotation guidelines should aid researchers seeking to best detect, classify, and characterize the myriad viruses 'hidden' in diverse sequence datasets.

Viruses infecting a warm water picoeukaryote shed light on spatial co-occurrence dynamics of marine viruses and their hosts.

The marine picoeukaryote Bathycoccus prasinos has been considered a cosmopolitan alga, although recent studies indicate two ecotypes exist, Clade BI (B. prasinos) and Clade BII. Viruses that infect Bathycoccus Clade BI are known (BpVs), but not that infect BII. We isolated three dsDNA prasinoviruses from the Sargasso Sea against Clade BII isolate RCC716. The BII-Vs do not infect BI, and two (BII-V2 and BII-V3) have larger genomes (~210 kb) than BI-Viruses and BII-V1. BII-Vs share ~90% of their proteins, and between 65% to 83% of their proteins with sequenced BpVs. Phylogenomic reconstructions and PolB analyses establish close-relatedness of BII-V2 and BII-V3, yet BII-V2 has 10-fold higher infectivity and induces greater mortality on host isolate RCC716. BII-V1 is more distant, has a shorter latent period, and infects both available BII isolates, RCC716 and RCC715, while BII-V2 and BII-V3 do not exhibit productive infection of the latter in our experiments. Global metagenome analyses show Clade BI and BII algal relative abundances correlate positively with their respective viruses. The distributions delineate BI/BpVs as occupying lower temperature mesotrophic and coastal systems, whereas BII/BII-Vs occupy warmer temperature, higher salinity ecosystems. Accordingly, with molecular diagnostic support, we name Clade BII Bathycoccus calidus sp. nov. and propose that molecular diversity within this new species likely connects to the differentiated host-virus dynamics observed in our time course experiments. Overall, the tightly linked biogeography of Bathycoccus host and virus clades observed herein supports species-level host specificity, with strain-level variations in infection parameters.

The microbial community from the early-plant colonizer (Baccharis linearis) is required for plant establishment on copper mine tailings.

Plants must deal with harsh environmental conditions when colonizing abandoned copper mine tailings. We hypothesized that the presence of a native microbial community can improve the colonization of the pioneer plant, Baccharis linearis, in soils from copper mining tailings. Plant growth and microbial community compositions and dynamics were determined in cultivation pots containing material from two abandoned copper mining tailings (Huana and Tambillos) and compared with pots containing fresh tailings or surrounding agricultural soil. Controls without plants or using irradiated microbe-free substrates, were also performed. Results indicated that bacteria (Actinobacteria, Gammaproteobacteria, and Firmicutes groups) and fungi (Glomus genus) are associated with B. linearis and may support plant acclimation, since growth parameters decreased in both irradiated (transiently without microbial community) and fresh tailing substrates (with a significantly different microbial community). Consistently, the composition of the bacterial community from abandoned copper mining tailings was more impacted by plant establishment than by differences in the physicochemical properties of the substrates. Bacteria located at B. linearis rhizoplane were clearly the most distinct bacterial community compared with those of fresh tailings, surrounding soil and non-rhizosphere abandoned tailings substrates. Beta diversity analyses showed that the rhizoplane bacterial community changed mainly through species replacement (turnover) than species loss (nestedness). In contrast, location/geographical conditions were more relevant than interaction with the plants, to explain fungal community differences.

Genome-resolved viral ecology in a marine oxygen minimum zone.

Oxygen minimum zones (OMZs) are critical to marine nitrogen cycling and global climate change. While OMZ microbial communities are relatively well-studied, little is known about their viruses. Here, we assess the viral community ecology of 22 deeply sequenced viral metagenomes along a gradient of oxygenated to anoxic waters (<0.02 µmol/l O2 ) in the Eastern Tropical South Pacific (ETSP) OMZ. We identified 46 127 viral populations (≥5 kb), which augments the known viruses from ETSP by 10-fold. Viral communities clustered into six groups that correspond to oceanographic features. Oxygen concentration was the predominant environmental feature driving viral community structure. Alpha and beta diversity of viral communities in the anoxic zone were lower than in surface waters, which parallels the low microbial diversity seen in other studies. ETSP viruses were largely endemic, with the majority of shared viruses (87%) also present in other OMZ samples. We detected 543 putative viral-encoded auxiliary metabolic genes (AMGs), of which some have a distribution that reflects physico-chemical characteristics across depth. Together these findings provide an ecological baseline for viral community structure, drivers and population variability in OMZs that will help future studies assess the role of viruses in these climate-critical environments.

Characterization of pHeBE7, an IncFII-type virulence-resistance plasmid carrying blaCTX-M-98b, blaTEM-1, and rmtB genes, detected in Escherichia coli from a chicken isolate in China.

Recently, a novel variant of the CTX-M enzyme, CTX-M-98, was detected in Escherichia coli isolates from food animals. However, few plasmids carrying blaCTX-M-98 have been fully characterized. In this study, we sequenced the complete pHeBE7 plasmid, an 86,015-bp plasmid that contains the blaCTX-M-98b, blaTEM-1, rmtB, and traT genes, using whole-genome sequencing. The backbone of pHeBE7 shows a high similarity (>99%) to pMC-NDM, which carries the blaNDM-1 gene, however its mosaic regions remain relatively unique among sequenced plasmids. We discovered that a typical ISEcp1-blaCTX-M-IS903 element in the mosaic region harbors the blaCTX-M-98b gene. Conjugation and growth competition assays indicate that pHeBE7 can be easily transmitted and that it confers a limited fitness cost to the recipient cell. The genetic characterization of pHeBE7 may improve our knowledge of how antibiotic resistance disseminates in enterobacteria.