Isolation of phages infecting the abundant freshwater Actinobacteriota order 'Ca. Nanopelagicales'.

Low-GC Actinobacteriota of the order 'Ca. Nanopelagicales' (also known as acI or hgcI clade) are abundant in freshwaters around the globe. Extensive predation pressure by phages has been assumed to be the reason for their high levels of microdiversity. So far, however, only a few metagenome-assembled phages have been proposed to infect them and no phages have been isolated. Taking advantage of recent advances in the cultivation of 'Ca. Nanopelagicales' we isolated a novel species of its genus 'Ca. Planktophila'. Using this isolate as bait, we cultivated the first two phages infecting this abundant bacterial order. Both genomes contained a whiB-like transcription factor and a RNA polymerase sigma-70 factor, which might aid in manipulating their host's metabolism. Both phages encoded a glycosyltransferase and one an anti-restriction protein, potential means to evade degradation of their DNA by nucleases present in the host genome. The two phage genomes shared only 6% of their genome with their closest relatives, with whom they form a previously uncultured family of actinophages within the Caudoviricetes. Read recruitment analyses against globally distributed metagenomes revealed the endemic distribution of this group of phages infecting 'Ca. Nanopelagicales'. The recruitment pattern against metagenomes from the isolation site and the modular distribution of shared genes between the two phages indicate high levels of horizontal gene transfer, likely mirroring the microdiversity of their host in the evolutionary arms race between host and phage.

High-resolution metagenomic reconstruction of the freshwater spring bloom.

BACKGROUND: The phytoplankton spring bloom in freshwater habitats is a complex, recurring, and dynamic ecological spectacle that unfolds at multiple biological scales. Although enormous taxonomic shifts in microbial assemblages during and after the bloom have been reported, genomic information on the microbial community of the spring bloom remains scarce. RESULTS: We performed a high-resolution spatio-temporal sampling of the spring bloom in a freshwater reservoir and describe a multitude of previously unknown taxa using metagenome-assembled genomes of eukaryotes, prokaryotes, and viruses in combination with a broad array of methodologies. The recovered genomes reveal multiple distributional dynamics for several bacterial groups with progressively increasing stratification. Analyses of abundances of metagenome-assembled genomes in concert with CARD-FISH revealed remarkably similar in situ doubling time estimates for dominant genome-streamlined microbial lineages. Discordance between quantitations of cryptophytes arising from sequence data and microscopic identification suggested the presence of hidden, yet extremely abundant aplastidic cryptophytes that were confirmed by CARD-FISH analyses. Aplastidic cryptophytes are prevalent throughout the water column but have never been considered in prior models of plankton dynamics. We also recovered the first metagenomic-assembled genomes of freshwater protists (a diatom and a haptophyte) along with thousands of giant viral genomic contigs, some of which appeared similar to viruses infecting haptophytes but owing to lack of known representatives, most remained without any indication of their hosts. The contrasting distribution of giant viruses that are present in the entire water column to that of parasitic perkinsids residing largely in deeper waters allows us to propose giant viruses as the biological agents of top-down control and bloom collapse, likely in combination with bottom-up factors like a nutrient limitation. CONCLUSION: We reconstructed thousands of genomes of microbes and viruses from a freshwater spring bloom and show that such large-scale genome recovery allows tracking of planktonic succession in great detail. However, integration of metagenomic information with other methodologies (e.g., microscopy, CARD-FISH) remains critical to reveal diverse phenomena (e.g., distributional patterns, in situ doubling times) and novel participants (e.g., aplastidic cryptophytes) and to further refine existing ecological models (e.g., factors affecting bloom collapse). This work provides a genomic foundation for future approaches towards a fine-scale characterization of the organisms in relation to the rapidly changing environment during the course of the freshwater spring bloom. Video Abstract.

The Evolutionary Kaleidoscope of Rhodopsins.

Rhodopsins are widely distributed across all domains of life where they perform a plethora of functions through the conversion of electromagnetic radiation into physicochemical signals. As a result of an extensive survey of available genomic and metagenomic sequencing data, we reported the existence of novel clades and exotic sequence motifs scattered throughout the evolutionary radiations of both Type-1 and Type-3 rhodopsins that will likely enlarge the optogenetics toolbox. We expanded the typical rhodopsin blueprint by showing that a highly conserved and functionally important arginine residue (i.e., Arg82) was substituted multiple times during evolution by an extensive amino acid spectrum. We proposed the umbrella term Alt-rhodopsins (AltRs) for all such proteins that departed Arg82 orthodoxy. Some AltRs formed novel clades in the rhodopsin phylogeny and were found in giant viruses. Some newly uncovered AltRs were phylogenetically close to heliorhodopsins, which allowed a closer examination of the phylogenetic border between Type-1 rhodopsins and heliorhodopsins. Comprehensive phylogenetic trees and ancestral sequence reconstructions allowed us to advance the hypothesis that proto-heliorhodopsins were a eukaryotic innovation before their subsequent diversification into the extant Type-3 rhodopsins. IMPORTANCE The rhodopsin scaffold is remarkably versatile and widespread, coupling light availability to energy production and other light-dependent cellular responses with minor alterations to critical residues. We described an unprecedented spectrum of substitutions at one of the most conserved amino acids in the rhodopsin fold, Arg82. We denoted such phylogenetically diverse rhodopsins with the umbrella name Alt-rhodopsins (AltR) and described a distinct branch of AltRs in giant viruses. Intriguingly, some AltRs were the closest phylogenetic neighbors to Heliorhodopsins (HeRs) whose origins have remained enigmatic. Our analyses of HeR origins in the light of AltRs led us to posit a most unusual evolutionary trajectory that suggested a eukaryotic origin for HeRs before their diversification in prokaryotes.

Expanded Diversity and Metabolic Versatility of Marine Nitrite-Oxidizing Bacteria Revealed by Cultivation- and Genomics-Based Approaches.

Nitrite-oxidizing bacteria (NOB) are ubiquitous and abundant microorganisms that play key roles in global nitrogen and carbon biogeochemical cycling. Despite recent advances in understanding NOB physiology and taxonomy, currently very few cultured NOB or representative NOB genome sequences from marine environments exist. In this study, we employed enrichment culturing and genomic approaches to shed light on the phylogeny and metabolic capacity of marine NOB. We successfully enriched two marine NOB (designated MSP and DJ) and obtained a high-quality metagenome-assembled genome (MAG) from each organism. The maximum nitrite oxidation rates of the MSP and DJ enrichment cultures were 13.8 and 30.0 µM nitrite per day, respectively, with these optimum rates occurring at 0.1 mM and 0.3 mM nitrite, respectively. Each enrichment culture exhibited a different tolerance to various nitrite and salt concentrations. Based on phylogenomic position and overall genome relatedness indices, both NOB MAGs were proposed as novel taxa within the Nitrospinota and Nitrospirota phyla. Functional predictions indicated that both NOB MAGs shared many highly conserved metabolic features with other NOB. Both NOB MAGs encoded proteins for hydrogen and organic compound metabolism and defense mechanisms for oxidative stress. Additionally, these organisms may have the genetic potential to produce cobalamin (an essential enzyme cofactor that is limiting in many environments) and, thus, may play an important role in recycling cobalamin in marine sediment. Overall, this study appreciably expands our understanding of the Nitrospinota and Nitrospirota phyla and suggests that these NOB play important biogeochemical roles in marine habitats.IMPORTANCE Nitrification is a key process in the biogeochemical and global nitrogen cycle. Nitrite-oxidizing bacteria (NOB) perform the second step of aerobic nitrification (converting nitrite to nitrate), which is critical for transferring nitrogen to other organisms for assimilation or energy. Despite their ecological importance, there are few cultured or genomic representatives from marine systems. Here, we obtained two NOB (designated MSP and DJ) enriched from marine sediments and estimated the physiological and genomic traits of these marine microbes. Both NOB enrichment cultures exhibit distinct responses to various nitrite and salt concentrations. Genomic analyses suggest that these NOB are metabolically flexible (similar to other previously described NOB) yet also have individual genomic differences that likely support distinct niche distribution. In conclusion, this study provides more insights into the ecological roles of NOB in marine environments.

Correction to: Phage-centric ecological interactions in aquatic ecosystems revealed through ultra-deep metagenomics.

Following publication of the original article [1], the authors reported that an affiliation of the first author was missing.

Phage-centric ecological interactions in aquatic ecosystems revealed through ultra-deep metagenomics.

The persistent inertia in the ability to culture environmentally abundant microbes from aquatic ecosystems represents an obstacle in disentangling the complex web of ecological interactions spun by a diverse assortment of participants (pro- and eukaryotes and their viruses). In aquatic microbial communities, the numerically most abundant actors, the viruses, remain the most elusive, and especially in freshwaters their identities and ecology remain unknown. Here, using ultra-deep metagenomic sequencing from pelagic freshwater habitats, we recovered complete genomes of > 2000 phages, including small "miniphages" and large "megaphages" infecting iconic freshwater prokaryotic lineages. For instance, abundant freshwater Actinobacteria support infection by a very broad size range of phages (13-200 Kb). We describe many phages encoding genes that likely afford protection to their host from reactive oxygen species (ROS) in the aquatic environment and in the oxidative burst in protist phagolysosomes (phage-mediated ROS defense). Spatiotemporal abundance analyses of phage genomes revealed evanescence as the primary dynamic in upper water layers, where they displayed short-lived existences. In contrast, persistence was characteristic for the deeper layers where many identical phage genomes were recovered repeatedly. Phage and host abundances corresponded closely, with distinct populations displaying preferential distributions in different seasons and depths, closely mimicking overall stratification and mixis.

ATLANTIC BATS: a data set of bat communities from the Atlantic Forests of South America.

Bats are the second most diverse mammal order and they provide vital ecosystem functions (e.g., pollination, seed dispersal, and nutrient flux in caves) and services (e.g., crop pest suppression). Bats are also important vectors of infectious diseases, harboring more than 100 different virus types. In the present study, we compiled information on bat communities from the Atlantic Forests of South America, a species-rich biome that is highly threatened by habitat loss and fragmentation. The ATLANTIC BATS data set comprises 135 quantitative studies carried out in 205 sites, which cover most vegetation types of the tropical and subtropical Atlantic Forest: dense ombrophilous forest, mixed ombrophilous forest, semideciduous forest, deciduous forest, savanna, steppe, and open ombrophilous forest. The data set includes information on more than 90,000 captures of 98 bat species of eight families. Species richness averaged 12.1 per site, with a median value of 10 species (ranging from 1 to 53 species). Six species occurred in more than 50% of the communities: Artibeus lituratus, Carollia perspicillata, Sturnira lilium, Artibeus fimbriatus, Glossophaga soricina, and Platyrrhinus lineatus. The number of captures divided by sampling effort, a proxy for abundance, varied from 0.000001 to 0.77 individuals·h-1 ·m-2 (0.04 ± 0.007 individuals·h-1 ·m-2 ). Our data set reveals a hyper-dominance of eight species that together that comprise 80% of all captures: Platyrrhinus lineatus (2.3%), Molossus molossus (2.8%), Artibeus obscurus (3.4%), Artibeus planirostris (5.2%), Artibeus fimbriatus (7%), Sturnira lilium (14.5%), Carollia perspicillata (15.6%), and Artibeus lituratus (29.2%).