Microorganisms in the terrestrial deep biosphere host up to 20% of the earth's biomass and are suggested to be sustained by the gases hydrogen and carbon dioxide. A metagenome analysis of three deep ...subsurface water types of contrasting age (from <20 to several thousand years) and depth (171 to 448 m) revealed phylogenetically distinct microbial community subsets that either passed or were retained by a 0.22 μm filter. Such cells of <0.22 μm would have been overlooked in previous studies relying on membrane capture. Metagenomes from the three water types were used for reconstruction of 69 distinct microbial genomes, each with >86% coverage. The populations were dominated by Proteobacteria, Candidate divisions, unclassified archaea and unclassified bacteria. The estimated genome sizes of the <0.22 μm populations were generally smaller than their phylogenetically closest relatives, suggesting that small dimensions along with a reduced genome size may be adaptations to oligotrophy. Shallow 'modern marine' water showed community members with a predominantly heterotrophic lifestyle. In contrast, the deeper, 'old saline' water adhered more closely to the current paradigm of a hydrogen-driven deep biosphere. The data were finally used to create a combined metabolic model of the deep terrestrial biosphere microbial community.
Viruses are fundamental to ecosystems ranging from oceans to humans, yet our ability to study them is bottlenecked by the lack of ecologically relevant isolates, resulting in “unknowns” dominating ...culture-independent surveys. Here we present genomes from 31 phages infecting multiple strains of the aquatic bacterium Cellulophaga baltica (Bacteroidetes) to provide data for an underrepresented and environmentally abundant bacterial lineage. Comparative genomics delineated 12 phage groups that (i) each represent a new genus, and (ii) represent one novel and four well-known viral families. This diversity contrasts the few well-studied marine phage systems, but parallels the diversity of phages infecting human-associated bacteria. Although all 12 Cellulophaga phages represent new genera, the podoviruses and icosahedral, nontailed ssDNA phages were exceptional, with genomes up to twice as large as those previously observed for each phage type. Structural novelty was also substantial, requiring experimental phage proteomics to identify 83% of the structural proteins. The presence of uncommon nucleotide metabolism genes in four genera likely underscores the importance of scavenging nutrient-rich molecules as previously seen for phages in marine environments. Metagenomic recruitment analyses suggest that these particular Cellulophaga phages are rare and may represent a first glimpse into the phage side of the rare biosphere. However, these analyses also revealed that these phage genera are widespread, occurring in 94% of 137 investigated metagenomes. Together, this diverse and novel collection of phages identifies a small but ubiquitous fraction of unknown marine viral diversity and provides numerous environmentally relevant phage–host systems for experimental hypothesis testing.
CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut
. The genomes of all crAss-like phages encode a large virion-packaged ...protein
that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)
. Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited state, which probably occurs before virion packaging. This conformation is attained with the help of a cleft-blocking domain that interacts with the active site and occupies the cavity in which the RNA-DNA hybrid binds. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs that are involved in RNA interference
, although most of the phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of the protein fold space. Considering this structural similarity, we propose that eukaryal RNA interference polymerases have their origins in phage, which parallels the emergence of the mitochondrial transcription apparatus
.
The continental subsurface is suggested to contain a significant part of the earth's total biomass. However, due to the difficulty of sampling, the deep subsurface is still one of the least ...understood ecosystems. Therefore, microorganisms inhabiting this environment might profoundly influence the global nutrient and energy cycles. In this study,
fixed RNA transcripts from two deep continental groundwaters from the Äspö Hard Rock Laboratory (a Baltic Sea-influenced water with a residence time of <20 years, defined as "modern marine," and an "old saline" groundwater with a residence time of thousands of years) were subjected to metatranscriptome sequencing. Although small subunit (SSU) rRNA gene and mRNA transcripts aligned to all three domains of life, supporting activity within these community subsets, the data also suggested that the groundwaters were dominated by bacteria. Many of the SSU rRNA transcripts grouped within newly described candidate phyla or could not be mapped to known branches on the tree of life, suggesting that a large portion of the active biota in the deep biosphere remains unexplored. Despite the extremely oligotrophic conditions, mRNA transcripts revealed a diverse range of metabolic strategies that were carried out by multiple taxa in the modern marine water that is fed by organic carbon from the surface. In contrast, the carbon dioxide- and hydrogen-fed old saline water with a residence time of thousands of years predominantly showed the potential to carry out translation. This suggested these cells were active, but waiting until an energy source episodically becomes available.
A newly designed sampling apparatus was used to fix RNA under
conditions in the deep continental biosphere and benchmarks a strategy for deep biosphere metatranscriptomic sequencing. This apparatus enabled the identification of active community members and the processes they carry out in this extremely oligotrophic environment. This work presents for the first time evidence of eukaryotic, archaeal, and bacterial activity in two deep subsurface crystalline rock groundwaters from the Äspö Hard Rock Laboratory with different depths and geochemical characteristics. The findings highlight differences between organic carbon-fed shallow communities and carbon dioxide- and hydrogen-fed old saline waters. In addition, the data reveal a large portion of uncharacterized microorganisms, as well as the important role of candidate phyla in the deep biosphere, but also the disparity in microbial diversity when using standard microbial 16S rRNA gene amplification versus the large unknown portion of the community identified with unbiased metatranscriptomes.
Ribonucleotide reductases (RNR) are essential enzymes for all known life forms. Their current taxonomic distribution suggests extensive horizontal gene transfer e.g., by processes involving viruses. ...To improve our understanding of the underlying processes, we characterized a monomeric class II RNR (NrdJm) enzyme from a Thermus virus, a subclass not present in any sequenced
spp. genome. Phylogenetic analysis revealed a distant origin of the
gene with the most closely related sequences found in mesophiles or moderate thermophiles from the Firmicutes phylum. GC-content, codon usage and the ratio of coding to non-coding substitutions (dN/dS) suggest extensive adaptation of the gene in the virus in terms of nucleotide composition and amino acid sequence. The NrdJm enzyme is a monomeric B
-dependent RNR with nucleoside triphosphate specificity. It exhibits a temperature optimum at 60-70 °C, which is in the range of the growth optimum of
spp. Experiments in combination with the
thioredoxin system show that the enzyme is able to retrieve electrons from the host NADPH pool via host thioredoxin and thioredoxin reductases. This is different from other characterized viral RNRs such as T4 phage RNR, where a viral thioredoxin is present. We hence show that the monomeric class II RNR, present in Thermus viruses, was likely transferred from an organism phylogenetically distant from the one they were isolated from, and adapted to the new host in genetic signature and amino acids sequence.
Bacteriophage control of harmful or pathogenic bacteria has aroused growing interest, largely due to the rise of antibiotic resistance. The objective of this study was to test phages as potential ...agents for the biocontrol of an opportunistic pathogen Pseudomonas aeruginosa in water. Two P. aeruginosa bacteriophages (vB_PaeM_V523 and vB_PaeM_V524) were isolated from wastewater and characterized physically and functionally. Genomic and morphological characterization showed that both were myoviruses within the Pbunavirus genus. Both had a similar latent period (50–55 min) and burst size (124–134 PFU/infected cell), whereas there was variation in the host range. In addition to these environmental phages, a commercial Pseudomonas phage, JG003 (DSM 19870), was also used in the biocontrol experiments. The biocontrol potential of the three phages in water was tested separately and together as a cocktail against two P. aeruginosa strains; PAO1 and the environmental strain 17V1507. With PAO1, all phages initially reduced the numbers of the bacterial host, with phage V523 being the most efficient (>2.4 log10 reduction). For the environmental P. aeruginosa strain (17V1507), only the phage JG003 caused a reduction (1.2 log10) compared to the control. The cocktail of three phages showed a slightly higher decrease in the level of the hosts compared to the use of individual phages. Although no synergistic effect was observed in the host reduction with the use of the phage cocktail, the cocktail-treated hosts did not appear to acquire resistance as rapidly as hosts treated with a single phage. The results of this study provide a significant step in the development of bacteriophage preparations for the control of pathogens and harmful microbes in water environments.
Bacteria impact humans, industry and nature, but do so under viral constraints. Problematically, knowledge of viral infection efficiencies and outcomes derives from few model systems that ...over-represent efficient lytic infections and under-represent virus-host natural diversity. Here we sought to understand infection efficiency regulation in an emerging environmental Bacteroidetes-virus model system with markedly different outcomes on two genetically and physiologically nearly identical host strains. For this, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout both infections. While phage transcriptomes were similar, transcriptional differences between hosts suggested host-derived regulation of infection efficiency. Specifically, the alternative host overexpressed DNA degradation genes and underexpressed translation genes, which seemingly targeted phage DNA particle production, as experiments revealed they were both significantly delayed (by >30 min) and reduced (by >50%) in the inefficient infection. This suggests phage failure to repress early alternative host expression and stress response allowed the host to respond against infection by delaying phage DNA replication and protein translation. Given that this phage type is ubiquitous and abundant in the global oceans and that variable viral infection efficiencies are central to dynamic ecosystems, these data provide a critically needed foundation for understanding and modeling viral infections in nature.
The deep biosphere contains members from all three domains of life along with viruses. Here we investigate the deep terrestrial virosphere by sequencing community nucleic acids from three ...groundwaters of contrasting chemistries, origins, and ages. These viromes constitute a highly unique community compared to other environmental viromes and sequenced viral isolates. Viral host prediction suggests that many of the viruses are associated with Firmicutes and Patescibacteria, a superphylum lacking previously described active viruses. RNA transcript-based activity implies viral predation in the shallower marine water-fed groundwater, while the deeper and more oligotrophic waters appear to be in 'metabolic standby'. Viral encoded antibiotic production and resistance systems suggest competition and antagonistic interactions. The data demonstrate a viral community with a wide range of predicted hosts that mediates nutrient recycling to support a higher microbial turnover than previously anticipated. This suggests the presence of 'kill-the-winner' oscillations creating slow motion 'boom and burst' cycles.
PDF
