Toxic antiphage defense proteins inhibited by intragenic antitoxin proteins.

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here, we report that these proteins are toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic RpnL. The crystal structure of RpnAS revealed a dimerization interface encompassing α helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document that plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose that many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms.

Evolutionary history and origins of Dsr-mediated sulfur oxidation.

Microorganisms play vital roles in sulfur cycling through the oxidation of elemental sulfur and reduction of sulfite. These metabolisms are catalyzed by dissimilatory sulfite reductases (Dsr) functioning in either the reductive or reverse, oxidative direction. Dsr-mediated sulfite reduction is an ancient metabolism proposed to have fueled energy metabolism in some of Earth's earliest microorganisms, whereas sulfur oxidation is believed to have evolved later in association with the widespread availability of oxygen on Earth. Organisms are generally believed to carry out either the reductive or oxidative pathway, yet organisms from diverse phyla have been discovered with gene combinations that implicate them in both pathways. A comprehensive investigation into the metabolisms of these phyla regarding Dsr is currently lacking. Here, we selected one of these phyla, the metabolically versatile candidate phylum SAR324, to study the ecology and evolution of Dsr-mediated metabolism. We confirmed that diverse SAR324 encode genes associated with reductive Dsr, oxidative Dsr, or both. Comparative analyses with other Dsr-encoding bacterial and archaeal phyla revealed that organisms encoding both reductive and oxidative Dsr proteins are constrained to a few phyla. Further, DsrAB sequences from genomes belonging to these phyla are phylogenetically positioned at the interface between well-defined oxidative and reductive bacterial clades. The phylogenetic context and dsr gene content in these organisms points to an evolutionary transition event that ultimately gave way to oxidative Dsr-mediated metabolism. Together, this research suggests that SAR324 and other phyla with mixed dsr gene content are associated with the evolution and origins of Dsr-mediated sulfur oxidation.

Viromes vs. mixed community metagenomes: choice of method dictates interpretation of viral community ecology.

BACKGROUND: Viruses, the majority of which are uncultivated, are among the most abundant biological entities on Earth. From altering microbial physiology to driving community dynamics, viruses are fundamental members of microbiomes. While the number of studies leveraging viral metagenomics (viromics) for studying uncultivated viruses is growing, standards for viromics research are lacking. Viromics can utilize computational discovery of viruses from total metagenomes of all community members (hereafter metagenomes) or use physical separation of virus-specific fractions (hereafter viromes). However, differences in the recovery and interpretation of viruses from metagenomes and viromes obtained from the same samples remain understudied. RESULTS: Here, we compare viral communities from paired viromes and metagenomes obtained from 60 diverse samples across human gut, soil, freshwater, and marine ecosystems. Overall, viral communities obtained from viromes had greater species richness and total viral genome abundances than those obtained from metagenomes, although there were some exceptions. Despite this, metagenomes still contained many viral genomes not detected in viromes. We also found notable differences in the predicted lytic state of viruses detected in viromes vs metagenomes at the time of sequencing. Other forms of variation observed include genome presence/absence, genome quality, and encoded protein content between viromes and metagenomes, but the magnitude of these differences varied by environment. CONCLUSIONS: Overall, our results show that the choice of method can lead to differing interpretations of viral community ecology. We suggest that the choice of whether to target a metagenome or virome to study viral communities should be dependent on the environmental context and ecological questions being asked. However, our overall recommendation to researchers investigating viral ecology and evolution is to pair both approaches to maximize their respective benefits. Video Abstract.

Viromes vs. mixed community metagenomes: choice of method dictates interpretation of viral community ecology.

Background: Viruses, the majority of which are uncultivated, are among the most abundant biological entities on Earth. From altering microbial physiology to driving community dynamics, viruses are fundamental members of microbiomes. While the number of studies leveraging viral metagenomics (viromics) for studying uncultivated viruses is growing, standards for viromics research are lacking. Viromics can utilize computational discovery of viruses from total metagenomes of all community members (hereafter metagenomes) or use physical separation of virus-specific fractions (hereafter viromes). However, differences in the recovery and interpretation of viruses from metagenomes and viromes obtained from the same samples remain understudied. Results: Here, we compare viral communities from paired viromes and metagenomes obtained from 60 diverse samples across human gut, soil, freshwater, and marine ecosystems. Overall, viral communities obtained from viromes were more abundant and species rich than those obtained from metagenomes, although there were some exceptions. Despite this, metagenomes still contained many viral genomes not detected in viromes. We also found notable differences in the predicted lytic state of viruses detected in viromes vs metagenomes at the time of sequencing. Other forms of variation observed include genome presence/absence, genome quality, and encoded protein content between viromes and metagenomes, but the magnitude of these differences varied by environment. Conclusions: Overall, our results show that the choice of method can lead to differing interpretations of viral community ecology. We suggest that the choice of whether to target a metagenome or virome to study viral communities should be dependent on the environmental context and ecological questions being asked. However, our overall recommendation to researchers investigating viral ecology and evolution is to pair both approaches to maximize their respective benefits.

Toxic anti-phage defense proteins inhibited by intragenic antitoxin proteins.

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here we report these proteins are new toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C -terminal domains (Rpn S ), which are translated separately from the full-length proteins (Rpn L ), directly block the activities of the toxic full-length proteins. The crystal structure of RpnA S revealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document plasmid-encoded RpnP2 L protects Escherichia coli against certain phages. We propose many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms. Significance: Here we document the function of small genes-within-genes, showing they encode antitoxin proteins that block the functions of the toxic DNA endonuclease proteins encoded by the longer rpn genes. Intriguingly, a sequence present in both long and short protein shows extensive variation in the number of four amino acid repeats. Consistent with a strong selection for the variation, we provide evidence that the Rpn proteins represent a phage defense system.