Summary Prions are a paradigm-shifting mechanism of inheritance in which phenotypes are encoded by self-templating protein conformations rather than nucleic acids. Here we examine the breadth of protein-based inheritance across the yeast proteome by assessing the ability of nearly every open reading frame (∼5,300 ORFs) to induce heritable traits. Transient overexpression of nearly 50 proteins created traits that remained heritable long after their expression returned to normal. These traits were beneficial, had prion-like patterns of inheritance, were common in wild yeasts, and could be transmitted to naïve cells with protein alone. Most inducing proteins were not known prions and did not form amyloid. Instead, they are highly enriched in nucleic acid binding proteins with large intrinsically disordered domains that have been widely conserved across evolution. Our data thus establish a common type of protein-based inheritance through which intrinsically disordered proteins can drive the emergence of new traits and adaptive opportunities.

Small proteins are traditionally overlooked due to computational and experimental difficulties in detecting them. To systematically identify small proteins, we carried out a comparative genomics study on 1,773 human-associated metagenomes from four different body sites. We describe >4,000 conserved protein families, the majority of which are novel; $30% of these protein families are predicted to be secreted or transmembrane. Over 90% of the small protein families have no known domain and almost half are not represented in reference genomes. We identify putative housekeeping, mammalian-specific, defense-related, and protein families that are likely to be horizontally transferred. We provide evidence of transcription and translation for a subset of these families. Our study suggests that small proteins are highly abundant and those of the human microbiome, in particular, may perform diverse functions that have not been previously reported.

Osmotic diarrhea is a prevalent condition in humans caused by food intolerance, malabsorption, and widespread laxative use. Here, we assess the resilience of the gut ecosystem to osmotic perturbation at multiple length and timescales using mice as model hosts. Osmotic stress caused reproducible extinction of highly abundant taxa and expansion of less prevalent members in human and mouse microbiotas. Quantitative imaging revealed decimation of the mucus barrier during osmotic perturbation, followed by recovery. The immune system exhibited temporary changes in cytokine levels and a lasting IgG response against commensal bacteria. Increased osmolality prevented growth of commensal strains in vitro, revealing one mechanism contributing to extinction. Environmental availability of microbiota members mitigated extinction events, demonstrating how species reintroduction can affect community resilience. Our findings (1) demonstrate that even mild osmotic diarrhea can cause lasting changes to the microbiota and host and (2) lay the foundation for interventions that increase system-wide resilience.

No method exists to measure large-scale translation of genes in uncultured organisms in microbiomes. To overcome this limitation, we develop MetaRibo-Seq, a method for simultaneous ribosome profiling of tens to hundreds of organisms in microbiome samples. MetaRibo-Seq was benchmarked against gold-standard Ribo-Seq in a mock microbial community and applied to five different human fecal samples. Unlike RNA-Seq, Ribo-Seq signal of a predicted gene suggests it encodes a translated protein. We demonstrate two applications of this technique: First, MetaRibo-Seq identifies small genes, whose identification until now has been challenging. For example, MetaRibo-Seq identifies 2,091 translated, previously unannotated small protein families from five fecal samples, more than doubling the number of small proteins predicted to exist in this niche. Second, the combined application of RNA-Seq and MetaRibo-Seq identifies differences in the translation of transcripts. In summary, MetaRibo-Seq enables comprehensive translational profiling in microbiomes and identifies previously unannotated small proteins.