One guanosine determines transcript fate: Transcripts of the HIV-1 RNA genome can be either spliced and translated into viral proteins or packaged into new virions as a progeny genome. The path taken depends on whether the transcript contains one guanosine at the 5′ terminus (1G) rather than two or three (2G or 3G). Brown et al. used nuclear magnetic resonance spectroscopy to show that 1G transcripts adopt a dimeric structure that sequesters a terminal cap required for translation and splicing but exposes sites that bind to the HIV-1 Gag protein, which recruits the genome during viral assembly. Conversely, 2G or 3G transcripts have the cap accessible, but Gag-binding sites are sequestered. Therefore, a single guanosine acts as a conformational switch to determine the fate of HIV-1 transcripts. Science , this issue p. 413

Despite the importance of the cervicovaginal microbiome, the mechanisms that govern its composition and drive its impact on host physiology remain poorly understood. This study expands our understanding of the function and ecology of the vaginal microbiome using VIRGO2, an enhanced non-redundant gene catalog comprising over 1.7 million well-annotated genes from body-site specific microbes and viruses. Analyses using VIRGO2 revealed novel insights, including the identification of previously uncharacterized vaginal bacteria, features of the vaginal mycobiome and phageome, and differential expression of bacterial carbohydrate catabolic genes. Constructed from over 2,500 metagenomes and 4,000 bacterial genomes, VIRGO2 broadens geographic representation and microbial diversity compared to its predecessor. This updated catalog enables more precise profiling of taxonomic and functional composition from metagenomic and metatranscriptomic datasets. VIRGO2 is a critical resource for integrative analyses of vaginal microbial communities and their interactions with host tissues, thereby enhancing our mechanistic understanding of vaginal health and disease.

Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems—a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR.

Despite the importance of the cervicovaginal microbiome, the mechanisms that govern its composition and drive its impact on host physiology remain poorly understood. With the aim to expand our understanding of the function and ecology of the vaginal microbiome, we present VIRGO2, an enhanced non-redundant gene catalog comprising over 1.7 million well-annotated genes from body-site specific microbes and viruses. Analyses using VIRGO2 reveal insights such as including the identification of previously uncharacterized vaginal bacteria, features of the vaginal mycobiome and phageome, and differential expression of bacterial carbohydrate catabolic genes. Constructed from over 2500 metagenomes and 4000 bacterial genomes, VIRGO2 broadens geographic representation and microbial diversity compared to its predecessor. This updated catalog enables more precise profiling of taxonomic and functional composition from metagenomic and metatranscriptomic datasets. VIRGO2 is a critical resource for integrative analyses of vaginal microbial communities and their interactions with host tissues, thereby enhancing our mechanistic understanding of vaginal health and disease.

Biofilm formation is a highly regulated process that contributes to the environmental fitness of microorganisms, including pathogenic bacteria. The second messenger c-di-GMP is a critical regulator of biofilm formation whose cellular levels are tightly regulated by the abundance and activity of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). These enzymes synthesize and degrade c-di-GMP, respectively. The Vibrio cholerae VpvABC system encodes a DGC and is critical for biofilm formation; however, much remains unknown about its regulation. Here we demonstrate that the vpvABC system is transcriptionally regulated by c-di-GMP and the master biofilm regulators VpsT and VpsR. However, we also identify the alternative sigma factor RpoS as a positive regulator of vpvABC. RpoS is involved in the regulation of many c-di-GMP metabolism genes and plays a role in biofilm architecture, likely mediated in part through vpvC. In mature biofilms, vpvA transcription was highest near the biofilm substratum and VpsT, VpsR, and RpoS were critical for vpvABC transcription. Overall, our genetic dissection reveals the vpvABC system is regulated by two parallel circuits: a c-di-GMP sensing-circuit acting through VpsT and VpsR and a stationary growth phase circuit via RpoS. These findings underscore the multilayered regulatory mechanisms that precisely govern biofilm formation by a pathogen.

Motivation: Accurate detection and quantification of bacterial strains in clinical samples is necessary to measure their colonization and persistence. Past methods to achieve this relied either on strain-specific qPCR assays, or shotgun metagenomic read mapping approaches. The resident microbial community is a major source of interference in both assays because it can contain conspecific strains bearing similarity to the focal strain(s). Results: We present kSanity, a k-mer based application for the detection and quantification of targeted bacterial strains in shotgun metagenomic data. Because kSanity uses exact string matches between the reads and reference, it is less sensitive to interference by conspecific strains. We test the performance of kSanity using a combination of in silico spike-in experiments, and in vivo observational data. Our results demonstrate that kSanity provides precise and accurate quantification of targeted bacterial strains, even when they are present at low sequence coverage in the metagenome. Availability and implementation: kSanity is available at: https://github.com/ravel-lab/kSanity