Project description:Viperin is an interferon-induced cellular protein conserved in animals. It was shown to inhibit the replication of multiple viruses by producing a ribonucleotide called 3’-deoxy-3’4’-didehydro-CTP (ddhCTP), which acts as a chain terminator for the viral RNA polymerase. Here we show that the eukaryotic viperin has originated from a clade of bacterial and archaeal proteins that protect against phage infection. Prokaryotic viperins (pVips) produce a set of modified ribonucleotides that include, in addition to ddhCTP, also ddhGTP and ddhUTP. We further provide evidence that pVips protect against T7 phage infection by inhibiting viral polymerase-dependent transcription, suggesting a conserved mechanism of action shared between pVips and the animal viperin. Our results unveil a potential repository of natural antiviral compounds produced by bacterial immune systems.
Project description:Viperin is an interferon-induced cellular protein that is conserved in animals1. It has previously been shown to inhibit the replication of multiple viruses by producing the ribonucleotide 3'-deoxy-3',4'-didehydro (ddh)-cytidine triphosphate (ddhCTP), which acts as a chain terminator for viral RNA polymerase2. Here we show that eukaryotic viperin originated from a clade of bacterial and archaeal proteins that protect against phage infection. Prokaryotic viperins produce a set of modified ribonucleotides that include ddhCTP, ddh-guanosine triphosphate (ddhGTP) and ddh-uridine triphosphate (ddhUTP). We further show that prokaryotic viperins protect against T7 phage infection by inhibiting viral polymerase-dependent transcription, suggesting that it has an antiviral mechanism of action similar to that of animal viperin. Our results reveal a class of potential natural antiviral compounds produced by bacterial immune systems.
Project description:The cyclic oligonucleotide-based anti-phage signalling system (CBASS) is a typeof innate prokaryotic immune system. Composed of a cyclic GMP-AMP synthase (cGAS) and CBASS-associated proteins, CBASS utilizes cyclic oligonucleotides to activate antiviral immunity. One major class of CBASS contains a homolog of eukaryotic ubiquitin-conjugating enzymes, which is either an E1-E2 fusion or a single E2. However, the functions of single E2s in CBASS remain elusive. Here, we report that a bacterial E2 enzyme regulates cGAS by imitating the ubiquitination cascade. This includes the processing of the cGAS C-terminus, conjugation of cGAS to a cysteine residue, ligation of cGAS to a lysine residue, cleavage of the isopeptide bond, and poly-cGASylation. The poly-cGASylation activates cGAS to produce cGAMP, which acts as an antiviral signal and leads to cell death. Thus, our findings reveal a unique regulatory role of E2 in CBASS.
Project description:Conventional prokaryotic RNA labeling method usually requires large amounts of starting materials and tends to generate high background signals. Recently, two novel methods based on amplification systems were introduced. Here, we compared three alternative strategies: direct labeling method, ployadenylation-involved oligo-dT priming amplification method and random priming amplification method (hereafter referred to as DL, PAOD and RPA method in this article) for prokaryotic RNA labeling employing the expression profiling investigation in Escherichia coli (E. coli) heat shock model.
Project description:Bacteriophage – host dynamics and interactions are important for microbial community composition and ecosystem function. Nonetheless, empirical evidence in engineered environment is scarce. Here, we examined phage and prokaryotic community composition of four anaerobic digestors in full-scale wastewater treatment plants (WWTPs) across China. Despite relatively stable process performance in biogas production, both phage and prokaryotic groups fluctuated monthly over a year of study period. Nonetheless, there were significant correlations in their α- and β-diversities between phage and prokaryotes. Phages explained 40.6% of total prokaryotic community composition, much higher than the explainable power by abiotic factors (14.5%). Consequently, phages were significantly (P<0.010) linked to parameters related to process performance including biogas production and volatile solid concentrations. Association network analyses showed that phage-prokaryote pairs were deeply rooted, and two network modules were exclusively comprised of phages, suggesting a possibility of co-infection. Those results collectively demonstrate phages as a major biotic factor in controlling bacterial composition. Therefore, phages may play a larger role in shaping prokaryotic dynamics and process performance of WWTPs than currently appreciated, enabling reliable prediction of microbial communities across time and space.
Project description:We present Prokaryotic Expression-profiling by Tagging RNA In Situ and sequencing (PETRI-seq), a high-throughput prokaryotic scRNA-seq pipeline. We demonstrated that PETRI-seq effectively barcoded single bacterial cells in a species-mixing experiment with E. coli (MG1655) and S. aureus (USA300). Within the S. aureus population, we found rare prophage induction in 0.04% of cells. We further demonstrated that PETRI-seq was able to distinguish between E. coli growth phases based on mRNA expression patterns by combining stationary E. coli with exponential E. coli in multiple experiments.
Project description:tRNA modifications help maintain tRNA structure and facilitate stress responses. Found in all three kingdoms of life, m1A tRNA modification occurs in the T loop of many tRNAs and stabilizes tertiary tRNA structure and impacts translation. M1A in T loop is known to be reversible by three mammalian demethylase enzymes, which bypasses the need of turning over the tRNA molecule to adjust their m1A levels in cells. However, no prokaryotic tRNA demethylase enzyme has been identified. Using Streptomyces venezuelae as a model organism, we confirmed the presence and quantitative m1A tRNA signatures using mass spectrometry and high throughput tRNA sequencing. We identified two RNA demethylases that can remove m1A in tRNA and confirmed the activity of a previously annotated tRNA m1A writer. Using single gene knockouts of these erasers and the m1A writer, we subjected these strains to stress conditions and found dynamic changes to m1A levels in many tRNAs. Phenotypic characterization highlighted changes to their growth and altered antibiotic production. Our identification of the first prokaryotic tRNA demethylase enzyme paves the way for investigating new mechanisms in global translational regulation in bacteria.