RESUMO
Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.
Assuntos
Bactérias , Bacteriófagos , Bactérias/genética , Bactérias/virologia , Bacteriófagos/genética , Sistemas CRISPR-Cas , Proteínas Virais/metabolismo , Mutação , Fenômenos Fisiológicos BacterianosRESUMO
Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it "guards" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed.
Assuntos
Bactérias/genética , Bactérias/imunologia , Bacteriófagos/fisiologia , RNA não Traduzido/genética , DNA Polimerase Dirigida por RNA/genética , Bactérias/virologia , Ilhas de CpG/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/virologia , Proteínas de Escherichia coli/metabolismo , FilogeniaRESUMO
Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.
Assuntos
Bacteriófagos , Microscopia Crioeletrônica , NAD , NAD/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/imunologia , Hidrólise , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , Sistemas Toxina-Antitoxina/genética , Escherichia coli/virologia , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismoRESUMO
Overcoming lysogenization defect (OLD) proteins are a conserved family of ATP-powered nucleases that function in anti-phage defense. Recent bioinformatic, genetic, and crystallographic studies have yielded new insights into the structure, function, and evolution of these enzymes. Here we review these developments and propose a new classification scheme to categorize OLD homologs that relies on gene neighborhoods, biochemical properties, domain organization, and catalytic machinery. This taxonomy reveals important similarities and differences between family members and provides a blueprint to contextualize future in vivo and in vitro findings. We also detail how OLD nucleases are related to PARIS and Septu anti-phage defense systems and discuss important mechanistic questions that remain unanswered.
Assuntos
Bactérias , Bacteriófagos , Esterases , Bacteriófagos/fisiologia , Bactérias/enzimologia , Bactérias/virologia , Esterases/química , Exodesoxirribonuclease V , Adenosina Trifosfatases/químicaRESUMO
Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.
Assuntos
Sistemas CRISPR-Cas/genética , DNA de Cadeia Simples/genética , Resistência Microbiana a Medicamentos/genética , Engenharia Genética , Genoma Bacteriano/genética , Alelos , DNA de Cadeia Simples/biossíntese , Escherichia coli/genética , Biblioteca Gênica , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala , Saccharomyces cerevisiae/genética , Biologia SintéticaRESUMO
Prokaryotic genomes harbour a plethora of uncharacterized reverse transcriptases (RTs). RTs phylogenetically related to those encoded by group-II introns have been found associated with type III CRISPR-Cas systems, adjacent or fused at the C-terminus to Cas1. It is thought that these RTs may have a relevant function in the CRISPR immune response mediating spacer acquisition from RNA molecules. The origin and relationships of these RTs and the ways in which the various protein domains evolved remain matters of debate. We carried out a large survey of annotated RTs in databases (198,760 sequences) and constructed a large dataset of unique representative sequences (9,141). The combined phylogenetic reconstruction and identification of the RTs and their various protein domains in the vicinity of CRISPR adaptation and effector modules revealed three different origins for these RTs, consistent with their emergence on multiple occasions: a larger group that have evolved from group-II intron RTs, and two minor lineages that may have arisen more recently from Retron/retron-like sequences and Abi-P2 RTs, the latter associated with type I-C systems. We also identified a particular group of RTs associated with CRISPR-cas loci in clade 12, fused C-terminally to an archaeo-eukaryotic primase (AEP), a protein domain (AE-Prim_S_like) forming a particular family within the AEP proper clade. Together, these data provide new insight into the evolution of CRISPR-Cas/RT systems.
Assuntos
Sistemas CRISPR-Cas , DNA Polimerase Dirigida por RNA/genética , Mapeamento Cromossômico , Ligação Genética , Variação Genética , Íntrons , Filogenia , Células Procarióticas/metabolismo , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
Multi-copy single-stranded DNA (msDNA) is composed of covalently bound single-stranded DNA and RNA, and synthesized by retron-encoded reverse transcriptase. msDNA-synthesizing systems are thought to be a recent acquisition by Escherichia coli because, to date, only seven types of msDNA, which differ markedly in their primary nucleotide sequences, have been found in a small subset of E. coli strains. The wide use of E. coli in molecular research means that it is important to understand more about these stable, covalently bound, single-stranded DNA or RNA compounds. The present review provides insights into the molecular biosynthesis, distribution and function of E. coli msDNA to raise awareness about these special molecules.
Assuntos
DNA Bacteriano/fisiologia , DNA de Cadeia Simples/fisiologia , Escherichia coli/genética , RNA Bacteriano/fisiologia , Sequência de Bases , Elementos de DNA Transponíveis/genética , DNA Bacteriano/biossíntese , DNA Bacteriano/química , DNA Bacteriano/genética , DNA de Cadeia Simples/biossíntese , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Escherichia coli/enzimologia , Conformação de Ácido Nucleico , RNA Bacteriano/biossíntese , RNA Bacteriano/química , RNA Bacteriano/genética , DNA Polimerase Dirigida por RNA/metabolismoRESUMO
Retron library recombineering (RLR) is a powerful tool in the field of genome editing that exceeds the scope and specificity of the CRISPR/Cas technique. In RLR, single-stranded DNA produced in vivo by harnessing the in-built potential of bacterial retrons is used for replication-dependent genome editing. RLR introduces several genomic variations at once, resulting in pooled and barcoded variant libraries, thus permitting multiplexed applications. Retron-generated RT-DNA has already shown promise for use in genome editing. Thus, this new tool will result in fresh, intriguing, and surprising developments in molecular biology and its juncture with other disciplines of research, including medicine, agriculture, and microbiology. In this review, we discuss the current state of this brand-new tool that could eventually boost genome editing research.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Edição de Genes/métodos , Sistemas CRISPR-Cas/genética , DNA , Bactérias/genética , DNA de Cadeia SimplesRESUMO
DNAzymes are catalytically active single-stranded DNAs in which DNAzyme 10-23 (Dz 10-23) consists of a catalytic core and a substrate-binding arm that reduces gene expression through sequence-specific mRNA cleavage. However, the in vivo application of Dz 10-23 depends on exogenous delivery, which leads to its inability to be synthesized and stabilized in vivo, thus limiting its application. As a unique reverse transcription system, the bacterial retron system can synthesize single-stranded DNA in vivo using ncRNA msr/msd as a template. The objective of this work is to reduce target gene expression using Dz 10-23 generated in vivo by the retron system. In this regard, we successfully generated Dz 10-23 by cloning the Dz 10-23 coding sequence into the retron msd gene and tested its ability to reduce specific gene expression by examining the mRNA levels of cfp encoding cyan fluorescence protein and other functional genes such as mreB and ftsZ. We found that Dz had different repressive effects when targeting different mRNA regions, and in general, the repressive effect was stronger when targeting downstream of mRNAs. Our results also suggested that the reduction effect was due to cleavage of the substrate mRNA by Dz 10-23 rather than the antisense effect of the substrate-binding arm. Therefore, this study not only provided a retron-based method for the intracellular generation of Dz 10-23 but also demonstrated that Dz 10-23 could reduce gene expression by cleaving target mRNAs in cells. We believe that this new strategy would have great potential in the regulation of gene expression.
Assuntos
DNA Catalítico , DNA Catalítico/genética , DNA Catalítico/metabolismo , DNA Bacteriano/genética , RNA Bacteriano/genética , Sequência de Bases , DNA de Cadeia Simples/genética , RNA Mensageiro/genéticaRESUMO
Genome editing technologies have the potential to transform our understanding of how genetic variation gives rise to complex traits through the systematic engineering and phenotypic characterization of genetic variants. However, there has yet to be a system with sufficient efficiency, fidelity, and throughput to comprehensively identify causal variants at the genome scale. Here we explored the ability of templated CRISPR editing systems to install natural variants genome-wide in budding yeast. We optimized several approaches to enhance homology-directed repair (HDR) with donor DNA templates, including donor recruitment to target sites, single-stranded donor production by bacterial retrons, and in vivo plasmid assembly. We uncovered unique advantages of each system that we integrated into a single superior system named MAGESTIC 3.0. We used MAGESTIC 3.0 to dissect causal variants residing in 112 quantitative trait loci across 32 environmental conditions, revealing an enrichment for missense variants and loci with multiple causal variants. MAGESTIC 3.0 will facilitate the functional analysis of the genome at single-nucleotide resolution and provides a roadmap for improving template-based genome editing systems in other organisms.
RESUMO
Retrons are a class of retroelements that produce multicopy single-stranded DNA (ssDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the overproduction of ssDNA, genome engineering and directed evolution in bacteria, yeast and mammalian cells. Retron-mediated ssDNA production in plants could unlock their potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Here, we demonstrated retron-mediated overproduction of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated clustered regularly interspaced short palindromic repeats-retron-coupled ssDNA overproduction and targeted HDR in N. benthamiana. Overall, we present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications. Graphical Abstract.
RESUMO
Recombineering is an important tool in gene editing, enabling fast, precise and highly specific in vivo modification of microbial genomes. Oligonucleotide-mediated recombineering via the in vivo production of single-stranded DNA can overcome the limitations of traditional recombineering methods that rely on the exogenous delivery of editing templates. By modifying a previously reported plasmid-based system for fully in vivo single-stranded DNA recombineering, we demonstrate iterative editing of independent loci by utilizing a temperature-sensitive origin of replication for easy curing of the editing plasmid from recombinant cells. Optimization of the promoters driving the expression of the system's functional components, combined with targeted counterselection against unedited cells with Cas9 nuclease, enabled editing efficiencies of 90-100%. The addition of a dominant-negative mutL allele to the system allowed single-nucleotide edits that were otherwise unachievable due to mismatch repair. Finally, we tested alternative recombinases and found that efficiency significantly increased for some targets. Requiring only a single cloning step for retargeting, our system provides an easy-to-use method for rapid, efficient construction of desired mutants. Graphical Abstract.
RESUMO
We developed a clustered regularly interspaced short palindromic repeats (CRISPR)/retron system for multiplexed generation of substitution mutations by coutilization of a retron system that continuously expresses donor DNA and a CRISPR/Cas9 cassette that induces cleavage at target genomic loci. Our system efficiently introduces substitution mutation in the Escherichia coli genome in a high-throughput manner. These substitution mutations can be tracked by analysis of retron plasmid sequences without laborious amplification of individual edited loci. We demonstrated that our CRISPR/retron system can introduce thousands of mutations in a single experiment and be used for screening phenotypes related to chemical responses or fitness changes. We expect that our system could facilitate genome-scale substitution screenings.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Escherichia coli/genética , Biblioteca Gênica , Genoma Bacteriano , Mutação , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
While several genome editing methods exist, few are suitable for the continuous evolution of targeted sequences. Here we develop bacterial retroelements known as "retrons" for the dynamic, in vivo editing and mutagenesis of targeted genes. We first optimized retrons' ability to introduce preprogrammed mutations, optimizing both their expression and the host machinery that interacts with them to increase the incorporation frequency of mutations 78-fold over rates previously reported in synthetic systems. The optimized system is capable of simultaneously overwriting 13 separate positions spanning a 31-base length, and is for the first time shown to yield targeted deletions and insertions. To engineer retrons as a tool to introduce novel, unprogrammed mutations in specific targeted regions, we expressed them under a mutagenic T7 RNA polymerase. This coupled mutagenic T7 RNA polymerase-retron system enabled the evolution of diverse variants of environmentally selected antibiotic resistance genes, producing mutation rates in the targeted region 190-fold higher than background cellular mutation rates, potentially enabling the dynamic, continuous self-evolution of selected phenotypes.
Assuntos
Evolução Molecular Direcionada , Edição de Genes/métodos , Retroelementos/genética , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Mutagênese , Regiões Promotoras Genéticas , Proteínas Virais/genéticaRESUMO
It has been long thought that reverse transcriptases are unique to the eukaryotes. However, through our research on a peculiar single stranded DNA called msDNA in Myxococcus xanthus, it was predicted that its synthesis requires reverse transcriptases. Subsequently, Lim and Maas as well as our group demonstrated the existence of reverse transcriptases for the production of msDNA. In this review, I describe how the discovery of msDNA led to the discovery of reverse transcriptases in bacteria and discuss the evolutionary significance of the discovery of revise transcriptases in bacteria.
Assuntos
DNA de Cadeia Simples , Myxococcus xanthus/genética , RNA Bacteriano , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/história , Bactérias/enzimologia , Bactérias/genética , DNA Bacteriano/genética , Evolução Molecular , História do Século XX , Myxococcus xanthus/enzimologiaRESUMO
A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.
RESUMO
Exonuclease VII (ExoVII) of Escherichia coli is a single strandspecific DNA nuclease composed of two different subunits: the large subunit, XseA, and the small subunit, XseB. In this study, we found that multicopy single-stranded DNAs (msDNAs), Ec83 and Ec78, are the in vivo substrates of ExoVII; the enzyme cuts the phosphodiester bond between the fourth and fifth nucleotides from the 5'end. We used this msDNA cleavage to assess ExoVII activity in vivo. Both subunits were required for enzyme activity. Expression of XseA without XseB caused cell death, even though no ExoVII activity was detected. The lethality caused by XseA was rescued by surplus XseB. In XseA-induced death, cells were elongated and multinucleated, and their chromosomes were fragmented and condensed; these are the morphological hallmarks of apoptotic cell death in bacteria. A putative caspase recognition sequence (FVAD) was found in XseA, and its hypothetical caspase product with 257 amino acids was as active as the intact protein in inducing cell death. We propose that under ordinary conditions, XseA protects chromosome as a component of the ExoVII enzyme, but in some conditions, the protein causes cell death; the destruction of cell is probably carried out by the amino terminal fragment derived from the cleavage of XseA by caspase-like enzyme.