RESUMO
Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.
Assuntos
Bactérias/genética , Biofilmes , DNA Bacteriano/química , Matriz Extracelular/metabolismo , Espaço Extracelular/química , Animais , Especificidade de Anticorpos , Proteínas de Bactérias/metabolismo , Linhagem Celular , Chinchila , DNA Cruciforme , Desoxirribonucleases/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Industrialization has impacted the human gut ecosystem, resulting in altered microbiome composition and diversity. Whether bacterial genomes may also adapt to the industrialization of their host populations remains largely unexplored. Here, we investigate the extent to which the rates and targets of horizontal gene transfer (HGT) vary across thousands of bacterial strains from 15 human populations spanning a range of industrialization. We show that HGTs have accumulated in the microbiome over recent host generations and that HGT occurs at high frequency within individuals. Comparison across human populations reveals that industrialized lifestyles are associated with higher HGT rates and that the functions of HGTs are related to the level of host industrialization. Our results suggest that gut bacteria continuously acquire new functionality based on host lifestyle and that high rates of HGT may be a recent development in human history linked to industrialization.
Assuntos
Bactérias/genética , Microbioma Gastrointestinal , Transferência Genética Horizontal , Bactérias/classificação , Bactérias/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Fezes/microbiologia , Genoma Bacteriano , Humanos , Filogenia , População Rural , Análise de Sequência de DNA , População Urbana , Sequenciamento Completo do GenomaRESUMO
All cells fold their genomes, including bacterial cells, where the chromosome is compacted into a domain-organized meshwork called the nucleoid. How compaction and domain organization arise is not fully understood. Here, we describe a method to estimate the average mesh size of the nucleoid in Escherichia coli. Using nucleoid mesh size and DNA concentration estimates, we find that the cytoplasm behaves as a poor solvent for the chromosome when the cell is considered as a simple semidilute polymer solution. Monte Carlo simulations suggest that a poor solvent leads to chromosome compaction and DNA density heterogeneity (i.e., domain formation) at physiological DNA concentration. Fluorescence microscopy reveals that the heterogeneous DNA density negatively correlates with ribosome density within the nucleoid, consistent with cryoelectron tomography data. Drug experiments, together with past observations, suggest the hypothesis that RNAs contribute to the poor solvent effects, connecting chromosome compaction and domain formation to transcription and intracellular organization.
Assuntos
Cromossomos Bacterianos/química , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Solventes/química , Transcrição Gênica , Aminoglicosídeos/farmacologia , Simulação por Computador , DNA Bacteriano/química , Difusão , Escherichia coli/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Tamanho da Partícula , RNA Bacteriano/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , Transcrição Gênica/efeitos dos fármacosRESUMO
The three-dimensional structures of chromosomes are increasingly being recognized as playing a major role in cellular regulatory states. The efficiency and promiscuity of phage Mu transposition was exploited to directly measure in vivo interactions between genomic loci in E. coli. Two global organizing principles have emerged: first, the chromosome is well-mixed and uncompartmentalized, with transpositions occurring freely between all measured loci; second, several gene families/regions show "clustering": strong three-dimensional co-localization regardless of linear genomic distance. The activities of the SMC/condensin protein MukB and nucleoid-compacting protein subunit HU-α are essential for the well-mixed state; HU-α is also needed for clustering of 6/7 ribosomal RNA-encoding loci. The data are explained by a model in which the chromosomal structure is driven by dynamic competition between DNA replication and chromosomal relaxation, providing a foundation for determining how region-specific properties contribute to both chromosomal structure and gene regulation.
Assuntos
Bacteriófago mu/genética , Cromossomos Bacterianos/genética , Elementos de DNA Transponíveis , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/química , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Conformação de Ácido Nucleico , Transposases/genética , Transposases/metabolismoRESUMO
When DNA is unwound during replication, it becomes overtwisted and forms positive supercoils in front of the translocating DNA polymerase. Unless removed or dissipated, this superhelical tension can impede replication elongation. Topoisomerases, including gyrase and topoisomerase IV in bacteria, are required to relax positive supercoils ahead of DNA polymerase but may not be sufficient for replication. Here, we find that GapR, a chromosome structuring protein in Caulobacter crescentus, is required to complete DNA replication. GapR associates in vivo with positively supercoiled chromosomal DNA, and our biochemical and structural studies demonstrate that GapR forms a dimer-of-dimers that fully encircles overtwisted DNA. Further, we show that GapR stimulates gyrase and topo IV to relax positive supercoils, thereby enabling DNA replication. Analogous chromosome structuring proteins that locate to the overtwisted DNA in front of replication forks may be present in other organisms, similarly helping to recruit and stimulate topoisomerases during DNA replication.
Assuntos
Cromossomos Bacterianos/fisiologia , DNA Bacteriano/química , DNA Super-Helicoidal/metabolismo , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Caulobacter crescentus/fisiologia , Estruturas Cromossômicas/fisiologia , Cromossomos Bacterianos/metabolismo , DNA/fisiologia , Replicação do DNA/fisiologia , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo II/fisiologia , DNA Bacteriano/fisiologia , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , CinéticaRESUMO
Conjugative transposition drives the emergence of multidrug resistance in diverse bacterial pathogens, yet the mechanisms are poorly characterized. The Tn1549 conjugative transposon propagates resistance to the antibiotic vancomycin used for severe drug-resistant infections. Here, we present four high-resolution structures of the conserved Y-transposase of Tn1549 complexed with circular transposon DNA intermediates. The structures reveal individual transposition steps and explain how specific DNA distortion and cleavage mechanisms enable DNA strand exchange with an absolute minimum homology requirement. This appears to uniquely allow Tn916-like conjugative transposons to bypass DNA homology and insert into diverse genomic sites, expanding gene transfer. We further uncover a structural regulatory mechanism that prevents premature cleavage of the transposon DNA before a suitable target DNA is found and generate a peptide antagonist that interferes with the transposase-DNA structure to block transposition. Our results reveal mechanistic principles of conjugative transposition that could help control the spread of antibiotic resistance genes.
Assuntos
DNA Bacteriano/metabolismo , Transposases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Clivagem do DNA , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , Farmacorresistência Bacteriana , Enterococcus faecalis/genética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Transposases/antagonistas & inibidores , Transposases/química , Transposases/genéticaRESUMO
Relaxases play essential roles in conjugation, the main process by which bacteria exchange genetic material, notably antibiotic resistance genes. They are bifunctional enzymes containing a trans-esterase activity, which is responsible for nicking the DNA strand to be transferred and for covalent attachment to the resulting 5'-phosphate end, and a helicase activity, which is responsible for unwinding the DNA while it is being transported to a recipient cell. Here we show that these two activities are carried out by two conformers that can both load simultaneously on the origin of transfer DNA. We solve the structure of one of these conformers by cryo electron microscopy to near-atomic resolution, elucidating the molecular basis of helicase function by relaxases and revealing insights into the mechanistic events taking place in the cell prior to substrate transport during conjugation.
Assuntos
Conjugação Genética , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , Escherichia coli/genética , Microscopia Crioeletrônica , DNA Helicases/química , DNA Bacteriano/química , DNA Bacteriano/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos MolecularesRESUMO
Transcript termination is essential for accurate gene expression and the removal of RNA polymerase (RNAP) at the ends of transcription units. In bacteria, two mechanisms are responsible for proper transcript termination: intrinsic termination and Rho-dependent termination. Intrinsic termination is mediated by signals directly encoded within the DNA template and nascent RNA, whereas Rho-dependent termination relies upon the adenosine triphosphate-dependent RNA translocase Rho, which binds nascent RNA and dissociates the elongation complex. Although significant progress has been made in understanding these pathways, fundamental details remain undetermined. Among those that remain unresolved are the existence of an inactivated intermediate in the intrinsic termination pathway, the role of Rho-RNAP interactions in Rho-dependent termination, and the mechanisms by which accessory factors and nucleoid-associated proteins affect termination. We describe current knowledge, discuss key outstanding questions, and highlight the importance of defining the structural rearrangements of RNAP that are involved in the two mechanisms of transcript termination.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Fatores de Alongamento de Peptídeos/genética , Fator Rho/genética , Fatores de Transcrição/genética , Terminação da Transcrição Genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Transporte Proteico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Fator Rho/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.
Assuntos
Proteínas Associadas a CRISPR/química , Cristalografia por Raios X , Endonucleases/química , RNA Bacteriano/química , Streptococcus pyogenes/química , Sequência de Aminoácidos , Bactérias/enzimologia , Proteínas Associadas a CRISPR/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Endonucleases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA Bacteriano/metabolismo , Alinhamento de Sequência , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/metabolismo , Pequeno RNA não TraduzidoRESUMO
Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.
Assuntos
DNA Bacteriano , RNA Polimerases Dirigidas por DNA , Escherichia coli , RNA Bacteriano , Terminação da Transcrição Genética , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/ultraestrutura , Regiões Terminadoras Genéticas/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestruturaRESUMO
Rho is a general transcription termination factor playing essential roles in RNA polymerase (RNAP) recycling, gene regulation, and genomic stability in most bacteria. Traditional models of transcription termination postulate that hexameric Rho loads onto RNA prior to contacting RNAP and then translocates along the transcript in pursuit of the moving RNAP to pull RNA from it. Here, we report the cryoelectron microscopy (cryo-EM) structures of two termination process intermediates. Prior to interacting with RNA, Rho forms a specific "pre-termination complex" (PTC) with RNAP and elongation factors NusA and NusG, which stabilize the PTC. RNA exiting RNAP interacts with NusA before entering the central channel of Rho from the distal C-terminal side of the ring. We map the principal interactions in the PTC and demonstrate their critical role in termination. Our results support a mechanism in which the formation of a persistent PTC is a prerequisite for termination.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Alongamento de Peptídeos/química , Fatores de Transcrição/química , Terminação da Transcrição Genética , Fatores de Elongação da Transcrição/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD1, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins4. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.
Assuntos
Adenosina Trifosfatases , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , RNA Bacteriano , Adenosina Trifosfatases/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Especificidade por Substrato , Transposases/metabolismoRESUMO
CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.
Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , Endodesoxirribonucleases/genética , Genoma Bacteriano/imunologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Bactérias/classificação , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Filogenia , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
Active matter consists of units that generate mechanical work by consuming energy1. Examples include living systems (such as assemblies of bacteria2-5 and biological tissues6,7), biopolymers driven by molecular motors8-11 and suspensions of synthetic self-propelled particles12-14. A central goal is to understand and control the self-organization of active assemblies in space and time. Most active systems exhibit either spatial order mediated by interactions that coordinate the spatial structure and the motion of active agents12,14,15 or the temporal synchronization of individual oscillatory dynamics2. The simultaneous control of spatial and temporal organization is more challenging and generally requires complex interactions, such as reaction-diffusion hierarchies16 or genetically engineered cellular circuits2. Here we report a simple technique to simultaneously control the spatial and temporal self-organization of bacterial active matter. We confine dense active suspensions of Escherichia coli cells and manipulate a single macroscopic parameter-namely, the viscoelasticity of the suspending fluid- through the addition of purified genomic DNA. This reveals self-driven spatial and temporal organization in the form of a millimetre-scale rotating vortex with periodically oscillating global chirality of tunable frequency, reminiscent of a torsional pendulum. By combining experiments with an active-matter model, we explain this behaviour in terms of the interplay between active forcing and viscoelastic stress relaxation. Our findings provide insight into the influence of bacterial motile behaviour in complex fluids, which may be of interest in health- and ecology-related research, and demonstrate experimentally that rheological properties can be harnessed to control active-matter flows17,18. We envisage that our millimetre-scale, tunable, self-oscillating bacterial vortex may be coupled to actuation systems to act a 'clock generator' capable of providing timing signals for rhythmic locomotion of soft robots and for programmed microfluidic pumping19, for example, by triggering the action of a shift register in soft-robotic logic devices20.
Assuntos
Escherichia coli/fisiologia , Reologia , Análise Espaço-Temporal , Substâncias Viscoelásticas/química , Substâncias Viscoelásticas/metabolismo , DNA Bacteriano/análise , DNA Bacteriano/química , Difusão , Escherichia coli/citologia , Escherichia coli/isolamento & purificação , Microfluídica , Peso Molecular , Movimento , Robótica , SuspensõesRESUMO
ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2-4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6-9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT-DarG toxin-antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP-HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT-DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.
Assuntos
ADP-Ribosilação , Proteínas de Bactérias/metabolismo , DNA/química , DNA/metabolismo , Timina/química , Timina/metabolismo , Adenosina Difosfato Ribose/metabolismo , Antitoxinas , Proteínas de Bactérias/química , Toxinas Bacterianas , Sequência de Bases , Biocatálise , DNA/genética , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA , Reparo do DNA , Elementos de DNA Transponíveis/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Modelos Moleculares , Mycobacterium/enzimologia , Mycobacterium/genética , Nitrogênio/química , Nitrogênio/metabolismo , Poli(ADP-Ribose) Polimerases/química , Origem de Replicação/genética , Especificidade por Substrato , Thermus/enzimologia , Timidina/química , Timidina/metabolismoRESUMO
In cells, dedicated AAA+ ATPases deposit hexameric, ring-shaped helicases onto DNA to initiate chromosomal replication. To better understand the mechanisms by which helicase loading can occur, we used cryo-EM to determine sub-4-Å-resolution structures of the E. coli DnaBâ DnaC helicaseâ loader complex with nucleotide in pre- and post-DNA engagement states. In the absence of DNA, six DnaC protomers latch onto and crack open a DnaB hexamer using an extended N-terminal domain, stabilizing this conformation through nucleotide-dependent ATPase interactions. Upon binding DNA, DnaC hydrolyzes ATP, allowing DnaB to isomerize into a topologically closed, pre-translocation state competent to bind primase. Our data show how DnaC opens the DnaB ring and represses the helicase prior to DNA binding and how DnaC ATPase activity is reciprocally regulated by DnaB and DNA. Comparative analyses reveal how the helicase loading mechanism of DnaC parallels and diverges from homologous AAA+ systems involved in DNA replication and transposition.
Assuntos
Replicação do DNA , DNA Bacteriano/biossíntese , DnaB Helicases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , DNA Primase/genética , DNA Primase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DnaB Helicases/química , DnaB Helicases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Hidrólise , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Endonuclease V (EndoV) cleaves the second phosphodiester bond 3' to a deaminated adenosine (inosine). Although highly conserved, EndoV homologs change substrate preference from DNA in bacteria to RNA in eukaryotes. We have characterized EndoV from six different species and determined crystal structures of human EndoV and three EndoV homologs from bacteria to mouse in complex with inosine-containing DNA/RNA hybrid or double-stranded RNA (dsRNA). Inosine recognition is conserved, but changes in several connecting loops in eukaryotic EndoV confer recognition of 3 ribonucleotides upstream and 7 or 8 bp of dsRNA downstream of the cleavage site, and bacterial EndoV binds only 2 or 3 nt flanking the scissile phosphate. In addition to the two canonical metal ions in the active site, a third Mn2+ that coordinates the nucleophilic water appears necessary for product formation. Comparison of EndoV with its homologs RNase H1 and Argonaute reveals the principles by which these enzymes recognize RNA versus DNA.
Assuntos
Proteínas de Bactérias/metabolismo , Reparo do DNA , DNA Bacteriano/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Evolução Molecular , Inosina/metabolismo , RNA/metabolismo , Ribonuclease H/metabolismo , Animais , Proteínas Argonautas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , DNA Bacteriano/química , DNA Bacteriano/genética , Desoxirribonuclease (Dímero de Pirimidina)/química , Desoxirribonuclease (Dímero de Pirimidina)/genética , Humanos , Magnésio/metabolismo , Manganês/metabolismo , Camundongos , Conformação de Ácido Nucleico , Conformação Proteica , RNA/química , RNA/genética , Ribonuclease H/química , Ribonuclease H/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)-guided detection and nuclease-mediated elimination of invading nucleic acids. Here, we present the cryo-electron microscopy (cryo-EM) structure of the type I-F crRNA-guided surveillance complex (Csy complex) from Pseudomonas aeruginosa bound to a double-stranded DNA target. Comparison of this structure to previously determined structures of this complex reveals a â¼180-degree rotation of the C-terminal helical bundle on the "large" Cas8f subunit. We show that the double-stranded DNA (dsDNA)-induced conformational change in Cas8f exposes a Cas2/3 "nuclease recruitment helix" that is structurally homologous to a virally encoded anti-CRISPR protein (AcrIF3). Structural homology between Cas8f and AcrIF3 suggests that AcrIF3 is a mimic of the Cas8f nuclease recruitment helix.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/metabolismo , Mimetismo Molecular , Pseudomonas aeruginosa/enzimologia , RNA Bacteriano/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Virais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/imunologia , Microscopia Crioeletrônica , DNA Bacteriano/química , DNA Bacteriano/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , Relação Estrutura-Atividade , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Since the nucleoid was isolated from bacteria in the 1970s, two fundamental questions emerged and are still in the spotlight: how bacteria organize their chromosomes to fit inside the cell and how nucleoid organization enables essential biological processes. During the last decades, knowledge of bacterial chromosome organization has advanced considerably, and today, such chromosomes are considered to be highly organized and dynamic structures that are shaped by multiple factors in a multiscale manner. Here we review not only the classical well-known factors involved in chromosome organization but also novel components that have recently been shown to dynamically shape the 3D structuring of the bacterial genome. We focus on the different functional elements that control short-range organization and describe how they collaborate in the establishment of the higher-order folding and disposition of the chromosome. Recent advances have opened new avenues for a deeper understanding of the principles and mechanisms of chromosome organization in bacteria.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA , Bactérias/genética , Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Genoma BacterianoRESUMO
Bacterial chromosomes are folded to compact DNA and facilitate cellular processes. Studying model bacteria has revealed aspects of chromosome folding that are applicable to many species. Primarily controlled by nucleoid-associated proteins, chromosome folding is hierarchical, from large-scale macrodomains to smaller-scale structures that influence DNA transactions, including replication and transcription. Here we review the environmentally regulated, architectural and regulatory roles of nucleoid-associated proteins and the implications for bacterial cell biology. We also highlight similarities and differences in the chromosome folding mechanisms of bacteria and eukaryotes.