RESUMO
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
Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. Although this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphology similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-associated proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphology in a genomically minimal cell.
Assuntos
Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Genoma Bacteriano , Mycoplasma/genética , Biologia Sintética/métodos , Proteínas de Bactérias/antagonistas & inibidores , Sistemas CRISPR-Cas , Engenharia GenéticaRESUMO
Tn7-like transposons have co-opted CRISPR systems, including class 1 type I-F, I-B, and class 2 type V-K. Intriguingly, although these CRISPR-associated transposases (CASTs) undergo robust CRISPR RNA (crRNA)-guided transposition, they are almost never found in sites targeted by the crRNAs encoded by the cognate CRISPR array. To understand this paradox, we investigated CAST V-K and I-B systems and found two distinct modes of transposition: (1) crRNA-guided transposition and (2) CRISPR array-independent homing. We show distinct CAST systems utilize different molecular mechanisms to target their homing site. Type V-K CAST systems use a short, delocalized crRNA for RNA-guided homing, whereas type I-B CAST systems, which contain two distinct target selector proteins, use TniQ for RNA-guided DNA transposition and TnsD for homing to an attachment site. These observations illuminate a key step in the life cycle of CAST systems and highlight the diversity of molecular mechanisms mediating transposon homing.
Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Elementos de DNA Transponíveis/fisiologia , DNA Bacteriano/metabolismo , RNA Guia de Cinetoplastídeos , Transposases/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/genética , Edição de Genes , Recombinação Genética , Transposases/genéticaRESUMO
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
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
Metagenomic inferences of bacterial strain diversity and infectious disease transmission studies largely assume a dominant, within-individual haplotype. We hypothesize that within-individual bacterial population diversity is critical for homeostasis of a healthy microbiome and infection risk. We characterized the evolutionary trajectory and functional distribution of Staphylococcus epidermidis-a keystone skin microbe and opportunistic pathogen. Analyzing 1,482 S. epidermidis genomes from 5 healthy individuals, we found that skin S. epidermidis isolates coalesce into multiple founder lineages rather than a single colonizer. Transmission events, natural selection, and pervasive horizontal gene transfer result in population admixture within skin sites and dissemination of antibiotic resistance genes within-individual. We provide experimental evidence for how admixture can modulate virulence and metabolism. Leveraging data on the contextual microbiome, we assess how interspecies interactions can shape genetic diversity and mobile gene elements. Our study provides insights into how within-individual evolution of human skin microbes shapes their functional diversification.
Assuntos
Evolução Molecular , Transferência Genética Horizontal , Interações entre Hospedeiro e Microrganismos/genética , Microbiota/genética , Polimorfismo de Nucleotídeo Único , Pele/microbiologia , Staphylococcus epidermidis/genética , Adulto , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Staphylococcus epidermidis/isolamento & purificação , Staphylococcus epidermidis/patogenicidade , Virulência/genética , Adulto JovemRESUMO
During cell division, newly replicated DNA is actively segregated to the daughter cells. In most bacteria, this process involves the DNA-binding protein ParB, which condenses the centromeric regions of sister DNA molecules into kinetochore-like structures that recruit the DNA partition ATPase ParA and the prokaroytic SMC/condensin complex. Here, we report the crystal structure of a ParB-like protein (PadC) that emerges to tightly bind the ribonucleotide CTP. The CTP-binding pocket of PadC is conserved in ParB and composed of signature motifs known to be essential for ParB function. We find that ParB indeed interacts with CTP and requires nucleotide binding for DNA condensation in vivo. We further show that CTP-binding modulates the affinity of ParB for centromeric parS sites, whereas parS recognition stimulates its CTPase activity. ParB proteins thus emerge as a new class of CTP-dependent molecular switches that act in concert with ATPases and GTPases to control fundamental cellular functions.
Assuntos
Proteínas de Bactérias/química , Citidina Trifosfato/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Motivos de Nucleotídeos , Ligação ProteicaRESUMO
Between 5,000 and 6,000 years ago, many Neolithic societies declined throughout western Eurasia due to a combination of factors that are still largely debated. Here, we report the discovery and genome reconstruction of Yersinia pestis, the etiological agent of plague, in Neolithic farmers in Sweden, pre-dating and basal to all modern and ancient known strains of this pathogen. We investigated the history of this strain by combining phylogenetic and molecular clock analyses of the bacterial genome, detailed archaeological information, and genomic analyses from infected individuals and hundreds of ancient human samples across Eurasia. These analyses revealed that multiple and independent lineages of Y. pestis branched and expanded across Eurasia during the Neolithic decline, spreading most likely through early trade networks rather than massive human migrations. Our results are consistent with the existence of a prehistoric plague pandemic that likely contributed to the decay of Neolithic populations in Europe.
Assuntos
Peste/história , Yersinia pestis/classificação , Yersinia pestis/patogenicidade , Evolução Biológica , DNA Bacteriano/genética , Europa (Continente) , Genoma Bacteriano , História Antiga , Humanos , Pandemias , FilogeniaRESUMO
The scaling of organelles with cell size is thought to be exclusive to eukaryotes. Here, we demonstrate that similar scaling relationships hold for the bacterial nucleoid. Despite the absence of a nuclear membrane, nucleoid size strongly correlates with cell size, independent of changes in DNA amount and across various nutrient conditions. This correlation is observed in diverse bacteria, revealing a near-constant ratio between nucleoid and cell size for a given species. As in eukaryotes, the nucleocytoplasmic ratio in bacteria varies greatly among species. This spectrum of nucleocytoplasmic ratios is independent of genome size, and instead it appears linked to the average population cell size. Bacteria with different nucleocytoplasmic ratios have a cytoplasm with different biophysical properties, impacting ribosome mobility and localization. Together, our findings identify new organizational principles and biophysical features of bacterial cells, implicating the nucleocytoplasmic ratio and cell size as determinants of the intracellular organization of translation.
Assuntos
Estruturas Celulares/metabolismo , Estruturas Celulares/fisiologia , Biossíntese de Proteínas/fisiologia , Bactérias/genética , Proteínas de Bactérias/metabolismo , Tamanho Celular , Citoplasma/fisiologia , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Organelas/metabolismo , Células Procarióticas/metabolismo , Células Procarióticas/fisiologia , Ribossomos/metabolismoRESUMO
Genes are often transcribed by multiple RNA polymerases (RNAPs) at densities that can vary widely across genes and environmental conditions. Here, we provide in vitro and in vivo evidence for a built-in mechanism by which co-transcribing RNAPs display either collaborative or antagonistic dynamics over long distances (>2 kb) through transcription-induced DNA supercoiling. In Escherichia coli, when the promoter is active, co-transcribing RNAPs translocate faster than a single RNAP, but their average speed is not altered by large variations in promoter strength and thus RNAP density. Environmentally induced promoter repression reduces the elongation efficiency of already-loaded RNAPs, causing premature termination and quick synthesis arrest of no-longer-needed proteins. This negative effect appears independent of RNAP convoy formation and is abrogated by topoisomerase I activity. Antagonistic dynamics can also occur between RNAPs from divergently transcribed gene pairs. Our findings may be broadly applicable given that transcription on topologically constrained DNA is the norm across organisms.
Assuntos
DNA Bacteriano/genética , DNA Super-Helicoidal/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/química , Regulação Bacteriana da Expressão Gênica/genética , Glucose/farmacologia , Glicosídeos/farmacologia , Isopropiltiogalactosídeo/farmacologia , Cinética , Óperon Lac/efeitos dos fármacos , Óperon Lac/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , Reação em Cadeia da Polimerase em Tempo Real , Rifampina/farmacologiaRESUMO
Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. However, the idea that replication forks would form at origins of DNA replication and proceed without impairment to copy the chromosomes has proven naive. It is now clear that replication forks stall frequently as a result of encounters between the replication machinery and template damage, slow-moving or paused transcription complexes, unrelieved positive superhelical tension, covalent protein-DNA complexes, and as a result of cellular stress responses. These stalled forks are a major source of genome instability. The cell has developed many strategies for ensuring that these obstructions to DNA replication do not result in loss of genetic information, including DNA damage tolerance mechanisms such as lesion skipping, whereby the replisome jumps the lesion and continues downstream; template switching both behind template damage and at the stalled fork; and the error-prone pathway of translesion synthesis.
Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Instabilidade Genômica , Humanos , Modelos BiológicosRESUMO
Spatial organization is a hallmark of all living systems. Even bacteria, the smallest forms of cellular life, display defined shapes and complex internal organization, showcasing a highly structured genome, cytoskeletal filaments, localized scaffolding structures, dynamic spatial patterns, active transport, and occasionally, intracellular organelles. Spatial order is required for faithful and efficient cellular replication and offers a powerful means for the development of unique biological properties. Here, we discuss organizational features of bacterial cells and highlight how bacteria have evolved diverse spatial mechanisms to overcome challenges cells face as self-replicating entities.
Assuntos
Bactérias/genética , Cromossomos Bacterianos/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Bactérias/citologia , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular/genética , Replicação do DNA/genética , DNA Bacteriano/genéticaRESUMO
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
In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles.
Assuntos
DNA Bacteriano , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Transcrição Gênica , Proteínas da Membrana Bacteriana Externa/metabolismo , Enzimas de Restrição do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Holoenzimas/metabolismo , Microscopia de Fluorescência , Poliestirenos/química , Proteoma , Análise de Sequência de RNA , Estresse Mecânico , TranscriptomaRESUMO
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
Microbial nucleic acids are major signatures of invading pathogens, and their recognition by various host pattern recognition receptors (PRRs) represents the first step toward an efficient innate immune response to clear the pathogens. The nucleic acid-sensing PRRs are localized at the plasma membrane, the cytosol, and/or various cellular organelles. Sensing of nucleic acids and signaling by PRRs involve recruitment of distinct signaling components, and PRRs are intensively regulated by cellular organelle trafficking. PRR-mediated innate immune responses are also heavily regulated by posttranslational modifications, including phosphorylation, polyubiquitination, sumoylation, and glutamylation. In this review, we focus on our current understanding of recognition of microbial nucleic acid by PRRs, particularly on their regulation by organelle trafficking and posttranslational modifications. We also discuss how sensing of self nucleic acids and dysregulation of PRR-mediated signaling lead to serious human diseases.
Assuntos
Interações Hospedeiro-Patógeno/genética , Imunidade Inata/genética , Ácidos Nucleicos/genética , Receptores de Reconhecimento de Padrão/genética , Bactérias/genética , Bactérias/patogenicidade , Citoplasma/imunologia , Citoplasma/microbiologia , DNA Bacteriano/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ácidos Nucleicos/imunologia , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais/genéticaRESUMO
The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium's restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium's DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world.
Assuntos
Sistema Livre de Células , Metilação de DNA , Biossíntese de Proteínas , Transcrição Gênica , Escherichia coli/genética , Escherichia coli/metabolismo , Transformação Bacteriana , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Metilases de Modificação do DNA/metabolismo , Metilases de Modificação do DNA/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
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.