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1.
Nat Commun ; 14(1): 294, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36653393

RESUMO

Conjugation is a contact-dependent mechanism for the transfer of plasmid DNA between bacterial cells, which contributes to the dissemination of antibiotic resistance. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugative transfer of F-plasmid in E. coli, in real time. We show that the transfer of plasmid in single-stranded form (ssDNA) and its subsequent conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ssDNA-to-dsDNA conversion determines the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent conversion into dsDNA turns off leading gene expression, and activates the expression of other plasmid genes under the control of conventional double-stranded promoters. This molecular strategy allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.


Assuntos
Conjugação Genética , Escherichia coli , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos/genética , DNA , DNA de Cadeia Simples/genética , Transferência Genética Horizontal
2.
Mol Microbiol ; 119(1): 19-28, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36565252

RESUMO

Transcription is a noisy and stochastic process that produces sibling-to-sibling variations in physiology across a population of genetically identical cells. This pattern of diversity reflects, in part, the burst-like nature of transcription. Transcription bursting has many causes and a failure to remove the supercoils that accumulate in DNA during transcription elongation is an important contributor. Positive supercoiling of the DNA ahead of the transcription elongation complex can result in RNA polymerase stalling if this DNA topological roadblock is not removed. The relaxation of these positive supercoils is performed by the ATP-dependent type II topoisomerases DNA gyrase and topoisomerase IV. Interference with the action of these topoisomerases involving, inter alia, topoisomerase poisons, fluctuations in the [ATP]/[ADP] ratio, and/or the intervention of nucleoid-associated proteins with GapR-like or YejK-like activities, may have consequences for the smooth operation of the transcriptional machinery. Antibiotic-tolerant (but not resistant) persister cells are among the phenotypic outliers that may emerge. However, interference with type II topoisomerase activity can have much broader consequences, making it an important epigenetic driver of physiological diversity in the bacterial population.


Assuntos
DNA Girase , DNA , DNA Girase/genética , DNA Girase/metabolismo , DNA Topoisomerase IV/genética , Bactérias/genética , Bactérias/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Trifosfato de Adenosina/metabolismo , Epigênese Genética , DNA Super-Helicoidal , DNA Bacteriano/genética , DNA Bacteriano/metabolismo
3.
Elife ; 112022 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-36533901

RESUMO

Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.


Assuntos
Bacteriófagos , Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Exodesoxirribonuclease V/genética , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Recombinases/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , DNA Bacteriano/metabolismo
4.
Int J Mol Sci ; 23(23)2022 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-36499040

RESUMO

Viability PCR (vPCR) uses a DNA intercalating dye to irreversibly bind double-stranded DNA from organisms with compromised cell membranes. This allows the selective amplification of DNA from intact cells. An optimized vPCR protocol should minimize false positives (DNA from compromised cells not fully removed) and false negatives (live cell DNA bound by the dye). We aimed to optimize a vPCR protocol using PMAxx™ as the intercalating agent and Salmonella Enteritidis as the target organism. To do this, we studied (1) single vs. sequential PMAxx™ addition; (2) a wash step post-PMAxx™ treatment; (3) a change of tube post-treatment before DNA extraction. The single vs. sequential PMAxx™ addition showed no difference. Results signified that PMAxx™ potentially attached to polypropylene tube walls and bound the released DNA from PMA-treated live cells when lysed in the same tube. A wash step was ineffective but transfer of the treated live cells to a new tube minimized these false-negative results. Our optimized protocol eliminated 108 CFU/mL heat-killed cell DNA in the presence of different live cell dilutions without compromising the amplification of the live cells, minimizing false positives. With further improvements, vPCR has great potential as a culture-independent diagnostic tool.


Assuntos
Azidas , Salmonella enteritidis , Propídio , Viabilidade Microbiana , Reação em Cadeia da Polimerase em Tempo Real/métodos , Salmonella enteritidis/genética , Salmonella enteritidis/metabolismo , DNA Bacteriano/metabolismo
5.
Sci Adv ; 8(46): eadd3912, 2022 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-36383666

RESUMO

The soil bacterium Agrobacterium tumefaciens is a preferred gene vector not only for plants but also for fungi. Agrobacterium delivers a small set of virulence proteins into host cells concomitantly with transferred DNA (T-DNA) to support the transformation process. Here, we find that expression of one of these proteins, called VirD5, in yeast host cells causes replication stress and DNA damage. This can result in both genomic rearrangements and local mutations, especially small deletions. Delivery of VirD5 during cocultivation with Agrobacterium led to mutations in the yeast genome that were unlinked to the integration of T-DNA. This load of mutations can be prevented by using a virD5 mutant for genome engineering, but this leads to a lower transformation frequency.


Assuntos
Agrobacterium tumefaciens , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Virulência/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Mutação , Dano ao DNA , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
6.
Elife ; 112022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36374535

RESUMO

The faithful segregation and inheritance of bacterial chromosomes and low-copy number plasmids requires dedicated partitioning systems. The most common of these, ParABS, consists of ParA, a DNA-binding ATPase and ParB, a protein that binds to centromeric-like parS sequences on the DNA cargo. The resulting nucleoprotein complexes are believed to move up a self-generated gradient of nucleoid-associated ParA. However, it remains unclear how this leads to the observed cargo positioning and dynamics. In particular, the evaluation of models of plasmid positioning has been hindered by the lack of quantitative measurements of plasmid dynamics. Here, we use high-throughput imaging, analysis and modelling to determine the dynamical nature of these systems. We find that F plasmid is actively brought to specific subcellular home positions within the cell with dynamics akin to an over-damped spring. We develop a unified stochastic model that quantitatively explains this behaviour and predicts that cells with the lowest plasmid concentration transition to oscillatory dynamics. We confirm this prediction for F plasmid as well as a distantly-related ParABS system. Our results indicate that ParABS regularly positions plasmids across the nucleoid but operates just below the threshold of an oscillatory instability, which according to our model, minimises ATP consumption. Our work also clarifies how various plasmid dynamics are achievable in a single unified stochastic model. Overall, this work uncovers the dynamical nature of plasmid positioning by ParABS and provides insights relevant for chromosome-based systems.


Assuntos
Adenosina Trifosfatases , Cromossomos Bacterianos , Plasmídeos/genética , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Adenosina Trifosfatases/metabolismo , Centrômero/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo
7.
J Biomed Sci ; 29(1): 97, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376872

RESUMO

BACKGROUND: The complement system functions primarily as a first-line host defense against invading microbes, including viruses. However, the interaction of Hepatitis B virus (HBV) with the complement-components during chronic HBV infection remains largely unknown. We investigated the mechanism by which HBV inhibits the formation of cytolytic complement membrane-attack complex (MAC) and studied its impact on MAC-mediated microbicidal activity and disease pathogenesis. METHODS: Blood/liver tissues were collected from chronically HBV-infected patients and controls. HepG2hNTCP cells were infected with HBV particles and Huh7 cells were transfected with full-length linear HBV-monomer or plasmids containing different HBV-ORFs and expression of complement components or other host genes were evaluated. Additionally, ELISA, Real-time PCR, Western blot, bioinformatics analysis, gene overexpression/knock-down, mutagenesis, chromatin immunoprecipitation, epigenetic studies, immunofluorescence, and quantification of serum HBV-DNA, bacterial-DNA and endotoxin were performed. RESULTS: Among the MAC components (C5b-C9), significant reduction was noted in the expression of C9, the major constituent of MAC, in HBV-infected HepG2hNTCP cells and in Huh7 cells transfected with full-length HBV as well as HBX. C9 level was also marked low in sera/liver of chronic hepatitis B (CHB) and Immune-tolerant (IT) patients than inactive carriers and healthy controls. HBX strongly repressed C9-promoter activity in Huh7 cells but CpG-island was not detected in C9-promoter. We identified USF-1 as the key transcription factor that drives C9 expression and demonstrated that HBX-induced hypermethylation of USF-1-promoter is the leading cause of USF-1 downregulation that in turn diminished C9 transcription. Reduced MAC formation and impaired lysis of HBV-transfected Huh7 and bacterial cells were observed following incubation of these cells with C9-deficient CHB sera but was reversed upon C9 supplementation. Significant inverse correlation was noted between C9 concentration and HBV-DNA, bacterial-DNA and endotoxin content in HBV-infected patients. One-year Tenofovir therapy resulted in improvement in C9 level and decline in viral/bacterial/endotoxin load in CHB patients. CONCLUSION: Collectively, HBX suppressed C9 transcription by restricting the availability of USF-1 through hypermethylation of USF-1-promoter and consequently hinder the formation and lytic functions of MAC. Early therapy is needed for both CHB and IT to normalize the aberrant complement profile and contain viral and bacterial infection and limit disease progression.


Assuntos
Vírus da Hepatite B , Hepatite B Crônica , Humanos , Complemento C9/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , DNA Bacteriano/metabolismo , Endotoxinas/metabolismo , Células Hep G2 , Vírus da Hepatite B/genética , Hepatite B Crônica/patologia , Transativadores/genética , Proteínas Virais Reguladoras e Acessórias
8.
PLoS One ; 17(11): e0277819, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36413541

RESUMO

The epigenetics of bacteria, and bacteria with a reduced genome in particular, is of great interest, but is still poorly understood. Mycoplasma gallisepticum, a representative of the class Mollicutes, is an excellent model of a minimal cell because of its reduced genome size, lack of a cell wall, and primitive cell organization. In this study we investigated DNA modifications of the model object Mycoplasma gallisepticum and their roles. We identified DNA modifications and methylation motifs in M. gallisepticum S6 at the genome level using single molecule real time (SMRT) sequencing. Only the ANCNNNNCCT methylation motif was found in the M. gallisepticum S6 genome. The studied bacteria have one functional system for DNA modifications, the Type I restriction-modification (RM) system, MgaS6I. We characterized its activity, affinity, protection and epigenetic functions. We demonstrated the protective effects of this RM system. A common epigenetic signal for bacteria is the m6A modification we found, which can cause changes in DNA-protein interactions and affect the cell phenotype. Native methylation sites are underrepresented in promoter regions and located only near the -35 box of the promoter, which does not have a significant effect on gene expression in mycoplasmas. To study the epigenetics effect of m6A for genome-reduced bacteria, we constructed a series of M. gallisepticum strains expressing EGFP under promoters with the methylation motifs in their different elements. We demonstrated that m6A modifications of the promoter located only in the -10-box affected gene expression and downregulated the expression of the corresponding gene.


Assuntos
Mycoplasma gallisepticum , Tenericutes , Mycoplasma gallisepticum/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Enzimas de Restrição-Modificação do DNA/genética , Tenericutes/genética , Metilação de DNA
9.
Adv Exp Med Biol ; 1389: 21-43, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36350505

RESUMO

The genomes of bacteria, archaea, and phage contain small amounts of C5-methylcytosine, N4-methylcytosine, and N6-methyladenine. Base methylation takes place after DNA replication and is catalyzed by DNA methyltransferases that recognize specific target sequences. Prokaryotic DNA methyltransferases can be classified into two main types: (1) belonging to restriction-modification systems and (2) solitary (or "orphan") enzymes that lack a restriction enzyme partner. All known roles of DNA methylation involve control of interactions between DNA-binding proteins and their cognate sites. Such roles include protection from DNA restriction, strand discrimination during mismatch repair, cell cycle control, and regulation of transcription. DNA methylation often affects the interaction of bacterial pathogens with their hosts, raising the possibility of epigenetic therapies for infectious diseases.


Assuntos
Metilação de DNA , Células Procarióticas , Células Procarióticas/metabolismo , Metilases de Modificação do DNA/genética , Epigenômica , Bactérias/metabolismo , DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo
10.
Sci Rep ; 12(1): 19232, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36357494

RESUMO

Polychlorinated biphenyl (PCB)-contaminated soils represent a major treat for ecosystems health. Plant biostimulation of autochthonous microbial PCB degraders is a way to restore polluted sites where traditional remediation techniques are not sustainable, though its success requires the understanding of site-specific plant-microbe interactions. In an historical PCB contaminated soil, we applied DNA stable isotope probing (SIP) using 13C-labeled 4-chlorobiphenyl (4-CB) and 16S rRNA MiSeq amplicon sequencing to determine how the structure of total and PCB-degrading bacterial populations were affected by different treatments: biostimulation with Phalaris arundinacea subjected (PhalRed) or not (Phal) to a redox cycle and the non-planted controls (Bulk and BulkRed). Phal soils hosted the most diverse community and plant biostimulation induced an enrichment of Actinobacteria. Mineralization of 4-CB in SIP microcosms varied between 10% in Bulk and 39% in PhalRed soil. The most abundant taxa deriving carbon from PCB were Betaproteobacteria and Actinobacteria. Comamonadaceae was the family most represented in Phal soils, Rhodocyclaceae and Nocardiaceae in non-planted soils. Planted soils subjected to redox cycle enriched PCB degraders affiliated to Pseudonocardiaceae, Micromonosporaceae and Nocardioidaceae. Overall, we demonstrated different responses of soil bacterial taxa to specific rhizoremediation treatments and we provided new insights into the populations active in PCB biodegradation.


Assuntos
Actinomycetales , Bifenilos Policlorados , Poluentes do Solo , Solo/química , Bifenilos Policlorados/metabolismo , Poluentes do Solo/metabolismo , Microbiologia do Solo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Ecossistema , Biodegradação Ambiental , Bactérias , Plantas/metabolismo , Actinomycetales/genética , Isótopos/metabolismo , DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo
11.
Proc Natl Acad Sci U S A ; 119(41): e2204042119, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36206370

RESUMO

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.


Assuntos
Bacillus subtilis , Cromossomos Bacterianos , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos
12.
Microbiologyopen ; 11(5): e1316, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36314749

RESUMO

Antibiotic resistance is becoming increasingly prevalent amongst bacterial pathogens and there is an urgent need to develop new types of antibiotics with novel modes of action. One promising strategy is to develop resistance-breaker compounds, which inhibit resistance mechanisms and thus resensitize bacteria to existing antibiotics. In the current study, we identify bacterial DNA double-strand break repair as a promising target for the development of resistance-breaking co-therapies. We examined genetic variants of Escherichia coli that combined antibiotic-resistance determinants with DNA repair defects. We observed that defects in the double-strand break repair pathway led to significant resensitization toward five bactericidal antibiotics representing different functional classes. Effects ranged from partial to full resensitization. For ciprofloxacin and nitrofurantoin, sensitization manifested as a reduction in the minimum inhibitory concentration. For kanamycin and trimethoprim, sensitivity manifested through increased rates of killing at high antibiotic concentrations. For ampicillin, repair defects dramatically reduced antibiotic tolerance. Ciprofloxacin, nitrofurantoin, and trimethoprim induce the promutagenic SOS response. Disruption of double-strand break repair strongly dampened the induction of SOS by these antibiotics. Our findings suggest that if break-repair inhibitors can be developed they could resensitize antibiotic-resistant bacteria to multiple classes of existing antibiotics and may suppress the development of de novo antibiotic-resistance mutations.


Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Humanos , Escherichia coli/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Nitrofurantoína/metabolismo , Nitrofurantoína/farmacologia , Reparo do DNA , Ciprofloxacina/farmacologia , Testes de Sensibilidade Microbiana , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Trimetoprima/metabolismo , Trimetoprima/farmacologia
13.
Environ Sci Technol ; 56(22): 15791-15804, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36215406

RESUMO

Monochloramine (MCA) is a widely used secondary disinfectant to suppress microbial growth in drinking water distribution systems. In monochloraminated drinking water, a significant amount of extracellular DNA (eDNA) has been reported, which has many implications ranging from obscuring DNA-based drinking water microbiome analyses to posing potential health concerns. To address this, it is imperative for us to know the origin of the eDNA in drinking water. Using Pseudomonas aeruginosa as a model organism, we report for the first time that MCA induces the release of nucleic acids from both biofilms and planktonic cells. Upon exposure to 2 mg/L MCA, massive release of DNA from suspended cells in both MilliQ water and 0.9% NaCl was directly visualized using live cell imaging in a CellASIC ONIX2 microfluidic system. Exposing established biofilms to MCA also resulted in DNA release from the biofilms, which was confirmed by increased detection of eDNA in the effluent. Intriguingly, massive release of RNA was also observed, and the extracellular RNA (eRNA) was also found to persist in water for days. Sequencing analyses of the eDNA revealed that it could be used to assemble the whole genome of the model organism, while in the water, certain fragments of the genome were more persistent than others. RNA sequencing showed that the eRNA contains non-coding RNA and mRNA, implying its role as a possible signaling molecule in environmental systems and a snapshot of the past metabolic state of the bacterial cells.


Assuntos
Água Potável , RNA , Cloraminas , Biofilmes , DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo
14.
Microbiology (Reading) ; 168(10)2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36301085

RESUMO

Replication and segregation of the genetic information is necessary for a cell to proliferate. In Bacillus subtilis, the Par system (ParA/Soj, ParB/Spo0J and parS) is required for segregation of the chromosome origin (oriC) region and for proper control of DNA replication initiation. ParB binds parS sites clustered near the origin of replication and assembles into sliding clamps that interact with ParA to drive origin segregation through a diffusion-ratchet mechanism. As part of this dynamic process, ParB stimulates ParA ATPase activity to trigger its switch from an ATP-bound dimer to an ADP-bound monomer. In addition to its conserved role in DNA segregation, ParA is also a regulator of the master DNA replication initiation protein DnaA. We hypothesized that in B. subtilis the location of the Par system proximal to oriC would be necessary for ParA to properly regulate DnaA. To test this model, we constructed a range of genetically modified strains with altered numbers and locations of parS sites, many of which perturbed chromosome origin segregation as expected. Contrary to our hypothesis, the results show that regulation of DNA replication initiation by ParA is maintained when a parS site is separated from oriC. Because a single parS site is sufficient for proper control of ParA, the results are consistent with a model where ParA is efficiently regulated by ParB sliding clamps following loading at parS.


Assuntos
Bacillus subtilis , Cromossomos Bacterianos , Bacillus subtilis/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Replicação do DNA/genética , Segregação de Cromossomos , Origem de Replicação/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo
15.
Nucleic Acids Res ; 50(19): 11013-11027, 2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36243965

RESUMO

Escherichia coli cells treated with a combination of cyanide (CN) and hydrogen peroxide (HP) succumb to catastrophic chromosome fragmentation (CCF), detectable in pulsed-field gels as >100 double-strand breaks per genome equivalent. Here we show that CN + HP-induced double-strand breaks are independent of replication and occur uniformly over the chromosome,-therefore we used CCF to probe the nucleoid structure by measuring DNA release from precipitated nucleoids. CCF releases surprisingly little chromosomal DNA from the nucleoid suggesting that: (i) the nucleoid is a single DNA-protein complex with only limited stretches of protein-free DNA and (ii) CN + HP-induced breaks happen within these unsecured DNA stretches, rather than at DNA attachments to the central scaffold. Mutants lacking individual nucleoid-associated proteins (NAPs) release more DNA during CCF, consistent with NAPs anchoring chromosome to the central scaffold (Dps also reduces the number of double-strand breaks directly). Finally, significantly more broken DNA is released once ATP production is restored, with about two-thirds of this ATP-dependent DNA release being due to transcription, suggesting that transcription complexes act as pulleys to move DNA loops. In addition to NAPs, recombinational repair of double-strand breaks also inhibits DNA release by CCF, contributing to a dynamic and complex nucleoid structure.


Assuntos
Cromossomos Bacterianos , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Peróxido de Hidrogênio/metabolismo , Trifosfato de Adenosina/metabolismo
16.
Biophys J ; 121(21): 4153-4165, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36171726

RESUMO

All biological processes ultimately come from physical interactions. The mechanical properties of DNA play a critical role in transcription. RNA polymerase can over or under twist DNA (referred to as DNA supercoiling) when it moves along a gene, resulting in mechanical stresses in DNA that impact its own motion and that of other polymerases. For example, when enough supercoiling accumulates, an isolated polymerase halts, and transcription stops. DNA supercoiling can also mediate nonlocal interactions between polymerases that shape gene expression fluctuations. Here, we construct a comprehensive model of transcription that captures how RNA polymerase motion changes the degree of DNA supercoiling, which in turn feeds back into the rate at which polymerases are recruited and move along the DNA. Surprisingly, our model predicts that a group of three or more polymerases move together at a constant velocity and sustain their motion (forming what we call a polymeton), whereas one or two polymerases would have halted. We further show that accounting for the impact of DNA supercoiling on both RNA polymerase recruitment and velocity recapitulates empirical observations of gene expression fluctuations. Finally, we propose a mechanical toggle switch whereby interactions between genes are mediated by DNA twisting as opposed to proteins. Understanding the mechanical regulation of gene expression provides new insights into how endogenous genes can interact and informs the design of new forms of engineered interactions.


Assuntos
DNA Super-Helicoidal , Transcrição Genética , RNA Polimerases Dirigidas por DNA/metabolismo , DNA/genética , DNA Bacteriano/metabolismo
17.
Front Cell Infect Microbiol ; 12: 999737, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36081771

RESUMO

HU protein is a member of nucleoid-associated proteins (NAPs) and is an important regulator of bacterial virulence, pathogenesis and survival. NAPs are mainly DNA structuring proteins that influence several molecular processes by binding the DNA. HU´s indispensable role in DNA-related processes in bacteria was described. HU protein is a necessary bacterial transcription factor and is considered to be a virulence determinant as well. Less is known about its direct role in host-pathogen interactions. The latest studies suggest that HU protein may be secreted outside bacteria and be a part of the extracellular matrix. Moreover, HU protein can be internalized in a host cell after bacterial infection. Its role in the host cell is not well described and further studies are extremely needed. Existing results suggest the involvement of HU protein in host cell immune response modulation in bacterial favor, which can help pathogens resist host defense mechanisms. A better understanding of the HU protein's role in the host cell will help to effective treatment development.


Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA , Proteínas de Bactérias/genética , DNA/química , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Fatores de Virulência
18.
DNA Repair (Amst) ; 118: 103389, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36030574

RESUMO

DNA recombination repair systems are essential for organisms to maintain genomic stability. In recent years, we have improved our understanding of the mechanisms of RecBCD/AddAB family-mediated DNA double-strand break repair. In E. coli, it is RecBCD that plays a central role, and in Firmicute Bacillus subtilis it is the AddAB complex that functions. However, there are open questions about the mechanism of DNA repair in bacteria. For example, how bacteria containing crossover hotspot instigator (Chi) sites regulate the activity of proteins. In addition, we still do not know the exact process by which the RecB nuclease or AddA nuclease structural domains load RecA onto DNA. We also know little about the mechanism of DNA repair in the industrially important production bacterium Corynebacterium glutamicum (C. glutamicum). Therefore, exploring DNA repair mechanisms in bacteria may not only deepen our understanding of the DNA repair process in this species but also guide us in the targeted treatment of diseases associated with recombination defects, such as cancer. In this paper, we firstly review the classical proteins RecBCD and AddAB involved in DNA recombination repair, secondly focus on the novel helical nuclease AdnAB found in the genus Mycobacterium.


Assuntos
Escherichia coli , Exodesoxirribonucleases , Bacillus subtilis , DNA/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/genética , Exodesoxirribonuclease V/metabolismo , Exodesoxirribonucleases/metabolismo
19.
Nature ; 609(7926): 384-393, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36002573

RESUMO

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/metabolismo
20.
Nucleic Acids Res ; 50(16): 9149-9161, 2022 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-35950487

RESUMO

DNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of these enzymes with antibiotics leads to global supercoiling modifications and subsequent changes in global gene expression. In previous studies, genes responding to DNA relaxation induced by DNA gyrase inhibition were categorised as 'supercoiling-sensitive'. Here, we studied the opposite variation of DNA supercoiling in the phytopathogen Dickeya dadantii using the non-marketed antibiotic seconeolitsine. We showed that the drug is active against topoisomerase I from this species, and analysed the first transcriptomic response of a Gram-negative bacterium to topoisomerase I inhibition. We find that the responding genes essentially differ from those observed after DNA relaxation, and further depend on the growth phase. We characterised these genes at the functional level, and also detected distinct patterns in terms of expression level, spatial and orientational organisation along the chromosome. Altogether, these results highlight that the supercoiling-sensitivity is a complex feature, which depends on the action of specific topoisomerases, on the physiological conditions, and on their genomic context. Based on previous in vitro expression data of several promoters, we propose a qualitative model of SC-dependent regulation that accounts for many of the contrasting transcriptomic features observed after DNA gyrase or topoisomerase I inhibition.


Assuntos
DNA Girase , DNA Topoisomerases Tipo I , DNA Girase/genética , DNA Girase/metabolismo , DNA Topoisomerases Tipo I/metabolismo , DNA Super-Helicoidal/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Antibacterianos/farmacologia
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