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1.
Genome Med ; 13(1): 182, 2021 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-34784976

RESUMO

BACKGROUND: Clinical metagenomics (CMg) has the potential to be translated from a research tool into routine service to improve antimicrobial treatment and infection control decisions. The SARS-CoV-2 pandemic provides added impetus to realise these benefits, given the increased risk of secondary infection and nosocomial transmission of multi-drug-resistant (MDR) pathogens linked with the expansion of critical care capacity. METHODS: CMg using nanopore sequencing was evaluated in a proof-of-concept study on 43 respiratory samples from 34 intubated patients across seven intensive care units (ICUs) over a 9-week period during the first COVID-19 pandemic wave. RESULTS: An 8-h CMg workflow was 92% sensitive (95% CI, 75-99%) and 82% specific (95% CI, 57-96%) for bacterial identification based on culture-positive and culture-negative samples, respectively. CMg sequencing reported the presence or absence of ß-lactam-resistant genes carried by Enterobacterales that would modify the initial guideline-recommended antibiotics in every case. CMg was also 100% concordant with quantitative PCR for detecting Aspergillus fumigatus from 4 positive and 39 negative samples. Molecular typing using 24-h sequencing data identified an MDR-K. pneumoniae ST307 outbreak involving 4 patients and an MDR-C. striatum outbreak involving 14 patients across three ICUs. CONCLUSION: CMg testing provides accurate pathogen detection and antibiotic resistance prediction in a same-day laboratory workflow, with assembled genomes available the next day for genomic surveillance. The provision of this technology in a service setting could fundamentally change the multi-disciplinary team approach to managing ICU infections. The potential to improve the initial targeted treatment and rapidly detect unsuspected outbreaks of MDR-pathogens justifies further expedited clinical assessment of CMg.


Assuntos
COVID-19/patologia , Infecção Hospitalar/transmissão , Metagenômica , Antibacterianos/uso terapêutico , COVID-19/virologia , Coinfecção/tratamento farmacológico , Coinfecção/microbiologia , Corynebacterium/genética , Corynebacterium/isolamento & purificação , Infecção Hospitalar/microbiologia , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Humanos , Unidades de Terapia Intensiva , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , SARS-CoV-2/isolamento & purificação , Análise de Sequência de DNA , beta-Lactamases/genética
2.
Phys Rev Lett ; 127(13): 138101, 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34623846

RESUMO

The spatiotemporal organization of bacterial cells is crucial for the active segregation of replicating chromosomes. In several species, including Caulobacter crescentus, the ATPase ParA binds to DNA and forms a gradient along the long cell axis. The ParB partition complex on the newly replicated chromosome translocates up this ParA gradient, thereby contributing to chromosome segregation. A DNA-relay mechanism-deriving from the elasticity of the fluctuating chromosome-has been proposed as the driving force for this cargo translocation, but a mechanistic theoretical description remains elusive. Here, we propose a minimal model to describe force generation by the DNA-relay mechanism over a broad range of operational conditions. Conceptually, we identify four distinct force-generation regimes characterized by their dependence on chromosome fluctuations. These relay force regimes arise from an interplay of the imposed ParA gradient, chromosome fluctuations, and an emergent friction force due to chromosome-cargo interactions.


Assuntos
DNA Bacteriano/metabolismo , Modelos Biológicos , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Transporte Biológico , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos , DNA Primase/química , DNA Primase/genética , DNA Primase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética
3.
Molecules ; 26(19)2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34641320

RESUMO

The nucleoid-associated protein GapR found in Caulobacter crescentus is crucial for DNA replication, transcription, and cell division. Associated with overtwisted DNA in front of replication forks and the 3' end of highly-expressed genes, GapR can stimulate gyrase and topo IV to relax (+) supercoils, thus facilitating the movement of the replication and transcription machines. GapR forms a dimer-of-dimers structure in solution that can exist in either an open or a closed conformation. It initially binds DNA through the open conformation and then undergoes structural rearrangement to form a closed tetramer, with DNA wrapped in the central channel. Here, we show that the DNA binding domain of GapR (residues 1-72, GapRΔC17) exists as a dimer in solution and adopts the same fold as the two dimer units in the full-length tetrameric protein. It binds DNA at the minor groove and reads the spatial distribution of DNA phosphate groups through a lysine/arginine network, with a preference towards AT-rich overtwisted DNA. These findings indicate that the dimer unit of GapR has an intrinsic DNA binding preference. Thus, at the initial binding step, the open tetramer of GapR with two relatively independent dimer units can be more efficiently recruited to overtwisted regions.


Assuntos
Caulobacter crescentus/metabolismo , DNA Bacteriano/metabolismo , Transativadores/química , Transativadores/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Caulobacter crescentus/química , Caulobacter crescentus/genética , Cristalografia por Raios X , DNA Bacteriano/química , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Transativadores/genética
4.
PLoS One ; 16(10): e0258583, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34644346

RESUMO

Axenically cultured Liberibacter crescens (Lcr) is a closely related surrogate for uncultured plant pathogenic species of the genus Liberibacter, including 'Candidatus L. asiaticus' (CLas) and 'Ca. L. solanacearum' (CLso). All Liberibacters encode a completely conserved gene repertoire for both flagella and Tad (Tight Adherence) pili and all are missing genes critical for nucleotide biosynthesis. Both flagellar swimming and Tad pilus-mediated twitching motility in Lcr were demonstrated for the first time. A role for Tad pili in the uptake of extracellular dsDNA for food in Liberibacters was suspected because both twitching and DNA uptake are impossible without repetitive pilus extension and retraction, and no genes encoding other pilus assemblages or mechanisms for DNA uptake were predicted to be even partially present in any of the 35 fully sequenced Liberibacter genomes. Insertional mutations of the Lcr Tad pilus genes cpaA, cpaB, cpaE, cpaF and tadC all displayed such severely reduced growth and viability that none could be complemented. A mutation affecting cpaF (motor ATPase) was further characterized and the strain displayed concomitant loss of twitching, viability and reduced periplasmic uptake of extracellular dsDNA. Mutations of comEC, encoding the inner membrane competence channel, had no effect on either motility or growth but completely abolished natural transformation in Lcr. The comEC mutation was restored by complementation using comEC from Lcr but not from CLas strain psy62 or CLso strain RS100, indicating that unlike Lcr, these pathogens were not naturally competent for transformation. This report provides the first evidence that the Liberibacter Tad pili are dynamic and essential for both motility and DNA uptake, thus extending their role beyond surface adherence.


Assuntos
DNA Bacteriano/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citrus/microbiologia , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/fisiologia , Liberibacter/genética , Liberibacter/crescimento & desenvolvimento , Liberibacter/fisiologia , Mutagênese Sítio-Dirigida , Doenças das Plantas/microbiologia , Alinhamento de Sequência
5.
PLoS One ; 16(10): e0258500, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34644359

RESUMO

Chlamydial infections, caused by a group of obligate, intracellular, gram-negative bacteria, have health implications for animals and humans. Due to their highly infectious nature and zoonotic potential, staff at wildlife rehabilitation centers should be educated on the clinical manifestations, prevalence, and risk factors associated with Chlamydia spp. infections in raptors. The objectives of this study were to document the prevalence of chlamydial DNA shedding and anti-chlamydial antibodies in raptors admitted to five wildlife rehabilitation centers in California over a one-year period. Chlamydial prevalence was estimated in raptors for each center and potential risk factors associated with infection were evaluated, including location, species, season, and age class. Plasma samples and conjunctiva/choana/cloaca swabs were collected for serology and qPCR from a subset of 263 birds of prey, representing 18 species. Serologic assays identified both anti-C. buteonis IgM and anti-chlamydial IgY antibodies. Chlamydial DNA and anti-chlamydial antibodies were detected in 4.18% (11/263) and 3.14% (6/191) of patients, respectively. Chamydial DNA was identified in raptors from the families Accipitridae and Strigidae while anti-C.buteonis IgM was identified in birds identified in Accipitridae, Falconidae, Strigidae, and Cathartidae. Two of the chlamydial DNA positive birds (one Swainson's hawk (Buteo swainsoni) and one red-tailed hawk (Buteo jamaicensis)) were necropsied, and tissues were collected for culture. Sequencing of the cultured elementary bodies revealed a chlamydial DNA sequence with 99.97% average nucleotide identity to the recently described Chlamydia buteonis. Spatial clusters of seropositive raptors and raptors positive for chlamydial DNA were detected in northern California. Infections were most prevalent during the winter season. Furthermore, while the proportion of raptors testing positive for chlamydial DNA was similar across age classes, seroprevalence was highest in adults. This study questions the current knowledge on C. buteonis host range and highlights the importance of further studies to evaluate the diversity and epidemiology of Chlamydia spp. infecting raptor populations.


Assuntos
Doenças das Aves/epidemiologia , Infecções por Chlamydia/epidemiologia , Chlamydia/isolamento & purificação , Aves Predatórias/microbiologia , Animais , Animais Selvagens , Anticorpos Antibacterianos/sangue , Doenças das Aves/imunologia , Doenças das Aves/microbiologia , California/epidemiologia , Chlamydia/classificação , Chlamydia/genética , Chlamydia/imunologia , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Cloaca/microbiologia , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Imunoglobulina M/sangue , Imunoglobulinas/sangue , Filogenia , Prevalência , Centros de Reabilitação , Fatores de Risco , Análise de Sequência de DNA
6.
Nat Commun ; 12(1): 5293, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489448

RESUMO

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31P,1H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19F and 27Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.


Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , DNA Bacteriano/química , DnaB Helicases/química , Helicobacter pylori/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Compostos de Alumínio/química , Compostos de Alumínio/metabolismo , Arginina/química , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DnaB Helicases/genética , DnaB Helicases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Fluoretos/química , Fluoretos/metabolismo , Expressão Gênica , Helicobacter pylori/genética , Hidrólise , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica
7.
J Biol Chem ; 297(4): 101165, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34487761

RESUMO

The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1-P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix-turn-helix domain (amino acids I13-R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1-D12) did not bind. The N-terminal M1-P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1-M234) and Tnp26 M1-P56 fragment. These findings indicate that the helix-turn-helix and disordered domains of Tnp26 play a role in Tnp26-TIR complex formation. Both domains are conserved in all members of the IS26 family.


Assuntos
Elementos de DNA Transponíveis , DNA Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Sequências Repetidas Terminais , Transposases/química , Substituição de Aminoácidos , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação de Sentido Incorreto , Domínios Proteicos , Transposases/genética , Transposases/metabolismo
8.
Nat Microbiol ; 6(10): 1300-1308, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34518655

RESUMO

Staphylococcal pathogenicity islands (SaPIs) are a family of closely related mobile chromosomal islands that encode and disseminate the superantigen toxins, toxic shock syndrome toxin 1 and superantigen enterotoxin B (SEB). They are regulated by master repressors, which are counteracted by helper phage-encoded proteins, thereby inducing their excision, replication, packaging and intercell transfer. SaPIs are major components of the staphylococcal mobilome, occupying five chromosomal att sites, with many strains harbouring two or more. As regulatory interactions between co-resident SaPIs could have profound effects on the spread of superantigen pathobiology, we initiated the current study to search for such interactions. Using classical genetics, we found that, with one exception, their regulatory systems do not cross-react. The exception was SaPI3, which was originally considered defective because it could not be mobilized by any known helper phage. We show here that SaPI3 has an atypical regulatory module and is induced not by a phage but by many other SaPIs, including SaPI2, SaPIbov1 and SaPIn1, each encoding a conserved protein, Sis, which counteracts the SaPI3 repressor, generating an intracellular regulatory cascade: the co-resident SaPI, when conventionally induced by a helper phage, expresses its sis gene which, in turn, induces SaPI3, enabling it to spread. Using bioinformatics analysis, we have identified more than 30 closely related coancestral SEB-encoding SaPI3 relatives occupying the same att site and controlled by a conserved regulatory module, immA-immR-str'. This module is functionally analogous but unrelated to the typical SaPI regulatory module, stl-str. As SaPIs are phage satellites, SaPI3 and its relatives are SaPI satellites.


Assuntos
Ilhas Genômicas/genética , Staphylococcus aureus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Replicação do DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Transferência Genética Horizontal , Fagos de Staphylococcus/fisiologia , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/virologia , Ativação Transcricional
9.
Nat Commun ; 12(1): 5707, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588455

RESUMO

Bacillus subtilis can form structurally complex biofilms on solid or liquid surfaces, which requires expression of genes for matrix production. The transcription of these genes is activated by regulatory protein RemA, which binds to poorly conserved, repetitive DNA regions but lacks obvious DNA-binding motifs or domains. Here, we present the structure of the RemA homologue from Geobacillus thermodenitrificans, showing a unique octameric ring with the potential to form a 16-meric superstructure. These results, together with further biochemical and in vivo characterization of B. subtilis RemA, suggests that the protein can wrap DNA around its ring-like structure through a LytTR-related domain.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , DNA Bacteriano/metabolismo , Geobacillus/fisiologia , Fatores de Transcrição/metabolismo , Bacillus subtilis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/ultraestrutura
10.
Int J Mol Sci ; 22(18)2021 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-34576332

RESUMO

Campylobacter jejuni has a large adaptive potential due to enormous genetic exchange. Factors regulating natural transformation in this food-borne pathogen are largely unknown but of interest for the application of sustained reduction strategies in the food-processing industry. Using a single cell DNA uptake assay, we visualized that recognition of methylated C. jejuni DNA was essential for the first step of DNA uptake into a DNase resistant state. Transformation rates using a resistance marker correlated with the fraction of competent bacteria, harboring one to maximally four locations of active DNA uptake, not necessarily being located at the cell pole. Competence developed with rising pH between 6.5 and 7.5 under microaerobic conditions and was nearly insensitive towards growth temperatures between 32 °C and 42 °C, CO2 concentrations ranging from 0 to 50% and growth rates. However, competence development was abolished at pH 5 or under aerobic stress conditions, in which the bacteria ceased growth but fully survived. The DNA uptake machinery in competent bacteria shut down at slightly acidic pH and was reversibly switched on upon neutralization. It was dependent on the proton motive force and, in contrast to competence development, slightly enhanced under aerobic conditions. The results suggest that natural transformation in C. jejuni occurs in the neutral and microaerobic intestinal environment for enhanced genetic diversity and pre-adaption before host switch. In addition, highly competent bacteria might be shed into the environment, still able to acquire genetic material for increased survival.


Assuntos
Proteínas de Bactérias/metabolismo , Campylobacter jejuni/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Bactérias/genética , Campylobacter jejuni/genética , DNA Bacteriano/genética , Transformação Bacteriana/genética , Transformação Bacteriana/fisiologia
11.
Nature ; 597(7876): 426-429, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34471288

RESUMO

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3' single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.


Assuntos
DNA Bacteriano/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Modelos Biológicos , Recombinases Rec A/metabolismo , Reparo de DNA por Recombinação , Homologia de Sequência do Ácido Nucleico , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/metabolismo , Fatores de Tempo
12.
Nat Commun ; 12(1): 4710, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354070

RESUMO

Cyanophage S-2L is known to profoundly alter the biophysical properties of its DNA by replacing all adenines (A) with 2-aminoadenines (Z), which still pair with thymines but with a triple hydrogen bond. It was recently demonstrated that a homologue of adenylosuccinate synthetase (PurZ) and a dATP triphosphohydrolase (DatZ) are two important pieces of the metabolism of 2-aminoadenine, participating in the synthesis of ZTGC-DNA. Here, we determine that S-2L PurZ can use either dATP or ATP as a source of energy, thereby also depleting the pool of nucleotides in dATP. Furthermore, we identify a conserved gene (mazZ) located between purZ and datZ genes in S-2L and related phage genomes. We show that it encodes a (d)GTP-specific diphosphohydrolase, thereby providing the substrate of PurZ in the 2-aminoadenine synthesis pathway. High-resolution crystal structures of S-2L PurZ and MazZ with their respective substrates provide a rationale for their specificities. The Z-cluster made of these three genes - datZ, mazZ and purZ - was expressed in E. coli, resulting in a successful incorporation of 2-aminoadenine in the bacterial chromosomal and plasmidic DNA. This work opens the possibility to study synthetic organisms containing ZTGC-DNA.


Assuntos
DNA Bacteriano/genética , Genes Virais , Siphoviridae/genética , 2-Aminopurina/análogos & derivados , 2-Aminopurina/metabolismo , Adenilossuccinato Sintase/química , Adenilossuccinato Sintase/genética , Adenilossuccinato Sintase/metabolismo , Bacteriófagos , Pareamento de Bases , Cristalografia por Raios X , DNA Bacteriano/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Desoxiadenosinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Viral , Redes e Vias Metabólicas , Modelos Moleculares , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Podoviridae/classificação , Podoviridae/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Siphoviridae/classificação , Eletricidade Estática , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
13.
Nat Commun ; 12(1): 5166, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34453062

RESUMO

The efficient segregation of replicated genetic material is an essential step for cell division. Bacterial cells use several evolutionarily-distinct genome segregation systems, the most common of which is the type I Par system. It consists of an adapter protein, ParB, that binds to the DNA cargo via interaction with the parS DNA sequence; and an ATPase, ParA, that binds nonspecific DNA and mediates cargo transport. However, the molecular details of how this system functions are not well understood. Here, we report the cryo-EM structure of the Vibrio cholerae ParA2 filament bound to DNA, as well as the crystal structures of this protein in various nucleotide states. These structures show that ParA forms a left-handed filament on DNA, stabilized by nucleotide binding, and that ParA undergoes profound structural rearrangements upon DNA binding and filament assembly. Collectively, our data suggest the structural basis for ParA's cooperative binding to DNA and the formation of high ParA density regions on the nucleoid.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Vibrio cholerae/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Segregação de Cromossomos , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Bacteriano/genética , Conformação de Ácido Nucleico , Conformação Proteica , Vibrio cholerae/química , Vibrio cholerae/enzimologia , Vibrio cholerae/genética
14.
Science ; 373(6556): 768-774, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385391

RESUMO

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3 These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.


Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Cianobactérias/química , Elementos de DNA Transponíveis , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Microscopia Crioeletrônica , Cianobactérias/genética , Cianobactérias/metabolismo , DNA Bacteriano/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , RNA Bacteriano/metabolismo , Transposases/química , Transposases/metabolismo
15.
Elife ; 102021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34397383

RESUMO

Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA (Jalal et al., 2020). Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with reopening of the gates to release DNA and recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Segregação de Cromossomos/genética , Citidina Trifosfato/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Bactérias/genética , Caulobacter crescentus/metabolismo , Cristalização , Hidrólise , Ligação Proteica , Domínios Proteicos
16.
Sci Rep ; 11(1): 17387, 2021 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-34462504

RESUMO

Multi-drug (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) continues to be a global public health problem especially in high TB burden countries like Nigeria. Many of these cases are undetected and go on to infect high risk individuals. Clinical samples from positive rifampicin resistant Xpert®MTB/Rif assay were subjected to direct whole genome sequencing and bioinformatics analysis to identify the full antibiotics resistance and lineage profile. We report two (2) XDR TB samples also belonging to the East-Asian/Beijing family of lineage 2 Mycobacterium tuberculosis complex from clinical samples in Nigeria. Our findings further reveal the presence of mutations that confer resistance to first-line drugs (rifampicin, isoniazid, ethambutol and pyrazanimide), second-line injectables (capreomycin, streptomycin, kanamycin and/or amikacin) and at least one of the fluoroquinolones (ofloxacin, moxifloxacin, levofloxacin and/or ciprofloxacin) in both samples. The genomic sequence data from this study not only provide the first evidence of XDR TB in Nigeria and West Africa, but also emphasize the importance of WGS in accurately detecting MDR and XDR TB, to ensure adequate and proper management treatment regimens for affected individuals. This will greatly aid in preventing the spread of drug resistance TB in high burden countries.


Assuntos
Tuberculose Extensivamente Resistente a Medicamentos/diagnóstico , Mycobacterium tuberculosis/genética , Adulto , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Capreomicina/farmacologia , Capreomicina/uso terapêutico , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Tuberculose Extensivamente Resistente a Medicamentos/complicações , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Feminino , Fluoroquinolonas/farmacologia , Fluoroquinolonas/uso terapêutico , Infecções por HIV/complicações , Infecções por HIV/diagnóstico , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Nigéria , Filogenia , Rifampina/farmacologia , Rifampina/uso terapêutico , Sequenciamento Completo do Genoma , Adulto Jovem
17.
FEBS Lett ; 595(19): 2504-2521, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34387867

RESUMO

Nucleoid-associated proteins (NAPs) maintain bacterial nucleoid configuration through their architectural properties of DNA bending, wrapping, and bridging. However, the contribution of DNA structural alterations to DNA-NAP recognition at the genomic scale remains unresolved. Present work dissects the DNA sequence, shape and altered structural preferences at a genomic scale for six NAPs in Mycobacterium tuberculosis. Results suggest narrower minor groove width (MGW) and higher DNA rigidity are marked for the binding sites of EspR and Lsr2, while mIHF, MtHU and NapM have heterogeneous DNA structural predilections. In contrast, WhiB4-DNA-binding sites were characterized by wider MGW, highly deformable and less curved DNA. This work provides systematic insight into NAP-mediated genome organization as a function of DNA structural features.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Genômica , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Sítios de Ligação , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica
18.
Cells ; 10(8)2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34440707

RESUMO

Cells respond to genome damage by inducing restorative programs, typified by the SOS response of Escherichia coli. Streptococcus pneumoniae (the pneumococcus), with no equivalent to the SOS system, induces the genetic program of competence in response to many types of stress, including genotoxic drugs. The pneumococcal competence regulon is controlled by the origin-proximal, auto-inducible comCDE operon. It was previously proposed that replication stress induces competence through continued initiation of replication in cells with arrested forks, thereby increasing the relative comCDE gene dosage and expression and accelerating the onset of competence. We have further investigated competence induction by genome stress. We find that absence of RecA recombinase stimulates competence induction, in contrast to SOS response, and that double-strand break repair (RexB) and gap repair (RecO, RecR) initiation effectors confer a similar effect, implying that recombinational repair removes competence induction signals. Failure of replication forks provoked by titrating PolC polymerase with the base analogue HPUra, over-supplying DnaA initiator, or under-supplying DnaE polymerase or DnaC helicase stimulated competence induction. This induction was not correlated with concurrent changes in origin-proximal gene dosage. Our results point to arrested and unrepaired replication forks, rather than increased comCDE dosage, as a basic trigger of pneumococcal competence.


Assuntos
Proteínas de Bactérias/metabolismo , Dano ao DNA , Reparo do DNA , DNA Bacteriano/metabolismo , Streptococcus pneumoniae/metabolismo , Proteínas de Bactérias/genética , Replicação do DNA , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Óperon , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crescimento & desenvolvimento
19.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199760

RESUMO

Polymerase chain reaction (PCR) is the standard in nucleic acid amplification technology for infectious disease pathogen detection and has been the primary diagnostic tool employed during the global COVID-19 pandemic. Various PCR technology adaptations, typically using two-oligonucleotide dye-binding methods or three-oligonucleotide hydrolysis probe systems, enable real-time multiplex target detection or single-base specificity for the identification of single-nucleotide polymorphisms (SNPs). A small number of two-oligonucleotide PCR systems facilitating both multiplex detection and SNP identification have been reported; however, these methods often have limitations in terms of target specificity, production of variable or false-positive results, and the requirement for extensive optimisation or post-amplification analysis. This study introduces 3' Tth endonuclease cleavage PCR (3TEC-PCR), a two-oligonucleotide PCR system incorporating a modified primer/probe and a thermostable cleavage enzyme, Tth endonuclease IV, for real-time multiplex detection and SNP identification. Complete analytical specificity, low limits of detection, single-base specificity, and simultaneous multiple target detection have been demonstrated in this study using 3TEC-PCR to identify bacterial meningitis associated pathogens. This is the first report of a two-oligonucleotide, real-time multiplex PCR technology with single-base specificity using Tth endonuclease IV.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Alelos , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Haemophilus influenzae/genética , Humanos , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/microbiologia , Neisseria meningitidis/genética , Streptococcus pneumoniae/genética
20.
Biomolecules ; 11(7)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206477

RESUMO

The coordination of bacterial genomic transcription involves an intricate network of interdependent genes encoding nucleoid-associated proteins (NAPs), DNA topoisomerases, RNA polymerase subunits and modulators of transcription machinery. The central element of this homeostatic regulatory system, integrating the information on cellular physiological state and producing a corresponding transcriptional response, is the multi-subunit RNA polymerase (RNAP) holoenzyme. In this review article, we argue that recent observations revealing DNA topoisomerases and metabolic enzymes associated with RNAP supramolecular complex support the notion of structural coupling between transcription machinery, DNA topology and cellular metabolism as a fundamental device coordinating the spatiotemporal genomic transcription. We analyse the impacts of various combinations of RNAP holoenzymes and global transcriptional regulators such as abundant NAPs, on genomic transcription from this viewpoint, monitoring the spatiotemporal patterns of couplons-overlapping subsets of the regulons of NAPs and RNAP sigma factors. We show that the temporal expression of regulons is by and large, correlated with that of cognate regulatory genes, whereas both the spatial organization and temporal expression of couplons is distinctly impacted by the regulons of NAPs and sigma factors. We propose that the coordination of the growth phase-dependent concentration gradients of global regulators with chromosome configurational dynamics determines the spatiotemporal patterns of genomic expression.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Fatores de Transcrição/metabolismo , Transcrição Genética , Bactérias/genética , Proteínas de Bactérias/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Fatores de Transcrição/genética
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