RESUMO
Initiation of chromosomal replication requires dynamic nucleoprotein complexes. In most eubacteria, the origin oriC contains multiple DnaA box sequences to which the ubiquitous DnaA initiators bind. In Escherichia coli oriC, DnaA boxes sustain construction of higher-order complexes via DnaA-DnaA interactions, promoting the unwinding of the DNA unwinding element (DUE) within oriC and concomitantly binding the single-stranded (ss) DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, oriC sequences are highly diverse. The present study investigated the design of oriC (tma-oriC) from Thermotoga maritima, an evolutionarily ancient eubacterium. The minimal tma-oriC sequence includes a DUE and a flanking region containing five DnaA boxes recognized by the cognate DnaA (tmaDnaA). This DUE was comprised of two distinct functional modules, an unwinding module and a tmaDnaA-binding module. Three direct repeats of the trinucleotide TAG within DUE were essential for both unwinding and ssDUE binding by tmaDnaA complexes constructed on the DnaA boxes. Its surrounding AT-rich sequences stimulated only duplex unwinding. Moreover, head-to-tail oligomers of ATP-bound tmaDnaA were constructed within tma-oriC, irrespective of the directions of the DnaA boxes. This binding mode was considered to be induced by flexible swiveling of DnaA domains III and IV, which were responsible for DnaA-DnaA interactions and DnaA box binding, respectively. Phasing of specific tmaDnaA boxes in tma-oriC was also responsible for unwinding. These findings indicate that a ssDUE recruitment mechanism was responsible for unwinding and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria.
Assuntos
Proteínas de Ligação a DNA , Origem de Replicação , Thermotoga maritima , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismoRESUMO
Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre-lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.
Assuntos
Mycoplasma , Vacinas , Animais , Cabras , Mycoplasma/genética , Serina ProteasesRESUMO
This review summarizes current knowledge about the mechanisms of timely binding and dissociation of two nucleoid proteins, IHF and Fis, which play fundamental roles in the initiation of chromosomal DNA replication in Escherichia coli. Replication is initiated from a unique replication origin called oriC and is tightly regulated so that it occurs only once per cell cycle. The timing of replication initiation at oriC is rigidly controlled by the timely binding of the initiator protein DnaA and IHF to oriC. The first part of this review presents up-to-date knowledge about the timely stabilization of oriC-IHF binding at oriC during replication initiation. Recent advances in our understanding of the genome-wide profile of cell cycle-coordinated IHF binding have revealed the oriC-specific stabilization of IHF binding by ATP-DnaA oligomers at oriC and by an initiation-specific IHF binding consensus sequence at oriC. The second part of this review summarizes the mechanism of the timely regulation of DnaA activity via the chromosomal loci DARS2 (DnaA-reactivating sequence 2) and datA. The timing of replication initiation at oriC is controlled predominantly by the phosphorylated form of the adenosine nucleotide bound to DnaA, i.e., ATP-DnaA, but not ADP-ADP, is competent for initiation. Before initiation, DARS2 increases the level of ATP-DnaA by stimulating the exchange of ADP for ATP on DnaA. This DARS2 function is activated by the site-specific and timely binding of both IHF and Fis within DARS2. After initiation, another chromosomal locus, datA, which inactivates ATP-DnaA by stimulating ATP hydrolysis, is activated by the timely binding of IHF. A recent study has shown that ATP-DnaA oligomers formed at DARS2-Fis binding sites competitively dissociate Fis via negative feedback, whereas IHF regulation at DARS2 and datA still remains to be investigated. This review summarizes the current knowledge about the specific role of IHF and Fis in the regulation of replication initiation and proposes a mechanism for the regulation of timely IHF binding and dissociation at DARS2 and datA.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fatores Hospedeiros de Integração/genética , Fatores Hospedeiros de Integração/metabolismo , Origem de Replicação , Replicação do DNA , Ciclo Celular , Trifosfato de Adenosina/metabolismo , DNA Bacteriano/genética , Fator Proteico para Inversão de Estimulação/genética , Fator Proteico para Inversão de Estimulação/metabolismoRESUMO
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/metabolismoRESUMO
Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the replication of host chromosomes and how they operate. The initiator and replicator, proposed in the replicon hypothesis, are now well defined in both prokaryotes and eukaryotes. On the other hand, studies in prokaryotes and Archaea indicate alternative modes of initiation, which may not depend on an initiator. Here I summarize recent progress in the field of DNA replication and discuss the evolution of replication systems.
Assuntos
Replicação do DNA , Origem de Replicação , Escherichia coli/metabolismo , Proteínas de Ligação a DNA/metabolismo , Replicon , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos , DNA Bacteriano/genéticaRESUMO
The DNA replication protein DnaA in Escherichia coli constructs higher-order complexes on the origin, oriC, to unwind this region. DnaB helicase is loaded onto unwound oriC via interactions with the DnaC loader and the DnaA complex. The DnaB-DnaC complex is recruited to the DnaA complex via stable binding of DnaB to DnaA domain I. The DnaB-DnaC complex is then directed to unwound oriC via a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe46 in DnaA domain I binds to DnaB. Here, we searched for the DnaA domain I-binding site in DnaB. The DnaB L160A variant was impaired in binding to DnaA complex on oriC but retained its DnaC-binding and helicase activities. DnaC binding moderately stimulated DnaA binding of DnaB L160A, and loading of DnaB L160A onto oriC was consistently and moderately inhibited. In a helicase assay with partly single-stranded DNA bearing a DnaA-binding site, DnaA stimulated DnaB loading, which was strongly inhibited in DnaB L160A even in the presence of DnaC. DnaB L160A was functionally impaired in vivo On the basis of these findings, we propose that DnaB Leu160 interacts with DnaA domain I Phe46 DnaB Leu160 is exposed on the lateral surface of the N-terminal domain, which can explain unobstructed interactions of DnaA domain I-bound DnaB with DnaC, DnaG primase, and DnaA domain III. We propose a probable structure for the DnaA-DnaB-DnaC complex, which could be relevant to the process of DnaB loading onto oriC.
Assuntos
DnaB Helicases/metabolismo , Proteínas de Escherichia coli/metabolismo , Origem de Replicação , Sequência de Aminoácidos , Sítios de Ligação , DnaB Helicases/química , Proteínas de Escherichia coli/química , Modelos Moleculares , Domínios ProteicosRESUMO
The main roles of the DnaA protein are to bind the origin of chromosome replication (oriC), to unwind DNA and to provide a hub for the step-wise assembly of a replisome. DnaA is composed of four domains, with each playing a distinct functional role in the orisome assembly. Out of the four domains, the role of domain I is the least understood and appears to be the most species-specific. To better characterise Helicobacter pylori DnaA domain I, we have constructed a series of DnaA variants and studied their interactions with H. pylori bipartite oriC. We show that domain I is responsible for the stabilisation and organisation of DnaA-oriC complexes and provides cooperativity in DnaA-DNA interactions. Domain I mediates cross-interactions between oriC subcomplexes, which indicates that domain I is important for long-distance DnaA interactions and is essential for orisosme assembly on bipartite origins. HobA, which interacts with domain I, increases the DnaA binding to bipartite oriC; however, it does not stimulate but rather inhibits DNA unwinding. This suggests that HobA helps DnaA to bind oriC, but an unknown factor triggers DNA unwinding. Together, our results indicate that domain I self-interaction is important for the DnaA assembly on bipartite H. pylori oriC.
Assuntos
Proteínas de Bactérias , Cromossomos Bacterianos/metabolismo , Proteínas de Ligação a DNA , Helicobacter pylori , Complexo de Reconhecimento de Origem/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/química , Replicação do DNA , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Nucleoproteínas/química , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Ligação Proteica , Origem de ReplicaçãoRESUMO
BACKGROUND: The sustainability of Australian rural maternity services is under threat due to current workforce shortages. In July 2019, a new midwifery caseload model of care was implemented in rural South Australia to provide midwifery continuity of care and promote a sustainable workforce in the area. The model is unique as it brings together five birthing sites connecting midwives, doctors, nurses and community teams. A critical precursor to successful implementation requires those working in the model be ready to adopt to the change. We surveyed clinicians at the five sites transitioning to the new model of care in order to assess their organizational readiness to implement change. METHODS: A descriptive study assessing readiness for change was measured using the Organizational Readiness for Implementing Change scale (ORIC). The 12 item Likert scale measures a participant's commitment to change and change efficacy. All clinicians working within the model of care (midwives, nurses and doctors) were invited to complete an e-survey. RESULTS: Overall, 55% (56/102) of clinicians participating in the model responded. The mean ORIC score was 41.5 (range 12-60) suggesting collectively, midwives, nurses and doctors began the new model of care with a sense of readiness for change. Participants were most likely to agree on the change efficacy statements, "People who work here feel confident that the organization can get people invested in implementing this change and the change commitment statements "People who work here are determined to implement this change", "People who work here want to implement this change", and "People who work here are committed to implementing this change. CONCLUSION: Results of the ORIC survey indicate that clinicians transitioning to the new model of care were willing to embrace change and commit to the new model. The process of organizational change in health care settings is challenging and a continuous process. If readiness for change is high, organizational members invest more in the change effort and exhibit greater persistence to overcome barriers and setbacks. This is the first reported use of the instrument amongst midwives and nurses in Australia and should be considered for use in other national and international clinical implementation studies.
Assuntos
Tocologia , Austrália , Feminino , Humanos , Inovação Organizacional , Organizações , Gravidez , Austrália do Sul , Inquéritos e QuestionáriosRESUMO
oriC is a region of the bacterial chromosome at which the initiator protein DnaA interacts with specific sequences, leading to DNA unwinding and the initiation of chromosome replication. The general architecture of oriCs is universal; however, the structure of oriC and the mode of orisome assembly differ in distantly related bacteria. In this work, we characterized oriC of Helicobacter pylori, which consists of two DnaA box clusters and a DNA unwinding element (DUE); the latter can be subdivided into a GC-rich region, a DnaA-trio and an AT-rich region. We show that the DnaA-trio submodule is crucial for DNA unwinding, possibly because it enables proper DnaA oligomerization on ssDNA. However, we also observed the reverse effect: DNA unwinding, enabling subsequent DnaA-ssDNA oligomer formation-stabilized DnaA binding to box ts1. This suggests the interplay between DnaA binding to ssDNA and dsDNA upon DNA unwinding. Further investigation of the ts1 DnaA box revealed that this box, together with the newly identified c-ATP DnaA box in oriC1, constitute a new class of ATP-DnaA boxes. Indeed, in vitro ATP-DnaA unwinds H. pylori oriC more efficiently than ADP-DnaA. Our results expand the understanding of H. pylori orisome formation, indicating another regulatory pathway of H. pylori orisome assembly.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Helicobacter pylori/metabolismo , Complexo de Reconhecimento de Origem , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Helicobacter pylori/genética , MutaçãoRESUMO
Insects are frequently infected by bacterial symbionts that greatly affect their physiology and ecology. Most of these endosymbionts are, however, barely tractable outside their native host, rendering functional genetics studies difficult or impossible. Spiroplasma poulsonii is a facultative bacterial endosymbiont of Drosophila melanogaster that manipulates the reproduction of its host by killing its male progeny at the embryonic stage. S. poulsonii, although a very fastidious bacterium, is closely related to pathogenic Spiroplasma species that are cultivable and genetically modifiable. In this work, we present the transformation of S. poulsonii with a plasmid bearing a fluorescence cassette, leveraging techniques adapted from those used to modify the pathogenic species Spiroplasma citri We demonstrate the feasibility of S. poulsonii transformation and discuss approaches for mutant selection and fly colonization, which are persisting hurdles that must be overcome to allow functional bacterial genetics studies of this endosymbiont in vivoIMPORTANCE Dozens of bacterial endosymbiont species have been described and estimated to infect about half of all insect species. However, only a few them are tractable in vitro, which hampers our understanding of the bacterial determinants of the host-symbiont interaction. Developing a transformation method for S. poulsonii is a major step toward genomic engineering of this symbiont, which will foster basic research on endosymbiosis. This could also open the way to practical uses of endosymbiont engineering through paratransgenesis of vector or pest insects.
Assuntos
Drosophila melanogaster/microbiologia , Drosophila melanogaster/fisiologia , Spiroplasma/genética , Simbiose , Transformação Bacteriana , Animais , Feminino , Masculino , ReproduçãoRESUMO
DNA replication is controlled mostly at the initiation step. In bacteria, replication of the chromosome starts at a single origin of replication called oriC. The initiator protein, DnaA, binds to specific sequences (DnaA boxes) within oriC and assembles into a filament that promotes DNA double helix opening within the DNA unwinding element (DUE). This process has been thoroughly examined in model bacteria, including Escherichia coli and Bacillus subtilis, but we have a relatively limited understanding of chromosomal replication initiation in other species. Here, we reveal new details of DNA replication initiation in Streptomyces, a group of Gram-positive soil bacteria that possesses a long linear (8-10 Mbps) and GC-rich chromosome with a centrally positioned oriC. We used comprehensive in silico, in vitro and in vivo analyses to better characterize the structure of Streptomyces oriC. We identified 14 DnaA-binding motifs and determined the consensus sequence of the DnaA box. Unexpectedly, our in silico analysis using the WebSIDD algorithm revealed the presence of two putative Streptomyces DUEs (DUE1 and DUE2) located very near one another toward the 5' end of the oriC region. In vitro P1 nuclease assay revealed that DNA unwinding occurs at both of the proposed sites, but using an in vivo replication initiation point mapping, we were able to confirm only one of them (DUE2). The previously observed transcriptional activity of the Streptomyces oriC region may help explain the current results. We speculate that transcription itself could modulate oriC activity in Streptomyces by determining whether DNA unwinding occurs at DUE1 or DUE2.
Assuntos
DNA Bacteriano/metabolismo , DNA Super-Helicoidal/metabolismo , Origem de Replicação/genética , Streptomyces/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Cromossomos Bacterianos/genética , Sequência Consenso , Replicação do DNA , DNA Bacteriano/química , DNA Super-Helicoidal/química , Proteínas de Ligação a DNA/metabolismoRESUMO
Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.
Assuntos
Replicação do DNA , DNA Bacteriano/química , Escherichia coli/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência de Bases , Simulação por Computador , Replicação do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fatores Hospedeiros de Integração/química , Fatores Hospedeiros de Integração/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexo de Reconhecimento de Origem/química , Complexo de Reconhecimento de Origem/metabolismo , Domínios e Motivos de Interação entre ProteínasRESUMO
Bacteria of the genus Mycoplasma have recently attracted considerable interest as model organisms in synthetic and systems biology. In particular, Mycoplasma pneumoniae is one of the most intensively studied organisms in the field of systems biology. However, the genetic manipulation of these bacteria is often difficult due to the lack of efficient genetic systems and some intrinsic peculiarities such as an aberrant genetic code. One major disadvantage in working with M. pneumoniae is the lack of replicating plasmids that can be used for the complementation of mutants and the expression of proteins. In this study, we have analysed the genomic region around the gene encoding the replication initiation protein, DnaA, and detected putative binding sites for DnaA (DnaA boxes) that are, however, less conserved than in other bacteria. The construction of several plasmids encompassing this region allowed the selection of plasmid pGP2756 that is stably inherited and that can be used for genetic experiments, as shown by the complementation assays with the glpQ gene encoding the glycerophosphoryl diester phosphodiesterase. Plasmid-borne complementation of the glpQ mutant restored the formation of hydrogen peroxide when bacteria were cultivated in the presence of glycerol phosphocholine. Interestingly, the replicating plasmid can also be used in the close relative, Mycoplasma genitalium but not in more distantly related members of the genus Mycoplasma. Thus, plasmid pGP2756 is a valuable tool for the genetic analysis of M. pneumoniae and M. genitalium.
Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Peróxido de Hidrogênio/metabolismo , Mycoplasma pneumoniae/genética , Complexo de Reconhecimento de Origem/genética , Plasmídeos/genética , Sítios de Ligação/genética , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Mycoplasma pneumoniae/metabolismo , Diester Fosfórico Hidrolases/genética , Transformação BacterianaRESUMO
DNA replication is an important step in the life cycle of every cell that ensures the continuous flow of genetic information from one generation to the next. In all organisms, chromosome replication must be coordinated with overall cell growth. Helicobacter pylori growth strongly depends on its interaction with the host, particularly with the gastric epithelium. Moreover, H. pylori actively searches for an optimal microniche within a stomach, and it has been shown that not every microniche equally supports growth of this bacterium. We postulate that besides nutrients, H. pylori senses different, unknown signals, which presumably also affect chromosome replication to maintain H. pylori propagation at optimal ratio allowing H. pylori to establish a chronic, lifelong infection. Thus, H. pylori chromosome replication and particularly the regulation of this process might be considered important for bacterial pathogenesis. Here, we summarize our current knowledge of chromosome and plasmid replication in H. pylori and discuss the mechanisms responsible for regulating this key cellular process. The results of extensive studies conducted thus far allow us to propose common and unique traits in H. pylori chromosome replication. Interestingly, the repertoire of proteins involved in replication in H. pylori is significantly different to that in E. coli, strongly suggesting that novel factors are engaged in H. pylori chromosome replication and could represent attractive drug targets.
Assuntos
Antibacterianos/química , DNA Bacteriano/genética , Desenho de Fármacos , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Antibacterianos/farmacologia , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Replicação do DNA/efeitos dos fármacos , DNA Bacteriano/metabolismo , Helicobacter pylori/fisiologia , HumanosRESUMO
Spiroplasma eriocheiris, the cause of crab trembling disease, is a wall-less bacterium, related to Mycoplasmas, measuring 2.0-10.0 µm long. It features a helical cell shape and a unique swimming mechanism that does not use flagella; instead, it moves by switching the cell helicity at a kink traveling from the front to the tail. S. eriocheiris seems to use a novel chemotactic system that is based on the frequency of reversal swimming behaviors rather than the conventional two-component system, which is generally essential for bacterial chemotaxis. To identify the genes involved in these novel mechanisms, we developed a transformation system by using oriC plasmid harboring the tetracycline resistant gene, tetM, which is under the control of a strong promoter for an abundant protein, elongation factor-Tu. The transformation efficiency achieved was 1.6 × 10-5 colony forming unit (CFU) for 1 µg DNA, enabling the expression of the enhanced yellow fluorescent protein (EYFP).
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Luminescentes/genética , Spiroplasma/genética , Transformação Bacteriana , Microscopia de Fluorescência , Plasmídeos/genéticaRESUMO
Chromosome replication in Eubacteria is initiated by initiator protein(s) binding to specific sites within the replication origin, oriC. Recently, initiator protein binding to chromosomal regions outside the origin has attracted renewed attention; as such binding sites contribute to control the frequency of initiations. These outside-oriC binding sites function in several different ways: by steric hindrances of replication fork assembly, by titration of initiator proteins away from the origin, by performing a chaperone-like activity for inactivation- or activation of initiator proteins or by mediating crosstalk between chromosomes. Here, we discuss initiator binding to outside-oriC sites in a broad range of different taxonomic groups, to highlight the significance of such regions for regulation of bacterial chromosome replication. For Escherichia coli, it was recently shown that the genomic positions of regulatory elements are important for bacterial fitness, which, as we discuss, could be true for several other organisms.
Assuntos
Cromossomos Bacterianos/genética , Replicação do DNA/genética , Sequências Reguladoras de Ácido Nucleico , Origem de Replicação/genética , Sítios de Ligação , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genéticaRESUMO
The Escherichia coli chromosomal origin consists of a duplex-unwinding region and a region bearing a DNA-bending protein, IHF-binding site, and clusters of binding sites for the initiator protein DnaA. ATP-DnaA molecules form highly organized oligomers in a process stimulated by DiaA, a DnaA-binding protein. The resultant ATP-DnaA complexes promote local unwinding of oriC with the aid of IHF, for which specific interaction of DnaA with the single-stranded DNA is crucial. DnaA complexes also interact with DnaB helicases bound to DnaC loaders, promoting loading of DnaB onto the unwound DNA strands for bidirectional replication. Initiation of replication is strictly regulated during the cell cycle by multiple regulatory systems for oriC and DnaA. The activity of oriC is regulated by its methylation state, whereas that of DnaA depends on the form of the bound nucleotide. ATP-DnaA can be yielded from initiation-inactive ADP-DnaA in a timely manner depending on specific chromosomal DNA elements termed DARS (DnaA-reactivating sequences). After initiation, DnaA-bound ATP is hydrolyzed by two systems, yielding ADP-DnaA. In this review, these and other mechanisms of initiation and its regulation in E. coli are described.
Assuntos
Replicação do DNA/fisiologia , Escherichia coli/genética , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação/genética , Sítios de Ligação , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/genética , Complexo de Reconhecimento de Origem/genéticaRESUMO
Arrest of replication fork progression is one of the most common causes for increasing the genomic instability. In bacteria, PriA, a conserved DEXH-type helicase, plays a major role in recognition of the stalled forks and restart of DNA replication. We took advantage of PriA's ability to specifically recognize stalled replication forks to determine the genomic loci where replication forks are prone to stall on the Escherichia coli genome. We found that PriA binds around oriC upon thymine starvation which reduces the nucleotide supply and causes replication fork stalling. PriA binding quickly disappeared upon readdition of thymine. Furthermore, BrdU was incorporated at around oriC upon release from thymine starvation. Our results indicate that reduced supply of DNA replication precursors causes replication fork stalling preferentially in the 600 kb segment centered at oriC. This suggests that replication of the vicinity of oriC requires higher level of nucleotide precursors. The results also point to a possibility of slow fork movement and/or the presence of multiple fork arrest signals within this segment. Indeed, we have identified rather strong fork stall/pausing signals symmetrically located at â¼50 kb away from oriC. We speculate that replication pausing and fork-slow-down shortly after initiation may represent a novel checkpoint that ensures the presence of sufficient nucleotide supply prior to commitment to duplication of the entire genome.
Assuntos
DNA Helicases/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Instabilidade Genômica/genética , Origem de Replicação/genética , Sítios de Ligação , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Ligação Proteica , TiminaRESUMO
Initiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. This process is tightly controlled by modulation of the availability or activity of DnaA and oriC during development or stress conditions. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. We successfully arrested replication in B. subtilis by employing a clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach to specifically target the key DnaA boxes 6 and 7, preventing DnaA binding to oriC. In this way, other functions of DnaA, such as a transcriptional regulator, were not significantly affected. When replication initiation was halted by this specific artificial and early blockage, we observed that non-replicating cells continued translation and cell growth, and the initial replication arrest did not induce global stress conditions such as the SOS response.IMPORTANCEAlthough bacteria constantly replicate under laboratory conditions, natural environments expose them to various stresses such as lack of nutrients, high salinity, and pH changes, which can trigger non-replicating states. These states can enable bacteria to (i) become tolerant to antibiotics (persisters), (ii) remain inactive in specific niches for an extended period (dormancy), and (iii) adjust to hostile environments. Non-replicating states have also been studied because of the possibility of repurposing energy for the production of additional metabolites or proteins. Using clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeting bacterial replication initiation sequences, we were able to successfully control replication initiation in Bacillus subtilis. This precise approach makes it possible to study non-replicating phenotypes, contributing to a better understanding of bacterial adaptive strategies.
Assuntos
Bacillus subtilis , Proteínas de Ligação a DNA , Proteínas de Ligação a DNA/genética , Bacillus subtilis/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas de Bactérias/genética , Replicação do DNA/genéticaRESUMO
Background: Effective interventions need to be implemented successfully to achieve impact. Two theory-based measures exist for measuring the effectiveness of implementation strategies and monitor implementation progress. The Normalization MeAsure Development questionnaire (NoMAD) explores the four core concepts (Coherence, Cognitive Participation, Collective Action, Reflexive Monitoring) of the Normalization Process Theory. The Organizational Readiness for Implementing Change (ORIC) is based on the theory of Organizational Readiness for Change, measuring organization members' psychological and behavioral preparedness for implementing a change. We examined the measurement properties of the NoMAD and ORIC in a multi-national implementation effectiveness study. Method: Twelve mental health organizations in nine countries implemented Internet-based cognitive behavioral therapy (iCBT) for common mental disorders. Staff involved in iCBT service delivery (n = 318) participated in the study. Both measures were translated into eight languages using a standardized forward-backward translation procedure. Correlations between measures and subscales were estimated to examine convergent validity. The theoretical factor structures of the scales were tested using confirmatory factor analysis (CFA). Test-retest reliability was based on the correlation between scores at two time points 3 months apart. Internal consistency was assessed using Cronbach's alpha. Floor and ceiling effects were quantified using the proportion of zero and maximum scores. Results: NoMAD and ORIC measure related but distinct latent constructs. The CFA showed that the use of a total score for each measure is appropriate. The theoretical subscales of the NoMAD had adequate internal consistency. The total scale had high internal consistency. The total ORIC scale and subscales demonstrated high internal consistency. Test-retest reliability was suboptimal for both measures and floor and ceiling effects were absent. Conclusions: This study confirmed the psychometric properties of the NoMAD and ORIC in multi-national mental health care settings. While measuring on different but related aspects of implementation processes, the NoMAD and ORIC prove to be valid and reliable across different language settings.
Why was the study done?: Effective interventions need to be implemented successfully to achieve impact. Reliable measurement instruments are needed to determine if an implementation was successful or not. Two theory-based instruments exist for measuring the effectiveness of implementation strategies and monitor progress. The NoMAD measures aspects of normalization related to sense-making, willingness to implement, the work people do, and reflection. The Organizational Readiness for Implementing Change (ORIC) measures organization members' preparedness for implementing a change. What did we do?: This study examined whether the NoMAD and ORIC measure what they are supposed to measure. We translated the instruments from English to eight languages (Albanian, Danish, Dutch, French, German, Italian, and Spanish/Catalan) We applied various statistical methods to confirm the measurement properties, including correlations of scales, factor structures, testretest reliability, consistency and floor and ceiling effects. 318 mental health professionals from nine countries participated in the study. What did we find?: For both instruments, total scores can be used as well as the subscale scores. Internal consistency for ORIC was high and for NoMAD adequate. Testretest reliability was demonstrated, and floor and ceiling effects were rare. What does this mean?: NoMAD and ORIC are reliable instruments for measuring implementation processes and outcomes across mental health care settings in different countries and languages. They measure related but different aspects of implementation processes and outcomes. The measures are brief, and theory supported. However, more work is to be done on interpreting scores in relation to implementation success and regarding changes over time.