A Replisome’s journey through the bacterial chromosome

Genome duplication requires the coordinated activity of a multi-component machine, the replisome. In contrast to the background of metabolic diversity across the bacterial domain, the composition and architecture of the bacterial replisome seems to have suffered few changes during evolution. This immutability underlines the replisome’s efficiency in copying the genome. It also highlights the success of various strategies inherent to the replisome for responding to stress and avoiding problems during critical stages of DNA synthesis. Here we summarise current understanding of bacterial replisome architecture and highlight the known variations in different bacterial taxa. We then look at the mechanisms in place to ensure that the bacterial replisome is assembled appropriately on DNA, kept together during elongation, and disassembled upon termination. We put forward the idea that the architecture of the replisome may be more flexible that previously thought and speculate on elements of the replisome that maintain its stability to ensure a safe journey from origin to terminus

MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves

The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region

Frequent exchange of the DNA polymerase during bacterial chromosome replication

The replisome is a multiprotein machine that carries out DNA replication. In Escherichia coli, a single pair of replisomes is responsible for duplicating the entire 4.6 Mbp circular chromosome. In vitro studies of reconstituted E. coli replisomes have attributed this remarkable processivity to the high stability of the replisome once assembled on DNA. By examining replisomes in live E. coli with fluorescence microscopy, we found that the Pol III* subassembly frequently disengages from the replisome during DNA synthesis and exchanges with free copies from solution. In contrast, the DnaB helicase associates stably with the replication fork, providing the molecular basis for how the E. coli replisome can maintain high processivity and yet possess the flexibility to bypass obstructions in template DNA. Our data challenges the widely-accepted semidiscontinuous model of chromosomal replication, instead supporting a fully discontinuous mechanism in which synthesis of both leading and lagging strands is frequently interrupted

Replication-directed sister chromosome alignment in Escherichia coli

Non-replicating Escherichia coli chromosomes are organized as sausage-shaped structures with the left (L) and the right (R) chromosome arms (replichores) on opposite cell halves and the replication origin (oriC) close to midcell. The replication termination region (ter) therefore passes between the two outer edges of the nucleoid. Four alignment patterns of the two <LR> sister chromosomes within a cell have been detected in an asynchronous population, with the <LRLR> pattern predominating. We test the hypothesis that the minority <LRRL> and <RLLR> patterns arise because of pausing of DNA replication on the right and left replichores respectively. The data resulting from transient pausing or longer-term site-specific blocking of replication show that paused/blocked loci remain close to midcell and the normally replicated-segregated loci locate to the outer regions of the nucleoid, therefore providing experimental support for a direct mechanistic link between DNA replication and chromosome organization

Las relaciones entre el método de investigación acción participativa y el arte de interacción social. Alcances y riesgos. / The associations between the participative action research method and social interaction art. Range and risks.

En la segunda mitad del siglo XX, en América, surgió un método en las ciencias sociales conocido como investigación acción participativa, el cual busca desdibujar la barrera entre el investigador y el objeto de estudio para que la comunidad que tiene un problema sea beneficiada, no a través de la publicación de un texto científico, sino mediante la solución efectiva del problema a través de acciones y estrategias colectivas en las que participan especialistas y habitantes de esa población.De manera paralela, en Alemania, Joseph Beuys desarrolló el concepto de escultura social que argumentaba que toda persona debía convertirse en creador, en un escultor o arquitecto del organismo social. Gracias a la influencia de Beuys varios artistas y colectivos han elaborado proyectos de interacción social en zonas de la periferia, en donde los creadores detectan un problema y elaboran una pieza que implica la participación de los habitantes de una comunidad o vecindario en desventaja. Estas obras tienden a transformar el entorno y la sociedad.Ambos enfoques buscan empoderar a los individuos que no tienen los beneficios del resto de la sociedad. Sin embargo, es necesario distinguir las afinidades y las diferencias entre ellos, así como las relaciones que generan los gestos simbólicos y los cambios efectivos. También es importante analizar las oportunidades y los riesgos que presentan ambos modelos.The associations between the participative action research method and social interaction art. Range and risks.A method of social research known as participative action research emerged in the Americas during the second half of the 20th, aiming to erase the barrier between the researcher and his study object hence a community with a problem is improved not through scientific publications but by means of an effective solution of the problem activated by collective strategies used by specialists and locals. At the same time, in Germany, Joseph Beuys developed the social sculpture concept suggesting that everyone should be creator, a sculptor or architect of the social organism. After Beuys’ influence many artists and collectives have elaborated social interaction projects in peripheral areas where the artists detect a problem and make a piece that involves participation of the residents of a disadvantaged community or neighborhood. These works tend to transform the environment and society. Both perspectives empower individuals with no benefits from the rest of society. Nevertheless, it is necessary to differentiate the affinities and differences between them, and the relationships caused by the symbolic gestures and the effective changes. It is important, as well, to analyze the opportunities and the risks presented in both models

Early and Solid Protection Afforded by the Thiverval Vaccine Provides Novel Vaccination Alternatives Against Classical Swine Fever Virus

Classical swine fever virus (CSFV) remains a challenge for the porcine industry. Inefficient vaccination programs in some endemic areas may have contributed to the emergence of low and moderate virulence CSFV variants. This work aimed to expand and update the information about the safety and efficacy of the CSFV Thiverval-strain vaccine. Two groups of pigs were vaccinated, and a contact and control groups were also included. Animals were challenged with a highly virulent CSFV strain at 21- or 5-days post vaccination (dpv). The vaccine induced rapid and strong IFN-α response, mainly in the 5-day immunized group, and no vaccine virus transmission was detected. Vaccinated pigs showed humoral response against CSFV E2 and Erns glycoproteins, with neutralising activity, starting at 14 days post vaccination (dpv). Strong clinical protection was afforded in all the vaccinated pigs as early as 5 dpv. The vaccine controlled viral replication after challenge, showing efficient virological protection in the 21-day immunized pigs despite being housed with animals excreting high CSFV titres. These results demonstrate the high efficacy of the Thiverval strain against CSFV replication. Its early protection capacity makes it a useful alternative for emergency vaccination and a consistent tool for CSFV control worldwide.info:eu-repo/semantics/publishedVersio

The MatP/matS Site-Specific System Organizes the Terminus Region of the E. coli Chromosome into a Macrodomain

The organization of the Escherichia coli chromosome into insulated macrodomains influences the segregation of sister chromatids and the mobility of chromosomal DNA. Here, we report that organization of the Terminus region (Ter) into a macrodomain relies on the presence of a 13 bp motif called matS repeated 23 times in the 800-kb-long domain. matS sites are the main targets in the E. coli chromosome of a newly identified protein designated MatP. MatP accumulates in the cell as a discrete focus that colocalizes with the Ter macrodomain. The effects of MatP inactivation reveal its role as main organizer of the Ter macrodomain: in the absence of MatP, DNA is less compacted, the mobility of markers is increased, and segregation of Ter macrodomain occurs early in the cell cycle. Our results indicate that a specific organizational system is required in the Terminus region for bacterial chromosome management during the cell cycle

SMC is recruited to oriC by ParB and promotes chromosome segregation in Streptococcus pneumoniae

Segregation of replicated chromosomes is an essential process in all organisms. How bacteria, such as the oval-shaped human pathogen Streptococcus pneumoniae, efficiently segregate their chromosomes is poorly understood. Here we show that the pneumococcal homologue of the DNA-binding protein ParB recruits S. pneumoniae condensin (SMC) to centromere-like DNA sequences (parS) that are located near the origin of replication, in a similar fashion as was shown for the rod-shaped model bacterium Bacillus subtilis. In contrast to B. subtilis, smc is not essential in S. pneumoniae, and Δsmc cells do not show an increased sensitivity to gyrase inhibitors or high temperatures. However, deletion of smc and/or parB results in a mild chromosome segregation defect. Our results show that S. pneumoniae contains a functional chromosome segregation machine that promotes efficient chromosome segregation by recruitment of SMC via ParB. Intriguingly, the data indicate that other, as of yet unknown mechanisms, are at play to ensure proper chromosome segregation in this organism.