scholarly journals Direct observation of independently moving replisomes in Escherichia coli

2019 ◽  
Author(s):  
Aleksandre Japaridze ◽  
Christos Gogou ◽  
Jacob W. J. Kerssemakers ◽  
Huyen My Nguyen ◽  
Cees Dekker
Keyword(s):  
Origin Of Replication ◽  
Train Track ◽  
Cell Width ◽  
Circular Chromosome ◽  
E Coli ◽  
Dna Segregation ◽  
Direct Support ◽  
A Cell ◽  
Genomic Material ◽  
Segregation Of Chromosomes

AbstractThe replication and transfer of genomic material from a cell to its progeny are vital processes in all living systems. Here we visualize the process of chromosome replication in E. coli cells with an increased width. Monitoring the replication of single chromosomes yields clear examples of replication bubbles that reveal that the two replisomes move independently from the origin to the terminus of replication along each of the two arms of the circular chromosome, providing direct support for the so-called train-track model, and against a factory model for replisomes. The origin of replication duplicates near midcell, initially splitting to random directions and subsequently towards the poles. The probability of successful segregation of chromosomes significantly decreases with increasing cell width, indicating that chromosome confinement by the cell boundary is an important driver of DNA segregation. Our findings resolve long standing questions in bacterial chromosome organization.

scholarly journals Excess SeqA Leads to Replication Arrest and a Cell Division Defect in Vibrio cholerae

2008 ◽  
Vol 190 (17) ◽  
pp. 5870-5878 ◽  
Author(s):  
Djenann Saint-Dic ◽  
Jason Kehrl ◽  
Brian Frushour ◽  
Lyn Sue Kahng
Keyword(s):  
Cell Division ◽  
Vibrio Cholerae ◽  
Small Chromosome ◽  
Circular Chromosome ◽  
E Coli ◽  
Template Strand ◽  
Replication Arrest ◽  
Asynchronous Replication ◽  
A Cell ◽  
Timing Of Initiation

ABSTRACT Although most bacteria contain a single circular chromosome, some have complex genomes, and all Vibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome of Vibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome of Escherichia coli. While factors influencing the replication and segregation of both chromosomes have begun to be elucidated, much remains to be learned about the maintenance of this genome and of complex bacterial genomes generally. An important aspect of replicating any genome is the correct timing of initiation, without which organisms risk aneuploidy. During DNA replication in E. coli, newly replicated origins cannot immediately reinitiate because they undergo sequestration by the SeqA protein, which binds hemimethylated origin DNA. This DNA is already methylated by Dam on the template strand and later becomes fully methylated; aberrant amounts of Dam or the deletion of seqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins of V. cholerae, suggesting that these origins are also subject to sequestration. The overproduction of SeqA led to a loss of viability, the condensation of DNA, and a filamentous morphology. Cells with abnormal DNA content arose in the population, and replication was inhibited as determined by a reduced ratio of origin to terminus DNA in SeqA-overexpressing cells. Thus, excessive SeqA negatively affects replication in V. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.

scholarly journals Direct imaging of the circular chromosome in a live bacterium

2018 ◽  
Author(s):  
Fabai Wu ◽  
Aleksandre Japaridze ◽  
Xuan Zheng ◽  
Jacob W. J. Kerssemakers ◽  
Cees Dekker
Keyword(s):  
Spatial Organization ◽  
Quantitative Imaging ◽  
Origin Of Replication ◽  
Circular Chromosome ◽  
E Coli ◽  
Domain Boundaries ◽  
Transcription Regulators ◽  
And Function ◽  
Organizational Principles ◽  
Dna Density

New assays for quantitative imaging1–6 and sequencing7–11 have yielded great progress towards understanding the organizational principles of chromosomes. Yet, even for the well-studied model bacterium Escherichia coli, many basic questions remain unresolved regarding chromosomal (sub-)structure2,11, its mechanics1,2,12 and dynamics13,14, and the link between structure and function1,15,16. Here we resolve the spatial organization of the circular chromosome of bacteria by directly imaging the chromosome in live E. coli cells with a broadened cell shape. The chromosome was observed to exhibit a torus topology with a 4.2 μm toroidal length and 0.4 μm bundle thickness. On average, the DNA density along the chromosome shows dense right and left arms that branch from a lower-density origin of replication, and are connected at the terminus of replication by an ultrathin flexible string of DNA. At the single-cell level, the DNA density along the torus is found to be strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. We show that prominent domain boundaries at the terminus and origin of replication are induced by MatP proteins, while weaker transient domain boundaries are facilitated by the global transcription regulators HU and Fis. These findings provide an architectural basis for the understanding of the spatial organization of bacterial genomes.

scholarly journals The Location of Substitutions and Bacterial Genome Arrangements

2020 ◽  
Author(s):  
Daniella F Lato ◽  
G Brian Golding
Keyword(s):  
Sinorhizobium Meliloti ◽  
Bacterial Genome ◽  
Evolutionary Analysis ◽  
Origin Of Replication ◽  
Ancestral Reconstruction ◽  
Molecular Change ◽  
E Coli ◽  
A Genome ◽  
The Impact ◽  
Molecular Evolutionary Analysis

Abstract Increasing evidence supports the notion that different regions of a genome have unique rates of molecular change. This variation is particularly evident in bacterial genomes where previous studies have reported gene expression and essentiality tend to decrease, while substitution rates usually increases with increasing distance from the origin of replication. Genomic reorganization such as rearrangements occur frequently in bacteria and allow for the introduction and restructuring of genetic content, creating gradients of molecular traits along genomes. Here, we explore the interplay of these phenomena by mapping substitutions to the genomes of Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti, quantifying how many substitutions have occurred at each position in the genome. Preceding work indicates that substitution rate significantly increases with distance from the origin. Using a larger sample size and accounting for genome rearrangements through ancestral reconstruction, our analysis demonstrates that the correlation between the number of substitutions and distance from the origin of replication is often significant but small and inconsistent in direction. Some replicons had a significantly decreasing trend (E. coli and the chromosome of S. meliloti), while others showed the opposite significant trend (B. subtilis, Streptomyces, pSymA and pSymB in S. meliloti). dN, dS and ω were examined across all genes and there was no significant correlation between those values and distance from the origin. This study highlights the impact that genomic rearrangements and location have on molecular trends in some bacteria, illustrating the importance of considering spatial trends in molecular evolutionary analysis. Assuming that molecular trends are exclusively in one direction can be problematic.

scholarly journals Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery

BioTech ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 24
Author(s):  
Marina Snapyan ◽  
Sylvain Robin ◽  
Garabet Yeretssian ◽  
Michèle Lecocq ◽  
Frédéric Marc ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Genomic Sequence ◽  
Synthetic Activity ◽  
Translation System ◽  
Free System ◽  
Transcription Terminator ◽  
Cell Free System ◽  
E Coli ◽  
Bacterial Transcription ◽  
A Cell

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

scholarly journals Genome of Bacteriophage P1

2004 ◽  
Vol 186 (21) ◽  
pp. 7032-7068 ◽  
Author(s):  
Małgorzata B. Łobocka ◽  
Debra J. Rose ◽  
Guy Plunkett ◽  
Marek Rusin ◽  
Arkadiusz Samojedny ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Enteric Bacteria ◽  
Origin Of Replication ◽  
Protein Coding ◽  
Bacteriophage P1 ◽  
E Coli ◽  
Base Content ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Rna Polymerase Holoenzyme

ABSTRACT P1 is a bacteriophage of Escherichia coli and other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from σ70 promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by the E. coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication.

Supramolecular self-assemblies for bacterial cell agglutination driven by directional charge-transfer interactions

2018 ◽  
Vol 54 (23) ◽  
pp. 2922-2925 ◽  
Author(s):  
Dan Wu ◽  
Jie Shen ◽  
Hongzhen Bai ◽  
Guocan Yu
Keyword(s):  
Charge Transfer ◽  
Bacterial Cell ◽  
Cell Agglutination ◽  
E Coli ◽  
A Cell ◽  
Multivalent Interactions ◽  
Supramolecular Amphiphiles

Two supramolecular amphiphiles are fabricated through directional charge-transfer interactions, which self-assemble into nanofibers and nanoribbons. Due to the existence of galactose on their surface, these self-assemblies act as a cell glue to agglutinate E. coli, benefiting from multivalent interactions.

Photoreaktivierung von UV-inaktivierter Polyuridylsäure

1964 ◽  
Vol 19 (5) ◽  
pp. 406-408 ◽  
Author(s):  
Adolf Wacker ◽  
Makoto Ishimoto ◽  
Prakash Chandra ◽  
Reinhold Selzer
Keyword(s):  
Hydrogen Peroxide ◽  
Visible Light ◽  
Uv Irradiation ◽  
Trichloroacetic Acid ◽  
Uranyl Acetate ◽  
Free System ◽  
E Coli ◽  
Polyuridylic Acid ◽  
A Cell ◽  
The One

A study on the effect of UV-irradiated polyuridylic acid on the incorporation of phenylalanine into the polypeptide precipitable through trichloroacetic acid, in a cell-free system from E. coli was made. Attempts were made to reactivate the UV-inactivated polyuridylic acid through hydrogen peroxide, uranyl acetate and visible light. We could show that polyuridylic acid irradiated at a dose of 1.2 ×105 ergs/mm2 could be completely reactivated, while the one irradiated at a higher dose of 2.4 ×105 ergs/mm2 could not be completely reactivated under the conditions of our experiment. We have studied the effects of hydrogen peroxide and uranyl acetate on UV-irradiated polyuridylic acid chemically as well. Our results altogether show that the photoreactivating effect of uranyl acetate and hydrogen peroxide is due to their ability to split the uracil dimers formed during UV-irradiation.

scholarly journals The Acidaminococcus sp. Cas12a nuclease recognizes GTTV and GCTV as non-canonical PAMs

2019 ◽  
Vol 366 (8) ◽  
Author(s):  
Thomas Jacobsen ◽  
Chunyu Liao ◽  
Chase L Beisel
Keyword(s):  
Dna Cleavage ◽  
Mammalian Cells ◽  
Consensus Sequence ◽  
Wild Type ◽  
E Coli ◽  
Cleavage Efficiency ◽  
Protospacer Adjacent Motif ◽  
Short Palindromic Repeat ◽  
A Cell ◽  
Validation Experiments

ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) nuclease Acidaminococcus sp. Cas12a (AsCas12a, also known as AsCpf1) has become a popular alternative to Cas9 for genome editing and other applications. AsCas12a has been associated with a TTTV protospacer-adjacent motif (PAM) as part of target recognition. Using a cell-free transcription-translation (TXTL)-based PAM screen, we discovered that AsCas12a can also recognize GTTV and, to a lesser degree, GCTV motifs. Validation experiments involving DNA cleavage in TXTL, plasmid clearance in Escherichia coli, and indel formation in mammalian cells showed that AsCas12a was able to recognize these motifs, with the GTTV motif resulting in higher cleavage efficiency compared to the GCTV motif. We also observed that the -5 position influenced the activity of DNA cleavage in TXTL and in E. coli, with a C at this position resulting in the lowest activity. Together, these results show that wild-type AsCas12a can recognize non-canonical GTTV and GCTV motifs and exemplify why the range of PAMs recognized by Cas nucleases are poorly captured with a consensus sequence.

γ-Bestrahlte Ribosomen von Micrococcus radiodurans in einem System der zellfreien Proteinsynthese / γ-Irradiated Ribosomes from Micrococcus radiodurans in a Cell-Free Protein Synthesizing System

1979 ◽  
Vol 34 (7-8) ◽  
pp. 565-569 ◽  
Author(s):  
Roland Süssmuth ◽  
Annette Widmann
Keyword(s):  
Magnesium Content ◽  
Radiation Sensitivity ◽  
Γ Irradiation ◽  
Free Protein ◽  
E Coli ◽  
Directed Synthesis ◽  
A Cell

Abstract γ-irradiation inactivation of isolated ribosomes of Micrococcus radiodurans was studied by ex amining poly U directed synthesis of polyphenylalanine. Ribosomes of M . radiodurans did not show significant y-radiation sensitivity up to a dose of approx. 11.6k Gy. Cells of M . radiodurans take up more magnesium than E. coli cells unter the same conditions. The magnesium content of ribosomes of M . radiodurans was 18% higher than that of E. coli ribosomes. A possible relation between Mg2+-content and γ-resistance is discussed.

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