Increasing the effectiveness of intracerebral injections in adult and neonatal mice: a neurosurgical point of view

International audienceIntracerebral injections of tracers or viral constructs in rodents are now commonly used in the neurosciences and must be executed perfectly. The purpose of this article is to update existing protocols for intracerebral injections in adult and neonatal mice. Our procedure for stereotaxic injections in adult mice allows the investigator to improve the effectiveness and safety, and save time. Furthermore, for the first time, we describe a two-handed procedure for intracerebral injections in neonatal mice that can be performed by a single operator in a very short time. Our technique using the stereotaxic arm allows a higher precision than freehand techniques previously described. Stereotaxic injections in adult mice can be performed in 20 min and have >90% efficacy in targeting the injection site. Injections in neonatal mice can be performed in 5 min. Efficacy depends on the difficulty of precisely localizing the injection sites, due to the small size of the animal. We describe an innovative, effortless, and reproducible surgical protocol for intracerebral injections in adult and neonatal mice

Targeting the Replication Initiator of the Second Vibrio Chromosome: Towards Generation of Vibrionaceae-Specific Antimicrobial Agents

The Vibrionaceae is comprised of numerous aquatic species and includes several human pathogens, such as Vibrio cholerae, the cause of cholera. All organisms in this family have two chromosomes, and replication of the smaller one depends on rctB, a gene that is restricted to the Vibrionaceae. Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. Here, we carried out a high throughput cell-based screen to find small molecule inhibitors of RctB. We identified a compound that blocked growth of an E. coli strain bearing an rctB-dependent plasmid but did not influence growth of E. coli lacking this plasmid. This compound, designated vibrepin, had potent cidal activity against V. cholerae and inhibited the growth of all vibrio species tested. Vibrepin blocked RctB oriCII unwinding, apparently by promoting formation of large non-functional RctB complexes. Although vibrepin also appears to have targets other than RctB, our findings suggest that RctB is an attractive target for generation of novel antibiotics that only block growth of vibrios. Vibrio-specific agents, unlike antibiotics currently used in clinical practice, will not engender resistance in the normal human flora or in non-vibrio environmental microorganisms

Replication Fork Reversal after Replication–Transcription Collision

Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle

Long range chromosome organization in Escherichia coli: The position of the replication origin defines the non-structured regions and the Right and Left macrodomains

International audienceThe Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell

Activity of MukBEF for chromosome management in E. coli and its inhibition by MatP

International audienceWhile different features for the activity of the bacterial canonical SMC complex, Smc-ScpAB, have been described in different bacteria, not much is known about the way chromosomes in enterobacteria interact with their SMC complex, MukBEF. Here we used a number of in vivo assays in E. coli to reveal how MukBEF controls chromosome conformation and how the MatP/ matS system prevents MukBEF activity. Our results indicate that the loading of MukBEF occurs preferentially in newly replicated DNA, at multiple loci on the chromosome where it can promote long-range contacts in cis even though MukBEF can promote long-range contacts in the absence of replication. Using HiC and ChIP-seq analyses in strains with rearranged chromosomes, the prevention of MukBEF activity increases with the number of matS sites and this effect likely results from the unloading of MukBEF by MatP. Altogether, our results reveal how MukBEF operates to control chromosome folding and segregation in E. coli

Caractérisation génétique et fonctionnelle de PolY1 et PolY2 chez B. subtilis

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

The position of the replication origin changes the Right and Left boundaries.

<p>Histograms of recombination frequencies between <i>att</i>L and <i>att</i>R sequences in WT, and Inv strain. Recombination frequencies between different <i>attR/L</i> sites are indicated on the y-axis. These values are the average of at least 3 independent experiments and the error bars correspond to the standard deviation. For both panels, strains were grown in minimal medium and the recombinase production was obtained by shifting cultures at 38°C for 10’. Relative position of each <i>att</i> sequence used in the experiment is represented on the MD map on top of each panel. The name of the <i>att</i> sequence, and the MD which they belong to, are indicated on the x-axis. The genetic backgrounds are indicated below the histogram (A), or with a color code (B). * indicates that the recombination frequency obtained was repeatedly 0.</p

The interaction limit of the Ori MD.

<p>Graphical representation of the Ori MD (green box), NSR region (gray line) and Right MD (red box). Coordinates of the <i>attR/L</i> sequences are indicated in fonction of their distance from the zero pb reference of the MG1655 genome for both configuration WT and RT. Percentages of recombination between <i>attL</i> and <i>attR</i> obtained after induction of 20’ at 36°C or 10’ at 37°C are shown. The histograms correspond to an average of at least 3 independent experiments with standard-deviations.</p

Effect of Right and Left transposition on long distance DNA interactions.

<p>Histograms of recombination frequency between <i>attL</i> and <i>attR</i> sequences in WT, RT, and LT strains. The y-axis indicates the percentage of recombination between <i>attL</i> and <i>attR</i> sequences, obtained as described in Materials and Methods with 20’ induction at 36°C. The relative position of each <i>att</i> sequence used in the experiment is represented on the MD map on top of each panel. Histograms show the average of at least 3 independent experiments with their respective standard-deviation. Frequencies obtained in the WT strain are represented with the black bars, in the RT strain (A,C) or LT strain (B,D) with the gray bars and in the Δ<i>matP</i> RT strain (C), or Δ<i>matP</i> LT strain (D) with the light gray bars. * indicates that no <i>att</i>L<i>-att</i>R recombinant was obtained.</p

Position of chromosomal loci in the Ori and Right MD depending of <i>oriC</i> position.

<p>MD map of the <i>E</i>. <i>coli</i> chromosome are represented with the position of the Ori-4, Right-2, FROS-Tag in an Inv (A), or <i>oriCZ</i> (B) configurations. For each panel, the position of foci in cell containing 2 foci (cell with one focus are show in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006758#pgen.1006758.s004" target="_blank">S4 Fig</a>), are represent in function of the long axis of the cell (y-axis) and in function of the cell length (x-axis). Red dots and bars correspond to the localization of the Right-2 loci and green ones to the Ori-4 loci. (C) Histogram of the percentage of the population presenting different interfocal distance (<0.2 to > 0.5) between Ori-4 and Right-2 in the WT strain (blue), Inv strain (red) and <i>oriCZ</i> strain (green). Numbers on the first columns show the percentage of cells where both foci are co-localized.</p