Jesús Ramírez-Santos

Plasmid DNA Supercoiling and Gyrase Activity in Escherichia coli Wild-Type and rpoS Stationary-Phase Cells

Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available. Preexisting gyrase molecules in these cells were responsible for this recovery. Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.

Methylated cytosine at Dcm (CC T A GG) sites in Escherichia coli: Possible function and evolutionary implications

The frequency and distribution of methylated cytosine (5-MeC) at CCTAGG (Dcm sites) in 49 E. coli DNA loci (207,530 bp) were determined. Principal observations of this analysis were: (1) Dcm frequency was higher than expected from random occurrence but lower than calculated with Markov chain analysis; (2) CCTGG sites were found more frequently in coding than in noncoding regions, while the opposite was true for CCAGG sites; (3) Dcm site distribution does not exhibit any identifiably regular pattern on the chromosome; (4) Dcm sites at oriC are probably not important for accurate initiation of DNA replication; (5) 5-MeC in codons was more frequently found in first than in second and third positions; (6) there are probably few genes in which the mutation rate is determined mainly by DNA methylation. It is proposed that the function of Dcm methylase is to protect chromosomal DNA from restriction-enzyme EcoRII. The Dcm methylation contribution to determine frequency of oligonucleotides, mutation rate, and recombination level, and thus evolution of the E. coli genome, could be interpreted as a consequence of the acquisition of this methylation.

In vivo effect of DNA relaxation on the transcription of gene rpoH in Escherichia coli

The in vivo effect of Novobiocin, a gyrase inhibitor, on the transcription of gene rpoH which codes for sigma32, the main positive regulator of the heat-shock response, was studied. Novobiocin induced a three-fold increase and a slight decrease in the activity of the rpoH promoters P1 and P4, respectively. The Novobiocin-induced increase in the activity of promoter P1 correlates with an increase in the amount of proteins sigma32 and DnaK. These results suggest that the increase in expression of the heat-shock proteins induced by gyrase inhibitors is probably due to the increased activity of P1 on relaxed DNA.

Role of chaperones and ATP synthase in DNA gyrase reactivation in Escherichia coli stationary-phase cells after nutrient addition

Escherichia coli stationary-phase (SP) cells contain relaxed DNA molecules and recover DNA supercoiling once nutrients become available. In these cells, the reactivation of DNA gyrase, which is a DNA topoisomerase type IIA enzyme, is responsible for the recovery of DNA supercoiling. The results presented in this study show that DNA gyrase reactivation does not require cellular chaperones or polyphosphate. Glucose addition to SP cells induced a slow recovery of DNA supercoiling, whereas resveratrol, which is an inhibitor of ATP synthase, inhibited the enzyme reactivation. These results suggest that DNA gyrase, which is an ATP-dependent enzyme, remains soluble in SP cells, and that its reactivation occurs primarily due to a rapid increase in the cellular ATP concentration.

Differential vascular permeability along the forebrain ventricular neurogenic niche in the adult murine brain

Adult neurogenesis is influenced by blood‐borne factors. In this context, greater or lesser vascular permeability along neurogenic niches would expose differentially neural stem cells (NSCs), transit amplifying cells (TACs), and neuroblasts to such factors. Here we evaluate endothelial cell morphology and vascular permeability along the forebrain neurogenic niche in the adult brain. Our results confirm that the subventricular zone (SVZ) contains highly permeable, discontinuous blood vessels, some of which allow the extravasation of molecules larger than those previously reported. In contrast, the rostral migratory stream (RMS) and the olfactory bulb core (OBc) display mostly impermeable, continuous blood vessels. These results imply that NSCs, TACs, and neuroblasts located within the SVZ are exposed more readily to blood‐borne molecules, including those with very high molecular weights, than those positioned along the RMS and the OBc, subregions in which every stage of neurogenesis also takes place. These observations suggest that the existence of specialized vascular niches is not a precondition for neurogenesis to occur; specialized vascular beds might be essential for keeping high rates of proliferation and/or differential differentiation of neural precursors located at distinct domains.

Role of single-strand DNA 3′-5′ exonuclease ExoI and nuclease SbcCD in stationary-phase mutation in Escherichia coli K-12

In RecBCD+ cells, a mutated single-strand DNA 3′-5′ exonuclease ExoI (SbcB15) induced an increase in stationary-phase mutation. In sbcB15 cells, as in wild-type cells, these mutations partially required RecA, RecB, RecF, and expression of the LexA regulon. The absence of nuclease SbcCD in sbcB15 cells decreased stationary-phase mutation and induced an increase in the number of cell filaments. The absence of ExoI (Δxon) in wild-type or sbcC cells did not change significantly the stationary-phase mutation. Differences between the sbcB15 and ΔxonA cells suggest a correlation between level of SOS induction and the generation of stationary-phase mutations.