Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores

We use a suspended microchannel resonator to characterize the water and small-molecule permeability of Bacillus subtilis spores based on spores' buoyant mass in different solutions. Consistent with previous results, we found that the spore coat is not a significant barrier to small molecules, and the extent to which small molecules may enter the spore is size dependent. We have developed a method to directly observe the exchange kinetics of intraspore water with deuterium oxide, and we applied this method to wild-type spores and a panel of congenic mutants with deficiencies in the assembly or structure of the coat. Compared to wild-type spores, which exchange in approximately 1 s, several coat mutant spores were found to have relatively high water permeability with exchange times below the ∼200-ms temporal resolution of our assay. In addition, we found that the water permeability of the spore correlates with the ability of spores to germinate with dodecylamine and with the ability of TbCl₃ to inhibit germination with l-valine. These results suggest that the structure of the coat may be necessary for maintaining low water permeability.United States. Army Research Office (W911F-09-1-0286)United States. Army Research Office (W911NF-09-0001

Relevance of photobiological repair

From symposium on new trends in photobiology; Rio de Janeiro, Brazil (15 Jul 1973). A review is presented of photobiological and photochemical studies in relation to radiation damage and repair. Some topics discussed are: repair of damage induced in DNA by ultraviolet and gamma radiation; the repair of chemically induced damage to DNA; uv-induced skin cancer; enzymic photoreactivation of uv-irradiated DNA; and phoioreactivation of uv-induced tumors in fish. (HLW

Memory of Germinant Stimuli in Bacterial Spores

Bacterial spores, despite being metabolically dormant, possess the remarkable capacity to detect nutrients and other molecules in their environment through a biochemical sensory apparatus that can trigger spore germination, allowing the return to vegetative growth within minutes of exposure of germinants. We demonstrate here that bacterial spores of multiple species retain memory of transient exposures to germinant stimuli that can result in altered responses to subsequent exposure. The magnitude and decay of these memory effects depend on the pulse duration as well as on the separation time, incubation temperature, and pH values between the pulses. Spores of Bacillus species germinate in response to nutrients that interact with germinant receptors (GRs) in the spore’s inner membrane, with different nutrient types acting on different receptors. In our experiments, B. subtilis spores display memory when the first and second germinant pulses target different receptors, suggesting that some components of spore memory are downstream of GRs. Furthermore, nonnutrient germinants, which do not require GRs, exhibit memory either alone or in combination with nutrient germinants, and memory of nonnutrient stimulation is found to be more persistent than that induced by GR-dependent stimuli. Spores of B. cereus and Clostridium difficile also exhibit germination memory, suggesting that memory may be a general property of bacterial spores. These observations along with experiments involving strains with mutations in various germination proteins suggest a model in which memory is stored primarily in the metastable states of SpoVA proteins, which comprise a channel for release of dipicolinic acid, a major early event in spore germination

Elastic and inelastic light scattering from single bacterial spores in an optical trap allows the monitoring of spore germination dynamics

Raman scattering spectroscopy and elastic light scattering intensity (ESLI) were used to simultaneously measure levels of Ca-dipicolinic acid (CaDPA) and changes in spore morphology and refractive index during germination of individual B. subtilis spores with and without the two redundant enzymes (CLEs), CwlJ and SleB, that degrade sporesâ peptidoglycan cortex. Conclusions from these measurements include: 1) CaDPA release from individual wild-type germinating spores was biphasic; in a first heterogeneous slow phase, Tlag, CaDPA levels decreased â ¼15% and in the second phase ending at Trelease, remaining CaDPA was released rapidly; 2) in L-alanine germination of wild-type spores and spores lacking SleB: a) the ESLI rose â ¼2-fold shortly before Tlag at T1; b) following Tlag, the ESLI again rose â ¼2-fold at T2 when CaDPA levels had decreased â ¼50%; and c) the ESLI reached its maximum value at â ¼Trelease and then decreased; 3) in CaDPA germination of wild-type spores: a) Tlag increased and the first increase in ESLI occurred well before Tlag, consistent with different pathways for CaDPA and L-alanine germination; b) at Trelease the ESLI again reached its maximum value; 4) in L-alanine germination of spores lacking both CLEs and unable to degrade their cortex, the time Î Trelease (Treleaseâ Tlag) for excretion of â ¥75% of CaDPA was â ¼15-fold higher than that for wild-type or sleB spores; and 5) spores lacking only CwlJ exhibited a similar, but not identical ESLI pattern during L-alanine germination to that seen with cwlJ sleB spores, and the high value for Î Trelease. Originally published Analytical Chemistry, Vol. 81, No. 10, May 200

Pharmacokinetics of delta-9-tetrahydrocannabinol following acute cannabis smoke exposure in mice; effects of sex, age, and strain

Increased use of cannabis and cannabinoids for recreational and medical purposes has led to a growth in research on their effects in animal models. The majority of this work has employed cannabinoid injections; however, smoking remains the most common route of cannabis consumption. To better model real-world cannabis use, we exposed mice to cannabis smoke to establish the pharmacokinetics of Δ9THC and its metabolites in plasma and brain. To determine the time course of Δ9THC and two major metabolites [11-hydroxy-delta-9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (11-COOH-THC)], male and female C57BL/6J mice were exposed to smoke from sequentially burning 5 cannabis cigarettes. Following smoke exposure, trunk blood and brains were collected at 6 time points (10–240 min). Plasma and brain homogenates were analyzed for Δ9THC and metabolites using a validated ultraperformance liquid chromatography-tandem mass spectrometry method. To assess effects of age, sex, and mouse strain, we exposed mice of four strains (C57BL/6J, FVB, Swiss Webster, and 129S6/SvEv, aged 4–24 months) to cannabis using the same smoke regimen. Samples were collected 10 and 40 min following exposure. Lastly, to assess effects of dose, C57BL/6J mice were exposed to smoke from burning 3 or 5 cannabis cigarettes, with samples collected 40 min following exposure. The pharmacokinetic study revealed that maximum plasma Δ9THC concentrations (Cmax) were achieved at 10 and 40 min for males and females, respectively, while Cmax for brain Δ9THC was observed at 20 and 40 min for males and females, respectively. There were no age or strain differences in plasma Δ9THC concentrations at 10 or 40 min; however, 129S6/SvEv mice had significantly higher brain Δ9THC concentrations than FVB mice. Additionally, 3 cigarettes produced significantly lower plasma 11-COOH-THC concentrations compared to 5 cigarettes, although dose differences were not evident in plasma or brain concentrations of Δ9THC or 11-OH-THC. Across all experiments, females had higher levels of 11-COOH-THC in plasma compared to males. The results reveal robust sex differences in Δ9THC pharmacokinetics, and lay the groundwork for future studies using mice to model the pharmacodynamics of smoked cannabis

Emergence d’une spécialité scientifique dans l’espace - La réparation de l’ADN

International audienceIn the study of science, the specialty is seen as the ideal level of analysis to understand the genesis and development of scientific communities. This article uses bibliometric data to analyze the emergence of DNA repair by testing a hybrid method to identify the specialty’s appearance in geographical space by focusing on the geographical trajectories of the pioneers in this field. We try to identify the professional mobility of researchers using these bibliometric data, and if possible to highlight the structural networks of places during the emergence stage of the specialty. These networks determine places as much as they are built by individual trajectories. In this way, we try to make a place for the geography of science in the field of social studies of science.Dans l’étude des sciences, la spécialité est perçue comme le niveau d’analyse idéal pour comprendre la genèse et le développement des collectifs scientifiques. Cet article utilise des données bibliométriques pour analyser l’émergence de la Réparation de l’ADN en expérimentant une méthode mixte pour repérer son apparition dans l’espace géographique. En nous concentrant sur les trajectoires géographiques de pionniers dans cedomaine, nous tâchons de repérer leur mobilité professionnelle à l’aide de données bibliométriques dans la perspective de mettre en évidence les réseaux de lieux structurants dans la phase d’émergence de la spécialité. Ces réseaux de lieux déterminent autant qu’ils sont construits par les trajectoires individuelles. Nous essayons ainsi de faire une place à la géographie des sciences dans le domaine des études sociales des sciences

Single-cell analysis reveals individual spore responses to simulated space vacuum

Outer space is a challenging environment for all forms of life, and dormant spores of bacteria have been frequently used to study the survival of terrestrial life in a space journey. Previous work showed that outer space vacuum alone can kill bacterial spores. However, the responses and mechanisms of resistance of individual spores to space vacuum are unclear. Here, we examined spores’ molecular changes under simulated space vacuum (~10−5 Pa) using micro-Raman spectroscopy and found that this vacuum did not cause significant denaturation of spore protein. Then, live-cell microscopy was developed to investigate the temporal events during germination, outgrowth, and growth of individual Bacillus spores. The results showed that after exposure to simulated space vacuum for 10 days, viability of spores of two Bacillus species was reduced up to 35%, but all spores retained their large Ca2 +-dipicolinic acid depot. Some of the killed spores did not germinate, and the remaining germinated but did not proceed to vegetative growth. The vacuum treatment slowed spore germination, and changed average times of all major germination events. In addition, viable vacuum-treated spores exhibited much greater sensitivity than untreated spores to dry heat and hyperosmotic stress. Among spores’ resistance mechanisms to high vacuum, DNA-protective α/β−type small acid-soluble proteins, and non- homologous end joining and base excision repair of DNA played the most important roles, especially against multiple cycles of vacuum treatment. Overall, these results give new insight into individual spore’s responses to space vacuum and provide new techniques for microorganism analysis at the single-cell level

Active site substitutions delineate distinct classes of eubacterial flap endonuclease

FENs (flap endonucleases) play essential roles in DNA replication, pivotally in the resolution of Okazaki fragments. In eubacteria, DNA PolI (polymerase I) contains a flap processing domain, the N-terminal 5′→3′ exonuclease. We present evidence of paralogous FEN-encoding genes present in many eubacteria. Two distinct classes of these independent FEN-encoding genes exist with four groups of eubacteria, being identified based on the number and type of FEN gene encoded. The respective proteins possess distinct motifs hallmarking their differentiation. Crucially, based on primary sequence and predicted secondary structural motifs, we reveal key differences at their active sites. These results are supported by biochemical characterization of two family members - ExoIX (exonuclease IX) from Escherichia coli and SaFEN (Staphylococcus aureus FEN). These proteins displayed marked differences in their ability to process a range of branched and linear DNA structures. On bifurcated substrates, SaFEN exhibited similar substrate specificity to previously characterized FENs. In quantitative exonuclease assays, SaFEN maintained a comparable activity with that reported for PolI. However, ExoIX showed no observable enzymatic activity. A threaded model is presented for SaFEN, demonstrating the probable interaction of this newly identified class of FEN with divalent metal ions and a branched DNA substrate. The results from the present study provide an intriguing model for the cellular role of these FEN sub-classes and illustrate the evolutionary importance of processing aberrant DNA, which has led to their maintenance alongside DNA PolI in many eubacteria