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

Analysis of Clostridium beijerinckii NCIMB 8052's transcriptional response to ferulic acid and its application to enhance the strain tolerance

Background: Plant-based cellulose presents the best source of renewable sugars for biofuel production. However, the lignin associated with plant cellulose presents a hurdle as hydrolysis of this component leads to the production of inhibitory compounds, such as ferulic acid. Results: The impacts of ferulic acid, a phenolic compound commonly found in lignin hydrolysates, on the growth, solvent production, and transcriptional responses of Clostridium beijerinckii NCIMB 8052 were determined. Addition of ferulic acid to growing cultures resulted in a decrease in the growth and solvent production by 30% and 25%, respectively, when compared to the control cultures. To better understand the toxicity of this compound, microarray analyses were performed using samples taken from these cultures at three different growth states. Several gene ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified showing significant change at each status, including ATP-binding cassette (ABC) transporters, two component system, and oxidoreductase activity. Moreover, genes related with efflux systems and heat shock proteins were also strongly up-regulated. Among these, expression of the groESL operon was induced by more than fourfold and was consequently selected to improve C. beijerinckii tolerance to ferulic acid. Real-time quantitative PCR (RT-qPCR) analysis confirmed that C. beijerinckii harboring the plasmid, pSAAT-ptb_Gro, had a two-to fivefold increased groESL operon expression during growth of these cultures. Moreover, this strain was more tolerant to ferulic acid as the growth of this recombinant strain and its bioconversion of glucose into solvents were both improved. Conclusions: Using transcriptomics, we identified numerous genes that are differentially expressed when C. beijerinckii cultures were exposed to ferulic acid for varying amounts of time. The operon expressing groESL was consistently up-regulated, suggesting that this gene cluster may contribute to strain tolerance. This was confirmed as recombinant cultures showed both an enhanced growth and solvent yield in the presence of 0.5 g/L ferulic acidopen00

Impact of Radiation Biology on Fundamental in Biology

Research supported by OHER and its predecessors has as one of its major goals an understanding of the effects of radiation at low doses and dose rates on biological systems, so as to predict their effects on humans. It is not possible to measure such effects directly. They must be predicted from basic knowledge on how radiation affects cellular components such as DNA and membranes and how cells react to such changes. What is the probability of radiation producing human mutations and what are the probabilities of radiation producing cancer? The end results of such studies are radiation exposure standards for workers and for the general population. An extension of these goals is setting standards for exposure to chemicals involved in various energy technologies. This latter problem is much more difficult because chemical dosimetry is is a primitive state compared to radiation dosimetry

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

A model for transition of 5 '-nuclease domain of DNA polymerase I from inert to active modes

Bacteria contain DNA polymerase I (PolI), a single polypeptide chain consisting of similar to 930 residues, possessing DNA-dependent DNA polymerase, 3'-5' proofreading and 5'-3' exonuclease (also known as flap endonuclease) activities. PolI is particularly important in the processing of Okazaki fragments generated during lagging strand replication and must ultimately produce a double-stranded substrate with a nick suitable for DNA ligase to seal. PolI's activities must be highly coordinated both temporally and spatially otherwise uncontrolled 5'-nuclease activity could attack a nick and produce extended gaps leading to potentially lethal double-strand breaks. To investigate the mechanism of how PolI efficiently produces these nicks, we present theoretical studies on the dynamics of two possible scenarios or models. In one the flap DNA substrate can transit from the polymerase active site to the 5'-nuclease active site, with the relative position of the two active sites being kept fixed; while the other is that the 5'-nuclease domain can transit from the inactive mode, with the 5'-nuclease active site distant from the cleavage site on the DNA substrate, to the active mode, where the active site and substrate cleavage site are juxtaposed. The theoretical results based on the former scenario are inconsistent with the available experimental data that indicated that the majority of 5'-nucleolytic processing events are carried out by the same PolI molecule that has just extended the upstream primer terminus. By contrast, the theoretical results on the latter model, which is constructed based on available structural studies, are consistent with the experimental data. We thus conclude that the latter model rather than the former one is reasonable to describe the cooperation of the PolI's polymerase and 5'-3' exonuclease activities. Moreover, predicted results for the latter model are presented

Horizontal DNA transfer mechanisms of bacteria as weapons of intragenomic conflict

Horizontal DNA transfer (HDT) is a pervasive mechanism of diversification in many microbial species, but its primary evolutionary role remains controversial. Much recent research has emphasised the adaptive benefit of acquiring novel DNA, but here we argue instead that intragenomic conflict provides a coherent framework for understanding the evolutionary origins of HDT. To test this hypothesis, we developed a mathematical model of a clonally descended bacterial population undergoing HDT through transmission of mobile genetic elements (MGEs) and genetic transformation. Including the known bias of transformation toward the acquisition of shorter alleles into the model suggested it could be an effective means of counteracting the spread of MGEs. Both constitutive and transient competence for transformation were found to provide an effective defence against parasitic MGEs; transient competence could also be effective at permitting the selective spread of MGEs conferring a benefit on their host bacterium. The coordination of transient competence with cell-cell killing, observed in multiple species, was found to result in synergistic blocking of MGE transmission through releasing genomic DNA for homologous recombination while simultaneously reducing horizontal MGE spread by lowering the local cell density. To evaluate the feasibility of the functions suggested by the modelling analysis, we analysed genomic data from longitudinal sampling of individuals carrying Streptococcus pneumoniae. This revealed the frequent within-host coexistence of clonally descended cells that differed in their MGE infection status, a necessary condition for the proposed mechanism to operate. Additionally, we found multiple examples of MGEs inhibiting transformation through integrative disruption of genes encoding the competence machinery across many species, providing evidence of an ongoing "arms race." Reduced rates of transformation have also been observed in cells infected by MGEs that reduce the concentration of extracellular DNA through secretion of DNases. Simulations predicted that either mechanism of limiting transformation would benefit individual MGEs, but also that this tactic's effectiveness was limited by competition with other MGEs coinfecting the same cell. A further observed behaviour we hypothesised to reduce elimination by transformation was MGE activation when cells become competent. Our model predicted that this response was effective at counteracting transformation independently of competing MGEs. Therefore, this framework is able to explain both common properties of MGEs, and the seemingly paradoxical bacterial behaviours of transformation and cell-cell killing within clonally related populations, as the consequences of intragenomic conflict between self-replicating chromosomes and parasitic MGEs. The antagonistic nature of the different mechanisms of HDT over short timescales means their contribution to bacterial evolution is likely to be substantially greater than previously appreciated

Stress-Induced Reinstatement of Drug Seeking: 20 Years of Progress

In human addicts, drug relapse and craving are often provoked by stress. Since 1995, this clinical scenario has been studied using a rat model of stress-induced reinstatement of drug seeking. Here, we first discuss the generality of stress-induced reinstatement to different drugs of abuse, different stressors, and different behavioral procedures. We also discuss neuropharmacological mechanisms, and brain areas and circuits controlling stress-induced reinstatement of drug seeking. We conclude by discussing results from translational human laboratory studies and clinical trials that were inspired by results from rat studies on stress-induced reinstatement. Our main conclusions are (1) The phenomenon of stress-induced reinstatement, first shown with an intermittent footshock stressor in rats trained to self-administer heroin, generalizes to other abused drugs, including cocaine, methamphetamine, nicotine, and alcohol, and is also observed in the conditioned place preference model in rats and mice. This phenomenon, however, is stressor specific and not all stressors induce reinstatement of drug seeking. (2) Neuropharmacological studies indicate the involvement of corticotropin-releasing factor (CRF), noradrenaline, dopamine, glutamate, kappa/dynorphin, and several other peptide and neurotransmitter systems in stress-induced reinstatement. Neuropharmacology and circuitry studies indicate the involvement of CRF and noradrenaline transmission in bed nucleus of stria terminalis and central amygdala, and dopamine, CRF, kappa/dynorphin, and glutamate transmission in other components of the mesocorticolimbic dopamine system (ventral tegmental area, medial prefrontal cortex, orbitofrontal cortex, and nucleus accumbens). (3) Translational human laboratory studies and a recent clinical trial study show the efficacy of alpha-2 adrenoceptor agonists in decreasing stress-induced drug craving and stress-induced initial heroin lapse

Phosphorylated DegU Manipulates Cell Fate Differentiation in the <i>Bacillus subtilis</i> Biofilm<em/>

Cell differentiation is ubiquitous and facilitates division of labor and development. Bacteria are capable of multicellular behaviors that benefit the bacterial community as a whole. A striking example of bacterial differentiation occurs throughout the formation of a biofilm. During Bacillus subtilis biofilm formation, a subpopulation of cells differentiates into a specialized population that synthesizes the exopolysaccharide and the TasA amyloid components of the extracellular matrix. The differentiation process is indirectly controlled by the transcription factor Spo0A that facilitates transcription of the eps and tapA (tasA) operons. DegU is a transcription factor involved in regulating biofilm formation. Here, using a combination of genetics and live single-cell cytological techniques, we define the mechanism of biofilm inhibition at high levels of phosphorylated DegU (DegU∼P) by showing that transcription from the eps and tapA promoter regions is inhibited. Data demonstrating that this is not a direct regulatory event are presented. We demonstrate that DegU∼P controls the frequency with which cells activate transcription from the operons needed for matrix biosynthesis in favor of an off state. Subsequent experimental analysis led us to conclude that DegU∼P functions to increase the level of Spo0A∼P, driving cell fate differentiation toward the terminal developmental process of sporulation