Multidimensional Active Flux Schemes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106467/1/AIAA2013-2940.pd

Neutron induced background in the COMPTEL detector on the Gamma Ray Observatory

Interactions of neutrons in a prototype of the Compton imaging telescope (COMPTEL) gamma ray detector for the Gamma Ray Observatory were studied to determine COMPTEL's sensitivity as a neutron telescope and to estimate the gamma ray background resulting from neutron interactions. The IUCF provided a pulsed neutron beam at five different energies between 18 and 120 MeV. These measurements showed that the gamma ray background from neutron interactions is greater than previously expected. It was thought that most such events would be due to interactions in the upper detector modules of COMPTEL and could be distinguished by pulse shape discrimination. Rather, the bulk of the gamma ray background appears to be due to interactions in passive material, primarily aluminum, surrounding the D1 modules. In a considerable fraction of these interactions, two or more gamma rays are produced simultaneously, with one interacting in the D1 module and the other interacting in the module of the lower (D2) detector. If the neutron interacts near the D1 module, the D1 D2 time of flight cannot distinguish such an event from a true gamma ray event. In order to assess the significance of this background, the flux of neutrons in orbit has been estimated based on observed events with neutron pulse shape signature in D1. The strength of this neutron induced background is estimated. This is compared with the rate expected from the isotropic cosmic gamma ray flux

Definition of the σW regulon of Bacillus subtilis in the absence of stress

Bacteria employ extracytoplasmic function (ECF) sigma factors for their responses to environmental stresses. Despite intensive research, the molecular dissection of ECF sigma factor regulons has remained a major challenge due to overlaps in the ECF sigma factor-regulated genes and the stimuli that activate the different ECF sigma factors. Here we have employed tiling arrays to single out the ECF σW regulon of the Gram-positive bacterium Bacillus subtilis from the overlapping ECF σX, σY, and σM regulons. For this purpose, we profiled the transcriptome of a B. subtilis sigW mutant under non-stress conditions to select candidate genes that are strictly σW-regulated. Under these conditions, σW exhibits a basal level of activity. Subsequently, we verified the σW-dependency of candidate genes by comparing their transcript profiles to transcriptome data obtained with the parental B. subtilis strain 168 grown under 104 different conditions, including relevant stress conditions, such as salt shock. In addition, we investigated the transcriptomes of rasP or prsW mutant strains that lack the proteases involved in the degradation of the σW anti-sigma factor RsiW and subsequent activation of the σW-regulon. Taken together, our studies identify 89 genes as being strictly σW-regulated, including several genes for non-coding RNAs. The effects of rasP or prsW mutations on the expression of σW-dependent genes were relatively mild, which implies that σW-dependent transcription under non-stress conditions is not strictly related to RasP and PrsW. Lastly, we show that the pleiotropic phenotype of rasP mutant cells, which have defects in competence development, protein secretion and membrane protein production, is not mirrored in the transcript profile of these cells. This implies that RasP is not only important for transcriptional regulation via σW, but that this membrane protease also exerts other important post-transcriptional regulatory functions

Eukaryotic-like Ser/Thr Protein Kinases SpkC/F/K Are Involved in Phosphorylation of GroES in the Cyanobacterium Synechocystis

Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES

The bacterial stressosome:a modular system that has been adapted to control secondary messenger signaling

SummaryThe stressosome complex regulates downstream effectors in response to environmental signals. In Bacillus subtilis, it activates the alternative sigma factor σB, leading to the upregulation of the general stress regulon. Herein, we characterize a stressosome-regulated biochemical pathway in Moorella thermoacetica. We show that the presumed sensor, MtR, and the scaffold, MtS, form a pseudo-icosahedral structure like that observed in B. subtilis. The N-terminal domain of MtR is structurally homologous to B. subtilis RsbR, despite low sequence identity. The affinity of the switch kinase, MtT, for MtS decreases following MtS phosphorylation and not because of structural reorganization. Dephosphorylation of MtS by the PP2C type phosphatase MtX permits the switch kinase to rebind the stressosome to reset the response. We also show that MtT regulates cyclic di-GMP biosynthesis through inhibition of a GG(D/E)EF-type diguanylate cyclase, demonstrating that secondary messenger levels are regulated by the stressosome

Comparative proteomics using 2-D gel electrophoresis and mass spectrometry as tools to dissect stimulons and regulons in bacteria with sequenced or partially sequenced genomes

We propose two-dimensional gel electrophoresis (2-DE) and mass spectrometry to define the protein components of regulons and stimulons in bacteria, including those organisms where genome sequencing is still in progress. The basic 2-DE protocol allows high resolution and reproducibility and enables the direct comparison of hundreds or even thousands of proteins simultaneously. To identify proteins that comprise stimulons and regulons, peptide mass fingerprint (PMF) with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF-MS) analysis is the first option and, if results from this tool are insufficient, complementary data obtained with electrospray ionization tandem-MS (ESI-MS/MS) may permit successful protein identification. ESI-MS/MS and MALDI-TOF-MS provide complementary data sets, and so a more comprehensive coverage of a proteome can be obtained using both techniques with the same sample, especially when few sequenced proteins of a particular organism exist or genome sequencing is still in progress

Fluctuations in spo0A Transcription Control Rare Developmental Transitions in Bacillus subtilis

Phosphorylated Spo0A is a master regulator of stationary phase development in the model bacterium Bacillus subtilis, controlling the formation of spores, biofilms, and cells competent for transformation. We have monitored the rate of transcription of the spo0A gene during growth in sporulation medium using promoter fusions to firefly luciferase. This rate increases sharply during transient diauxie-like pauses in growth rate and then declines as growth resumes. In contrast, the rate of transcription of an rRNA gene decreases and increases in parallel with the growth rate, as expected for stable RNA synthesis. The growth pause-dependent bursts of spo0A transcription, which reflect the activity of the spo0A vegetative promoter, are largely independent of all known regulators of spo0A transcription. Evidence is offered in support of a “passive regulation” model in which RNA polymerase stops transcribing rRNA genes during growth pauses, thus becoming available for the transcription of spo0A. We show that the bursts are followed by the production of phosphorylated Spo0A, and we propose that they represent initial responses to stress that bring the average cell closer to the thresholds for transition to bimodally expressed developmental responses. Measurement of the numbers of cells expressing a competence marker before and after the bursts supports this hypothesis. In the absence of ppGpp, the increase in spo0A transcription that accompanies the entrance to stationary phase is delayed and sporulation is markedly diminished. In spite of this, our data contradicts the hypothesis that sporulation is initiated when a ppGpp-induced depression of the GTP pool relieves repression by CodY. We suggest that, while the programmed induction of sporulation that occurs in stationary phase is apparently provoked by increased flux through the phosphorelay, bet-hedging stochastic transitions to at least competence are induced by bursts in transcription

Disease proteomics

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62680/1/nature01514.pd