Characterization of transposon insertion mutants in desulfovibrio vulgaris hilderborough by sequencing genomic DNA [abstract]

Abstract only availableTn5 transposon mutagenesis occurs by a mechanism in which a segment of DNA (transposon) encoded in a plasmid is inserted into genomic DNA (the target) by a conservative (cut-and-paste) mechanism. When the insertion position is in a coding sequence or regulatory region of DNA, the insertion results in a mutation. The plasmid pRL27 encodes a mini-Tn5 transposon, Tn5 transposase, and kanamycin resistance, (Metcalf, William W. et al, 2002 Arch Microbiol 178 :193-201) and was used to transform Desulfovibrio vulgaris Hildenborough by electroporation. Transposon insertion mutants were identified by their ability to grow in the presence of kanamycin. To identify the insertion sites of the transposons, in theory one should be able to sequence from the transposon into chromosomal DNA and identify the mutation site by comparison with the known genome. Unlike sequencing of plasmid DNA or PCR products, direct genomic sequencing has a limited success rate. Direct genomic sequencing is sensitive to DNA quality, interference of secondary DNA structures, salt concentration, and the availability of primer binding sites. Because of these difficulties, in our attempts to identify insertion sites of mini-Tn5, we examined template DNA quality as well as modifying sequencing reaction conditions. Our objective is to develop an effective, reliable method for sequencing genomic DNA to identify where transposon insertion sites have occurred in each mutant.Department of Energy Genomics: Genomes to Life Progra

Rapid automated characterization of transposon insertion mutants in Desulfovibrio vulgaris Hildenborough by srnPCR [abstract]

Abstract only availableTn5 transposon mutagenesis occurs by a mechanism in which a segment of DNA (transposon) encoded in a plasmid is inserted into genomic DNA (the target) by a conservative (cut-and-paste) mechanism (Fig. 2). When the insertion position is in a coding sequence or regulatory region of DNA, the insertion results in a mutation. The plasmid pRL27 (a generous gift from Bill Metcalf) encodes a mini-Tn5 transposon, Tn5 transposase, and kanamycin resistance (neo), and was used to transform Desulfovibrio vulgaris Hildenborough by electroporation. Transposon insertion mutants were identified by their ability to grow in the presence of kanamycin. To locate the insertion site of the transposon, in theory, one should be able to directly sequence from the transposon into chromosomal DNA (Fig. 3.1) and identify the mutation site by comparison with the known genome BLAST. Unlike sequencing of plasmid DNA or PCR products, direct genomic sequencing has a limited success rate. Therefore, a method of enriching the transposon-flanking sequence is needed. Nested semi-random PCR (Fig. 3.2) is an efficient and cost effective enrichment method. Sequencing these enriched products allows us to identify the transposon insertion site. The factors that influence characterization success rate are: frequency and location of priming sites, reaction volume, and reaction conditions (annealing temperature, extension time, etc.). By varying these factors, we have developed an efficient and reliable method for characterizing transposon insertion mutants. Utilizing high-throughput robotics and nested semi-random PCR, we have generated single gene mutants that may provide valuable biological data.U.S. Department of Energy Genomes to Life gran

Differentiating EDRs from the Background Magnetopause Current Sheet: A Statistical Study

The solar wind is a continuous outflow of charged particles from the Sun's atmosphere into the solar system. At Earth, the solar wind's outward pressure is balanced by the Earth's magnetic field in a boundary layer known as the magnetopause. Plasma density and temperature differences across the boundary layer generate the Chapman-Ferraro current which supports the magnetopause. Along the dayside magnetopause, magnetic reconnection can occur in electron diffusion regions (EDRs) embedded into the larger ion diffusion regions (IDRs). These diffusion regions form when opposing magnetic field lines in the solar wind and Earth's magnetic field merge, releasing magnetic energy into the surrounding plasma. While previous studies have given us a general understanding of the structure of the diffusion regions, we still do not have a good grasp of how they are statistically differentiated from the non-diffusion region magnetopause. By investigating 251 magnetopause crossings from NASA's Magnetospheric Multiscale (MMS) Mission, we demonstrate that EDR magnetopause crossings show current densities an order of magnitude higher than regular magnetopause crossings - crossings that either passed through the reconnection exhausts or through the non-reconnecting magnetopause, providing a baseline for the magnetopause current sheet under a wide range of driving conditions. Significant current signatures parallel to the local magnetic field in EDR crossings are also identified, which is in contrast to the dominantly perpendicular current found in the regular magnetopause. Additionally, we show that the ion velocity along the magnetopause is highly correlated with a crossing's location, indicating the presence of magnetosheath flows inside the magnetopause

Structure of the Current Sheet in the 11 July 2017 Electron Diffusion Region Event.

The structure of the current sheet along the Magnetospheric Multiscale (MMS) orbit is examined during the 11 July 2017 Electron Diffusion Region (EDR) event. The location of MMS relative to the X-line is deduced and used to obtain the spatial changes in the electron parameters. The electron velocity gradient values are used to estimate the reconnection electric field sustained by nongyrotropic pressure. It is shown that the observations are consistent with theoretical expectations for an inner EDR in 2-D reconnection. That is, the magnetic field gradient scale, where the electric field due to electron nongyrotropic pressure dominates, is comparable to the gyroscale of the thermal electrons at the edge of the inner EDR. Our approximation of the MMS observations using a steady state, quasi-2-D, tailward retreating X-line was valid only for about 1.4 s. This suggests that the inner EDR is localized; that is, electron outflow jet braking takes place within an ion inertia scale from the X-line. The existence of multiple events or current sheet processes outside the EDR may play an important role in the geometry of reconnection in the near-Earth magnetotail

Energy Conversion and Partition in the Asymmetric Reconnection Diffusion Region

We investigate the energy conversion and partition in the asymmetric reconnection diffusion region using two-dimensional particle-in-cell simulations and Magnetosphere Multiscale (MMS) mission observations. Under an upstream condition with equal temperatures in the two inflow regions, the simulation analysis indicates that the energy partition between ions and electrons depends on the distance from the X-line. Within the central electron diffusion region (EDR), nearly all dissipated electromagnetic field energies are converted to electrons. From the EDR to the ion diffusion region (IDR) scales, the rate of the electron energy gain decreases to be lower than that of ions. A magnetopause reconnection event inside the IDR observed by MMS shows comparable ion and electron energy gains, consistent with the simulation result in the transition region from EDR to IDR. At the EDR scale, the electron energization is mainly by the reconnection electric field (E(sub r)); in-plane electric fields (E(sub xz)) provide additional positive contributions near the X-line and do negative work on electrons beyond the EDR. The guide field reduces the electron energization by both E(sub r) and E(sub xz) in the EDR. For ion energization, E(sub r) and E(sub xz) have comparable contributions near the time of the peak reconnection rate, while E(sub xz) dominants at later time. At the IDR scale, the guide field causes asymmetry in the amount of the energy gain and energization mechanisms between two exhausts but does not have significant effects on energy partition. Our study advances understanding of ion and electron energization in asymmetric reconnect IDRs

Near-Earth plasma sheet boundary dynamics during substorm dipolarization.

We report on the large-scale evolution of dipolarization in the near-Earth plasma sheet during an intense (AL ~ -1000 nT) substorm on August 10, 2016, when multiple spacecraft at radial distances between 4 and 15 R E were present in the night-side magnetosphere. This global dipolarization consisted of multiple short-timescale (a couple of minutes) B z disturbances detected by spacecraft distributed over 9 MLT, consistent with the large-scale substorm current wedge observed by ground-based magnetometers. The four spacecraft of the Magnetospheric Multiscale were located in the southern hemisphere plasma sheet and observed fast flow disturbances associated with this dipolarization. The high-time-resolution measurements from MMS enable us to detect the rapid motion of the field structures and flow disturbances separately. A distinct pattern of the flow and field disturbance near the plasma boundaries was found. We suggest that a vortex motion created around the localized flows resulted in another field-aligned current system at the off-equatorial side of the BBF-associated R1/R2 systems, as was predicted by the MHD simulation of a localized reconnection jet. The observations by GOES and Geotail, which were located in the opposite hemisphere and local time, support this view. We demonstrate that the processes of both Earthward flow braking and of accumulated magnetic flux evolving tailward also control the dynamics in the boundary region of the near-Earth plasma sheet.Graphical AbstractMultispacecraft observations of dipolarization (left panel). Magnetic field component normal to the current sheet (BZ) observed in the night side magnetosphere are plotted from post-midnight to premidnight region: a GOES 13, b Van Allen Probe-A, c GOES 14, d GOES 15, e MMS3, g Geotail, h Cluster 1, together with f a combined product of energy spectra of electrons from MMS1 and MMS3 and i auroral electrojet indices. Spacecraft location in the GSM X-Y plane (upper right panel). Colorcoded By disturbances around the reconnection jets from the MHD simulation of the reconnection by Birn and Hesse (1996) (lower right panel). MMS and GOES 14-15 observed disturbances similar to those at the location indicated by arrows