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

Constraining the structure of the non-spherical preprotostellar core L1544

A series of self-consistent, three-dimensional continuum radiative transfer models are constructed of the pre-protostellar core L1544, with the results compared with existing SCUBA and ISO data. The source is well-fit by a prolate spheroid, having an ellipsoidal power-law density distribution of index m ~ 2 (1.75 < m < 2.25) in to at least r ~ 1600AU. For r<1600 AU, the data are consistent with either an extension of the power law to smaller radii, or a flattened (Bonner-Ebert like) density distribtion. We can further constrain the optical depth along the short axis at 1300um to be ~ 5e-3, the central luminosity to be L < 1e-3 solar luminosities, the long axis diameter D ~ 0.1 pc, the axis ratio to be q ~ 2, and the external ISRF to be similar to that defined by Mathis, Mezger, & Panagia (1983) to within 50 per cent. The outer diameter and axis ratio may each be somewhat larger due to potential on-source chopping in the observations, and the projection of the long axis onto the plane of the sky. While these results are similar to those inferred directly from observations or spherical modeling due to the source transparency at submillimeter wavelengths, we infer a smaller size, lower mass, and higher optical depth / column density, exposed to a stronger external radiation field than previously assumed. Finally, we find that both the spectral energy distribution (SED) and surface brightness distribution are necessary to constrain the source properties in this way.Comment: 9 pages; 8 figures; accepted for publication in MNRA

Radio continuum observations of Class I protostellar disks in Taurus: constraining the greybody tail at centimetre wavelengths

We present deep 1.8 cm (16 GHz) radio continuum imaging of seven young stellar objects in the Taurus molecular cloud. These objects have previously been extensively studied in the sub-mm to NIR range and their SEDs modelled to provide reliable physical and geometrical parametres.We use this new data to constrain the properties of the long-wavelength tail of the greybody spectrum, which is expected to be dominated by emission from large dust grains in the protostellar disk. We find spectra consistent with the opacity indices expected for such a population, with an average opacity index of beta = 0.26+/-0.22 indicating grain growth within the disks. We use spectra fitted jointly to radio and sub-mm data to separate the contributions from thermal dust and radio emission at 1.8 cm and derive disk masses directly from the cm-wave dust contribution. We find that disk masses derived from these flux densities under assumptions consistent with the literature are systematically higher than those calculated from sub-mm data, and meet the criteria for giant planet formation in a number of cases.Comment: submitted MNRA

AMI-LA radio continuum observations of Spitzer c2d small clouds and cores: Perseus region

We present deep radio continuum observations of the cores identified as deeply embedded young stellar objects in the Perseus molecular cloud by the Spitzer c2d programme at a wavelength of 1.8 cm with the Arcminute Microkelvin Imager Large Array (AMI-LA). We detect 72% of Class 0 objects from this sample and 31% of Class I objects. No starless cores are detected. We use the flux densities measured from these data to improve constraints on the correlations between radio luminosity and bolometric luminosity, infrared luminosity and, where measured, outflow force. We discuss the differing behaviour of these objects as a function of protostellar class and investigate the differences in radio emission as a function of core mass. Two of four possible very low luminosity objects (VeLLOs) are detected at 1.8 cm.Comment: 18 pages, 9 figures, accepted MNRA

The clustering of galaxies in the SDSS-III Baryon Oscillation Spectroscopic Survey: single-probe measurements from CMASS anisotropic galaxy clustering

With the largest spectroscopic galaxy survey volume drawn from the SDSS-III Baryon Oscillation Spectroscopic Survey (BOSS), we can extract cosmological constraints from the measurements of redshift and geometric distortions at quasi-linear scales (e.g. above 50 $h^{-1}$Mpc). We analyze the broad-range shape of the monopole and quadrupole correlation functions of the BOSS Data Release 12 (DR12) CMASS galaxy sample, at the effective redshift $z=0.59$, to obtain constraints on the Hubble expansion rate $H(z)$, the angular-diameter distance $D_A(z)$, the normalized growth rate $f(z)\sigma_8(z)$, and the physical matter density $\Omega_mh^2$. We obtain robust measurements by including a polynomial as the model for the systematic errors, and find it works very well against the systematic effects, e.g., ones induced by stars and seeing. We provide accurate measurements $\{D_A(0.59)r_{s,fid}/r_s$ $\rm Mpc$, $H(0.59)r_s/r_{s,fid}$ $km s^{-1} Mpc^{-1}$, $f(0.59)\sigma_8(0.59)$, $\Omega_m h^2\}$ = $\{1427\pm26$, $97.3\pm3.3$, $0.488 \pm 0.060$, $0.135\pm0.016\}$, where $r_s$ is the comoving sound horizon at the drag epoch and $r_{s,fid}=147.66$ Mpc is the sound scale of the fiducial cosmology used in this study. The parameters which are not well constrained by our galaxy clustering analysis are marginalized over with wide flat priors. Since no priors from other data sets, e.g., cosmic microwave background (CMB), are adopted and no dark energy models are assumed, our results from BOSS CMASS galaxy clustering alone may be combined with other data sets, i.e., CMB, SNe, lensing or other galaxy clustering data to constrain the parameters of a given cosmological model. The uncertainty on the dark energy equation of state parameter, $w$, from CMB+CMASS is about 8 per cent. The uncertainty on the curvature fraction, $\Omega_k$, is 0.3 per cent. We do not find deviation from flat $\Lambda$CDM.Comment: 15 pages, 11 figures. The latest version matches and the accepted version by MNRAS. A bug in the first version has been identified and fixed in the new version. We have redone the analysis with newest data (BOSS DR12

Synergistic Effects of Climate and Land-Cover Change on Long-Term Bird Population Trends of the Western USA: A Test of Modeled Predictions

Climate and land-use change are predicted to lead to widespread changes in population dynamics, but quantitative predictions on the relative effects of these stressors have not yet been examined empirically. We analyzed historical abundance data of 110 terrestrial bird species sampled from 1983 to 2010 along 406 Breeding Bird Survey (BBS) across the northwestern USA. Using boosted-regression trees, we modeled bird abundance at the beginning of this interval as a function of (1) climate variables, (2) Landsat-derived landcover data, (3) the additive and interactive effects of climate and land-cover variables. We evaluated the capacity of each model set to predict observed 27-year bird population trends. On average, 45 species significantly declined over the period observed and only 8 increased (mean trend = -0.84%/year). Climate change alone significantly predicted observed abundance trends for 44/108 species (mean 0.37 ± 0.09 [SD]), land-cover changes alone predicted trends for 47/108 species (mean r = 0.36 ±0.09), and the synergistic effects predicted 59/108 species (mean r = 0.37 ±0.11). However, for 37 of these species, including information on land-cover change increased prediction success over climate data alone. Across stressors, species with trends that were predicted accurately were more likely to be in decline across the western USA. For instance, species with high correlations between predicted and observed abundances (> r = 0.6) were declining at rates that were on average >2%/ year. We then provide the first empirical evidence that abundance models based on land cover and climate have the capacity to predict the species most likely to be at risk from climate and land-use change. However, for many species there were substantial discrepancies between modeled and observed trends. Nevertheless, our results highlight that climate change is already influencing bird populations of the western U.S. and that such effects often operate synergistically with land-cover change to affect population decline

BLAST: The Mass Function, Lifetimes, and Properties of Intermediate Mass Cores from a 50 Square Degree Submillimeter Galactic Survey in Vela (l = ~265)

We present first results from an unbiased 50 deg^2 submillimeter Galactic survey at 250, 350, and 500 micron from the 2006 flight of the Balloon-borne Large Aperture Submillimeter Telescope (BLAST). The map has resolution ranging from 36 arcsec to 60 arcsec in the three submillimeter bands spanning the thermal emission peak of cold starless cores. We determine the temperature, luminosity, and mass of more than one thousand compact sources in a range of evolutionary stages and an unbiased statistical characterization of the population. From comparison with C^(18)O data, we find the dust opacity per gas mass, kappa r = 0.16 cm^2 g^(-1) at 250 micron, for cold clumps. We find that 2% of the mass of the molecular gas over this diverse region is in cores colder than 14 K, and that the mass function for these cold cores is consistent with a power law with index alpha = -3.22 +/- 0.14 over the mass range 14 M_sun < M < 80 M_sun. Additionally, we infer a mass-dependent cold core lifetime of t_c(M) = 4E6 (M/20 M_sun)^(-0.9) years - longer than what has been found in previous surveys of either low or high mass cores, and significantly longer than free fall or likely turbulent decay times. This implies some form of non-thermal support for cold cores during this early stage of star formation.Comment: Accepted for publication in the Astrophysical Journal. Maps available at http://blastexperiment.info

Precipitation and winter temperature predict long-term range-scale abundance changes in Western North American birds

Predicting biodiversity responses to climate change remains a difficult challenge, especially in climatically complex regions where precipitation is a limiting factor. Though statistical climatic envelope models are frequently used to project future scenarios for species distributions under climate change, these models are rarely tested using empirical data. We used long-term data on bird distributions and abundance covering five states in the western US and in the Canadian province of British Columbia to test the capacity of statistical models to predict temporal changes in bird populations over a 32-year period. Using boosted regression trees, we built presence-absence and abundance models that related the presence and abundance of 132 bird species to spatial variation in climatic conditions. Presence/absence models built using 1970–1974 data forecast the distributions of the majority of species in the later time period, 1998–2002 (mean AUC = 0.79 ± 0.01). Hindcast models performed equivalently (mean AUC = 0.82 ± 0.01). Correlations between observed and predicted abundances were also statistically significant for most species (forecast mean Spearman′s ρ = 0.34 ± 0.02, hindcast = 0.39 ± 0.02). The most stringent test is to test predicted changes in geographic patterns through time. Observed changes in abundance patterns were significantly positively correlated with those predicted for 59% of species (mean Spearman′s ρ = 0.28 ± 0.02, across all species). Three precipitation variables (for the wettest month, breeding season, and driest month) and minimum temperature of the coldest month were the most important predictors of bird distributions and abundances in this region, and hence of abundance changes through time. Our results suggest that models describing associations between climatic variables and abundance patterns can predict changes through time for some species, and that changes in precipitation and winter temperature appear to have already driven shifts in the geographic patterns of abundance of bird populations in western North America

Temporal, Spatial, and Genomic Analyses of Enterobacteriaceae Clinical Antimicrobial Resistance in Companion Animals Reveals Phenotypes and Genotypes of One Health Concern

BackgroundAntimicrobial resistance (AMR) is a globally important one health threat. The impact of resistant infections on companion animals, and the potential public health implications of such infections, has not been widely explored, largely due to an absence of structured population-level data.ObjectivesWe aimed to efficiently capture and repurpose antimicrobial susceptibility test (AST) results data from several veterinary diagnostic laboratories (VDLs) across the United Kingdom to facilitate national companion animal clinical AMR surveillance. We also sought to harness and genotypically characterize isolates of potential AMR importance from these laboratories.MethodsWe summarized AST results for 29,330 canine and 8,279 feline Enterobacteriaceae isolates originating from companion animal clinical practice, performed between April 2016 and July 2018 from four VDLs, with submissions from 2,237 United Kingdom veterinary practice sites.ResultsEscherichia coli (E. coli) was the most commonly isolated Enterobacteriaceae in dogs (69.4% of AST results, 95% confidence interval, CI, 68.7–70.0) and cats (90.5%, CI 89.8–91.3). Multi-drug resistance was reported in 14.1% (CI 13.5–14.8) of canine and 12.0% (CI 11.1–12.9) of feline E. coli isolates. Referral practices were associated with increased E. coli 3rd generation ≤ cephalosporin resistance odds (dogs: odds ratio 2.0, CI 1.2–3.4). We selected 95 E. coli isolates for whole genome analyses, of which seven belonged to sequence type 131, also carrying the plasmid-associated extended spectrum β-lactamase gene blaCTX–M–15. The plasmid-mediated colistin resistance gene mcr-9 was also identified for the first time in companion animals.ConclusionsLinking clinical AMR data with genotypic characterization represents an efficient means of identifying important resistance trends in companion animals on a national scale.</sec