Genome Editing Method for the Anaerobic Magnetotactic Bacterium Desulfovibrio magneticus RS-1.

Magnetosomes are complex bacterial organelles that serve as model systems for studying bacterial cell biology, biomineralization, and global iron cycling. Magnetosome biogenesis is primarily studied in two closely related Alphaproteobacteria of the genus Magnetospirillum that form cubooctahedral-shaped magnetite crystals within a lipid membrane. However, chemically and structurally distinct magnetic particles have been found in physiologically and phylogenetically diverse bacteria. Due to a lack of molecular genetic tools, the mechanistic diversity of magnetosome formation remains poorly understood. Desulfovibrio magneticus RS-1 is an anaerobic sulfate-reducing deltaproteobacterium that forms bullet-shaped magnetite crystals. A recent forward genetic screen identified 10 genes in the conserved magnetosome gene island of D. magneticus that are essential for its magnetic phenotype. However, this screen likely missed mutants with defects in crystal size, shape, and arrangement. Reverse genetics to target the remaining putative magnetosome genes using standard genetic methods of suicide vector integration have not been feasible due to the low transconjugation efficiency. Here, we present a reverse genetic method for targeted mutagenesis in D. magneticus using a replicative plasmid. To test this method, we generated a mutant resistant to 5-fluorouracil by making a markerless deletion of the upp gene that encodes uracil phosphoribosyltransferase. We also used this method for targeted marker exchange mutagenesis by replacing kupM, a gene identified in our previous screen as a magnetosome formation factor, with a streptomycin resistance cassette. Overall, our results show that targeted mutagenesis using a replicative plasmid is effective in D. magneticus and may also be applied to other genetically recalcitrant bacteria.IMPORTANCE Magnetotactic bacteria (MTB) are a group of organisms that form intracellular nanometer-scale magnetic crystals though a complex process involving lipid and protein scaffolds. These magnetic crystals and their lipid membranes, termed magnetosomes, are model systems for studying bacterial cell biology and biomineralization and are potential platforms for biotechnological applications. Due to a lack of genetic tools and unculturable representatives, the mechanisms of magnetosome formation in phylogenetically deeply branching MTB remain unknown. These MTB contain elongated bullet-/tooth-shaped magnetite and greigite crystals that likely form in a manner distinct from that of the cubooctahedral-shaped magnetite crystals of the genetically tractable MTB within the Alphaproteobacteria Here, we present a method for genome editing in Desulfovibrio magneticus RS-1, a cultured representative of the deeply branching MTB of the class Deltaproteobacteria This marks a crucial step in developing D. magneticus as a model for studying diverse mechanisms of magnetic particle formation by MTB

Paramagnon dispersion in β\beta-FeSe observed by Fe LL-edge resonant inelastic x-ray scattering

We report an Fe $L$-edge resonant inelastic x-ray scattering (RIXS) study of the unusual superconductor $\beta$-FeSe. The high energy resolution of this RIXS experiment ($\approx\,$55$\,$meV FWHM) made it possible to resolve low-energy excitations of the Fe $3d$ manifold. These include a broad peak which shows dispersive trends between 100-200$\,$meV along the $(\pi,0)$ and $(\pi,\pi)$ directions of the one-Fe square reciprocal lattice, and which can be attributed to paramagnon excitations. The multi-band valence state of FeSe is among the most metallic in which such excitations have been discerned by soft x-ray RIXS

Spin resonance in the superconducting state of Li1−x_{1-x}Fex_{x}ODFe1−y_{1-y}Se observed by neutron spectroscopy

We have performed inelastic neutron scattering measurements on a powder sample of the superconductor lithium iron selenide hydroxide Li$_{1-x}$Fe$_{x}$ODFe$_{1-y}$Se ($x \simeq 0.16, y \simeq 0.02$, $T_{\rm c} = 41$\,K). The spectrum shows an enhanced intensity below $T_{\rm c}$ over an energy range $0.64\times2\Delta < E < 2\Delta$, where $\Delta$ is the superconducting gap, with maxima at the wave vectors $Q_1 \simeq 1.46$\,\AA$^{-1}$ and $Q_2 \simeq 1.97$\,\AA$^{-1}$. The behavior of this feature is consistent with the spin resonance mode found in other unconventional superconductors, and strongly resembles the spin resonance observed in the spectrum of the molecular-intercalated iron selenide, Li$_{0.6}$(ND$_{2}$)$_{0.2}$(ND$_{3}$)$_{0.8}$Fe$_{2}$Se$_{2}$. The signal can be described with a characteristic two-dimensional wave vector $(\pi, 0.67\pi)$ in the Brillouin zone of the iron square lattice, consistent with the nesting vector between electron Fermi sheets

Evaluation der potenziellen Regeneratschädigung bei der Kallusumformung nach Distraktionsosteogenese der Mandibula: Eine experimentelle Untersuchung am Tiermodell

Zusammenfassung: Ziel: Bei der Korrektur dreidimensionaler Deformitäten des Gesichtsskelettes mit der Distraktionsosteogenese werden Umformungsvorgänge des Regenerates einerseits als Bestandteil des Behandlungsplans, andererseits im Falle eines Verlustes der Kontrolle über den Distraktionsvektor vorgenommen. Die vorliegende Untersuchung hatte das Ziel, die Grenzen der Kallusmanipulation zu beurteilen. Dazu wurden die Auswirkungen komprimierender sowie dehnender Einflüsse am gleichen Regenerat untersucht. Material und Methode: Bei 15Beagle-Hunden wurde mit speziell angefertigten bidirektionalen Distraktoren eine lineare Distraktion von 10mm beidseits im Unterkieferwinkel vorgenommen. Der neu gebildete Kallus wurde in einem Schritt um 20° anguliert, was im vorliegenden Modell einer Verkürzung/Verlängerung von ca. 35% der Ausgangslänge des Regenerates gleichkommt. Die Position des Rotationszentrums erlaubte es, das Regenerat gleichzeitig zu komprimieren und zu dehnen. Die Auswirkungen dieser mechanischen Einflüsse auf die Ossifikation des Regenerates wurden nach 6 bzw. 13Wochen beurteilt und mit einer Kontrollgruppe, bei der lediglich eine lineare Distraktion durchgeführt worden war, verglichen. Ergebnisse: Die radiologischen und histologischen Untersuchungen ergaben keinen statisch signifikanten Unterschied zwischen dem komprimierten und gedehnten Regenerat. Es zeigten sich jedoch im gedehnten Sektor des Kallus Zonen unvollständiger Ossifikation nach 6-wöchiger Konsolidierungszeit. Unter stabilen Verhältnissen wurde die verzögerte Knochenheilung im weiteren Verlauf kompensiert und eine vollständige Ossifikation nach 13Wochen erreicht. Schlussfolgerung: Unter stabilen Verhältnissen kann ein durch Distraktion gebildetes frisches Regenerat in einem beträchtlichen Ausmaß umgeformt werden, ohne die knöcherne Heilung bleibend zu kompromittieren. Die Dehnung des Kallus kann jedoch zu einer Verzögerung oder dem Ausbleiben des Ossifikationsprozesses führen und sollte vermieden werden. Dies lässt sich durch eine Überkorrektur der Regeneratlänge oder durch eine graduelle Angulation während des Distraktionsvorgangs erreiche

Comparative genomic assessment of Multi-Locus Sequence Typing: rapid accumulation of genomic heterogeneity among clonal isolates of Campylobacter jejuni

<p>Abstract</p> <p>Background</p> <p>Multi-Locus Sequence Typing (MLST) has emerged as a leading molecular typing method owing to its high ability to discriminate among bacterial isolates, the relative ease with which data acquisition and analysis can be standardized, and the high portability of the resulting sequence data. While MLST has been successfully applied to the study of the population structure for a number of different bacterial species, it has also provided compelling evidence for high rates of recombination in some species. We have analyzed a set of <it>Campylobacter jejuni </it>strains using MLST and Comparative Genomic Hybridization (CGH) on a full-genome microarray in order to determine whether recombination and high levels of genomic mosaicism adversely affect the inference of strain relationships based on the analysis of a restricted number of genetic loci.</p> <p>Results</p> <p>Our results indicate that, in general, there is significant concordance between strain relationships established by MLST and those based on shared gene content as established by CGH. While MLST has significant predictive power with respect to overall genome similarity of isolates, we also found evidence for significant differences in genomic content among strains that would otherwise appear to be highly related based on their MLST profiles.</p> <p>Conclusion</p> <p>The extensive genomic mosaicism between closely related strains has important implications in the context of establishing strain to strain relationships because it suggests that the exact gene content of strains, and by extension their phenotype, is less likely to be "predicted" based on a small number of typing loci. This in turn suggests that a greater emphasis should be placed on analyzing genes of clinical interest as we forge ahead with the next generation of molecular typing methods.</p

Gravitational Radiation from Gamma-Ray Burst Progenitors

We study gravitational radiation from various proposed gamma-ray burst (GRB) progenitor models, in particular compact mergers and massive stellar collapses. These models have in common a high angular rotation rate, and the final stage involves a rotating black hole and accretion disk system. We consider the in-spiral, merger and ringing phases, and for massive collapses we consider the possible effects of asymmetric collapse and break-up, as well bar-mode instabilities in the disks. We calculate the strain and frequency of the gravitational waves expected from various progenitors, at distances based on occurrence rate estimates. Based on simplifying assumptions, we give estimates of the probability of detection of gravitational waves by the advanced LIGO system from the different GRB scenarios.Comment: 26 pages, 5 figures, accepted for publication in Ap