Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni.

BACKGROUND: Campylobacter jejuni is a major cause of human gastroenteritis yet there is limited knowledge of how disease is caused. Molecular genetic approaches are vital for research into the virulence mechanisms of this important pathogen. Vectors that allow expression of genes in C. jejuni via recombination onto the chromosome are particularly useful for genetic complementation of insertional knockout mutants and more generally for expression of genes in particular C. jejuni host backgrounds. METHODS: A series of three vectors that allow integration of genes onto the C. jejuni chromosome were constructed by standard cloning techniques with expression driven from three different strong promoters. Following integration onto the C. jejuni chromosome expression levels were quantified by fluorescence measurements and cells visualized by fluorescence microscopy. RESULTS: We have created plasmid, pCJC1, designed for recombination-mediated delivery of genes onto the C. jejuni chromosome. This plasmid contains a chloramphenicol resistance cassette (cat) with upstream and downstream restriction sites, flanked by regions of the C. jejuni pseudogene Cj0223. Cloning of genes immediately upstream or downstream of the cat gene allows their subsequent introduction onto the C. jejuni chromosome within the pseudogene. Gene expression can be driven from the native gene promoter if included, or alternatively from the cat promoter if the gene is cloned downstream of, and in the same transcriptional orientation as cat. To provide increased and variable expression of genes from the C. jejuni chromosome we modified pCJC1 through incorporation of three relatively strong promoters from the porA, ureI and flaA genes of C. jejuni, Helicobacter pylori and Helicobacter pullorum respectively. These promoters along with their associated ribosome binding sites were cloned upstream of the cat gene on pCJC1 to create plasmids pCJC2, pCJC3 and pCJC4. To test their effectiveness, a green fluorescent protein (gfp) reporter gene was inserted downstream of each of the three promoters and following integration of promoter-gene fusions onto the C. jejuni host chromosome, expression levels were quantified. Expression from the porA promoter produced the highest fluorescence, from flaA intermediate levels and from ureI the lowest. Expression of gfp from the porA promoter enabled visualization by fluorescent microscopy of intracellular C. jejuni cells following invasion of HeLa cells. CONCLUSIONS: The plasmids constructed allow stable chromosomal expression of genes in C. jejuni and, depending on the promoter used, different expression levels were obtained making these plasmids useful tools for genetic complementation and high level expression

A Quantitative Model of Energy Release and Heating by Time-dependent, Localized Reconnection in a Flare with a Thermal Loop-top X-ray Source

We present a quantitative model of the magnetic energy stored and then released through magnetic reconnection for a flare on 26 Feb 2004. This flare, well observed by RHESSI and TRACE, shows evidence of non-thermal electrons only for a brief, early phase. Throughout the main period of energy release there is a super-hot (T>30 MK) plasma emitting thermal bremsstrahlung atop the flare loops. Our model describes the heating and compression of such a source by localized, transient magnetic reconnection. It is a three-dimensional generalization of the Petschek model whereby Alfven-speed retraction following reconnection drives supersonic inflows parallel to the field lines, which form shocks heating, compressing, and confining a loop-top plasma plug. The confining inflows provide longer life than a freely-expanding or conductively-cooling plasma of similar size and temperature. Superposition of successive transient episodes of localized reconnection across a current sheet produces an apparently persistent, localized source of high-temperature emission. The temperature of the source decreases smoothly on a time scale consistent with observations, far longer than the cooling time of a single plug. Built from a disordered collection of small plugs, the source need not have the coherent jet-like structure predicted by steady-state reconnection models. This new model predicts temperatures and emission measure consistent with the observations of 26 Feb 2004. Furthermore, the total energy released by the flare is found to be roughly consistent with that predicted by the model. Only a small fraction of the energy released appears in the super-hot source at any one time, but roughly a quarter of the flare energy is thermalized by the reconnection shocks over the course of the flare. All energy is presumed to ultimately appear in the lower-temperature T<20 MK, post-flare loops

Exploiting Laboratory and Heliophysics Plasma Synergies

Recent advances in space-based heliospheric observations, laboratory experimentation, and plasma simulation codes are creating an exciting new cross-disciplinary opportunity for understanding fast energy release and transport mechanisms in heliophysics and laboratory plasma dynamics, which had not been previously accessible. This article provides an overview of some new observational, experimental, and computational assets, and discusses current and near-term activities towards exploitation of synergies involving those assets. This overview does not claim to be comprehensive, but instead covers mainly activities closely associated with the authors’ interests and reearch. Heliospheric observations reviewed include the Sun Earth Connection Coronal and Heliospheric Investigation (SECCHI) on the National Aeronautics and Space Administration (NASA) Solar Terrestrial Relations Observatory (STEREO) mission, the first instrument to provide remote sensing imagery observations with spatial continuity extending from the Sun to the Earth, and the Extreme-ultraviolet Imaging Spectrometer (EIS) on the Japanese Hinode spacecraft that is measuring spectroscopically physical parameters of the solar atmosphere towards obtaining plasma temperatures, densities, and mass motions. The Solar Dynamics Observatory (SDO) and the upcoming Solar Orbiter with the Heliospheric Imager (SoloHI) on-board will also be discussed. Laboratory plasma experiments surveyed include the line-tied magnetic reconnection experiments at University of Wisconsin (relevant to coronal heating magnetic flux tube observations and simulations), and a dynamo facility under construction there; the Space Plasma Simulation Chamber at the Naval Research Laboratory that currently produces plasmas scalable to ionospheric and magnetospheric conditions and in the future also will be suited to study the physics of the solar corona; the Versatile Toroidal Facility at the Massachusetts Institute of Technology that provides direct experimental observation of reconnection dynamics; and the Swarthmore Spheromak Experiment, which provides well-diagnosed data on three-dimensional (3D) null-point magnetic reconnection that is also applicable to solar active regions embedded in pre-existing coronal fields. New computer capabilities highlighted include: HYPERION, a fully compressible 3D magnetohydrodynamics (MHD) code with radiation transport and thermal conduction; ORBIT-RF, a 4D Monte-Carlo code for the study of wave interactions with fast ions embedded in background MHD plasmas; the 3D implicit multi-fluid MHD spectral element code, HiFi; and, the 3D Hall MHD code VooDoo. Research synergies for these new tools are primarily in the areas of magnetic reconnection, plasma charged particle acceleration, plasma wave propagation and turbulence in a diverging magnetic field, plasma atomic processes, and magnetic dynamo behavior.United States. Office of Naval ResearchNaval Research Laboratory (U.S.

Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system.

N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system

CD28 Costimulation Regulates Genome-Wide Effects on Alternative Splicing

CD28 is the major costimulatory receptor required for activation of naïve T cells, yet CD28 costimulation affects the expression level of surprisingly few genes over those altered by TCR stimulation alone. Alternate splicing of genes adds diversity to the proteome and contributes to tissue-specific regulation of genes. Here we demonstrate that CD28 costimulation leads to major changes in alternative splicing during activation of naïve T cells, beyond the effects of TCR alone. CD28 costimulation affected many more genes through modulation of alternate splicing than by modulation of transcription. Different families of biological processes are over-represented among genes alternatively spliced in response to CD28 costimulation compared to those genes whose transcription is altered, suggesting that alternative splicing regulates distinct biological effects. Moreover, genes dependent upon hnRNPLL, a global regulator of splicing in activated T cells, were enriched in T cells activated through TCR plus CD28 as compared to TCR alone. We show that hnRNPLL expression is dependent on CD28 signaling, providing a mechanism by which CD28 can regulate splicing in T cells and insight into how hnRNPLL can influence signal-induced alternative splicing in T cells. The effects of CD28 on alternative splicing provide a newly appreciated means by which CD28 can regulate T cell responses

Domestication of Campylobacter jejuni NCTC 11168

Reference and type strains of well-known bacteria have been a cornerstone of microbiology research for decades. The sharing of well-characterized isolates among laboratories has run in parallel with research efforts and enhanced the reproducibility of experiments, leading to a wealth of knowledge about trait variation in different species and the underlying genetics. Campylobacter jejuni strain NCTC 11168, deposited at the National Collection of Type Cultures in 1977, has been adopted widely as a reference strain by researchers worldwide and was the first Campylobacter for which the complete genome was published (in 2000). In this study, we collected 23 C . jejuni NCTC 11168 reference isolates from laboratories across the UK and compared variation in simple laboratory phenotypes with genetic variation in sequenced genomes. Putatively identical isolates, identified previously to have aberrant phenotypes, varied by up to 281 SNPs (in 15 genes) compared to the most recent reference strain. Isolates also display considerable phenotype variation in motility, morphology, growth at 37 °C, invasion of chicken and human cell lines, and susceptibility to ampicillin. This study provides evidence of ongoing evolutionary change among C. jejuni isolates as they are cultured in different laboratories and highlights the need for careful consideration of genetic variation within laboratory reference strains. This article contains data hosted by Microreact

Modification of the Campylobacter jejuni flagellin glycan by the product of the Cj1295 homopolymeric-tract-containing gene

The Campylobacter jejuni flagellin protein is O-glycosylated with structural analogues of the nine-carbon sugar pseudaminic acid. The most common modifications in the C. jejuni 81-176 strain are the 5,7-di-N-acetylated derivative (Pse5Ac7Ac) and an acetamidino-substituted version (Pse5Am7Ac). Other structures detected include O-acetylated and N-acetylglutamine-substituted derivatives (Pse5Am7Ac8OAc and Pse5Am7Ac8GlnNAc, respectively). Recently, a derivative of pseudaminic acid modified with a di-O-methylglyceroyl group was detected in C. jejuni NCTC 11168 strain. The gene products required for Pse5Ac7Ac biosynthesis have been characterized, but those genes involved in generating other structures have not. We have demonstrated that the mobility of the NCTC 11168 flagellin protein in SDS-PAGE gels can vary spontaneously and we investigated the role of single nucleotide repeats or homopolymeric- tractcontaining genes from the flagellin glycosylation locus in this process. One such gene, Cj1295, was shown to be responsible for structural changes in the flagellin glycoprot ein. Mass spectrometry demonstrated that the Cj1295 gene is required for glycosylation with the di-O-methylglyceroyl-modified version of pseudaminic acid. © 2010 SGM

Functional analysis of the Helicobacter pullorum N-linked protein glycosylation system.

N-linked protein glycosylation systems operate in species from all three domains of life. The model bacterial N-linked glycosylation system from Campylobacter jejuni is encoded by pgl genes present at a single chromosomal locus. This gene cluster includes the pglB oligosaccharyltransferase responsible for transfer of glycan from lipid carrier to protein. Although all genomes from species of the Campylobacter genus contain a pgl locus, among the related Helicobacter genus only three evolutionarily related species (H. pullorum, H. canadensis and H. winghamensis) potentially encode N-linked protein glycosylation systems. Helicobacter putative pgl genes are scattered in five chromosomal loci and include two putative oligosaccharyltransferase-encoding pglB genes per genome. We have previously demonstrated the in vitro N-linked glycosylation activity of H. pullorum resulting in transfer of a pentasaccharide to a peptide at asparagine within the sequon (D/E)XNXS/T. In this study, we identified the first H. pullorum N-linked glycoprotein, termed HgpA. Production of histidine-tagged HgpA in the background of insertional knockout mutants of H. pullorum pgl/wbp genes followed by analysis of HgpA glycan structures demonstrated the role of individual gene products in the PglB1-dependent N-linked protein glycosylation pathway. Glycopeptide purification by zwitterionic-hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry identified six glycosites from five H. pullorum proteins, which was consistent with proteins reactive with a polyclonal antiserum generated against glycosylated HgpA. This study demonstrates functioning of a H. pullorum N-linked general protein glycosylation system

Science Objectives for an X-Ray Microcalorimeter Observing the Sun

We present the science case for a broadband X-ray imager with high-resolution spectroscopy, including simulations of X-ray spectral diagnostics of both active regions and solar flares. This is part of a trilogy of white papers discussing science, instrument (Bandler et al. 2010), and missions (Bookbinder et al. 2010) to exploit major advances recently made in transition-edge sensor (TES) detector technology that enable resolution better than 2 eV in an array that can handle high count rates. Combined with a modest X-ray mirror, this instrument would combine arcsecondscale imaging with high-resolution spectra over a field of view sufficiently large for the study of active regions and flares, enabling a wide range of studies such as the detection of microheating in active regions, ion-resolved velocity flows, and the presence of non-thermal electrons in hot plasmas. It would also enable more direct comparisons between solar and stellar soft X-ray spectra, a waveband in which (unusually) we currently have much better stellar data than we do of the Sun