Landscape of mobile genetic elements and their antibiotic resistance cargo in prokaryotic genomes

Prokaryotic Mobile Genetic Elements (MGEs) such as transposons, integrons, phages and plasmids, play important roles in prokaryotic evolution and in the dispersal of cargo functions like antibiotic resistance. However, each of these MGE types is usually annotated and analysed individually, hampering a global understanding of phylogenetic and environmental patterns of MGE dispersal. We thus developed a computational framework that captures diverse MGE types, their cargos and MGE-mediated horizontal transfer events, using recombinases as ubiquitous MGE marker genes and pangenome information for MGE boundary estimation. Applied to ∼84k genomes with habitat annotation, we mapped 2.8 million MGE-specific recombinases to six operational MGE types, which together contain on average 13% of all the genes in a genome. Transposable elements (TEs) dominated across all taxa (∼1.7 million occurrences), outnumbering phages and phage-like elements (<0.4 million). We recorded numerous MGE-mediated horizontal transfer events across diverse phyla and habitats involving all MGE types, disentangled and quantified the extent of hitchhiking of TEs (17%) and integrons (63%) with other MGE categories, and established TEs as dominant carriers of antibiotic resistance genes. We integrated all these findings into a resource (proMGE.embl.de), which should facilitate future studies on the large mobile part of genomes and its horizontal dispersal.     

Prediction of the pilot-scale recovery of a recombinant yeast enzyme using integrated models

This article describes the rapid prediction of recovery process performance for a new recombinant enzyme product on the basis of a broad portfolio of computer models and highly targeted experimentation. A process model for the recombinant system was generated by linking unit operation models in an integrated fashion, with required parameter estimation and physical property determination accomplished using data from scale-down studies. This enabled the generic modeling framework established for processing of a natural enzyme from bakers' yeast to be applied. An experimental study of the same operations at the pilot scale showed that the process model gave a conservative prediction of recombinant enzyme recovery. The model successfully captured interactions leading to a low overall product yield and indicated the need for further study of precipitate breakage in the feed zone of a disc stack centrifuge in order to improve performance. The utility of scale-down units as an aid to fast model generation and the advantage of integrating computer modeling and scale-down studies to accelerate bioprocess development are highlighted.     

The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface

Shigella flexneri is an intracellular pathogen that is able to move within the cytoplasm of infected cells by the continual assembly of actin onto one pole of the bacterium. IcsA, an outer membrane protein, is localized to the old pole of the bacterium and is both necessary and sufficient for actin assembly. IcsA is slowly cleaved from the bacterial surface by the protease IcsP (SopA). Absence of IcsP leads to an alteration in the distribution of surface IcsA, such that the polar cap is maintained and some IcsA is distributed along the lateral walls of the bacillus. The mechanism of unipolar localization of IcsA and the role of IcsP in its unipolar localization are incompletely understood. Here, we demonstrate that cleavage of IcsA occurs exclusively in the outer membrane and that IcsP is localized to the outer membrane. In addition, we show that IcsA at the old pole is susceptible to cleavage by IcsP and that native IcsP is active at the pole. Taken together, these data indicate that IcsP cleaves IcsA over the entire bacterial surface. Finally, we show that, immediately after induction from a tightly regulated promoter, IcsA is expressed exclusively at the old pole in both the icsP- icsA- and the icsA- background. These data demonstrate that unipolar localization of IcsA results from its direct targeting to the pole, followed by its diffusion laterally in the outer membrane.     

Attempted chemoprophylaxis of cryptosporidiosis in chickens, using diclazuril, toltrazuril, or garlic extract

Three battery tests were conducted to study the anticryptosporidial efficacy of the 2 commercially available anticoccidial triazinone derivates, diclazuril and toltrazuril, and a garlic extract. At the recommended level, diclazuril reduced the oocyst output of birds by 14.6%. The efficacy of toltrazuril was 52.1% at the recommended level, which could be moderately increased using 5 or 10 times the recommended dose. However, these doses resulted in significant weight gain reduction. The efficacy of garlic extract was 24.4%. It is concluded that none of the drugs can be recommended for chemoprophylaxis or therapy of cryptosporidiosis in chickens.     

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.     

Single cell lineage and regionalization of cell populations during Medaka neurulation

To study the movement of individual cells and development of cell grouping during neurogenesis, we labeled single cells in early Medaka gastrula at stage 13 [13 hours post-fertilization (hpf)] with a fluorescent vital dye, and analyzed cells and their descendants using time-lapse live recording up to stage 24 (44 hpf). At stage 13, all future neural cells were located in a dorsal 140 degrees sector of the embryo, and migrated toward the vegetal pole; but during stage 15 to 16, they converged towards the midline. Cells that contributed to later neural subdivisions initially formed overlapping populations, but after stage 16+ they formed non-overlapping cell groups having characteristics of tissue 'compartments', preceding development of morphologically distinct neural subdivisions. In early retinal development, a single compartment for future retinal cells was formed superficial to telencephalic and diencephalic compartments, but it was split into left and right eye components at stage 17 in parallel with anterodorsal movement of the diencephalic compartment. At stage 16+, when these compartments were established, Pax6 expression initiated, but only in the laterally located subpopulation of the retina precursor. These observations revise the current view of bilateral retinal development. Continuous live recording of labeled single precursor cells and computer graphics-assisted data analysis, which are presented for the first time in this study, provide excellent means with which to analyze essential cellular processes in organogenesis.     

Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids

Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect nonpolarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene luciferase (LUC) into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, which were most successful in initial LUC assays, were also tested for transfer of vectors bearing the reporter gene green fluorescent protein (GFP) and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function.     

dUTPase and nucleocapsid polypeptides of the Mason-Pfizer monkey virus form a fusion protein in the virion with homotrimeric organization and low catalytic efficiency

Betaretroviruses encode dUTPase, an essential factor in DNA metabolism and repair, in the pro open reading frame located between gag and pol. Ribosomal frame-shifts during expression of retroviral proteins provide a unique possibility for covalent joining of nucleocapsid (NC) and dUTPase within Gag-Pro polyproteins. By developing an antibody against the prototype betaretrovirus Mason-Pfizer monkey virus dUTPase, we demonstrate that i) the NC-dUTPase fusion protein exists both within the virions and infected cells providing the only form of dUTPase, and ii) the retroviral protease does not cleave NC-dUTPase either in the virion or in vitro. We show that recombinant betaretroviral NC-dUTPase and dUTPase are both inefficient catalysts compared with all other dUTPases. Dynamic light scattering and gel filtration confirm that the homotrimeric organization, common among dUTPases, is retained in the NC-dUTPase fusion protein. The betaretroviral dUTPase has been crystallized and single crystals contain homotrimers. Oligonucleotide and Zn2+ binding is well retained in the fusion protein, which is the first example of acquisition of a functional nucleic acid binding module by the DNA repair factor dUTPase. Binding of the hexanucleotide ACTGCC or the octanucleotide (TG)4 to NC-dUTPase modulates enzymatic function, indicating that the low catalytic activity may be compensated by adequate localization.