Risk mitigation of genetically modified bacteria and plants designed for bioremediation

While the possible advantages of bioremediation and phytoremediation, by both recombinant microbes and plants, have been extensively reviewed, the biosafety concerns have been less extensively treated. This article reviews the possible risks associated with the use of recombinant bacteria and plants for bioremediation, with particular emphasis on ways in which molecular genetics could contribute to risk mitigation. For example, genetic techniques exist that permit the site-specific excision of unnecessary DNA, so that only the transgenes of interest remain. Other mechanisms exist whereby the recombinant plants or bacteria contain conditional suicide genes that may be activated under certain conditions. These methods act to prevent the spread and survival of the transgenic bacteria or plants in the environment, and to prevent horizontal gene flow to wild or cultivated relatives. Ways in which these genetic technologies may be applied to risk mitigation in bioremediation and phytoremediation are discussed.     

Regulatory variation at glypican-3 underlies a major growth QTL in mice

The genetic basis of variation in complex traits remains poorly understood, and few genes underlying variation have been identified. Previous work identified a quantitative trait locus (QTL) responsible for much of the response to selection on growth in mice, effecting a change in body mass of approximately 20%. By fine-mapping, we have resolved the location of this QTL to a 660-kb region containing only two genes of known function, Gpc3 and Gpc4, and two other putative genes of unknown function. There are no non-synonymous polymorphisms in any of these genes, indicating that the QTL affects gene regulation. Mice carrying the high-growth QTL allele have approximately 15% lower Gpc3 mRNA expression in kidney and liver, whereas expression differences at Gpc4 are non-significant. Expression profiles of the two other genes within the region are inconsistent with a factor responsible for a general effect on growth. Polymorphisms in the 3′ untranslated region of Gpc3 are strong candidates for the causal sequence variation. Gpc3 loss-of-function mutations in humans and mice cause overgrowth and developmental abnormalities. However, no deleterious side-effects were detected in our mice, indicating that genes involved in Mendelian diseases also contribute to complex trait variation. Furthermore, these findings show that small changes in gene expression can have substantial phenotypic effects.     

Identification of the Bacillus anthracis (gamma) phage receptor

Bacillus anthracis, a gram-positive, spore-forming bacterium, is the etiological agent of anthrax. It belongs to the Bacillus cereus group, which also contains Bacillus cereus and Bacillus thuringiensis. Most B. anthracis strains are sensitive to phage gamma, but most B. cereus and B. thuringiensis strains are resistant to the lytic action of phage gamma. Here, we report the identification of a protein involved in the bacterial receptor for the gamma phage, which we term GamR (Gamma phage receptor). It is an LPXTG protein (BA3367, BAS3121) and is anchored by the sortase A. A B. anthracis sortase A mutant is not as sensitive as the parental strain nor as the sortase B and sortase C mutants, whereas the GamR mutant is resistant to the lytic action of the phage. Electron microscopy reveals the binding of the phage to the surface of the parental strain and its absence from the GamR mutant. Spontaneous B. anthracis mutants resistant to the phage harbor mutations in the gene encoding the GamR protein. A B. cereus strain that is sensitive to the phage possesses a protein similar (89% identity) to GamR. B. thuringiensis 97-27, a strain which, by sequence analysis, is predicted to harbor a GamR-like protein, is resistant to the phage but nevertheless displays phage binding.     

Epithelial distribution of neural receptors in the guinea pig small intestine

Neural and paracrine agents, such as dopamine, epinephrine, and histamine, affect intestinal epithelial function, but it is unclear if these agents act on receptors directly at the enterocyte level. The cellular localization and villus-crypt distribution of adrenergic, dopamine, and histamine receptors within the intestinal epithelium is obscure and needs to be identified. Single cell populations of villus or crypt epithelial cells were isolated from the jejunum of adult guinea pigs. Enterocytes were separated from intraepithelial lymphocytes by flow cytometry and specific binding was determined using fluorescent probes. Alpha1-adrenergic receptors were located on villus and crypt intraepithelial lymphocytes and enterocytes. Beta-adrenergic receptors were found on villus and crypt enterocytes. Dopamine receptors were found on all cell types examined, whereas histamine receptors were not detected (<10% for each cell population). These studies demonstrated that (1) receptors for epinephrine and dopamine exist on epithelial cells of the guinea pig jejunum, (2) beta-adrenergic receptors are found primarily on villus and crypt enterocytes and (3) intraepithelial lymphocytes contain alpha1-adrenergic, but have few beta-adrenergic, receptors. The presence of neural receptors suggests that these agents are acting, at least in part, at the enterocyte or intraepithelial lymphocyte levels to modulate intestinal and immune function.     

Determination and comparison of the baseline proteomes of the versatile microbe Rhodopseudomonas palustris under its major metabolic states

Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.     

Novel 5beta-hydroxyspirostan-6-ones ecdysteroid antagonists: synthesis and biological testing

Eight new 5beta-hydroxy-spirostan-6-ones bearing hydroxy and amino functions in the A ring, i.e., 3beta-OH, 3alpha-OH, 2beta,3beta-OH, 2alpha,3beta-OH, 3beta-NH2, 2alpha-NH2-3beta-OH, 2beta-NH2-3beta-OH, and 2beta-OH-3beta-NH2, were efficiently synthesized, and their antiecdysteroid activities were evaluated on the metamorphosis bioassay of mosquito Aedes aegypti. To our knowledge, these new steroids represent the first 5beta-hydroxy-spirostanes which have been tested for antiecdysteroid activity in mosquitoes. The higher antagonistic effect was found for compounds bearing the 3beta-hydroxy and 2beta,3beta-dihydroxy functionality, which show promise as environmental friendly insecticides.     

Immune reconstitution following hematopoietic stem-cell transplantation

BACKGROUND: Reconstitution of the immune system following allogeneic stem-cell transplantation is a complex process that requires successful engraftment of the hematopoietic stem cell, as well as adequate thymic function. As the majority of patients have reduced thymic function due to age, hormonal changes, as well as the damage caused by conditioning and GvHD, immune recovery is often delayed and incomplete. Following graft infusion there is rapid proliferation of natural killer (NK) cells that appear to proceed directly from the hematopoietic stem cell. B-cell function is dependent on specific maturation development in the BM micro-environment, as well as CD4 help. The CD8 population expands rapidly due to proliferation of many memory cells that react against Class I Ags, as well as viral molecules. Expansion of T-helper cells originates mainly from the memory pool that is present in the bone marrow graft. Naive cells require competent thymus hence the CD4 cell counts may be subnormal with clinical immunodeficiency. Controversy remains as to the capacity of the thymus to recover and thus extra thymic proliferation of T cells have been postulated. However these cells appear to have a limited capacity to expand and a fixed repertoire. DISCUSSION: Donor lymphocyte infusions may contribute a competent CD4 population that can cause GvHD, but have limitations in the capacity to respond to new antigens. Future research needs to be concentrated on improving the capacity of the thymus to reconstitute a functional naive population.     

Structure of the cyanobacterial Magnesium Chelatase H subunit determined by single particle reconstruction and small-angle X-ray scattering

The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg²⁺ into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.