Isolation and Maintenance.- Isolation and Differentiation of Medaka Embryonic Stem Cells.- Maintenance of Chicken Embryonic Stem Cells In Vitro.- Derivation and Culture of Mouse Trophoblast Stem Cells In Vitro.- Derivation, Maintenance, and Characterization of Rat Embryonic Stem Cells In Vitro.- Derivation, Maintenance, and Induction of the Differentiation In Vitro of Equine Embryonic Stem Cells.- Generation and Characterization of Monkey Embryonic Stem Cells.- Derivation and Propagation of Embryonic Stem Cells in Serum- and Feeder-Free Culture.- Signaling in Embryonic Stem Cell Differentiation.- Internal Standards in Differentiating Embryonic Stem Cells In Vitro.- Matrix Assembly, Cell Polarization, and Cell Survival.- Phosphoinositides, Inositol Phosphates, and Phospholipase C in Embryonic Stem Cells.- Cripto Signaling in Differentiating Embryonic Stem Cells.- The Use of Embryonic Stem Cells to Study Hedgehog Signaling.- Transfection and Promoter Analysis in Embryonic Stem Cells.- SAGE Analysis to Identify Embryonic Stem Cell-Predominant Transcripts.- Utilization of Digital Differential Display to Identify Novel Targets of Oct3/4.- Gene Silencing Using RNA Interference in Embryonic Stem Cells.- Genetic Manipulation of Embryonic Stem Cells.- Efficient Transfer of HSV-1 Amplicon Vectors Into Embryonic Stem Cells and Their Derivatives.- Lentiviral Vector-Mediated Gene Transfer in Embryonic Stem Cells.- Use of the Cytomegalovirus Promoter for Transient and Stable Transgene Expression in Mouse Embryonic Stem Cells.- Use of Simian Immunodeficiency Virus Vectors for Simian Embryonic Stem Cells.- Generation of Green Fluorescent Protein-Expressing Monkey Embryonic Stem Cells.- DNA Damage Response and Mutagenesis in Mouse Embryonic Stem Cells.- Ultraviolet-Induced Apoptosis in Embryonic Stem Cells In Vitro.- Use of Embryonic Stem Cells in Pharmacological and Toxicological Screens.- Use of Differentiating Embryonic Stem Cells in Pharmacological Studies.- Embryonic Stem Cells as a Source of Differentiated Neural Cells for Pharmacological Screens.- Use of Murine Embryonic Stem Cells in Embryotoxicity Assays.- Use of Chemical Mutagenesis in Mouse Embryonic Stem Cells.- Epigenetic Analysis of Embryonic Stem Cells.- Nuclear Reprogramming of Somatic Nucleus Hybridized With Embryonic Stem Cells by Electrofusion.- Methylation in Embryonic Stem Cells In Vitro.- Tumor-Like Properties.- Identification of Genes Involved in Tumor-Like Properties of Embryonic Stem Cells.- In Vivo Tumor Formation From Primate Embryonic Stem Cells.- Animal Models and Therapy.- Directed Differentiation and Characterization of Genetically Modified Embryonic Stem Cells for Therapy.- Use of Differentiating Embryonic Stem Cells in the Parkinsonian Mouse Model.

Isolation and characterization

Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

/pdf/cell-biology-and-molecular-basis-of-denitrification-red4l5iffz.pdf

Cell biology and molecular basis of denitrification.

Phosphate solubilizing bacteria and their role in plant growth promotion

Dissimilatory Fe(III) and Mn(IV) reduction.

Removal of nutrients in various types of constructed wetlands.

The evidence concerning aerobic denitrification over the past 100 years has been reviewed and the conclusion reached that the denitrification systems of some bacteria are inhibited by oxygen, other species are capable of aerobic denitrification, or co-respiration of nitrate and oxygen. Possible mechanisms and ecological implications are discussed.

/pdf/aerobic-denitrification-old-wine-in-new-bottles-2kerjkhkef.pdf

Aerobic denitrification ― old wine in new bottles?

The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.

/pdf/energy-transduction-by-electron-transfer-via-a-pyrrolo-202dk7zq7x.pdf

Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi).

A theoretical analysis has been made of carbon conversion efficiency during heterotrophic microbial growth. The expectation was that the maximal growth yield occurs when all the substrate is assimilated and the net flow of carbon through dissimilation is zero. This, however, is not identical to a 100% carbon conversion, since assimilatory pathways lead to a net production of CO2. It can be shown that the amount of CO2 produced by way of assimilatory processes is dependent upon the nature of the carbon source, but independent of its degree of reduction and varies between 12 and 29% of the substrate carbon. An analysis of published yield data reveals that nearly complete assimilation can occur during growth on substrates with a high energy content. This holds for substrates with a heat of combustion of ca. 550 kJ/mol C, or a degree of reduction higher than 5 (e.g. ethane, ethanol, and methanol). Complete assimilation can also be achieved on substrates with a lower energy content, provided that an auxiliary energy source is present that cannot be used as a carbon source. This is evident from the cell yields reported for Candida utilis grown on glucose plus formate and for Thiobacillus versutus grown on acetate plus thiosulfate. This evaluation of the carbon conversion efficiency during assimilation also made it possible to compare the energy content of the auxiliary energy substrate added with the quantity of the carbon source it had replaced. It will be shown that utilization of the auxiliary energy source may lead to extreme changes in the efficiency of dissimilatory processes.

/pdf/biochemical-limits-to-microbial-growth-yields-an-analysis-of-5aq5c2n8n3.pdf

Biochemical Limits to Microbial-Growth Yields - an Analysis of Mixed Substrate Utilization

A rotating annular reactor (Roto Torque) was used for qualitative and quantitative studied on biofilm heterogeneity. In contrast to the classic image of biofilms as smooth, homogeneous layers of biomass on a substratum, studies using various pure and mixed cultures consistently revealed more-dimensional structures that resembled dunes and ridges, among others. These heterogeneities were categorized and their underlying causes analyzed. Contrary to expectations, motility of the microorganisms not a decisive factor in determining biofilm homogeneity. Small Variations in substratum geometry homogeneity. Small variations in substratum geometry and flow patterns were clearly reflected in the biofilm pattern. Nonhomogeneous flow and shear patterns in the reactor, together with inadequate mixing resulted in significant, position-dependent differences in surface growth. It was therefore not possible to take representative samples of the attached biomass. Like many other types of reactors, the Roto Torque reactor is valuable for qualitative and morphological biofilm experiments but less suitable for quantitative physiological and kinetics studies using attached microorganisms. (c) 1994 John Wiley & Sons, Inc.

/pdf/heterogeneity-of-biofilms-in-rotating-annular-reactors-10j9wi7efv.pdf

Heterogeneity of biofilms in rotating annular reactors: Occurrence, structure, and consequences

The yield of Hyphomicrobium EG on dimethyl sulphoxide, dimethyl sulphide and methylamine, considering the metabolic pathways of these compounds, suggested that the organism gained energy from the oxidation of the sulphur moiety of the former compounds. Indeed, a comparison of chemostat cultures of Hyphomicrobium EG grown on methylamine in the presence and absence of sulphide or thiosulphate proved this obligate methylotroph to be a chemolithoheterotroph. The apparent Ysulphide and Ythiosulphate were comparable, being 8–10 g dry weight/mol. In batch cultures thiosulphate concentrations up to 10 mM had a stimulatory effect on the growth rate of Hyphomicrobium EG, whereas higher concentrations increased the organisms doubling time. Enzyme- and respiration data showed that the organism had constitutive enzymes for the breakdown of dimethyl sulphoxide although they were clearly regulated to need. Addition of sulphide or thiosulphate to methylamine-limited chemostat cultures of Hyphomicrobium EG not only resulted in the induction of enzymes necessary for their breakdown, but also caused the enzymes for dimethyl sulphoxide metabolism, especially methyl mercaptan oxidase, to be induced. The formation of H2O2, a product of the latter enzyme, was reflected in the relatively high catalase activities during growth on dimethyl sulphoxide and in the organisms inability to grow on this compound in the presence of a catalase inhibitor.

/pdf/chemolithotrophic-potential-of-a-hyphomicrobium-species-wbi0mzw9d1.pdf

J.G. Kuenen

Papers

Aerobic denitrification ― old wine in new bottles?

Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi).

Biochemical Limits to Microbial-Growth Yields - an Analysis of Mixed Substrate Utilization

Heterogeneity of biofilms in rotating annular reactors: Occurrence, structure, and consequences

Chemolithotrophic potential of a Hyphomicrobium species, capable of growth on methylated sulphur compounds