Structural and functional properties of a single domain hemoglobin from the food-borne pathogen Campylobactor jejuni

Campylobacter jejuni contains two globins, a truncated hemoglobin, Ctb, and a single domain hemoglobin, Cgb. The physiological function of Ctb remains unclear, whereas Cgb has been linked to NO detoxification. With resonance Raman scattering, the iron-histidine stretching mode of Cgb was identified at 251 cm(-1). This frequency is unusually high, suggesting an imidazolate character of the proximal histidine as a result of the H-bonding network linking the catalytic triad involving the F8His, H23Glu, and G5Tyr residues. In the CO-complex, two conformers were identified with the nuC-O/nuFe-CO at 529/1914 cm(-1) and 492/1963 cm(-1). The former is assigned to a "closed" conformation, in which the heme-bound CO is stabilized by the H-bond(s) donated from the B10Tyr-E7Gln residues, whereas the latter is assigned to an "open" conformer, in which the H-bonding interaction is absent. The presence of the two alternative conformations demonstrates the plasticity of the protein matrix. In the O2-complex, the iron-O2 stretching frequency was identified at 554 cm(-1), which is unusually low, indicating that the heme-bound O2 is stabilized by strong H-bond(s) donated by the B10Tyr-E7Gln residues. This scenario is consistent with its low O2 off-rate (0.87 s(-1)). Taken together the data suggest that the NO-detoxifying activity of Cgb is facilitated by the imidazolate character of the proximal F8His and the distal positive polar environment provided by the B10Tyr-E7Gln. They may offer electronic "push" and "pull," respectively, for the O-O bond cleavage reaction required for the isomerization of the presumed peroxynitrite intermediate to the product, nitrate.     

Effects of Abiotic Factors on the Phylogenetic Diversity of Bacterial Communities in Acidic Thermal Springs

Acidic thermal springs offer ideal environments for studying processes underlying extremophile microbial diversity. We used a carefully designed comparative analysis of acidic thermal springs in Yellowstone National Park to determine how abiotic factors (chemistry and temperature) shape acidophile microbial communities. Small-subunit rRNA gene sequences were PCR amplified, cloned, and sequenced, by using evolutionarily conserved bacterium-specific primers, directly from environmental DNA extracted from Amphitheater Springs and Roaring Mountain sediment samples. Energy-dispersive X-ray spectroscopy, X-ray diffraction, and colorimetric assays were used to analyze sediment chemistry, while an optical emission spectrometer was used to evaluate water chemistry and electronic probes were used to measure the pH, temperature, and E_h of the spring waters. Phylogenetic-statistical analyses found exceptionally strong correlations between bacterial community composition and sediment mineral chemistry, followed by weaker but significant correlations with temperature gradients. For example, sulfur-rich sediment samples contained a high diversity of uncultured organisms related to Hydrogenobaculum spp., while iron-rich sediments were dominated by uncultured organisms related to a diverse array of gram-positive iron oxidizers. A detailed analysis of redox chemistry indicated that the available energy sources and electron acceptors were sufficient to support the metabolic potential of Hydrogenobaculum spp. and iron oxidizers, respectively. Principal-component analysis found that two factors explained 95% of the genetic diversity, with most of the variance attributable to mineral chemistry and a smaller fraction attributable to temperature.     

Fossil and Genetic Evidence for the Polyphyletic Nature of the Planktonic Foraminifera "Globigerinoides", and Description of the New Genus Trilobatus

Planktonic foraminifera are one of the most abundant and diverse protists in the oceans. Their utility as paleo proxies requires rigorous taxonomy and comparison with living and genetically related counterparts. We merge genetic and fossil evidence of “Globigerinoides”, characterized by supplementary apertures on spiral side, in a new approach to trace their “total evidence phylogeny” since their first appearance in the latest Paleogene. Combined fossil and molecular genetic data indicate that this genus, as traditionally understood, is polyphyletic. Both datasets indicate the existence of two distinct lineages that evolved independently. One group includes “Globigerinoides” trilobus and its descendants, the extant “Globigerinoides” sacculifer, Orbulina universa and Sphaeroidinella dehiscens. The second group includes the Globigerinoides ruber clade with the extant G. conglobatus and G. elongatus and ancestors. In molecular phylogenies, the trilobus group is not the sister taxon of the ruber group. The ruber group clusters consistently together with the modern Globoturborotalita rubescens as a sister taxon. The re-analysis of the fossil record indicates that the first “Globigerinoides” in the late Oligocene are ancestral to the trilobus group, whereas the ruber group first appeared at the base of the Miocene with representatives distinct from the trilobus group. Therefore, polyphyly of the genus "Globigerinoides" as currently defined can only be avoided either by broadening the genus concept to include G. rubescens and a large number of fossil species without supplementary apertures, or if the trilobus group is assigned to a separate genus. Since the former is not feasible due to the lack of a clear diagnosis for such a broad genus, we erect a new genus Trilobatus for the trilobus group (type species Globigerina triloba Reuss) and amend Globoturborotalita and Globigerinoides to clarify morphology and wall textures of these genera. In the new concept, Trilobatus n. gen. is paraphyletic and gave rise to the Praeorbulina / Orbulina and Sphaeroidinellopsis / Sphaeroidinella lineages.     

Using the bacterium, Paracoccus denitrificans and other 'runaway mitochondria' as classroom models for respiratory electron transport studies.

Our suggestions for experiments demonstrating electron-transport-chain composition and reactions all exploit bacteria which can be prepared quickly, easily and cheaply from cells grown in Erlenmeyer flasks. While they have been designed from a cytochrome oxidase point of view using organisms of our own prejudice, strains containing mutations in other sites could be just as educational. Most bacteria that can grow aerobically have features in common with the mitochondrial respiratory chain. Because of the vital importance of oxygen utilization throughout most of evolution, and consequent conservation of electron-transport complexes and carriers, the teaching of bioenergetics, whether in the laboratory or lecture room, could benefit from the inclusion of micro-organisms in the curriculum.     

Rat tumour necrosis factor-alpha: expression in recombinant Pichia pastoris, purification, characterization and development of a novel ELISA.

Tumour necrosis factor-alpha is a pro-inflammatory cytokine involved in many aspects of acute phase and immune responses. Species specificity in the biological action and receptor binding of TNF-alpha make it desirable to use homologous reagents in experimental models, both in vivo and in vitro. As the rat is the model of choice in many investigations on fever, trauma and pathology, there is a need for specific rat reagents. In this paper, we describe the production of recombinant rat TNF-alpha in milligram quantities, using a methylotrophic yeast expression system, Pichia pastoris. Recombinant TNF-alpha was produced intracellularly in a soluble form, cells were lysed and the protein purified by ammonium sulphate precipitation, Sephadex G75 fractionation and finally, ion-exchange chromatography. The purified recombinant rat TNF-alpha had a molecular mass of 17401.38 +/- 0.38 Da, which is within 1 Da of the value predicted by the sequence data, taking into account N-acetylation of the initial methionine residue and a single disulphide bridge between amino acids 70 and 101. Recombinant rat TNF-alpha was shown to be 20 x fold more biologically active in the WEHI cytotoxicity assay, than the human standard preparation. Polyclonal antibodies were raised against purified recombinant rat TNF-alpha, these reagents were used to develop a novel enzyme-linked immunosorbant assay (ELISA). The ELISA was sensitive to 10 pg.ml- 1 rat TNF-alpha and was specific for TNF-alpha, showing no cross-reactivity with rat IL-1alpha, rat IL-1beta, rat IL-1Ra or rat IL-6. The ELISA was used to measure TNF-alpha in the plasma of rats injected with bacterial endotoxin and in cultures of rat white blood cells. The ELISA was shown to be a robust method suitable for use in assaying samples generated in both in vivo or in vitro experiments.     

Enzymatic detection of phospholipid biosynthetic enzymes after electroblotting.

The membrane-associated enzymes phosphatidylinositol synthase (CDPdiacylglycerol:myo-inositol 3-phosphatidyltransferase; EC 2.7.8.11) and phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase; EC 2.7.8.8) from Saccharomyces cerevisiae were detected enzymatically after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting. Enzyme activities were measured on nitrocellulose blots by using pure enzyme preparations as well as Triton X-100-solubilized membranes. Phosphatidylinositol synthase activity migrated to Mr 34,000, and phosphatidylserine synthase activity migrated to Mr 23,000.     

Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation

We examined bovine fetal epiphyseal and growth plate cartilages by immunofluorescence microscopy and immunoelectron microscopy using monospecific antibodies to a newly discovered cartilage-matrix calcium-binding protein that we now call chondrocalcin. Chondrocalcin was evenly distributed at relatively low concentration in resting fetal epiphyseal cartilage. In growth plate cartilage, it was absent from the extracellular matrix in the zone of proliferating chondrocytes but was present in intracellular vacuoles in proliferating, maturing and upper hypertrophic chondrocytes. The protein then disappeared from the lower hypertrophic chondrocytes and appeared in the adjoining extracellular matrix, where it was selectively concentrated in the longitudinal septa in precisely the same location where amorphous mineral was deposited in large amounts as demonstrated by von Kossa staining and electron microscopy. Mineral then spread out from these "nucleation sites" to occupy much of the surrounding matrix. Matrix vesicles were identified in this calcifying matrix but they bore no observable morphological relationship to these major sites of calcification where chondrocalcin was concentrated. Since chondrocalcin is a calcium-binding protein and has a strong affinity for hydroxyapatite, these observations suggest that chondrocalcin may play a fundamental role in the creation of nucleation sites for the calcification of cartilage matrix in endochondral bone formation.     

Modification of the conductance,selectivity and concentration-dependent saturation of Pseudomonas aeruginosa protein P channels by chemical acetylation

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the ?-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.