New Nitrogen-Fixing Microorganisms Detected in Oligotrophic Oceans by Amplification of Nitrogenase (nifH) Genes

Oligotrophic oceanic waters of the central ocean gyres typically have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated. Nitrogenase gene (nifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques. Nitrogenase genes derived from unicellular and filamentous cyanobacteria, as well as from the α and γ subdivisions of the class Proteobacteria, were found in both the Atlantic and Pacific oceans. nifH sequences that cluster phylogenetically with sequences from sulfate reducers or clostridia were found associated with planktonic crustaceans. Nitrogenase sequence types obtained from invertebrates represented phylotypes distinct from the phylotypes detected in the picoplankton size fraction. The results indicate that there are in the oceanic environment several distinct potentially nitrogen-fixing microbial assemblages that include representatives of diverse phylotypes.The productivity of the oceans controls the fluxes of many biogeochemically important compounds, including the rate of exchange of carbon dioxide between the open ocean and the atmosphere. In turn, oceanic carbon fixation is limited by the bioavailability of nutrients, including nitrogen, phosphorus, and iron (9, 10, 20). In contrast to the biogeochemical cycles of phosphorus and iron, nitrogen is present in relatively high concentrations in seawater as gaseous N₂. Gaseous nitrogen is available only to microorganisms with the capability of biological nitrogen fixation, the reduction of atmospheric N₂ to ammonium. Although large areas of the world’s oceans are virtually devoid of fixed dissolved inorganic nitrogen and primary production may be nitrogen limited, very few species of nitrogen-fixing organisms have been identified or isolated from the plankton. Trichodesmium, a filamentous aggregate-forming cyanobacterium, is an abundant diazotroph in tropical and subtropical waters (3, 5), but few other examples of diazotrophs from the open ocean are known (21, 35). The seeming low diversity of known nitrogen-fixing organisms in the open ocean stands in stark contrast to the presumptive nitrogen limitation in the world’s oceans and presents an evolutionary paradox.Recently, biological nitrogen fixation has gained recognition as an important source of nitrogen for supporting oceanic primary production (3, 11, 18, 22). The nitrogen budget for the Atlantic Ocean does not balance because a source of nitrogen cannot be accounted for by current knowledge of fluxes and pools of nitrogen, even after including nitrogen fixation by Trichodesmium (22). It is speculated that rates of nitrogen fixation by known diazotrophic organisms have been underestimated (17), or as yet unidentified diazotrophic organisms are active in the ocean (18). Conventional nitrogenase, the enzyme that catalyzes biological dinitrogen reduction to ammonium, is composed of two highly conserved proteins: the iron (Fe) protein (encoded by the nifH gene) and the molybdenum iron (MoFe) protein (encoded by the nifDK genes). The nitrogenase enzyme is present in diverse lineages of prokaryotes and is generally believed to be ancient (38). Evolutionarily conserved amino acid sequences within the nifH (which encodes the Fe protein component of nitrogenase) gene have been exploited to design PCR primers to detect the genetic potential for nitrogen fixation in the marine environment (39). With this approach, the diversity of nitrogen-fixing microorganisms in oceanic water and marine plankton was determined. This report shows that there are far more diverse nitrogen-fixing populations and diverse habitats which can support nitrogen fixation in the open ocean than previously documented.     

Intermittent access to preferred food reduces the reinforcing efficacy of chow in rats

Intermittent, extended access to preferred diets increases their intake. However, the effects of such access on the acceptance and reinforcing efficacy of otherwise satisfying alternatives is less known. To investigate the role of nonnutritional contributions to the hypophagia that follows removal of preferred food, male Wistar rats were fed a chow diet (Chow A/I), preferred to their regular chow (Chow), which was equally consumed under 1-choice conditions to an even more preferred chocolate-flavored, sucrose-rich diet (Preferred). Rats then learned to obtain Chow A/I pellets under a progressive ratio schedule of reinforcement and were assigned to two matched groups. Each week, one group (n = 15) was diet-cycled, receiving Chow A/I for 5 days followed by the Preferred diet for 2 days. Controls received Chow A/I daily (n = 14). Progressive ratio sessions were performed daily during the 5 days that all subjects received Chow A/I in the home cage. Across 5 wk, diet-cycled rats progressively ate less of the otherwise palatable Chow A/I diet. Hypophagia was not due to greater prior intake or weight gain, motor impairment, or facilitated satiation and was associated with changes in progressive ratio performance that suggested a reduced reinforcing efficacy of the Chow A/I diet in diet-cycled animals. By week 4, diet-cycled animals began to overeat the preferred diet, especially during the first 6 h of renewed access, resembling a deprivation effect. The results suggest that intermittent access to highly preferred food, as practiced by many restrained eaters, may progressively decrease the acceptability of less palatable foods, and may promote relapse to more rewarding alternatives.     

Microbial production of fructosyltransferases for synthesis of pre-biotics

Fructooligosaccharides (FOS) are prebiotic substances found in several vegetable or natural foods. The main commercial production of FOS comes from enzymatic transformation of sucrose by the microbial enzyme fructosyltransferase. The development of more efficient enzymes, with high activity and stability, is required and this has attracted the interest of biotechnologists and microbiologists with production by several microorganisms being studied. This article reviews and discusses FOS chemical structure, enzyme characteristics, the nomenclature, producer microorganisms and enzyme production both in solid state fermentation and submerged cultivation.     

MitoNuc: a database of nuclear genes coding for mitochondrial proteins. Update 2002

Mitochondria, besides their central role in energy metabolism, have recently been found to be involved in a number of basic processes of cell life and to contribute to the pathogenesis of many degenerative diseases. All functions of mitochondria depend on the interaction of nuclear and organelle genomes. Mitochondrial genomes have been extensively sequenced and analysed and data have been collected in several specialised databases. In order to collect information on nuclear coded mitochondrial proteins we developed MitoNuc, a database containing detailed information on sequenced nuclear genes coding for mitochondrial proteins in Metazoa. The MitoNuc database can be retrieved through SRS and is available via the web site http://bighost.area.ba.cnr.it/mitochondriome where other mitochondrial databases developed by our group, the complete list of the sequenced mitochondrial genomes, links to other mitochondrial sites and related information, are available. The MitoAln database, related to MitoNuc in the previous release, reporting the multiple alignments of the relevant homologous protein coding regions, is no longer supported in the present release. In order to keep the links among entries in MitoNuc from homologous proteins, a new field in the database has been defined: the cluster identifier, an alpha numeric code used to identify each cluster of homologous proteins. A comment field derived from the corresponding SWISS-PROT entry has been introduced; this reports clinical data related to dysfunction of the protein. The logic scheme of MitoNuc database has been implemented in the ORACLE DBMS. This will allow the end-users to retrieve data through a friendly interface that will be soon implemented.     

Parameters affecting plant defense pathway mediated recruitment of entomopathogenic nematodes

Entomopathogenic nematodes are natural enemies and effective biological control agents of subterranean insect herbivores. Interactions between herbivores, plants, and entomopathogenic nematodes are mediated by plant defense pathways. These pathways can induce release of volatiles and recruit entomopathogenic nematodes. Stimulation of these plant defense pathways for induced defense against belowground herbivory may enhance biological control in the field. Knowledge of the factors affecting entomopathogenic nematode behaviour belowground is needed to effectively implement such strategies. To that end, we explore the effect of elicitor, elicitor dose, mechanical damage, and entomopathogenic nematode release distance on recruitment of entomopathogenic nematode infective juveniles to corn seedlings. Increasing doses of methyl jasmonate and methyl salicylate elicitors recruited more entomopathogenic nematodes as did mechanical damage. Recruitment of entomopathogenic nematodes was higher at greater release distances. These results suggest entomopathogenic nematodes are highly tuned to plant status and present a strategy for enhancing biological control using elicitor-stimulated recruitment of entomopathogenic nematodes.     

Computational identification of protein coding potential of conserved sequence tags through cross-species evolutionary analysis

The identification of conserved sequence tags (CSTs) through comparative genome analysis may reveal important regulatory elements involved in shaping the spatio-temporal expression of genetic information. It is well known that the most significant fraction of CSTs observed in human–mouse comparisons correspond to protein coding exons, due to their strong evolutionary constraints. As we still do not know the complete gene inventory of the human and mouse genomes it is of the utmost importance to establish if detected conserved sequences are genes or not. We propose here a simple algorithm that, based on the observation of the specific evolutionary dynamics of coding sequences, efficiently discriminates between coding and non-coding CSTs. The application of this method may help the validation of predicted genes, the prediction of alternative splicing patterns in known and unknown genes and the definition of a dictionary of non-coding regulatory elements.     

Diversity and Detection of Nitrate Assimilation Genes in Marine Bacteria

A PCR approach was used to construct a database of nasA genes (called narB genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A nasA-specific PCR primer set that could be used to selectively amplify the nasA gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced nasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.     

Acute glutathione depletion restricts mitochondrial ATP export in cerebellar granule neurons

Decreases in GSH pools detected during ischemia sensitize neurons to excitotoxic damage. Thermodynamic analysis predicts that partial GSH depletion will cause an oxidative shift in the thiol redox potential. To investigate the acute bioenergetic consequences, neurons were exposed to monochlorobimane (mBCl), which depletes GSH by forming a fluorescent conjugate. Neurons transfected with redox-sensitive green fluorescent protein showed a positive shift in thiol redox potential synchronous with the formation of the conjugate. Mitochondria within neurons treated with mBCl for 1 h failed to hyperpolarize upon addition of oligomycin to inhibit their ATP synthesis. A decreased ATP turnover was confirmed by monitoring neuronal oxygen consumption in parallel with mitochondrial membrane potential (Deltapsi(m)) and GSH-mBCl formation. mBCl progressively decreased cell respiration, with no effect on mitochondrial proton leak or maximal respiratory capacity, suggesting adequate glycolysis and a functional electron transport chain. This approach to "state 4" could be mimicked by the adenine nucleotide translocator inhibitor bongkrekic acid, which did not further decrease respiration when administered after mBCl. The cellular ATP/ADP ratio was decreased by mBCl, and consistent with mitochondrial ATP export failure, respiration could not respond to an increased cytoplasmic ATP demand by plasma membrane Na(+) cycling; instead, mitochondria depolarized. More prolonged mBCl exposure induced mitochondrial failure, with Deltapsi(m) collapse followed by cytoplasmic Ca(2+) deregulation. The initial bioenergetic consequence of neuronal GSH depletion in this model is thus an inhibition of ATP export, which precedes other forms of mitochondrial dysfunction.     

Ionic content in plant extracts determined by ion chromatography with conductivity detection

A simple method is described for the determination of the ionic content of vegetable samples by ion chromatography with suppressed conductivity detection. Extracts of leaves of cucumber (Cucumis sativus), leaves and cotyledons of watermelon (Citrullus lanantus), cotyledons of zucchini (Cucurbitapepo), and leaves and roots of olive (Olea europaea) obtained at room temperature yielded chromatographic profiles with substantial differences in the relative contents of Cl-, NO3-, HPO4(2-) and SO4(2-) as well as of Na+, NH4+, K+, Mg2+ and Ca2+. Although NO3-, Cl- and K+ were common to each extracted sample and accounted for most of the ions present, two additional anion peaks (i.e. malate and oxalate) were detected. Among the vegetable tissues investigated, olive roots contained a considerable amount of oxalate (37 mg/g dry weight), while Na+, which is present in very low amount in extracted samples of leaves and cotyledons, represented ca. 30% of the cationic content of olive roots. In all the examined tissue extracts, K+ was the main cation (16-55 mg/g dry weight) and NO3-, Cl- and HPO4(2-) were the main inorganic anions.