Burkholderia uboniae sp. nov., L-arabinose-assimilating but different from Burkholderia thailandensis and Burkholderia vietnamiensis

A polar multitrichous gram-negative motile rod, EY 3383, originally identified as Burkholderia thailandensis, revealed a DNA-DNA reassociation rate of 36.7%, under stringent conditions, with the type strain of B. thailandensis, despite the 16S rDNA homology value between two type strains being as high as 97.9%. The strain was clearly differentiated from the type strain of B. thailandensis by physiological, bio-chemical, and nutritional characteristics, without significant difference in cellular fatty acid and lipid composition. Based on the results of 16S rDNA sequence analysis, DNA-DNA hybridization and phenotypic characterization, Burkholderia uboniae sp. nov. is herein proposed. The type strain is NCTC 13147=EY 3383, isolated on 8 December 1989 from surface soil along the roadside in Ubon Ratchathani, Thailand. Major respiratory quinone is ubiquinone-8(Q8). G+C content of DNA is 69.71%.     

Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants

Glutathione (gamma-glu-cys-gly; GSH) is usually present at high concentrations in most living cells, being the major reservoir of non-protein reduced sulfur. Because of its unique redox and nucleophilic properties, GSH serves in bio-reductive reactions as an important line of defense against reactive oxygen species, xenobiotics and heavy metals. GSH is synthesized from its constituent amino acids by two ATP-dependent reactions catalyzed by gamma-glutamylcysteine synthetase and glutathione synthetase. In yeast, these enzymes are found in the cytosol, whereas in plants they are located in the cytosol and chloroplast. In protists, their location is not well established. In turn, the sulfur assimilation pathway, which leads to cysteine biosynthesis, involves high and low affinity sulfate transporters, and the enzymes ATP sulfurylase, APS kinase, PAPS reductase or APS reductase, sulfite reductase, serine acetyl transferase, O-acetylserine/O-acetylhomoserine sulfhydrylase and, in some organisms, also cystathionine beta-synthase and cystathionine gamma-lyase. The biochemical and genetic regulation of these pathways is affected by oxidative stress, sulfur deficiency and heavy metal exposure. Cells cope with heavy metal stress using different mechanisms, such as complexation and compartmentation. One of these mechanisms in some yeast, plants and protists is the enhanced synthesis of the heavy metal-chelating molecules GSH and phytochelatins, which are formed from GSH by phytochelatin synthase (PCS) in a heavy metal-dependent reaction; Cd(2+) is the most potent activator of PCS. In this work, we review the biochemical and genetic mechanisms involved in the regulation of sulfate assimilation-reduction and GSH metabolism when yeast, plants and protists are challenged by Cd(2+).     

Hansenula polymorpha NMR2 and NMR4, two new loci involved in nitrogen metabolite repression

In the yeast Hansenula polymorpha (Pichia angusta) nitrate assimilation is tightly regulated and subject to a dual control: nitrogen metabolite repression (NMR), triggered by reduced nitrogen compounds, and induction, elicited by nitrate itself. In a previous paper [Serrani, F., Rossi, B. and Berardi, E (2001) Nitrogen metabolite repression in Hansenula polymorpha: the nmrl-l mutation. Curr. Genet. 40, 243-250], we identified five loci (NMR1-NMR5) involved in NMR, and characterised one of them (NMR1), which likely identifies a regulatory factor. Here, we describe two more mutants, namely nmr2-1 and nmr4-1. The first one possibly identifies a regulatory factor involved in nitrogen metabolite repression by various nitrogen sources alternative to ammonium. The second one, apparently involved in ammonium assimilation, probably has sensor functions.     

Expression of a ferredoxin-dependent glutamate synthase gene in mesophyll and vascular cells and functions of the enzyme in ammonium assimilation in Nicotiana tabacum (L.)

GLU1 encodes the major ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in Arabidopsis thaliana (ecotype Columbia). With the aim of providing clues on the role of Fd-GOGAT, we analyzed the expression of Fd-GOGAT in tobacco (Nicotiana tabacum L. cv. Xanthi). The 5′ flanking element of GLU1 directed the expression of the uidA reporter gene in the palisade and spongy parenchyma of mesophyll, in the phloem cells of vascular tissue and in the roots of tobacco. White light, red light or sucrose induced GUS expression in the dark-grown seedlings in a pattern similar to the GLU1 mRNA accumulation in Arabidopsis. The levels of GLU2 mRNA encoding the second Fd-GOGAT and NADH-glutamate synthase (NADH-GOGAT, EC 1.4.1.14) were not affected by light. Both in the light and in darkness, ¹⁵NH₄⁺ was incorporated into [5−¹⁵N]glutamine and [2−¹⁵N]glutamate by glutamine synthetase (GS, EC 6.3.1.2) and Fd-GOGAT in leaf disks of transgenic tobacco expressing antisense Fd-GOGAT mRNA and in wild-type tobacco. In the light, low level of Fd-glutamate synthase limited the [2−¹⁵N]glutamate synthesis in transgenic leaf disks. The efficient dark labeling of [2−¹⁵N]glutamate in the antisense transgenic tobacco leaves indicates that the remaining Fd-GOGAT (15–20% of the wild-type activity) was not the main limiting factor in the dark ammonium assimilation. The antisense tobacco under high CO₂ contained glutamine, glutamate, asparagine and aspartate as the bulk of the nitrogen carriers in leaves (62.5%), roots (69.9%) and phloem exudates (53.2%). The levels of glutamate, asparagine and aspartate in the transgenic phloem exudates were similar to the wild-type levels while the glutamine level increased. The proportion of these amino acids remained unchanged in the roots of the transgenic plants. Expression of GLU1 in mesophyll cells implies that Fd-GOGAT assimilates photorespiratory and primary ammonium. GLU1 expression in vascular cells indicates that Fd-GOGAT provides amino acids for nitrogen translocation. The nucleotide sequence data of the GLU1 gene reported in the present study is available from GenBank with the following accession number: AY189525     

Carbon Dioxide Assimilation and Methane Oxidation in Various Zones of the Rainbow Hydrothermal Field

Rates of carbon dioxide assimilation and methane oxidation were determined in various zones of the Rainbow Hydrothermal Field (36°N) of the Mid-Atlantic Ridge. In the plume above the hydrothermal field, anomalously high methane content was recorded, the microbial population density (up to 10⁵ cells/ml) was an order of magnitude higher than the background values, and the CO₂ assimilation rate varied from 0.01 to 1.1 g C/(l day). Based on the data on CO₂ assimilation, the production of organic carbon due to bacterial chemosynthesis in the plume was calculated to be 930 kg/day or 340 tons/year (about 29% of the organic carbon production in the photic zone). In the black smoke above active smokers, the microbial population density was as high as 10⁶ cells/ml, the rate of CO₂ assimilation made up 5–10 g C/(l day), the methane oxidation rate varied from 0.15 to 12.7 l/(l day), and the methane concentration ranged from 1.05 to 70.6 l/l. In bottom sediments enriched with sulfides, the rate of CO₂ assimilation was at least an order of magnitude higher than in oxidized metal-bearing sediments. At the base of an active construction, whitish sediment was found, which was characterized by a high methane content (92 l/dm³) and a high rate of methane oxidation (1.7 l/(dm³ day)).     

Gibberellic-acid-responsive protoplasts from mature aleurone of Himalaya barley

The role of oxygen in the photoinactivation of the photosynthetic apparatus of Spinacia oleracea L. was investigated. Moderate irradiation (1200 mol photons m^-2s^-1) of spinach leaves in an atmosphere of pure nitrogen caused strong inhibition of subsequently measured net CO₂ assimilation, whereas considerably less photoinhibition was observed in the presence of low partial pressures (10–20 mbar) of O₂. The decrease in activity caused by anaerobiosis in the light was not based on stomatal closure; the decline of assimilation represents a photoinhibition, as activity was not impaired by low irradiation (80 mol photos m^-2s^-1). In contrast, gassing with pure N₂ in the dark caused strong inhibition. Electron-transport rates and chlorophyll-fluorescence data of thylakoids isolated from photoinhibited leaves indicated damage to the electron-transport system, in particular to photosystem II reaction centers. In vitro, photoinhibition in isolated thylakoid membranes was also strongly promoted by anaerobiosis. Photoinhibition of electron-transport rates under anaerobic conditions was characterized by a pronounced increase in the initial fluorescence level, F₀, of chlorophyll-fluorescence induction, in contrast to photoinhibition under aerobic conditions. The results are discussed in terms of two mechanisms of photoinhibition, one that is suppressed and a second that is promoted by oxygen.Abbreviations Chl chlorophyll - DCMU 3-(3, 4-dichlorophenyl)-1,1-dimethylurea - PSI, II photosystem I, II     

Isolation and characterization of mutants of the facultative methylotroph Arthrobacter P1 blocked in one-carbon metabolism

Among methylamine and/or ethylamine minus mutants of Arthrobacter P1 four different classes were identified, which were blocked either in the methylamine transport system, amine oxidase, hexulose phosphate synthase or acetaldehyde dehydrogenase. The results indicated that a common primary amine oxidase is involved in the metabolism of methylamine and ethylamine. Growth on ethylamine, however, was not dependent on the presence of the methylamine transport system. In mutants lacking amine oxidase, methylamine was unable to induce the synthesis of hexulose phosphate synthase, thus confirming the view that the actual inducer for the latter enzyme is not methylamine, but its oxidation product formaldehyde. Contrary to expectation, when the formaldehyde fixing enzyme hexulose phosphate synthase was deleted (mutant Art 11), accumulation of formaldehyde during growth on choline or on glucose plus methylamine as a nitrogen source did not occur. Evidence was obtained to indicate that under these conditions formaldehyde may be oxidized to carbon dioxide via formate, a sequence in which peroxidative reactions mediated by catalase are involved. In addition, a specific NAD-dependent formaldehyde dehydrogenase was detected in choline-grown cells of wild type Arthrobacter P1 and strain Art 11. This enzyme, however, does not play a role in methylamine or formaldehyde metabolism, apparently because these compounds do not induce its synthesis.Abbreviations RuMP ribulose monophosphate - HPS hexulose phosphate synthase - HPI hexulose phosphate isomerase