Analysis of Community Composition during Moderately Thermophilic Bioleaching of Pyrite,Arsenical Pyrite,and Chalcopyrite.

An analysis of the community composition of three previously undefined mixed cultures of moderately thermophilic bioleaching bacteria grown at 45°C on pyrite, arsenical pyrite, and chalcopyrite has been carried out. The bacterial species present were identified by comparative sequence analysis of the 16S rRNA gene isolated from the bioleaching vessels and analyzed by denaturing gradient gel electrophoresis, cloning, and sequencing. The mixed cultures leached all three minerals, as shown by the increase in iron released from the mineral concentrates. The species identified from the mixed cultures during bioleaching of pyrite, arsenical pyrite, and chalcopyrite were clones closely related to Acidithiobacillus caldus C-SH12, Sulfobacillus thermosulfidooxidans AT-1, Sulfobacillus montserratensis L15, and an uncultured thermal soil bacterium YNP. It was also found that the same mixed culture maintained for over a year on chalcopyrite mineral selected approximately the same consortia of bacteria as the original mixed culture grown on chalcopyrite.     

Isolation of a strain of <Emphasis Type="Italic">Acidithiobacillus caldus</Emphasis> and its role in bioleaching of chalcopyrite

A moderately thermophilic and acidophilic sulfur-oxidizing bacterium named S₂, was isolated from coal heap drainage. The bacterium was motile, Gram-negative, rod-shaped, measured 0.4 to 0.6 by 1 to 2 μm, and grew optimally at 42–45°C and an initial pH of 2.5. The strain S₂ grew autotrophically by using elemental sulfur, sodium thiosulfate and potassium tetrathionate as energy sources. The strain did not use organic matter and inorganic minerals including ferrous sulfate, pyrite and chalcopyrite as energy sources. The morphological, biochemical, physiological characterization and analysis based on 16S rRNA gene sequence indicated that the strain S₂ is most closely related to Acidithiobacillus caldus (>99% similarity in gene sequence). The combination of the strain S₂ with Leptospirillum ferriphilum or Acidithiobacillus ferrooxidans in chalcopyrite bioleaching improved the copper-leaching efficiency. Scanning electron microscope (SEM) analysis revealed that the chalcopyrite surface in a mixed culture of Leptospirillum ferriphilum and Acidithiobacillus caldus was heavily etched. The energy dispersive X-ray (EDX) analysis indicated that Acidithiobacillus caldus has the potential role to enhance the recovery of copper from chalcopyrite by oxidizing the sulfur formed during the bioleaching progress.     

RFLP mapping of three new loci for resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) in barley

Three new major, race-specific, resistance genes to powdery mildew (Erysiphe graminis f. sp. hordei) were identified in three barley lines, RS42-6*O, RS137-28*E, and HSY-78*A, derived from crosses with wild barley (Hordeum vulgare ssp. spontaneum). The resistance gene origining from wild barley in line RS42-6*O, showed a recessive mode of inheritance, whereas the other wild barley genes were (semi)-dominant. RFLP mapping of these three genes was performed in segregating F₂ populations. The recessive gene in line RS42-6*O, was localized on barley chromosome 1S (7HS), while the (semi)-dominant genes in lines RS137-28*E, and HSY-78*A, were localized on chromosomes 1L (7HL) and 7L (5HL), respectively. Closely linked RFLP clones mapped at distances between 2.6cM and 5.3 cM. Hitherto, specific loci for powdery mildew resistance in barley had not been located on these chromosomes. Furthermore, tests for linkage to the unlocalized resistance gene Mlp revealed free segregation. Therefore, these genes represent new loci and new designations are suggested: mlt (RS42-6*O), Mlf (RS137-28*E), and Mlj (HSY-78*A). Comparisons with mapped QTLs for mildew resistance were made and are discussed in the context of homoeology among the genomes of barley (H-vulgare), wheat (Triticum aestivum), and rye (Secale cereale). Duplications of RFLP bands detected in the neighbourhood of Mlf and mlt might indicate an evolutionary interrelationship to the Mla locus for mildew resistance.     

Theβ-subunit of human chorionic gonadotropin containsN-glycosidic trisialo tri- and tri′-antennary carbohydrate chains

TheN-linked carbohydrate chains of the-subunit of highly purified urinary human chorionic gonadotropin have been re-investigated. The oligosaccharides were released enzymatically by peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F, and fractionated by a combination of FPLC and HPLC. As a result of the application of improved fractionation methods, apart from the earlier reported carbohydrate chains, also small amounts of trisialo tri- and tri-antennary oligosaccharides were found. The primary structures of the latter carbohydrate chains have been determined by 500-MHz¹H-NMR spectroscopy to beAbbreviations hCG human chorionic gonadotropin - hCG- -subunit - hCG- -subunit - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (E.C. 3.5.1.52) - endo-F endo--N-acetylglucosaminidase-F (E.C. 3.2.1.96) - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - CBB coomassie brilliant blue R 250 - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

A Comparison of the Lipopolysaccharides and O-Specific Polysaccharides of Azospirillum brasilense Sp245 and Its Omegon-Km Mutants KM018 and KM252

The lipopolysaccharides (LPSs) extracted from the outer membrane of Azospirillum brasilense Sp245 and its Omegon-Km mutants KM018 and KM252 with a hot aqueous solution of phenol were found to differ in the content of carbohydrates, glucosamine, and total phosphorus and in the proportion of octadecenoic and hexadecanoic acids in the lipid moieties of the LPSs. The carbohydrate moieties of the LPSs were heterogeneous in charge. The analysis of the O-specific polysaccharides (O-PSs) of the mutants KM018 and KM252 by gas–liquid chromatography, IR spectroscopy, and NMR spectroscopy showed that they are composed of the same linear pentasugar repeating units 2)--D-Rhap-(1 3)--D-Rhap-(1 3)--D-Rhap-(1 2)--D-Rhap-(1 2)--D-Rhap-(1 as the O-PSs of the parent strain Sp245. The reported differences in the biological activity of the LPSs of the parent and mutant strains can be due to their different chemical composition.     

Anaerobic metabolism of Nitrosomonas europaea

Nitrosomonas europaea is capable of maintaining an anaerobic metabolism, using pyruvate as an electron donor and nitrite as an electron acceptor; utilization of nitrite depends upon supply of both pyruvate and ammonia. The role of ammonia in this reaction was not determined. N europaea also assimilates CO₂ anaerobically into cell material in the presence of nitrite (0.5–1.0 mM), pyruvate and ammonia. This reaction was partially inhibited by nitrite which apparently competed with CO₂ for reducing power. Anaerobic nitrite respiration is sensitive to ionophores, FCCP being the most effective.Non-standard-abbreviations TCA trichloroacetic acid - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazon     

Comparative effects of sulfhydryl reagents on membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes. Evidence for partial involvement of a thiol group in the nucleotide sugar binding site of the solubilized enzyme

We have studied the inactivation of membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes by various types of sulfhydryl reagents. The strong inhibition of the membrane-bound form by the non-penetrant mercurial-type reagents clearly corroborated the fact that in sealed and right-side-out Golgi vesicles the ceramide glucosyltransferase is located on the cytoplasmic face. No significant differences in the susceptibility to the various sulfhydryl reagents were noted when solubilized enzyme was assayed, showing that solubilization does not reveal other critical SH groups. The different results obtained must be interpreted with regard to several thiol groups, essential for enzyme activity. No protection by the substrate UDP-glucose against mercurial-type reagents was obtained indicating that these thiol groups were not located in the nucleotide sugar binding domain. A more thorough investigation of the thiol inactivation mechanism was undertaken with NEM (N-ethylmaleimide), an irreversible reagent. The time dependent inactivation followed first order kinetics and provided evidence for the binding of 1 mol NEM per mol of enzyme. UDP-Glucose protected partially against NEM inactivation, indicating that the thiol groups may be situated in or near the substrate binding domain. Inactivation experiments with disulfide reagents showed that increased hydrophobicity led to more internal essential SH groups which are not obviously protected by the substrate UDP-glucose, thus not implicated in the substrate binding domain, but rather related to conformational changes of the enzyme during the catalytic process.Abbreviations Chaps 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate - Mops 4-morpholinepropanesulfonic acid - PC phosphatidylcholine - NEM N-ethylmaleimide - CPDS carboxypyridine disulfide (dithio-6,6-dinicotinic acid) - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - DTP dithiodipyridine - p-HMB para-hydroxymercuribenzoate - DTT dithiothreitol - BAL British anti-Lewisite (dimercaptopropanol) - Zw 3–14 Zwittergent 3–14     

Lactate conversion to acetate,CO2 and H2 in cell suspensions of Desulfovibrio vulgaris (Marburg): indications for the involvement of an energy driven reaction

Cell suspensions of Desulfovibrio vulgaris were found to catalyze, in the absence of sulfate, the complete conversion of 1 lactate to 1 acetate, 1 CO₂, and 2 H₂ (G₀=-8.8 kJ/mol) and of 1 pyruvate to 1 acetate, 1 CO₂, and 1 H₂ (G₀=-52 kJ/mol). Protonophores, the proton translocating ATPase inhibitor N,N-dicyclohexylcarbodiimide, and arsenate specifically inhibited H₂ formation from lactate but not from pyruvate. The results suggest that lactate oxidation to pyruvate and H₂ (G ₀=+43.2 kJ/mol) is energy driven.     

Effect of phytotoxic metabolites ofFusarium culmorum on barley root and root-hair development

Summary A mixture of gluconic acid and two unidentified organic acids, isolated from the culture filtrate ofFusarium culmorum, grown in glucose-limited continuous culture, were found to be highly toxic to growth of barley seedlings. A concentration of 900 g ml^–1 of the acid totally inhibited germination of barley seeds, with lower concentrations greatly inhibiting growth and development of germinated seeds. Root extension and root-hair production were inhibited at a concentration of 450 g ml^–1 of the acids, but the number of roots per seedling was increased. Toxicity of the acids was promoted at low pH.     

Monosomic analysis of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond in cell culture was examined in crosses between the Wichita monosomic series and a highly regenerable line, ND7532. Segregation in disomic controls and 13 monosomic families showed a good fit to a monogenic ratio indicating a qualitative mode of inheritance. Segregation in the cross involving monosomic 2D showed a high frequency of regeneration (93.6%) and high callus growth rate (1.87 g/90 days) indicating that 2D is a critical chromosome. Modifying genes may be located on other chromosomes. Substitution of chromosomes from a low regenerable cultivar Vona further indicated that the group 2 chromosomes, in particular chromosome 2D, possess genetic factors promoting callus growth and regeneration.     

Bacterial and chemical leaching pattern on copper ores of sandstone and limestone type

Thin polished sections of copper sulphide ore were placed as an energy source in stationary cultures of wild strains and Thiobacillus neapolitanus at pH 7.5. Scanning electron microscopy revealed characteristic leaching patterns that depended on the type of leaching process and time of bioleaching. In some cases, a biological film on the ore surface was observed. Close contact between bacterial cells and ore seems necessary for leaching in some cases.M. Ostrowski and A. Skodowska are with the Department of Photo and Image Information, University of Warsaw, ul.Nowy wiat 67, 00-046 Warsaw, Poland.     

Effect of aluminium on yield and plant chemical concentrations of some temperate legumes

The aluminium (Al) tolerance of 34 temperate legume species (143 genotypes, including 57 from Trifolium repens) was determined in 60 experiments over a 3 year period in a low ionic strength (2.7 × 10^-3 M) solution culture. For each genotype, the relationship between solution Al³⁺ activity (M) and relative yield was determined and the Al³⁺ activity associated with a 50% reduction in yield (Al_RY50) calculated. In addition, plant chemical concentrations were determined in at least one genotype from most species. For white clover, Al_RY50 over all genotypes had an approximately normal distribution with mean of 1.31 M for the tops and 1.51 M for the roots, and a standard deviation of about 0.4. This suggested that Al tolerance had a polygenic inheritance. For the other species tested, Al_RY50 ranged from 0.15 to 4.53 M in the tops and from 0.21 to 4.89 M in the roots. In the tops and roots, 37% and 26% respectively of the genotypes had an Al_RY50 less than 1 M, including all species tested in the genera Melilotus and Medicago. Only 8% or 23% of the genotypes, based on the tops and roots respectively, had an Al_RY50 greater than 2, including all genotypes in the species Lotus pedunculatus. Except for Lotus, there were no consistent differences between genera in plant chemical concentrations. In Lotus, concentrations of Ca, Zn, Mn and Cu in the tops and of all elements except B in the roots were lower than that of the other species. The Al_RY50 of the species was not related to plant chemical concentrations in the absence of Al. Depending on the plant element, increasing solution Al concentrations had no significant effect on plant chemical concentrations for 56–94% of the species. When a significant effect did occur, increasing Al in solution generally decreased S and K concentrations and increased Mn, Zn, Cu Fe, B and Al concentrations in the tops and roots and decreased Ca concentrations in the tops. Plant P concentrations decreased in the tops but increased in the roots. Increasing Al in solution increase plant Al at the average rate of 44 g g^-1 M ^-1 (range 20–87) in the tops and 333 g M ^-1 (range 162–616) in the roots.     

Phytotoxic activity of selected water-soluble metabolites of Fusarium against Lemna minor L. (Duckweed)

Phytotoxicity and inhibitory effects of the fusarial toxins fumonisin B₁ (FB₁) [m.p. 103–105 °C], fusaric acid [m.p. 106–107 °C], butenolide (4-acetamido-4-hydroxy-2-butenoic acid lactone) [116–117 °C], 9, 10-dihydroxyfusaric acid [m.p. 150–155 ° C], and moniliformin on chlorophyll synthesis in the aquatic macrophyte Lemna minor (duckweed) were examined. FB₁ proved to be most active, reducing the growth of L. minor fronds and their ability to synthesize chlorophyll by 53% and 59%, respectively, at 0.7 g/ml. The growth rate of L. minor was reduced 59% by 6.7 g/ml fusaric acid, 62% by 66.7 g/ml butenolide, and 22% by 66.7 g/ml 9,10-dihydroxyfusaric acid. Moniliformin was the least phytotoxic to L. minor, with only a 16% suppression of growth rate and a 54% reduction in chlorophyll at 66.7 g/ml.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Ultrastructure and behaviour of the spindle pole body of the aphid-pathogenic fungusErynia neoaphidis

Summary A detailed account of the ultrastructure and behaviour of the spindle pole body (SPB) of the entomophthoraceous fungusErynia neoaphidis is presented for the first time.The SPB consists of extranuclear (ENC) and intranuclear (INC) components. The ENC is a saucepan-shaped structure which lies in a pocket of the nuclear envelope. It is composed of a forked, fibrillar handle and a shallow, cylindrical pan. The pan has a wall of two layers, both of which are thickened with a regular periodicity so that they appear to be beaded. It is postulated that the pan is formed from rough endoplasmic reticulum and that it synthesizes the amorphous, electron-dense material coating the ENC.The INC is a saucer-shaped, electron-dense plaque in which the ends of the spindle microtubules terminate. During metaphase, a clear zone separates the INC from the nuclear envelope and persists until telophase. The roles of the amorphous, electron-dense material and the clear zone as well as the method of SPB replication are discussed.     

Uptake of phosphate by symbiotic and free-living dinoflagellates

Uptake of phosphate in the light by Amphidinium carterae, Amphidinium klebsii, cultured and symbiotic Gymnodinium microadriaticum conformed to Michaelis-Menten type saturation kinetics with all organisms showing similar K _m values, namely 0.005 to 0.016 M phosphorus. V _max values were 0.009–0.32 nmol phosphorus · 10⁵ cells^-1 · 10 min^-1. Phosphate uptake by all the dinoflagellates was greater in the dark than in the light. The metabolic inhibitor 3-(3,4-dichlorophenyl) 1,1-dimethylurea stimulated phosphate uptake in the light by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. Carbonylcyanide 3-chlorophenylhydrazone (CCCP) inhibited phosphate uptake by A. carterae and A. klebsii under both light and dark conditions. Uptake of phosphate by cultured and symbiotic G. microadriaticum in the light, but not in the dark, was inhibited by CCCP. Low concentrations of arsenate (5 g As · l^-1) stimulated phosphate by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. High concentrations of arsenate (100 g As · l^-1) did not affect uptake of phosphate by A. carterae and A. klebsii.     

β-Carotene synthesis in isolated chromoplasts from Narcissus pseudonarcissus

A system has been established from isolated intact chromoplasts of Narcissus pseudonarcissus flowers that synthesizes geranylgeraniol, an unknown polyprenoid alcohol, phytoene, and -carotene from [1-¹⁴C]isopentenyl pyrophosphate in a good yeild. Long chain pyrophosphates are not accumulated. San 6706 inhibits the dehydrogenation of phytoene, whereas nicotine does not lead to an accumulation of lycopene. Separation and identification of polyprenoid lipids was performed by HPLC. The properties and advantages of the chromoplast system are discussed.Abbreviations IPP isopentenyl pyrophosphate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - GC gas chromatography - Sau 6706 4-chloro-5-(dimethylamino)-2-,,-(trifluoro-m-tolyl)3(2H)-pyridazinone     

Bioleaching of chalcopyrite by pure and mixed cultures of Acidithiobacillus spp. and Leptospirillum ferriphilum

Bioleaching is an economical method for the recovery of metals that requires low investment and operation costs. Furthermore, it is generally more environmentally friendly than many physicochemical metal extraction processes. The bioleaching of chalcopyrite in shake flasks was investigated with pure and mixed cultures of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Acidithiobacillus caldus, and Leptospirillum ferriphilum. The mixed cultures containing both iron- and sulfur-oxidizing bacteria were more efficient than the pure culture alone. The presence of sulfur-oxidizing bacteria positively increased the dissolution rate and the percentage recovery of copper from chalcopyrite. Mixed cultures consisting of moderately thermophilic L. ferriphilum and A. caldus leached chalcopyrite more effectively than mesophilic A. ferrooxidans pure and mixed cultures. The decrease of the chalcopyrite dissolution rate in leaching systems containing A. ferrooxidans after 12–16 days coincided with the formation of jarosite precipitation as a passivation layer on the mineral surface during bioleaching. Low pH significantly reduces jarosite formation in pure and mixed cultures of L. ferriphilum and A. caldus.     

A physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome

A combined physical and genetic map of theCorynebacterium glutamicum ATCC 13032 chromosome was constructed using pulsed-field gel electrophoresis (PFGE) and hybridizations with cloned gene probes. Total genomic DNA was digested with the meganucleasesSwaI (5-ATTTAAAT-3),PacI (5-TTAATTAA-3), andPmeI (5-GTTTAAAC-3) yielding 26, 27, and 23 fragments, respectively. The chromosomal restriction fragments were then separated by PFGE. By summing up the lengths of the fragments generated with each of the three enzymes, a genome size of 3082 +/- 20 kb was determined. To identify adjacentSwaI fragments, a genomic cosmid library ofC. glutamicum was screened for chromosomal inserts containingSwaI sites. Southern blots of the PFGE gels were hybridized with these linking clones to connect theSwaI fragments in their natural order. By this method, about 90% of the genome could be ordered into three contigs. Two of the remaining gaps were closed by cross-hybridization of blottedSwaI digests using as probesPacI andPmeI fragments isolated from PFGE gels. The last gap in the chromosomal map was closed by hybridization experiments using partialSwaI digestions, thereby proving the circularity of the chromosome. By hybridization of gene probes toSwaI fragments separated by PFGE about 30 genes, including rRNA operons, IS element and transposon insertions were localized on the physical map.     

Antibiotic effect of linolenic acid fromChlorococcum strain HS-101 andDunaliella primolecta on methicillin-resistantStaphylococcus aureus

Methanol extracts fromChlorococcum strain HS-101 andDunaliella primolecta strongly inhibited the growth of a strain of methicillin-resistantStaphylococcus aureus (MRSA), which is causing serious problems in Japanese hospitals. So that the anti-MRSA substance(s) could be purified and identified, the growth medium was improved for antibiotic production. When the two strains were cultured in their improved media, antibiotic production byChlorococcum strain HS-101 was 1.8-fold that in the standard BG-11 medium, and production byD. primolecta was 2.3-fold. The activity pattern of fractions eluted by silica-gel or gel-permeation chromatography suggested that both strains produced two antibiotic substances. Identification of the purified substances by NMR and GC-MS showed that one of the active substances in both strains was-linolenic acid. Ten fatty acids from other sources were tested, and it was found that unsaturated fatty acids had antibiotic activity against MRSA, with the highest activity that of -linolenic acid.     

Biodegradation of beta-cyfluthrin by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain S1

-Cyfluthrin [-cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate] pesticide has been in agricultural use in the recent years for controlling Lepidopteran pests affecting solanaceous crops. The extensive use of synthetic pyrethroids like -cyfluthrin has resulted in wide spread environmental contamination. The purpose of this study was to isolate bacteria from soil and to determine their ability to degrade -cyfluthrin and identify the intermediates in culture broth using spectroscopy. An aerobic bacterium capable of degrading -cyfluthrin was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain S1) had 100% identity to the sequence from Pseudomonas stutzeri. Finally products formed during degradation of -cyfluthrin have been identified as -cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (M.W. 341); 4-fluoro-3-phenoxy--cyanobenzyl alcohol (M.W. 243) and 3(2,2-dichlorovinyl)-2,2-dimethyl cyclopropanecarboxylic acid (M.W. 208).