Morphology of bacterial leaching patterns by Thiobacillus ferrooxidans on synthetic pyrite

Thiobacillus ferrooxidans has been cultivated on synthetic pyrite (FeS₂) single crystals as the only energy source and the pyrite interface investigated with respect to characteristic morphological changes using scanning electron microscopy. Corrosion patterns of bacterial size were identified in different stages of development and correlated with bacterial activity. It appears that bacterial attack of the sulfide interface starts by secretion of organic substances around the contact area between the bacterial cell and the sulfide energy source. They might either be part of a pseudo capsule which shields the contact area or may form a sulfur absorbing and transporting organic film. Degradation of the sulfide occurs in the contact area below the bacterial cell leading to a corrosion pit which the bacterium may abandon after it has reached a depth of bacterial dimension. Electron spectroscopic (XPS) and X-ray fluorescence studies indicate a layer of organic substances covering the sulfide surface under bacterial leaching conditions, which is sufficiently thick for consideration in interfacial chemical mechanisms.     

Proteolysis in the obligate methylotroph Methylophilus methylotrophus

This report represents the first demonstration of degradation of intracellular protein in the obligate methylotroph, Methylophilus methylotrophus. Proteolysis in batch culture was followed by a pulse-chase protocol which included chloramphenicol during the chase period to prevent re-incorporation of the radio-label, l-[4,5-³H] isoleucine. Starvation for a nitrogen source mildly stimulated proteolysis whereas starvation for the carbon source (0.5% v/v methanol) inhibited proteolysis by over 50%. Respiratory inhibitors (e.g. 2,4-DNP) caused a rapid decline in both intracellular ATP concentration and protein catabolism. Proteins synthesized after the addition of methanol (5% v/v) and ethanol (5% v/v) to the growth medium were subject to rapid degradation. Breakdown of abnormal proteins generated by treatment with dihydrostreptomycin and puromycin was also inhibited by inhibitors of respiration and deprivation of carbon source. The stability of an heterologous gene product, interferon -2, was also investigated; loss of immunoreactivity was reduced in the absence of methanol but not prevented.     

Nitrogen fixation by the hydrogen-oxidizing bacterium Alcaligenes latus

The aerobic hydrogen-oxidizing bacterium Alcaligenes latus represented by three strains was found to be able to grow with dinitrogen as the sole nitrogen source: The doubling time of total (Kjeldahl) nitrogen during growth on glucose at 30°C under an atmosphere containing 2% (v/v) oxygen in dinitrogen amounted to 39 h, while that in the presence of ammonium was 3 h. Nitrogen fixation did apparently not occur under air. During diazotrophic growth the cells accumulated up to 75% (w/dry weight) poly--hydroxybutyric acid. The efficiency of nitrogen fixation varied between 10 and 15 mg N per g glucose utilized. The specific nitrogenase activity measured in the acetylene reduction assay amounted to 5–17 nmol C₂H₄ formed per min and mg protein.     

Purification and Characterization of a Novel α-Agarase from a Thalassomonas sp.

An agar-degrading Thalassomonas bacterium, strain JAMB-A33, was isolated from the sediment off Noma Point, Japan, at a depth of 230 m. A novel -agarase from the isolate was purified to homogeneity from cultures containing agar as a carbon source. The molecular mass of the purified enzyme, designated as agaraseA33, was 85 kDa on both SDS-PAGE and gel-filtration chromatography, suggesting that it is a monomer. The optimal pH and temperature for activity were about 8.5 and 45°C, respectively. The enzyme had a specific activity of 40.7 U/mg protein. The pattern of agarose hydrolysis showed that the enzyme is an endo-type -agarase, and the final main product was agarotetraose. The enzyme degraded not only agarose but also agarohexaose, neoagarohexaose, and porphyran.     

Mass production of poly-β-hydroxybutyric acid by fully automatic fed-batch culture of methylotroph

Summary Fifty-one methylotrophs were checked with respect to their ability of poly--hydroxybutyric acid (PHB) production from methanol. One of them, Pseudomonas sp. K, was chosen from its good growth on a minimum synthetic medium. Optimal temperature and pH for its growth were 30° C and 7.0, respectively. Concentrations of PO ₄ ^3- and NH ₄ ⁺ in the medium should be kept at low levels. PHB formation was stimulated by deficiency of nutrient such as NH ₄ ⁺ , SO ₄ ^2- , Mg²⁺, Fe²⁺ or Mn²⁺. Among them, nitrogen deficiency was chosen from its effectiveness and easiness for PHB accumulation.The microorganism was cultivated to produce a large amount of poly--hydroxybutyric acid (PHB) from methanol by means of microcomputer-aided fully automatic fed-batch culture technique. During the cultivation, temperature, dissolved oxygen concentration (DO), and methanol concentration in the culture broth were maintained at 30° C 2.5±0.5 ppm and 0.5±0.2 g/l, respectively. Other nutrients, nitrogen source and mineral ions, were also controlled to maintain their initial concentrations in the medium during cell growth phase. When the high cell concentration was achieved (160 g/l), feedings of ammonia and minerals were stopped and only methanol was supplied successively to accumulate PHB. At 175 h, high concentration of PHB (136 g/l) was obtained and total cell concentration became 206 g/l. DO must be maintained above the critical level during the PHB formation phase, too. PHB yield from methanol (g PHB/g methanol) was 0.18 and the maximum PHB content reached 66% of dry weight. Solid PHB produced by the strain had the melting point of 176° C and the average molecular weight of 3.0x10⁵.     

An endoglucanase from an isolated strain of Bacillus circulans

Summary A cellulolytic bacterium was isolated from a carboxymethylcellulose production plant, where it caused drametic damage due to its high cellulolytic activity. It was identified as Bacillus circulans, and found to produce endo--1,4-glucanase with pH and temperature optima of 7.8 and 50° C respectively. It also showed good activity towards native cellulose. Conditions for optimum endoglucanase production were a medium containing 8 g/l sugar cane bagasse, 5 g/l peptone, 2 g/l yeast extract and 5 g/l NaCl, at a pH of 7.6 and incubation temperature of 30° C. Diauxic growth and increase in endoglucanase activity throughout the fermentation were observed on this medium in a 1-1 fermentor. The bacterium showed excellent endoglucanase activity, but would have to be used in conjunction with other enzymes to degrade native cellulose completely.     

Improvement in cell yield of Methylobacterium sp. by reducing the inhibition of medium components for poly-β-hydroxybutyrate production

The inhibitory effect of the concentrations of medium components on the growth of Methylobacterium sp. for poly--hydroxybutyrate production was investigated by measuring the specific growth rates for various concentrations of each medium component. When the methanol concentration was increased, the cell growth decreased and was strongly inhibited above 6% (v/v) methanol. Ammonia, calcium and iron ion did not significantly inhibit the cell growth while there were some inhibitory effects at high concentrations of sodium, potassium, and magnesium. In particular, phosphate gave most significant inhibition at concentrations higher than 75 mM. By using an automatic feeding control system of methanol, ammonia, phosphate, and minerals, their concentrations were maintained within the level necessary to reduce the inhibition of medium components. The finial dry cell weight of Methylobacterium sp. in such a system was 172 g/l at 84 h.     

Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeastHansenula polymorpha

Summary Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeastHansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures ofH. polymorpha, equivalent to 35–40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract. it became clear that the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8–9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinantH. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20–22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production.Part of these results have been presented at the Deuxième Congrès de la Société Française de Microbiologie, Strasbourg, France, September 1989.     

d-Xylanase produced by Schizophyllum radiatum

Summary d-Xylanase (1,4--xylan xylanohydrolase, EC 3.2.1.8) was obtained from mycelial submerged culture of the mushroom Schizophyllum radiatum, grown on wheat straw pretreated with steam explosion as the substrate. The enzyme was purified 192-fold (specific activity 455 IU mg^-1 protein), with 37% yield with respect to total d-xylanase activity. Polyacrylamide electrophoresis of the d-xylanase peak showed a single band of protein whose molecular weight, calculated by electrophoretic mobility, was 25 700. The enzyme exhibited maximum activity at pH 4.9 and 55°C. d-Xylanase was stable from pH 5.0 to 7.5; its half-life was 12 h at 45°C. The Michaelis constant was 9.5 mg ml^-1 and V _max 0.37 mole min^-1. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylobiose, d-xylotriose, d-xylotetraose, and d-xylopentose showing the mode of action of an endo-type enzyme.     

Characterization of an acetate-decarboxylating,non-hydrogen-oxidizing methane bacterium

A methanogenic bacterium, commonly seen in digested sludge and referred to as the fat rod or Methanobacterium soehngenii, has been enriched to a monoculture and is characterized. Cells are gramnegative, non-motile and appear as straight rods with flat ends. They form filaments which can grow to great lengths. The structure of the outer cell envelop is similar to Methanospirillum hungatii. The organism grows on a mineral salt medium with acetate as the only organic component. Acetate is the energy source, and methane is formed exclusively from the methyl group. Acetate and carbon dioxide act as sole carbon source and are assimilated in a molar ratio of about 1.9:1. The reducing equivalents necessary to build biomass from these two precursors are obtained from the total oxidation of some acetate. Hydrogen is not used for methane formation and is not needed for growth. Formate is cleaved into hydrogen and carbon dioxide. Coenzyme M was found to be present at levels of 0.35 nmol per mg of dry cells and F₄₂₀ amounted to 0.55 g per mg protein. The mean generation time was 9 days at 33°C.     

Microbial oxidation of alpha-pinene by Serratia marcescens

Summary A strain of the bacterium Serratia marcescens, isolated from sewage sludge, can oxidise the terpene hydrocarbon -pinene to produce rans-verbenol as the major product, with verbeone and trans-sobrerol as minor products. A change in nitrogen source and inclusion of glucose as a second carbon source caused the bacterium to produce -terpineol as the major oxidation product. Products were identified by gas liquid chromatography and mass spectrometry.     

Isolation and characterization of a cyanide-utilizing Burkholderia cepacia strain

A bacterium that utilizes cyanide as a nitrogen source was isolated from soil after enrichment in a liquid medium containing potassium cyanide (10mM) and glucose (1.0%, w/v). The strain could tolerate and grow in potassium cyanide at concentrations of up to 25mM. It could also utilize potassium cyanate, potassium thiocyanate, linamarin and a range of aliphatic and aromatic nitriles. The isolate was tentatively identified as Burkholderia cepacia strain C-3. Ammonia and formic acid were found in the culture supernatant of the strain grown on fructose and potassium cyanide, no formamide was detected, suggesting a hydrolytic pathway for the degradation of cyanide. The cyanide-degrading activity was higher in early and the stationary phase cells. Crude cell extracts of strain C-3 grown on nutrient broth exhibited cyanide-degrading activity. The characteristics of strain C-3 suggest that it would be useful in the bioremediation of cyanide-containing waste.     

Genetic transformation of Pseudomonas oleovorans by electroporation

An electroporation procedure for the transformation of Pseudomonas oleovorans was developed using a model plasmid, pCN51. The optimal electrotransformation was achieved with cells harvested at 45 to 60 min of growth and concentrated to a cell density of 5 OD600nm, plasmid concentration of 6 g per 100 l of cell suspension, and a 0.1-cm gap-width cuvette. Electroporation was performed at the settings of 250 , 25F and 2.5 kV. Transformation yields in the order of 103 colony-forming-unit per electroporation sample were obtained. This is a first report of the electroporation of the commercially valuable bacterium Ps. oleovorans. © Rapid Science Ltd. 1998     

Histochemistry of 11-beta-hydroxysteroid dehydrogenase in rat submandibular gland. Effect of cortisol stimulation.

Synopsis The activity pattern of NAD/NADP-linked 11-hydroxysteroid dehydrogenase (HSD) in the submandibular gland of the rat was re-evaluated using several control experiments. The incubation time needed for the initial appearance of red and blue formazans was used to investigate the activity of NAD-dependent 11-HSD in control and cortisol-treated rats. The following results were obtained. (1) Prefixation of small tissue blocks with 1% w/v methanol-free formaldehyde (pH 7.2) for up to 20 min preserved morphological integrity and maximal enzyme activity. The substantivity of formazans was enhanced. (2) The substantivity of Nitro BT was highly variable. The implication of this forin situ localization of enzymes was analysed. (3) Pretreatment with acetone and application of phenanthroline was necessary to avoid a false positive reaction caused by alcohol dehydrogenase. (4) No diffusion of 11-HSD was noticed within 30 min of incubation, nor was rediffusion of reduced intermediates seen. (5) With either NAD or NADP as coenzyme, 11-HSD was localized in the striated, intralobular, interlobular, interlobar, and main duct. (6) 11-HSD was found to be primarily NAD-dependent. (7) With DMF or DMSO as solvent, the rate of utilization of substrates was as follows: Cortisol=11-hydroxyandrostendione>11-hydroxyprogesterone. Aldosterone was utilized very poorly, if at all. (8) After injection (i.p.) of a single pharmacological dose of cortisol, the activity of NAD-linked 11-HSD was significantly increased 24 h later. (9) NADH-tetrazolium reductase was not inhibited by levamisole. (10) Distinct NADPH-tetrazolium reductase activity was localized in the apical part of cells (or cell membranes) of the interlobar ducts and the main duct.     

A 43 kD light-regulated chloroplast RNA-binding protein interacts with the psbA 5′ non-translated leader RNA

Expression of the chloroplast psbA gene coding for the D1 protein of Photosystem II is subject to regulation at different levels in higher plants, including control of mRNA accumulation and translation. In dicots, the conserved 5 non-translated leader (5-UTR) of the psbA mRNA is sufficient to direct the light-dependent translation of the D1 protein. In this report we show that the psbA mRNA 5-UTR forms a stem-loop structure and binds a 43 kD chloroplast protein (43RNP). Binding of the 43RNP is sensitive to competition with poly(U), but insensitive to high concentrations of tRNA, the RNA homopolymers poly(A), poly(G), poly(C), or poly(A):poly(U) as a double-strand RNA. The 43RNP does not bind efficiently to the psbA mRNA 3 non-translated region, although the RNA sequence is U-rich and folds into a stem-loop. A deletion mutant of the psbA 5-UTR RNA in which 5 sequences of the stem-loop are removed does not affect 43RNP binding. Together, these properties suggest that the 43RNP binds most effectively to a specific single-strand U-rich sequence preceding the AUG start codon in the psbA mRNA. Binding of the 43RNP is not detectable in plastid protein extracts from 5-day-old dark-grown seedlings, but is detectable in light-grown seedlings as well as mature plants in the light and after shifted to the dark. The 43RNP is therefore a candidate for a regulatory RNA-binding protein that may control the accumulation and/or translation of the psbA mRNA during light-dependent seedling development.Abbreviations DMS dimethylsulfate - psb Photosystem II genes - RNP ribonucleoprotein - UTR non-translated leader - UV crosslinking ultra-violet light crosslinking     

Partial purification of endo- and exo-β-D-glucanase enzymes from Zea mays L. seedlings and their involvement in cell-wall autohydrolysis

The proteins dissociated from isolated Zea seedling cell wall using high-ionic-strength salt solutions have been found to include a number of enzymes which appear to participate in autolytic reactions of the cell wall. These enzymes caused extensive degradation of enzymatically inactive cell wall, liberating as much as 100 g/mg dry weight over a 48-h period. Lithium chloride (3M) was shown to be most effective in yielding protein and wall-degrading activities.Molecular-sieve chromatography of the cell-wall protein resolved endo--D-glucanase and exo--1,3-glucanase (EC 3.2.1.58) activities when Avena glucan and laminarin, respectively, were employed as substrates. The exoenzyme (molecular weight around 60,000) was strongly inhibited by inorganic mercury at a concentration which suppressed the release of monosaccharide from autolytically active cell wall. The endo--D-glucanase (MW around 26,000), which showed a marked preference for substrates of mixed-linkage, exhibited features indicating that it initiates the autolytic solubilization of wall glucan.Cell-wall -D-glucan, recovered as a component of an alkali-soluble cell-wall fraction, served as a substrate for the purified glucanases. Their hydrolysis pattern, assessed using gel exclusion chromatography and product analysis, confirmed that they hydrolyze -D-glucan. The products generated by the endoglucanase were similar in molecular-size distribution to those liberated from autolytically active-wall. Exoglucanase activity was required for extensive hydrolysis of -D-glucan in vitro.During coleoptile development the autolytic activity of the cell wall increased dramatically. This increased activity, however, did not parallel the growth potential of the tissue, but more closely reflected an increase in cell-wall -D-glucan, the primary substrate for autolytic reactions.These results were presented, in part, as papers at the annual meeting of the American Society of Plant Physiologists. Ohio State University, Columbus, in August 1979     

Characterization of calcium-dependent forms of protein kinase C in adult rat ventricular myocytes

The presence and subcellular localization of the Ca2+-dependent protein kinase C (PKC) isoforms and were investigated in freshly isolated adult rat cardiac ventricular myocytes. PKC activity was measured in cytosolic and particulate fractions prepared from control myocytes and those treated with either phorbol ester (phorbol 12-myristate 13-acetate, PMA) or a permeant synthetic diacylglycerol analog (1-oleoyl-2-acetylglycerol, OAG) in the absence or presence of an inhibitor of diacylglycerol kinase activity, compound R59022. Preliminary studies detected no Ca2+-/phospholipid-dependent histone kinase activity in either subcellular fraction. To reproducibly observe Ca2+-/phospholipid-dependent protein kinase activity, partial purification using a MonoQ HR 5/5 column and the presence of the peptide inhibitor of the cAMP-dependent protein kinase were essential. MonoQ chromatography of cytosolic and particulate fractions resulted in three peaks of Ca2+/phospholipid-dependent protein kinase activity. In the cytosolic fraction a large peak of activity eluted at 230-300 mM NaCl. Isoform-specific antisera indicated both PKC and PKC were present. In the particulate fraction two peak of Ca2+-/phospholipid-dependent protein kinase activity, both containing PKCa immunoreactivity, were observed. The larger peak eluted at 230-300 mM NaCl. In addition, a peak eluting at lower salt concentrations contained a Ca2+-/phospholipid-independent histone kinase activity. This peak of kinase activity contained PKC immunoreactive bands of 80- and 50-kDa. The 80-kDa band was the holoenzyme of PKC whereas the band of lower molecular mass was likely a proteolytic fragment. In both cytosolic and particulate fractions, the peak of kinase activity eluting at 230-300 mM NaCl contained PKC in the form of an 80-kDa doublet; this suggested the presence of autophosphorylated PKC. Incubation of the myocytes with PMA, but not OAG, resulted in translocation of PKC from the cytosolic to the particulate fraction. Curiously, a transient decrease in PKC activity was observed in both subcellular fractions following treatment with either OAG or ethanol (1%). Results from this study show that freshly isolated adult rat cardiac ventricular myocytes contain both PKC and PKC, and that these isoforms translocate to the particulate fraction in response to treatment with PMA, but not OAG. (Mol Cell Biochem 166: 11-23, 1997)     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

Synthesis and activity of α-glucosidase produced byBifidobacterium pseudolongum

Bifidobacterium pseudolongum NCFB 2244 grew on starch as sole source of carbon and energy, but cell yields and specific growth rates were considerably lower than on glucose (=0.19±0.04 and 0.38±0.09 respectively). Amylase activity was not detected in cultures of this bacterium, but cell-associated -glucosidase was constitutively produced. Analysis of -glucosidase activity from cell extracts by preparative isoelectric focusing gave two peaks of activity with apparent isoelectric points of 3.9 (Enzyme I) and 4.2 (Enzyme II), corresponding to threefold and fourfold purification factors respectively. No -glucosidase activity was detected with Enzyme I after gel-filtration chromatography on Sephadex G150. However, activity was recovered in samples containing Enzyme II, indicating the protein had a molecular mass of approximately 126 kDa. This was subsequently confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). These results show that the restricted ability ofB. pseudolongum to utilize starch as a carbohydrate source is owing to synthesis of at least one, and possibly two, -glucosidase(s).     

The major part of polar carotenoids of the aerobic bacteria Roseococcus thiosulfatophilus RB3 and Erythromicrobium ramosum E5 is not bound to the bacteriochlorophyll a-complexes of the photosynthetic apparatus

The obligate aerobic bacteria Roseococcus thiosulfatophilus RB3 and Erythromicrobium ramosum E5 contain numerous polar carotenoids. The major carotenoid of the strain RB3 was the C₃₀ carotene-dioate (4,4-diapocarotene-4,4-dioate) and the respective diglycosyl ester which have never been isolated before from a bacteriochlorophyll containing bacterium. Strain E5 contains the very polar erythroxanthin sulphate. The major carotenoid bound to reaction center and light-harvesting complexes is bacteriorubixanthinal. Most of the carotenoids of both strains are not bound to the pigment-protein complexes of the photosynthetic apparatus but to the envelope fraction (cytoplasmic membrane and cell wall).Abbreviations Bchl bacteriochlorophyll - MeOH methanol