Ammonia assimilation enzymes in a thermophilicBacillus sp. of marine origin

The physiology of ammonia assimilation enzymes was examined inBacillus sp. FE-1, a thermophilic marine bacterium. Glutamine synthetase (GS) and glutamate synthase (GOGAT) activities varied with the nitrogen source present in the medium, ranging as much as 10-fold for the former and 2.5-fold for the latter. Glutamate dehydrogenase (GDH) was detected but, under the growth conditions studied, levels were not affected by the nitrogen source. Anaerobic growth in the presence of nitrate yielded enzyme levels that were not significantly different from those measured under aerobic growth. Partially purified GS exhibited a temperature optimum between 65° and 75°C. The enzyme's Mn²⁺-dependent reverse transferase activity was stimulated by K₂SO₄ and demonstrated some tolerance to NaCl. Hyperbolic kinetics were observed for ammonium, with an apparentK _M of 1.0mm.     

Clostridium autoethanogenum,sp. nov., an anaerobic bacterium that produces ethanol from carbon monoxide

A strictly anaerobic, gram-positive, sporeforming, rod-like, motile bacterium was enriched from rabbit feces, and isolated using carbon monoxide as sole source of energy and carbon. The isolate metabolizes CO with ethanol, acetate and CO₂ as end-products. Other substrates used as carbon and energy sources include CO₂ plus H₂, pyruvate, xylose, arabinose, fructose, rhamnose, and l-glutamate. The optimum temperature for growth is 37°C. The optimum pH for chemolithotrophic growth lies around 5.8 to 6.0 Sulfate is not reduced. Growth is inhibited either by penicillin, chloramphenicol, tetracyclin or ampicillin, each at 100 g per ml. The isolate has a DNA-base composition of 25.9±0.6% guanine plus cytosine. The isolate represents a new species of Clostridium for which the name Clostridium autoethanogenum is proposed. The type strain is strain JA1-1     

Propionigenium modestum gen. nov. sp. nov. a new strictly anaerobic,nonsporing bacterium growing on succinate

From marine and freshwater mud samples and from human saliva new strictly anaerobic, Gram-negative, nonsporeforming bacteria were isolated growing with succinate as sole source of carbon and energy. All strains grew in defined mineral media containing at least 1% sodium chloride. Succinate was stoichiometrically transformed to propionate und carbon dioxide; the growth yield varied between 2.1 and 2.4 g cell dry weight per mol of succinate fermented. In addition to succinate, only fumarate, l-aspartate, l-malate, oxaloacetate and pyruvate, were utilized and were stoichiometrically fermented to propionate and acetate. Yeast extract was not fermented but enhanced growth rates and yields. Neither sulfate, sulfur, nor nitrate were reduced. The DNA base ratio was 33.9±0.3 mol % guanine plus cytosine. A marine isolate, strain Gra Succ 2, is described as type strain of a new species, Propionigenium modestum gen. nov. sp. nov., in the family Bacteroidaceae.     

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK ₁=0.67±0.16 m,k ₂=1.6±0.7 sec^–1,k _–2=0.17±0.09 sec^–1,K ₁=6.3±1.7 m,k ₂=9±4 sec^–1 andk _–2=7±3 sec^–1. The apparent dissociation constants of chloride from band 3,K _Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.     

Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

Summary In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands ofSqualus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170mm KCl gradient was initially about five-fold higher than in the presence of a 170mm KNO₃ gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4mm bumetanide and 40% by 0.8mm N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170mm NaCl gradient than in the presence of a 170mm NaNO₃ gradient. The effect of chloride was completely abolished by 0.4mm bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound³[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparentK _D of 3.5×10^–6 m and a number of sitesn of 104 pmol/mg protein. At 0.8 m, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600mm or when 1mm bumetanide or furosemide were added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.     

Isolation and characterization of a novel polysaccharide fromBacillus licheniformis NCIB 11634

Summary A polysaccharide producing strain ofBacillus licheniformis was isolated from exudate of raffia palm,Raffia vinifera. The optimum conditions for growth and polysaccharide production have been investigated and established. No appreciable polysaccharide was formed on glucose. It grew best in Czapek-Dox media with sucrose as the carbon source. The polysaccharide has been characterized as a heteropolymer containingd-glucose,d-mannose andd-xylose.     

Evaluation ofDrosophila melanogaster as an alternative animal for studying the neurotoxicity of heavy metals

Heavy metals cause irreversible neurobehavioral damage in many developing mammals, but the mechanisms of this damage are unknown. The influence of three heavy metal compounds, triethyllead chloride, lead acetate and cadmium chloride, on lethality, development, behavior and learning was studied using the fruit fly,Drosophila melanogaster. This animal was used because it allows hundreds of subjects to be assayed very easily in individual experiments and because it is a system in which toxicological questions might be answered by using the techniques of modern molecular genetics. When triethyllead chloride, lead acetate or cadmium chloride was placed in the medium, the larval LC₅₀ (± standard error) was found to be 0.090±0.004, 6.60±0.64 and 0.42±0.04mm, respectively. Each of the tested compounds produced a dose-related delay in development. In particular, they caused a delay in the development of larvae to pupae. When larvae were reared on medium containing triethyllead chloride (0.06mm), lead acetate (3.07mm) or cadmium chloride (0.11mm), phototaxis, locomotion and learning were not inhibited. Since significant neurobehavioral effects were not observed under the experimental conditions used,Drosophila does not appear to be an appropriate animal for the genetic dissection of such effects of heavy metals during development.     

Effect of culture conditions on astaxanthin production by a mutant of Phaffia rhodozyma in batch and chemostat culture

Temperature and pH had only a slight effect on the astaxanthin content of a Phaffia rhodozyma mutant, but influenced the maximum specific growth rate and cell yield profoundly. The optimum conditions for astaxanthin production were 22°C at pH 5.0 with a low concentration of carbon source. Astaxanthin production was growth-associated, and the volumetric astaxanthin concentration gradually decreased after depletion of the carbon source. The biomass concentration decreased rapidly during the stationary growth phase with a concomitant increase in the cellular content of astaxanthin. Sucrose hydrolysis exceeded the assimilation rates of D-glucose and D-fructose and these sugars accumulated during batch cultivation. D-Glucose initially delayed D-fructose uptake, but D-fructose utilization commenced before glucose depletion. In continuous culture, the highest astaxanthin content was obtained at the lowest dilution rate of 0.043 h^–1. The cell yield reached a maximum of 0.48 g cells·g^–1 glucose utilized between dilution rates of 0.05 h^–1 and 0.07 h^–1 and decreased markedly at higher dilution rates. Correspondence to: J. C. Du Preez     

Isolation,identification and characterisation of a soil bacterium producing an enzyme with l-phenylalanine oxidase activity

A gram-positive, mesophilic bacterium which assimilated l-phenylalanine but which failed to utilise l-tyrosine was isolated from soil. The isolate, identified as a strain of Bacillus carotarum, converted l-phenylalanine to phenylpyruvate with the initial step catalysed by an inducible, intracellular enzyme which possessed l-phenylalanine oxidase activity. Phenylalanine oxidase has not been previously reported in Gram-positive bacteria, although there are a few examples of non-specific l-amino acid oxidases with activity towards l-phenylalanine. The isolate grew abundantly on complex media but failed to synthesise significant amounts of the enzyme in the absence of l-phenylalanine. The highest enzyme levels were achieved in a chemically defined minimal salts medium containing the amino acid at 10 g/l as the primary carbon and energy source.     

Ionic dependence of bumetanide binding to the rabbit parotid Na/K/Cl cotransporter

Summary The Na/K/Cl-dependent component of the binding of the loop diuretic bumetanide to basolateral membrane vesicles from the rabbit parotid is studied. A Scatchard analysis indicates that this binding is due to a single high-affinity site withK _D =3.2±0.3 m (n=9) at 100mm sodium, 100mm potassium and 5mm chloride. When KCl-dependent²²Na transport and tracer [³H]-bumetanide binding are monitored simultaneously as a function of (unlabeled) bumetanide concentration it is found that theK _0.5 for bumetanide inhibition of both processes are identical indicating that the high-affinity bumetanide binding site studied here is identical with a bumetanide-inhibitory site on the Na/K/Cl cotransport system previously identified in this preparation (R.J. Turner, J.N. George and B.J. Baum,J. Membrane Biol. 94:143–152, 1986). High-affinity bumetanide binding exhibits a hyperbolic dependence on both [Na] and [K] consistent with Na/bumetanide and K/bumetanide binding stoichiometries of 11 andK _0.5 values of approximately 33mm for sodium and 23mm for potassium. In contrast, the dependence on [Cl] is biphasic, with bumetanide binding increasing from 0 to 5mm chloride and decreasing toward baseline levels thereafter. Scatchard analysis of this latter inhibitory effect of chloride indicates a competitive interaction with bumetanide in agreement with earlier indications that bumetanide inhibits Na/K/Cl cotransport at a chloride site. However, studies of the effects of various anions on bumetanide binding and²²Na transport show a poor correlation between the specificities of these two processes, suggesting that the inhibitory chloride site is not a chloride transport site.     

Phosphate requirements of the nitrifying bacteria

The influence of the phosphate concentration on the specific growth rate and the duration of lag has been studied inNitrobacter winogradskyi andNitrosomonas europaea.The optimum phosphate concentration range for the specific growth rate was 10 to 30mm forNitrobacter and 10 to 100mm forNitrosomonas. In this range the lag was least. Depletion of the cell-P does not affect the relation between specific growth rate and phosphate concentration while the lag seems to increase as cell-P depletion proceeds.     

Some effects of organic acid supplementation on utilization of ammonium compounds by three pathogenic fungi

Utilization of ammonium nitrogen byFusarium moniliforme Sheld.,Curvularia verruciformis Agarwal &Sahni andSclerotium rolfsii Sacc., with and without organic acid supplementation was studied. Ammonium utilization by these organisms generally improved with small amounts of organic acid supplementation except in the case ofC. verruciformis on ammonium chloride where fumaric acid was observed to suppress utilization. Effects of supplementation were seen to be dependent on the nature of the organic acid, the ammonium source used and the organism employed. Further evidence obtained by varying the quantities of succinic acid in ammonium chloride media showed that higher levels of supplementation gave increased growth of the fungi and prevented the pH from falling. It seems that these acids play a dual role as pH buffers as well as nutritional compounds.     

Characterization of an extremely thermophilic sulphur-metabolizing archaebacterium belonging to the Thermococcales

An extremely thermophilic, obligately anaerobic, sulphur-metabolizing archaebacterium of the order Thermococcales, previously isolated from a thermal pool at Kuirau Park, Rotorua, New Zealand, partially described, and designated isolate ANI, Thermococcales was further characteized. The isolate was a regular coccus of 0.5–2.0 mm in diameter, was strictly anaerobic, chemoorganotrophic, and fermentative. Peptone, yeast extract, or casein served as carbon and nitrogen source, and a variety of amino acids and glucose, but not organic acids, carbohydrates, or other sugars supported growth in the presence of peptone (0.1%). Major metabolic end products were H₂, sulphide, acetate, isobutyrate, and isovalerate/2-methylbutyrate. Isolate ANI had a temperature optimum of 75–80°C, a pH optimum of 7.4, and a sodium chloride concentration optimum of 50mM. No growth was observed in the absence of sodium chloride (or lithium chloride) and sulphur (or cystine or oxidized glutathione).     

Isolation and Growth Characteristics of Chromium(VI) and Pentachlorophenol Tolerant Bacterial Isolate from Treated Tannery Effluent for its Possible Use in Simultaneous Bioremediation

The bacterial strains resistant to pentachlorophenol (PCP) and hexavalent chromium [Cr(VI)] were isolated from treated tannery effluent of a common effluent treatment plant. Most of the physico-chemical parameters analyzed were above permissible limits. Thirty-eight and four bacterial isolates, respectively were found resistant to >50 μg/ml concentration of [Cr(VI)] and the same level of PCP. Out of the above 42 isolates, only one was found simultaneously tolerant to higher levels of both PCP (500 μg/ml) and Cr(VI) (200 μg/ml), and hence was selected for further studies. To the best of our knowledge, this is the first report in which a native bacterial isolate simultaneously tolerant to such a high concentrations of Cr(VI) and PCP has been reported. The culture growth was best at 0.4% (w/v) glucose as an additional carbon source and 0.2% (w/v) ammonium chloride as a nitrogen source. The growth results with cow urine as a nitrogen source were comparable with the best nitrogen source ammonium chloride. The isolate exhibited resistance to multiple heavy metals (Pb, As, Hg, Zn, Co & Ni) and to antibiotics nalidixic acid and polymixin-B. The efficacy of bacterial isolate for growth, PCP degradation (56.5%) and Cr(VI) bioremediation (74.5%) was best at 48 h incubation. The isolate was identified as Bacillus sp. by morphological and biochemical tests. The 16S rDNA sequence analysis revealed 98% homology with Bacillus cereus. However, further molecular analysis is underway to ascertain its likelyhood of a novel species.     

Isolation and Characterization of a Hyphomicrobium Species and its Polysaccharide Formation from Methanol

A microorganism which could utilize methanol as the sole source of carbon and excreted a new polysaccharide was isolated from soil. This isolate was a stalked bacterium which reproduced by a budding process, and could grow on only methanol, formaldehyde or methylamines as the carbon source. The most suitable nitrogen source for growth was the ammonium ion. The optimum pH and temperature for growth were about 7.0 and 30°C, respectively. The cell growth was inhibited by blue light irradiation. Amino acid composition and fatty acid composition of the cells and electrophoretic behavior of methanol dehydrogenase were also studied. On the basis of these properties as well as taxonomical studies, the isolate (strain JTS-811) was identified as belonging to the genus Hyphomicrobium. This strain had different characteristics as compared to those described for other Hyphomicrobium isolates. At present, it is difficult to give a specific name to this strain, because classification of hyphomicrobia is not clear.     

Evaluation of alternative nitrogen and carbon sources for sugarbeet suspension culture platings in development of cell selection schemes

Summary Low molecular weight nitrogenous impurity compounds as well as raffinose are negative quality factors that interfere with efficient processing of sugarbeet (Beta vulgaris L.) for sucrose. In order to identify nutrient media for cell selection of biochemical mutants or transgenics that might have reduced levels of these processing impurities, the ability of 10 endogenous compounds to serve as sole nitrogen or carbon source for suspension plating and subculture callus growth was evaluated. The most productive concentrations of nitrate, ammonium, l-glutamine, l-glutamate, urea, and l-proline as sole nitrogen sources supported plating callus growth at 106, 159, 233, 167, 80, and 52%, respectively, as well as the historical 60 mM mix of nitrate and ammonium in Murashige-Skoog medium. Glycine betaine and choline did not support growth. d(+) Raffinose and d(+) galactose supported plating callus growth only 67 and 25%, respectively, as well as sucrose as sole carbohydrate source. No callus growth occurred on glutamine, glutamate, or glycine betaine as the sole carbon or carbon plus nitrogen source. Platings on either nitrate or ammonium as sole nitrogen source did not differ in sensitivity to the nitrate uptake inhibitor phenylglyoxal, suggesting that phenylglyoxal lacks the specificity for use in selection for mutants of nitrate uptake. The ability of raffinose to be used as the carbon source, and glutamine or glutamate as the nitrogen source, may preclude their use for selection of genetic variants accumulating less of these processing impurities. However, mutants or transgenics able to utilize either glutamine, glutamate, or glycine betaine might be selectable on media containing any one of these as carbon, nitrogen, or carbon plus nitrogen source, respectively, that is incapable of supporting wild-type cell growth.     

Characterization of a peptidase fromDiplococcus pneumoniae

Some properties of a purified peptidase fromDiplococcus pneumoniae have been studied. The enzyme has a broad pH optimum between 6 and 8 and a K_m (onl-leucylglycylglycine) of 2.8mm. It is activated by low levels of Hg⁺⁺ and is inhibited by Mn⁺⁺, Co⁺⁺, β-mercaptoethanol and EDTA. Substrate specificity studies show that the enzyme is an exopeptidase of the aminopeptidase type, most active on tripeptide substrates bearing bulky substituents at the NH₂ ⁻ terminal end.     

<Emphasis Type="Italic">Thermincola ferriacetica</Emphasis> sp. nov<Emphasis Type="Italic">.,</Emphasis> a new anaerobic,thermophilic, facultatively chemolithoautotrophic bacterium capable of dissimilatory Fe(III) reduction

A moderately thermophilic, sporeforming bacterium able to reduce amorphous Fe(III)-hydroxide was isolated from ferric deposits of a terrestrial hydrothermal spring, Kunashir Island (Kurils), and designated as strain Z-0001. Cells of strain Z-0001 were straight, Gram-positive rods, slowly motile. Strain Z-0001 was found to be an obligate anaerobe. It grew in the temperature range from 45 to 70°C with an optimum at 57–60°C, in a pH range from 5.9 to 8.0 with an optimum at 7.0–7.2, and in NaCl concentration range 0–3.5% with an optimum at 0%. Molecular hydrogen, acetate, peptone, yeast and beef extracts, glycogen, glycolate, pyruvate, betaine, choline, N-acetyl-d-glucosamine and casamino acids were used as energy substrates for growth in presence of Fe(III) as an electron acceptor. Sugars did not support growth. Magnetite, Mn(IV) and anthraquinone-2,6-disulfonate served as the alternative electron acceptors, supporting the growth of isolate Z-0001 with acetate as electron donor. Formation of magnetite was observed when amorphous Fe(III) hydroxide was used as electron acceptor. Yeast extract, if added, stimulated growth, but was not required. Isolate Z-0001 was able to grow chemolithoautotrophicaly with molecular hydrogen as the only energy substrate, Fe(III) as electron acceptor and CO₂ as the carbon source. Isolate Z-0001 was able to grow with 100% CO as the sole energy source, producing H₂ and CO₂, requiring the presence of 0.2 g l⁻¹ of acetate as the carbon source. The G+C content of strain Z-0001^T DNA G+C was 47.8 mol%. Based on 16S rRNA sequence analyses strain Z-0001 fell into the cluster of family Peptococcaceae, within the low G+C content Gram-Positive bacteria, clustering with Thermincola carboxydophila (98% similarity). DNA–DNA hybridization with T. carboxydophila was 27%. On the basis of physiological and phylogenetic data it is proposed that strain Z-0001^T (=DSMZ 14005, VKM B-2307) should be placed in the genus Thermincola as a new species Thermincola ferriacetica sp. nov. The GenBank accession number for the sequence reported in the paper is AY 631277.     

Isolation and characterization of a novel mesophilic,fresh-water methanogen from river sediment Methanoculleus oldenburgensis sp. nov.

A new mesophilic, irregular coccoid methanogen isolated from a river sediment is described. Hydrogen plus carbon dioxide or formate served as substrates for methanogenesis in a mineral salt medium. For growth acetate is strictly required. Elevated levels of sodium chloride were not required and were inhibitory at concentrations above 1.5% (w/v). The optimal growth temperature was at 45°C. The DNA base ratio was 48.6±1 mol% G+C. The polar lipid pattern and the polyamine content were similar to that found in several Methanoculleus species. The new isolate CB-1 was assigned as Methanoculleus oldenburgensis (DSM 6216).     

The utilization of the mixtures of monosaccharides by some mucorales

Summary The utilization of mixtures of monosaccharides byBlakeslea trispora Thaxter,Choanephora circinans (Naganish &Kawakami)Hesseltine &Benjamin,Gilbertella persicaria var.indica Mehrotra &Mehrotra andHelicostylum piriforme Bainier was studied. The effect of sorbose on the utilization of other sugars present in the mixtures was also studied. It was found that all the mixtures of sugars in combination with asparagine or ammonium chloride were valueless for all the organisms exceptHelicostylum piriforme. Growth ofHelicostylum piriforme on the mixtures with asparagine as the nitrogen source was better than on the mixtures with ammonium chloride as the source of nitrogen. Asparagine being a favourable source counteractecd sorbose inhibition, while ammonium chloride failed to do so. On the other hand, both of the nitrogen sources failed to counteract sorbose inhibition in the rest of the organisms. None of the organisms could finish sorbose and rhamnose from any of the mixtures within the specified period.