Ilyobacter delafieldii sp. nov., a metabolically restricted anaerobic bacterium fermenting PHB

Enrichments from an estuarine sediment with crotonate as substrate resulted in the isolation of a motile, gram-negative, obligately anaerobic rod with pointed ends, designated strain 10cr1. The organism was asporogenous, did not reduce sulfur, sulfate, thiosulfate, nitrate, oxygen or fumarate, and had a mol %G+C ratio of 29. Strain 10cr1 was able to ferment crotonate, 3-hydroxybutyrate, lactate, pyruvate, and poly--hydroxybutyric acid (PHB). Acetate, propionate, butyrate, CO₂ and H₂ were the fermentation products. When grown on PHB there was accumulation of 3-hydroxybutyrate once growth had ceased, indicating degradation of PHB to the monomer. The 3-hydroxybutyrate formed during growth of the culture was fermented to acetate, butyrate and H₂. Experimental evidence suggested the production of an extracellular PHB depolymerase. The cells were not attached to the PHB granules. This is the first isolation of an anaerobic bacterium capable of degrading exogenous PHB. This strain is described as a new species, Ilyobacter delafieldii sp. nov., and strain 10cr1 (=DSM 5704) is designated as the type (and at present, only) strain.Abbreviations G+C guanine plus cytosine - OD optical density - PHB poly--hydroxybutyric acid - specific growth rate - HPLC high-performance liquid chromatography - YE yeast extract     

Acetone production by methylobacteria

An accumulation of acetone was observed during the metabolism of ethane and products of ethane oxidation by washed suspensions of Methylosinus trichosporium OB3B. This strain possessed an acetoacetate decarboxylase and 3-hydroxybutyrate dehydrogenase, and a decline in poly--hydroxybutyric acid occurred under the same conditions as acetone formation. A pathway of acetone production from poly--hydroxybutyric acid via 3-hydroxybutyrate and acetoacetate was suggested.     

Adenine nucleotide contents and energy charge of Azotobacter vinelandii grown at low phosphate concentration

The effect of a low phosphate concentration on intracellular adenine nucleotide content, oxygen consumption and poly--hydroxybutyrate deposition was investigated with N-free and NH ₄ ⁺ batch cultures of Azotobacter vinelandii. When the microorganisms were cultured under low-phosphate concentrations the cells contained much larger amounts of poly--hydroxybutyrate, but displayed lower oxygen consumption activities and energy charge values than did control cells. Also, the ratio ATP to ADP was much higher in control cells and the intracellular levels of ATP were lower in low-phosphate cells.     

Competition between β-ketothiolase and citrate synthase during poly(β-hydroxybutyrate) synthesis inMethylobacterium rhodesianum

The enzymes -ketothiolase and citrate synthase from the facultatively methylotrophicMethylobacterium rhodesianum MB 126, which uses the serine pathway, were purified and characterized. The -ketothiolase had a relatively highK _m for acetyl-CoA (0.5 mM) and was strongly inhibited by CoA (K _i 0.02 mM). The citrate synthase had a much higher affinity for acetyl-CoA (K _m 0.07 mM) and was significantly inhibited by NADH (K _i 0.15 mM). The intracellular concentration of CoA metabolites and nucleotides was determined inM. rhodesianum MB 126 during growth on methanol. The level of CoA decreased from about 0.6 nmol (mg dry mass)^-1 during growth to the detection limit when poly(-hydroxybutyrate) (PHB) accumulated. Nearly unchanged intracellular concentrations of NADH, NADPH, and acetyl-CoA of about 0.5, 0.6–0.7, and 1.0 nmol (mg dry mass)^-1, respectively, were determined during growth and PHB synthesis. During growth, the -ketothiolase was almost completely inhibited by CoA, and acetyl-CoA was principally consumed by the citrate synthase. During PHB accumulation, the -ketothiolase had about 75% of its maximum activity and showed much higher activity than citrate synthase, which at the actual NADH concentration was about 75% inhibited. NADPH concentration was sufficiently high to allow the unlimited activity of acetoacetyl-CoA reductase (K _mNADPH 18 M). PHB synthesis is probably mainly controlled by the CoA concentration inM. rhodesianum MB 126.Abbreviation PHB Poly(-hydroxybutyrate)     

Metabolism of Poly-β-Hydroxybutyric Acid in Yersinia pseudotuberculosis and Listeria monocytogenes Cultivated at Different Temperatures

A comparative investigation of the intracellular content of poly--hydroxybutyric acid showed that Yersinia pseudotuberculosis strains accumulated, on the average, lower amounts of this reserve substance than Listeria monocytogenes strains. The intracellular pool of poly--hydroxybutyric acid was responsible for the growth of the bacteria at low temperatures (4–6°C) in the absence of any exogenous carbon and energy source.     

Regulation of polyphosphate metabolism in Acinetobacter strain 210A grown in carbon- and phosphate-limited continuous cultures

The response of Acinetobacter strain 210A to low phosphate concentrations was investigated in P- or C-limited chemostat cultures. The organism accumulated poly--hydroxybutyric acid under P-deprivation, at phosphate concentrations ranging from 0.1 to 0.7 mM. The amount of biomass was proportional to the phosphate concentration in the medium and no polyphosphate was formed. When shifting a culture from P- to C-limitation phosphate was accumulated as polyphosphate. No poly--hydroxybutyrate could be detected in these cells. The amount of polyphosphate in the cell showed a hysteresis. When cultures were shifted from low to high phosphate concentrations, polyphosphate reached a maximum of about 60 mg P per gram of dry weight at about 3 times excess phosphate (ca. 2.5 mM P_i). It decreased to 45 mg P per gram dry weight at approximately 5 times the phosphate needed for growth (ca. 3.5 mM P_i). In the reverse case (high to low) polyphosphate did never exceed 45 mg P per gram dry weight. The specific activities of alkaline phosphatase and the phosphate uptake system were induced at residual P_i concentrations below the detection limit (<10 M). The specific uptake rate followed also a hysteresis. The specific activities of polyphosphatase and polyphosphate: AMP phosphotransferase increased when polyphosphate formation was possible.Abbreviations HPP High polymeric polyphosphates - PHB Poly--hydroxybutyric acid - PP_n Polyphosphate - PQQ Pyrrolo-quinoline quinone - U 1 mol product formed · min^-1     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Metabolite concentrations in Alcaligenes eutrophus H 16 and a mutant defective in poly-β-hydroxybutyrate synthesis

Intracellular concentrations of hexose phosphates, phosphoenolpyruvate, pyruvate, NAD(H) and NADP(H) as well as the protein and poly--hydroxybutyrate (PHB) content were measured in suspensions of autotrophically grown cells of Alcaligenes eutrophus H 16 and compared with those in a mutant unable to synthesize poly--hydroxybutyrate. The parent strain was subjected to successive changes in conditions, and new steady states were rapidly (20 min) attained. When the parent strain was provided with carbon and energy but no nitrogen source, it fixed CO₂ and accumulated large amounts of PHB. When the mutant PHB^-4 was exposed to identical conditions, no accumulation of PHB occurred, but pyruvate, malate and citrate were excreted, and a 6-fold accumulation of hexose monophosphate (over the levels in the parent) was observed: in contrast, cofactors in intermediates between fructose-1,6-phosphate and phosphoenolpyruvate reached steady state as in the parent strain. When ammonium ion was then supplied, growth started and the metabolite concentrations in the mutant returned to the levels observed in the parent strain.     

Enzymatic synthesis of poly-β-hydroxybutyrate inZoogloea ramigera

The enzyme activity synthesizing poly--hydroxybutyrate (PHB) was mainly localized in the PHB-containing particulate fraction ofZoogloea ramigera I-16-M, when it grew flocculatedly in a medium supplemented with glucose. On the other hand, the enzyme activity remained in the soluble fraction, when the bacterium grew dispersedly in a glucose-starved medium.The soluble PHB synthase activity became associated with the particulate fraction as PHB synthesis was initiated on the addition of glucose to the dispersed culture. Conversely, the enzyme activity was released from the PHB-containing granules to the soluble fraction when the flocculated culture was kept incubated without supplementing the medium with glucose.PHB synthase was also incorporated into the newly formed PHB fraction when partially purified soluble PHB synthase was incubated withd(-)--hydroxybutyryl CoA in vitro.Although attempts to solubilize the particulate enzyme were unsuccessful, and the soluble enzyme became extremely unstable in advanced stages of purification, both PHB synthases had the same strict substrate specificity ford(-)--hydroxybutyryl CoA, and showed the same pH optimum at 7.0.Non-Standard Abbreviations PHB poly--hydroxybutyrate     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Nucleotide sequence of the Clostridium thermocellum bgIB gene encoding thermostable beta-glucosidase B: homology to fungal beta-glucosidases

Summary The nucleotide sequence of the bglB gene, coding for the thermostable -glucosidase B of Clostridium thermocellum was determined. The coding region of 2265 bp was identified by comparison with the N-terminal amino acid sequence of -glucosidase B purified from Escherichia coli. The derived amino acid sequence corresponding to a polypeptide of M_r 84100 was confirmed by sequencing of the C-terminal peptide generated by cleavage with cyanogen bromide. The protein bears no resemblance to other bacterial -glucosidase sequences. However, extensive regions of homology were identified between the C. thermocellum enzyme and fungal -glucosidases. The N-terminal homologous region contains an amino acid sequence very similar to the active site of -glucosidase A₃ from Aspergillus wentii. The striking sequence similarities between C. thermocellum -glucosidase B and Kluyveromyces fragilis -glucosidase suggest the possibility of a genetic exchange between thermophilic anaerobic bacteria and yeasts.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

The linear tetrasaccharide,Galβ1-4GlcNAcβ1-6Galβ1-4GlcNAc,isolated from radiolabeled teratocarcinoma poly-N-acetyllactosaminoglycan resists the action ofE. freundii endo-β-galactosidase

A novel linear tetrasaccharide, Gal1-4GlcNAc1-6Gal1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAc1-6Gal1-4GlcNAc, resisted the action of endo--galactosidase (EC 3.2.1.103) fromE. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Gal1-4GlcNAc1-3Gal1-4GlcNAc and GlcNAc1-3Gal1-4GlcNAc were cleaved in the expected manner.Abbreviations WGA wheat germ agglutinin - BSA bovine serum albumin - [³H]GlcNAc1-4-GlcNAc1-4GlcNAcOL N,N,NN'-triacetylchitotriose reduced with NaB³H₄     

An NADP-linked acetoacetyl CoA reductase from Zoogloea ramigera

Zoogloea ramigera I-16-M was found to contain two stereospecific acetoacetyl CoA reductases; one was NADP⁺-linked and d(-)--hydroxybutyryl CoA specific and the other was NAD⁺-linked and l(+)-isomer specific. The NADP⁺-linked enzyme, purified approximately 150-fold, had a pH optimum for the reduction of acetoacetyl CoA at 8.1, but no definite pH optimum for the oxidation of -hydroxybutyryl CoA. The apparent Michaelis constants for acetoacetyl CoA and NADPH were 8.3 and 21 M, respectively. The enzyme was markedly inhibited by acetoacetyl CoA at concentrations higher than 10 M.The incorporation of [1-¹⁴C]acetyl CoA into poly--hydroxybutyrate (PHB) by bacterial crude extract (containing -ketothiolase, acetoacetyl CoA reductases, enoyl CoA hydratases and PHB synthases) or by a system reconstituted from purified preparations of -ketothiolase, acetoacetyl CoA reductase and PHB synthase, was observed only in the presence of NADPH, but not NADH. Among various enzymes involved in PHB metabolism, only the specific activity of glucose 6-phosphate dehydrogenase was elevated 5-fold within 2 h after the addition of glucose to the cells grown in the basal medium.These findings suggest that, in Z. ramigera I-16-M, acetoacetyl CoA is directly reduced to d(-)--hydroxybutyryl CoA by the NADP⁺-dependent reductase, and PHB synthesis is at least partially controled by NADPH availability through glucose 6-phosphate dehydrogenase.Non-Standard Abbreviation PHB poly--hydroxybutyrate     

Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L.

In contrast to undifferentiated cell suspension cultures of Digitalis lanata, photomixotrophic shoot cultures of Digitalis purpurea accumulate cardiac glycosides in substantial concentrations. They are used to investigate enzymes of the cardenolide pathway. All cardenolides are 5-configurated. The progesterone 5-reductase and the 3-hydroxysteroid-5-oxidoreductase are present in shoot cultures but not in undifferentiated cell cultures. These enzymes provide precursors for cardenolides, whereas the presence of the progesterone 5-reductase, also present in shoot cultures, is discussed with regard to its role in phytosterol biosynthesis and may be attributed to the general steroid pathway. The progesterone 5-reductase had an activity maximum during the early growth period seven days after onset of cultivation, whereas the corresponding progesterone 5-reductase activity was highest on day 11. The maximum cardenolide accumulation was after 24 days. The enzyme activities present in crude extracts from shoot cultures were characterized with regard to their requirements for NADPH and NADH, pH-optimum, temperature optimum, affinity to their substrates and their localization in the cell. The progesterone 5-reductase was purified 769-fold.Abbreviations DW dry weight - FW fresh weight - PVP polyvinylpyrrolidone     

β-Adrenoceptors in the Tree Shrew Brain. II. Time-Dependent Effects of Chronic Psychosocial Stress on [125I]Iodocyanopindolol Binding Sites

1. Stress is known to affect the functioning of the central noradrenergic system in a region-specific manner. The aim of the present investigation was to understand better the consequences of recurrent stressful experiences on central -adrenoceptors.2. Alterations in the central nervous -adrenoceptor system resulting from different periods of psychosocial stress (PSS) were analyzed in male tree shrews (Tupaia belangeri) which were submitted to subordination stress for varying time periods.3. In the first experiment, the whole number of -adrenoceptors was analyzed in the forebrains of subordinate animals and controls by in vitro autoradiography using ¹²⁵I-iodocyanopindolol (¹²⁵ICYP), while nonspecific binding of the radioligand to serotonin receptors was blocked with 100 M 5HT.4. PSS affects -adrenoceptors in a time-dependent manner. A decrease in receptor affinity occurred after just 21 days of PSS in cortical areas and in the hippocampus, indicating stress effects on the conformation of -adrenoceptors. After 30 days of PSS,the numbers of -adrenoceptors were significantly decreased in several cortical regions and in the olfactory area.5. In the second experiment, we investigated the influence of PSS on both ₁- and ₂-adrenoceptors separately. ¹²⁵ICYP binding was quantified in the presence of either ICI188.551 to block ₂-adrenoceptors or in the presence of CGP20712A to block ₁-adrenoceptors.6. After 2, 10, 21,and 28 days of PSS, it become apparent that the two -adrenoceptor subtypes are regulated independently. ₁-Adrenoceptors were transiently down-regulated after 2 days of PSS in the prefrontal cortex and in the olfactory area and were decreased after 28 days of PSS in the parietal cortex and the hippocampus. A transient up-regulation of ₁-adrenoceptors occurred in the pulvinar nucleus after 10 days of PSS. ₂-Adrenoceptors were transiently down-regulated after 2 days of PSS in the prefrontal cortex and up-regulated in the pulvinar nucleus after 28 days of PSS.7. These data demonstrate that chronic psychosocial stress in subordinate tree shrews leads to time-dependent changes in the central nervous -adrenoceptors system.8. The high regional variability in stress-induced -adrenoceptor regulation is supposed to be due to the complex mechanisms of intracellular -adrenoceptor sequestration, which includes down-regulation and/or reinsertion of receptors into the plasma membrane. These mechanisms may be important components of the regulatory apparatus which enables the individual to adapt to situations of recurrent stressful experiences by balancing the central nervous adrenoceptor number.     

Subunit structures of multiple hemoglobins in carp

Three hemoglobin components in carp designated CI, CII, and CIII, were isolated by DEAE-Toyopearl ion-exchange chromatography. Constituent globin chains, ¹, ², ¹ and ², were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography with a reversed-phase column. Tryptic peptide mapping indicated that the -globin chains of the three hemoglobin components have slightly different structures. In addition, N-terminal amino acid sequence analysis indicated that the ¹-globin chain has a primary structure different from that of the ²-chain. A series of hybridization experiments between isolated hemoglobins, together with such structural properties of globin chains, suggested that the three hemoglobins have the following compositions: CI (^{1 2} ₂ ¹ ), CII (^{1 2 1 2}), and CIII (^{1 2} ₂ ² ). Hemoglobin CII was a hybrid between the two types each of - and -chain and could be constructed in vitro from two hemoglobin components CI and CIII.Abbreviations a-a amino acid - Hb hemoglobin - HPLC high performance liquid chromatography - P ₅₀ oxygen pressure at half saturation - PAGE polyacrylamide gel electrophoresis - TFA trifluoroacetic acid     

Activities ofβ-glucanases andβ-glucosidases during blastospore formation inSaccharomycopsis fibuligera

Summary The activities of three glycosidases, -glucosidase and (1,3)- and (1,6)-glucanases have been monitored during growth and blastospore formation inSaccharomycopsis fibuligera. The assays were carried out on the cell-free culture and in a cell-free extract and a wall autolysate preparation from the growing cells. In complex medium containing 1% glucose an increase in the level of all three enzymes was associated with the transition from mycelium to blastospores. When the level of glucose was increased to 5% blastospore formation was repressed and the level of -glucanases only increased at the end of the fermentation. The -glucosidase activity increased during the growth phase. In a defined medium in which slow growth in a wholly yeast-like form was observed, growth was not associated with a high level of -glucanase activity.