N2 fixation and NH 4 + assimilation in the thermophilic anaerobes Clostridium thermosaccharolyticum and Clostridium thermoautotrophicum

Inorganic nitrogen metabolism in the obligate anaerobic thermophiles Chlostridium thermosaccharolyticum and Clostridium thermoautotrophicum differs in several respects. C. thermosaccharolyticum contains a nitrogenase as inferred from NH ₄ ⁺ repressible C₂H₂ reduction, a glutamine synthetase which is partially repressed by ammonium, very labile glutamate synthase activities with both NADH and NADPH, NADPH-dependent glutamate dehydrogenase, and NH ₄ ⁺ -dependent asparagine synthetase. C. thermoautotrophicum contains no nitrogenase, but glutamine synthetase, no glutamate synthase, no glutamate dehydrogenase, but a NADH-dependent alanine dehydrogenase and a NH ₄ ⁺ -dependent asparagine synthetase.Abbreviation GOGAT glutamine-oxoglutarate amidotransferase amidotransferase (glutamate synthase)     

Surface properties from the S-layer of Clostridium thermosaccharolyticum D120-70 and Clostridium thermohydrosulfuricum L111-69

Labelling experiments using a positively charged topographical marker for electron microscopy, polycationized ferritin, showed that the S-layers of two closely related clostridia Clostridium thermohydrosulfuricum L111-69 and C. thermosaccharolyticum D120-70 do not exhibit a net negative charge, as usually observed for bacterial cell surfaces. Chemical modification of reactive sites confirmed that amino and carboxyl groups are exposed on the S-layer surface of both strains. Amino-specific, bifunctional agents crosslinked both S-layer lattices. Studies with carbodiimides revealed that only the S-layer surface of C. thermohydrosulfuricum L111-69 had amino and carboxyl groups closely enough aligned to permit electrostatic interactions between the constituent protomers. The regular structure of this S-layer lattice was lost upon converting the carboxyl groups into neutral groups by amidation. Disintegration of both S-layer lattices occurred upon N-acetylation or N-succinylation of the free amino groups. Adhesion experiments showed that in neutral and weakly alkaline environment whole cells of C. thermosaccharolyticum D120-70 exhibited a stronger tendency to bind to charged surfaces than whole cells of C. thermohydrosulfuricum L111-69, but showed a lower tendency to bind to hydrophobic materials.     

Cellular organization of the embryonic lobster heart

Summary The cellular organization of the embryonic heart of the lobster Homarus americanus was examined in 6-week and 6-month-old animals. The heart wall consists of an outer adventitial layer of fibroblast cells and an inner layer of transversely striated myocardial cells. Present in close association with the myocardium are cardiac neurons, hemocytes and so-called storage cells.Adjacent fibroblasts form fasciae adhaerentes and gap junctions. Adherent junctions also occur between fibroblasts and myocardial cells. Intercalated discs and differentiated membrane regions of close apposition (4 nm) occur between adjacent myocardial cells.The cardiac neurons form a ganglion that contains four small and five large somata. Regions of neuropil are present. Motor axons arising from the cardiac ganglion form neuromuscular synapses with the myocardial cells.The storage cells contain large inclusions and form gap junctions with the myocardial cells. They may supply nutritive material to the developing myocardium.The heart at 6 weeks is about 200 m long and 160 m wide. At 6 months, it is about 300 m long and 250 m wide. The myocardium at 6 weeks is one cell layer thick, and the cells are from 2–6 m in maximum width. At 6 months the myocardium is 2–4 cells thick, and the cells are from 6–12 m in width. Therefore, the myocardium grows by an increase in the number and size of the myocardial cells.     

Patterns of cortical and perinuclear microtubule organization in meristematic root cells ofAdiantum capillus veneris

Summary The interphase meristematic root cells ofAdiantum capillus venerispossess a well developed cytoskeleton of cortical microtubules (Mts), which disappear at prophase. The preprophase-prophase cells display a well organized preprophase microtubule band (PMB) and a perinuclear Mt system. The observations favour the suggestion that the cell edges included in the PMB cortical zone possess a Mt organizing capacity and thus play an important role in PMB formation. The perinuclear Mts are probably organized on the nuclear surface. The preprophase-prophase nuclei often form protrusions towards the PMB cortical zone and the spindle poles, assuming a conical or rhomboid shape. Mts may be involved in this nuclear shaping.Reinstallation of cortical Mts in dividing cells begins about the middle of cytokinesis with the reappearance of short Mts on the cell surface. When cytokinesis terminates, numerous Mts line the postcytokinetic daughter wall. Many of them converge or form clusters in the cytoplasm occupying the junctions of the new and the old walls. In the examined fern, the cortical Mt arrays seem to be initiated in the cortex of post-cytokinetic root cells. A transitory radial perinuclear Mt array, comparable to that found in post-telophase root cells of flowering plants, was not observed inA. capillus veneris.     

Changes in the cell wall of Clostridium species following passage in animals

Morphological changes in clostridial isolates after animal passage with other flora in mixed infections were studied by utilizing a subcutaneous abscess model in mice. We used 26 isolates of 7 clostridial species, and one isolate each of Bacteroides fragilis and Klebsiella pneumoniae. Abscesses were induced by all 7 Clostridium perfringens and 3 Clostridium butyricum isolates and by some of the other isolates. A thick granular wall prior to animal inoculation was shown only in C. perfringens, C. butyricum, and C. difficile. This structure was observed in other clostridia only following their animal passage alone or when co-inoculated with K. pneumoniae or B. fragilis.     

Medium optimization for glucoamylase production by a yeast,Pichia subpelliculosa ABWF-64, in submerged cultivation

An amylolytic yeast strain Pichia subpelliculosawas shown to produce glucoamylase in submerged cultivation. The yeast strain produced the enzyme optimally at 30 °C and pH 5.6 in shake flasks agitated at 200 rev min^–1 in the optimized glucoamylase production medium containing 1% starch, 0.2% yeast extract, 0.4% K₂HPO₄, 0.035% NaCl and 0.1% MgCl₂. Maximum enzyme production was attained during early growth of 11 h in shake flasks, and 6 h in a laboratory fermenter. By optimizing media components and cultivation parameters, a 15-fold increase in glucoamylase secretion was achieved.     

High-level expression of his-tagged clostridial collagenase in Clostridium perfringens

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-D: -Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn(2+) instead of Ni(2+), a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.     

Accumulation and incorporation of 185W-tungsten into proteins of Clostridium acidiurici and Clostridium cylindrosporum

Uptake of tungstate by growing cells was unaffected by the presence of molybdate in Clostridium cylindrosporum, whereas in C. acidiurici the accumulation was decreased by molybdate at 10^-6 mol/l tungstate and higher concentrations. The labelling pattern of soluble proteins by ¹⁸⁵W-tungsten indicated after gel chromatography the presence of three different tungstoproteins in both bacteria. Formate dehydrogenase activity always eluted at a maximum of tungsten labelling. The incorporation of tungsten into formate dehydrogenase containing fractions and a possible tungsten-binding-storage protein was independent of the presence of excess molydate pointing to a genuine role for tungstate in these bacteria.     

Effect of carbohydrate source on alpha-amylase and glucoamylase formation byClostridium acetobutylicum SA-1

Summary An examination into the effect of different carbohydrate sources indicated that the production of extracellular alpha-amylase and glucoamylase was under similar biosynthetic control inClostridium acetobutylicum SA-1. Cell-associated starch-hydrolytic enzymes may be more important than extracellular enzymes in the processing of the starch molecule.     

Utilization of (E)-2-butenoate (Crotonate) by Clostridium kluyveri and some other Clostridium species

Clostridium La 1 obtained from a Clostridium kluyveri culture was compared with a typical C. kluyvery strain (DSM 555). The former grows on crotonate and is unable to use ethanol-acetate as carbon sources. The latter grows on crotonate only after long adaptation periods. Resting cells of both strains show also pronounced differences in the fermentation of crotonate. This holds even for C. kluyveri grown on crotonate. Besides several other differences the most striking is that there is no hybridization between the DNA of both strains.Crotonate seems not to be a very special carbon source since C. butyricum and C. pasteurianum grow on crotonate medium supplemented by peptone and yeast extract.Non Standard Abbreviations EA-medium ethanol and acetate as carbon source - C-medium crotonate as carbon source - DSM Deutsche Sammlung von Mikroorganismen     

Structure and stability of glucoamylase II fromAspergillus niger: A circular dichroism study

Glucoamylase II (EC 3.2.1.3) fromAspergillus niger has 31 % α-helix, 36 %Β- structure and rest aperiodic structure at pH 4.8 as analysed by the method of Provencher and Glockner (1981,Biochemistry, 20,33). In the near ultra-violet circular dichroism spectrum the enzyme exhibits peaks at 304, 289, 282 and 257 nm and troughs at 285, 277 and 265 nm respectively. The enzyme activity and structure showed greater stability at pH 4.8 than at pH 7.0, were highly sensitive to alkaline pH but less sensitive to acid pH values. The enzyme retained most of its catalytic activity and structure even on partial removal of carbohydrate moieties by periodate treatment but was less stable at higher temperatures and storage at 30‡C. Reduction of the periodate treated enzyme did not reverse the loss of stability. Binding of the synthetic substrate,p-nitrophenyl-α-D-glucoside, perturbed the environment around aromatic amino acids and caused a decrease in the ordered structure.     

Electron microscopic study on the quaternary structure of the isolated particulate alcohol-acetaldehyde dehydrogenase complex and on its identity with the polygonal bodies of Clostridium kluyveri

The alcohol-acetaldehyde dehydrogenase complex of Clostridium kluyveri has been separated from contaminating -hydroxybutyryl-CoA dehydrogenase by repeated precipitation with manganese and ammonium sulfate. Mn⁺⁺ was required for maximum alcohol dehydrogenase activity.The molecular weight of the enzyme complex was 194,000 as determined by sucrose density gradient centrifugation. The enzyme complex has been shown to contain two types of subunits with molecular weights of 55,000±2,600 and 42,000±1,200, respectively which are arranged in H-shaped particles.In solutions with an ionic strength above 25 mM the enzyme complex precipitated in the form of lumps as has been shown with specific ferritin-conjugated antibodies. These lumps are assumed to be aggregated polygonal bodies present in C. kluyveri.     

Cloning, Expression and Characterization of a Thiolase Gene from Clostridium pasteurianum

A thl gene encoding the thiolase (EC 2.3.1.9) of Clostridium pasteurianum was cloned by thermal asymmetric interlaced (TAIL) PCR. It consists of 1179 bp with 36.8% GC content and encodes 392 amino acids with a deduced molecular mass of 40,954 Da and shows 77% identity and 88% similarity to that of Clostridium tetani E88 and should be classified as a biosynthetic thiolase with three conserved residues Cys89, Cys382 and His352. The gene was over-expressed in Escherichia coli and the thiolase was purified with Ni-NTA agarose column to homogeneity. The K _m of this thiolase for acetoacetyl-CoA is 0.13 mM with 0.06 mM CoASH at pH 8.2, 25°C and a V _max value of 46 μmol min⁻¹ mg⁻¹.     

Multiple forms of a glucoamylase inhibitory factor fromAspergillus niger and its elution after adsorption onto raw wheat starch

An inhibitory factor (IF) fromAspergillus niger, that inhibited the action of glucoamylase on raw starch, was adsorbed tightly onto raw starch but was almost completely desorbed by 0.02m sodium borate. The IF was a glycoprotein and was partially purified by ion exchange chromatography into three active fractions.     

The influence of tapioca on the growth,the activity of glucoamylase and pigment production of <Emphasis Type="Italic">Monascus purpureus</Emphasis> UKSW 40 in soybean-soaking wastewater

The present study evaluates the usefulness of tapioca starch as additional carbon source for the growth of Monascus purpureus in soybean-soaking wastewater (SSW). The result revealed that M. purpureus grown on 2.0% (w/v) tapioca starch in SSW produced significantly (P < 0.05) higher amounts of biomass and production of the pigments (OD₄₀₀ and OD₅₀₀) when compared to those grown on glucose-or maltose-containing media. However, the glucoamylase activity of M. purpureus grown on the tapioca-SSW medium was not significantly increased when compared to those from the glucose-containing medium.     

Improvement in enzymatic desizing of starched cotton cloth using yeast codisplaying glucoamylase and cellulose-binding domain

To utilize glucoamylase-displaying yeast cells for enzymatic desizing of starched cotton cloth, we constructed yeast strains that codisplayed Rhizopus oryzae glucoamylase and two kinds of Trichoderma reesei cellulose-binding domains (CBD1, CBD of cellobiohydrolase I (CBHI); and CBD2, CBD of cellobiohydrolase II (CBHII)). In this study, we aimed to obtain a high efficiency of enzymatic desizing of starched cotton cloth. Yeast cells that codisplayed glucoamylase and CBD had higher activity on starched cotton cloth than yeast cells that displayed only glucoamylase. Glucoamylase and double CBDs (CBD1 and CBD2) codisplaying yeast cells exhibited the highest activity ratio (4.36-fold), and glucoamylase and single CBD (CBD1 or CBD2) codisplaying yeast cells had higher relative activity ratios (2.78- and 2.99-fold, respectively) than glucoamylase single-displaying cells. These results indicate that the glucoamylase activity of glucoamylase-displaying cells would be affected by the binding ability of CBD codisplayed on the cell surface to starched cotton cloth. These novel strains might play useful roles in the enzymatic desizing of starched cotton cloth in the textile industry.     

Molecular cloning, overexpression in Escherichia coli, and purification of 6x his-tagged C-terminal domain of Clostridium difficile toxins A and B

Genomic DNA from ribotype-01 and -17 Clostridium difficile strains was used for amplification of the sequences encoding the carboxy-terminal domain of toxins A (TcdA) and B (TcdB). The deduced C-terminal TcdB ribotype-01 and -17 domains share 99.5% amino acid sequence identity while TcdA ribotype-17 comprises a 607 amino acid deletion compared to TcdA-01. When compared to previously sequenced C. difficile toxins, 99.3% amino acid identity was found between TcdA-01 and TcdA from strain VPI10643 and 98.8% identity between TcdA-17 and TcdA from strain F-1470. The obtained sequences were fused in 3' to a sequence encoding a hexahistidine tag and cloned into an Escherichia coli expression vector. The recombinant proteins were expressed in E. coli and purified using single-step metal-chelate chromatography. The recombinant carboxy-terminal domain of TcdA-01 was purified from the soluble E. coli lysate fraction whereas TcdA-17 and TcdB-17 carboxy-terminal domains were purified from inclusion bodies. At least 40 mg of each protein was purified per liter of bacterial culture. The recombinant toxin domains were detected specifically by Western blot and ELISA with antibodies against native C. difficile toxins. This study demonstrated that the carboxy-terminal domains of TcdA and TcdB can be produced using an E. coli expression system and easily purified. These recombinant, stable, and non-toxic proteins provide a convenient source for use in the diagnosis of C. difficile infections, instead of native toxins, as controls and calibrators in immunoassay kits and to obtain specific monoclonal antibodies.