Amylolytic Isoenzymes of Thermoactinomyces vulgaris

Thermoactinomyces vulgaris strain 5 produced two electrophoretically different alpha-amylases. Precipitation with ammonium sulfate and acetone did not alter the electrophoretic mobilities of either amylase isoenzyme. Patterns of the hydrolysis products of amylose by the two amylase isoenzymes were essentially identical.     

Phages of lysogenic Thermoactinomyces vulgaris

Summary Two phages isolated from Thermoactinomyces vulgaris multiplied optimally at 55–60°, and were inactivated at 80°. The two isolates had similar growth characteristics, host-range, serology and morphology. Tadpole-shaped with an elongated head, they resemble other actinophages, with long tail lacking contractile sheath and they seem specific to T. vulgaris.     

Crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris.

The crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the three-dimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed beta sheet. The active site is located at the C-edge of the central (parallel) part of the beta sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.     

Viable endospores of Thermoactinomyces vulgaris in lake sediments as indicators of agricultural history.

Bacteria of the genus Thermoactinomyces form endospores with an extreme longevity in natural habitats. We isolated Thermoactinomyces sacchari from 9,000-year-old varved (annually laminated) sediment; thus, T. sacchari is probably one of the oldest known living organisms. More importantly, we tested and verified the hypothesis that there is a relationship between concentrations of dormant, viable endospores of T. vulgaris in lake sediments and the extent of agriculture in the catchments of the lakes. In surface sediments, low concentrations were recorded in forest lakes and the concentrations increased with increasing areas of cultivated land around the lakes. In varved sediment cores from three lakes, we found a temporal relationship between records of T. vulgaris endospores and the pollen of plants indicating agriculture. Endospores were very rare in sediments deposited before agriculture, ca. 1100 A.D. From then to between 1300 and 1700 A.D., a period with restricted cultivation, low but more regular rates of accumulation of endospores were recorded. High endospore accumulation rates were found with the subsequent agricultural expansion. This investigation confirms suggestions that this bacterium could be used as a paleoindicator for agricultural activity and be complementary to pollen analyses. Viable bacteria in continuous records of lake sediments are also potential material for evolutionary studies.     

Development of Thermoactinomyces vulgaris on a synthetic medium]

A synthetic medium was used to obtain the dormant spores of Thermoactinomyces vulgaris. The fraction of the dormant spores depended on the amino acid composition of the growth medium. The rate of growth and development of the organism on the synthetic medium is lower as compared to the routinely employed complex medium.     

Lipoxygenase of Thermoactinomyces vulgaris,purification and characterization of reaction products

• 1.1. A lipoxygenase preparation was obtained from Thermoactinomyces vulgaris and was purified by affinity chromatography on a linoleyl aminoethyl sepharose column.
• 2.2. Two active fractions were obtained.
• 3.3. The fraction obtained by elution with 100 mM borate buffer pH 9.0 was used in the subsequent work.
• 4.4. Th. vulgaris lipoxygenase oxidized linoleic acid into two products: 13-HPOD and 9-HPOD at a ratio of 44 to 56, respectively.
• 5.5. The identification and characterization of the isomers was done by HPLC, I.R. and mass spectrometry.
• 6.6. When arachidonic acid was used as substrate, 15-HPETE and 15-HETE were found to be the main enzymatic products.

     

Properties of Thermoactinomyces vulgaris phage Ta1 and its extracted DNA

The virulent phage Ta1 was obtained in good yields from infected cultures of Thermoactinomyces vulgaris 1227. The purified phage was found to sediment with a single band, the sedimentation constant being (519 +/- 14)S, and to exhibit a typical nucleoprotein behaviour in UV-spectrophotometric and CD experiments. The Ta1 phage consists of a hexagonal head about 0.056 micrometers in diameter and a very short tail. It is morphologically similar to the temperate Salmonella phage P22. The nucleic acid extracted from the phage was found to be a double-stranded linear DNA with a G+C content of 42 mole-% as deduced both from its melting temperature and buoyant density in CsCl. Analytical sedimentation revealed a high degree of molecular homogeneity of Ta1 Dna. the sedimentation constant of this DNA amounts to (35.9 +/- 0.3)S, corresponding to a DNA molecular weight of about 29 millions daltons. The biological activity of Ta1 DNA was indicated by its ability to infect the mycelium of the components T. vulgaris strain 1227 and to give rise to mature phages.     

Adenylate energy charge during batch culture of Thermoactinomyces vulgaris 42

The adenylate energy charge of thermophilic actinomycete Thermoactinomyces vulgaris 42 cells growing exponentially on mineral medium with glucose and casein was around 0.95. After the glucose exhausion, the energy charge fell steadily to a value of 0.5 and remained constant for at least 5 h. The ATP content was found to be a suitable gowth monitoring parameter for Thermoactinomyces vulgaris 42.Abbreviations TCA trichloroacetic acid     

Fibrinolytic and thrombolytic properties of thiol-dependent serine proteinase from Thermoactinomyces vulgaris in vitro

Fibrinolytic and thrombolytic properties of the subtilisin-like thiol-dependent serine proteinase were studied. At concentrations from 50 to 4000 micrograms/ml the enzyme causes lysis of fibrin plates and activates plasminogen. At concentrations above 100 micrograms/ml it shows a pronounced thrombolytic effect on the clots formed in vitro from both plasma and human and rat blood. Plasma inhibitors partly inactivate the thiol-dependent serine proteinase. The enzyme hydrolyses also fibrinogen, thrombin, plasmin and plasminogen.     

Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine

The 3D structure of recombinant bacterial carboxypeptidase T (CPT) in complex with N-BOC-L-leucine was determined at 1.38 Å resolution. Crystals for the X-ray study were grown in microgravity using the counter-diffusion technique. N-BOC-L-leucine and SO ₄ ^2? ion bound in the enzyme active site were localized in the electron density map. Location of the leucine side chain in CPT-N-BOC-L-leucine complex allowed identification of the S1 subsite of the enzyme, and its structure was determined. Superposition of the structures of CPT-N-BOC-L-leucine complex and complexes of pancreatic carboxypeptidases A and B with substrate and inhibitors was carried out, and similarity of the S1 sub-sites in these three carboxypeptidases was revealed. It was found that SO ₄ ^2? ion occupies the same position in the S1’ subsite as the C-terminal carboxy group of the substrate.