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.     

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.     

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.     

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     

Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion.

Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like swarms of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.     

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.     

Crystal structure of thermitase from Thermoactinomyces vulgaris at 2.2 A resolution

The crystal structure of thermitase from Thermoactinomyces vulgaris has been determined by x-ray diffraction at 2.2 A resolution. The structure was solved by a combination of single isomorphous replacement and molecular replacement methods. The structure was refined to a conventional R factor of 0.24 using restrained least square procedures CORELS and PROLSQ. The tertiary structure of thermitase is similar to that of subtilsin BPN'. The greatest differences between these structures are related to the insertions and deletions in the sequence.     

Importance of carbon dioxide for the cultivation of Thermoactinomyces vulgaris from spores

Thermoactinomyces vulgaris has been used for the production of the serine protease Thermitase [EC 3.4.21.14] by fermentation. Spore germination of this strain is influenced by the gas composition of the medium. Since the outgrowth of the germ tubes occurs only in the presence of a raised CO₂ concentration, the cultivation in aerated stirred fermenters is not reproducible. However, if cultivation takes place in a closed fermentation system, initially unaerated, in which the CO₂ formed by spore respiration accumulates, reproducible germination can be obtained. From an O₂-balance, indirect conclusions can be drawn with regard to the effect of CO₂, and the process of germination can be described. The cultivation method described permits almost complete germination of all inoculated spores and reproducible rates of biomass and enzyme production in the subsequent fermentation. Thus, stabilization of the procedure with increased enzyme yields is ensured.     

Phosphate utilization and constitutive synthesis of phosphatases in Thermoactinomyces vulgaris Tsilinsky.

Thermoactinomyces vulgaris utilized both organic and inorganic phosphates with equal efficiency for its growth. The specific activities of the thermophilic acid and alkaline phosphatases were found to be maximum at 1 mM concentration of each phosphate source. All the phosphatase isoenzymes (three alkaline and one acidic) were observed irrespective of the substrate source and concentration, suggesting constitutive synthesis of the enzymes. During growth and differentiation, both acid and alkaline phosphatases exhibited uniformly stable patterns of isoenzymes with slight variations in their specific activities.     

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.     

Structures of Thermoactinomyces vulgaris R-47 alpha-amylase II complexed with substrate analogues

The structures of Thermoactinomyces vulgaris R-47 alpha-amylase II mutant (d325nTVA II) complexed with substrate analogues, methyl beta-cyclodextrin (m beta-CD) and maltohexaose (G6), were solved by X-ray diffraction at 3.2 A and 3.3 A resolution, respectively. In d325nTVA II-m beta-CD complex, the orientation and binding-position of beta-CD in TVA II were identical to those in cyclodextin glucanotransferase (CGTase). The active site residues were essentialy conserved, while there are no residues corresponding to Tyr89, Phe183, and His233 of CGTase in TVA II. In d325nTVA II-G6 complex, the electron density maps of two glucosyl units at the non-reducing end were disordered and invisible. The four glucosyl units of G6 were bound to TVA II as in CGTase, while the others were not stacked and were probably flexible. The residues of TVA II corresponding to Tyr89, Lys232, and His233 of CGTase were completely lacking. These results suggest that the lack of the residues related to alpha-glucan and CD-stacking causes the functional distinctions between CGTase and TVA II.     

Isolation of amylolytic strains of Thermoactinomyces vulgaris and production of thermophilic actinomycete amylases

Kuo, M. J. (Iowa State University, Ames), and P. A. Hartman. Isolation of amylolytic strains of Thermoactinomyces vulgaris and production of thermophilic actinomycete amylases. J. Bacteriol. 92:723-726. 1966.-Of 759 isolates obtained from dung, compost, and soil samples, a culture of Thermoactinomyces vulgaris (strain 5) was selected for further study on the basis of quantities of amylase produced in synthetic and nonsynthetic media, rapid growth and sporulation, culture stability upon prolonged storage at 5 C, and growth temperature range. Inoculum preparation, temperature optimum for amylase formation, and the effects of various kinds and levels of carbon and nitrogen sources on amylase production were studied with T. vulgaris strain 5. An optimal procedure for production of T. vulgaris amylases is proposed.