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Despite numerous attempts to discover genetic variants associated with elite athletic performance, injury predisposition and elite/world-class athletic status, there has been limited progress to date. Past reliance on candidate gene studies predominantly focusing on genotyping a limited number of single nucleotide polymorphisms (SNPs) or the insertion/deletion variants in small, often heterogeneous cohorts have not generated the kind of results that could offer solid opportunities to bridge the gap between basic research in exercise sciences and deliverables in biomedicine. A retrospective view of genetic association studies with complex disease traits indicates that transition to hypothesis-free genome-wide approaches will be more fruitful. In studies of complex disease, it is well recognized that the magnitude of genetic associations is often smaller than initially anticipated and, as such, large sample sizes are required to identify them robustly. Thus, alternative approaches involving large-scale, collaborative efforts, within which high-resolution genome-wide data is generated and interrogated using advanced bioinformatics approaches, are likely necessary for meaningful progress to be made. Accordingly, a symposium was held on the Greek island of Santorini from 14-17th May 2015 to review the main findings in exercise genetics and genomics and to explore promising trends and possibilities. The symposium offered a forum for the development of a position stand. Among the participants, many were involved in ongoing collaborative studies. A consensus emerged among participants that it would be advantageous to bring together all current studies and those recently launched into one new large collaborative initiative, which was subsequently named the Athlome Project Consortium. Sin financiación 3.044 JCR (2016) Q2, 26/84 Physiology, 68/167 Genetics and Heredity; Q3, 104/190 Cell Biology 1.448 SJR (2016) Q2, 106/351 Genetics, 47/191 Physiology No data IDR 2016 UEM

The gene encoding for Bacillus intermedius serine proteinase was cloned and the complete nucleotide sequence was determined. Gene expression was explored in the protease-deficient strain Bacillus subtilis AJ73 during different stages of growth. Catabolite repression involved in control of proteinase expression during transition state and onset of sporulation was not efficient at the late stationary phase. Salt stress leads to induction of serine proteinase production during B. subtilis AJ73(pCS9) post-exponential growth. Expression of proteinase in B. subtilis deg-mutants may be controlled by DegU regulator. B. subtilis spo0-mutants failed to accomplish B. intermedius proteinase production. These data suggest complex network regulation of B. intermedius serine proteinase expression, including the action of spo0, degU, catabolite repression and demonstrate changes in control of enzyme biosynthesis at different stages of growth. © 2006 Elsevier GmbH. All rights reserved.

We studied the biosynthesis of bacillus intermedius glutamyl endopeptidase in the recombinant bacillus subtilis strain AJ73 Δ58.21 during the stationary growth phase. We optimized the composition of the culture medium to favor effective enzyme production during the stationary growth phase and found that the nutritional requirements for glutamyl endopeptidase synthesis were different in the stationary phase and the growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During the final stages of the culture growth, the enzyme production was stimulated by Ca2+, Mn2+, and Co2+ and inhibited by Zn2+, Fe2+, and Cu2+. The synthesis of glutamyl endopeptidase in the late stationary phase was not inhibited by glucose, unlike that in the trophophase during proliferation. We conclude that the regulatory mechanisms of proteinase synthesis during vegetative growth and sporulation are different. © 2005 Pleiades Publishing, Inc.

B. intermedius have been shown previously to secrete two serine proteinases: glutamyl endopeptidase 2 and subtilisin 2 during the late stationary phase, with maximal levels of the enzymes activities recorded at the 40th and 44th hours of growth, respectively. In the current study, we analyzed the impact of various culture medium components on biosynthesis of these proteinases. Yeast extract and gelatin did not stimulate the enzymes biosynthesis. However, on the medium containing 0.1% casein subtilisin 2 production increased to reach 140%. Biosynthesis of both serine proteinases, produced by B. intermedius at the late stationary phase, were found to be inhibited by individual amino acids, and to be insensitive to catabolite repression. In order to maximise enzyme production, the presence of Ca 2+ and Mg2+ at concentration of 5 mM was shown to be necessary. Based on the results of this work, the composition of a complex culture media for the effective production of late stationary phase proteinases by B. intermedius was developed.

The growth of the recombinant Bacillus subtilis strain AJ73 carrying the Bacillus intermedius 3-19 glutamyl endopeptidase gene on a multicopy plasmid and the effect of some nutrients on the efficiency of extracellular glutamyl endopeptidase production in the stationary growth phase were studied. In this phase, the concentration of glutamyl endopeptidase in the culture liquid peaked at the 48th and 78th hours of cultivation and depended on the composition of the cultivation medium. Unlike the synthesis of glutamyl endopeptidase in the trophophase (i.e., during vegetative growth), which was suppressed by glucose, the synthesis of this enzyme during sporulation was resistant to glucose present in the cultivation medium. A multifactorial experimental design allowed optimal proportions between the concentrations of major nutrients (peptone and inorganic phosphate) to be determined. Inorganic phosphate and ammonium ions augmented the production of glutamyl endopeptidase by 30-150%, and complex organic substrates, such as casein and gelatin, enhanced the production of glutamyl endopeptidase by 50-100%. During sporulation, the production of glutamyl endopeptidase was stimulated by some bivalent cations (Ca2+, Mg2+, and Co2+) and inhibited by others (Zn2+, Fe2+, and Cu2+). The inference is drawn that the regulatory mechanisms of glutamyl endopeptidase synthesis during vegetative growth and sporulation are different.

A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and k cat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating β-galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites.

Proteolytic proteins solubilized from the membrane of Bacillus intermedius were studied by electrophoresis. The content of membrane-bound proteinases was lower in cells grown in the presence of glucose. Proteinase enzymograms revealed four molecular forms of subtilisin and four molecular forms of glutamyl endopeptidase. The electrophoretic mobility of one of the molecular forms was similar to those of the mature extracellular proteinases. Chromatography of membrane proteins on a MonoS column yielded four protein fractions that caused hydrolysis of Z-Glu-pNA and four fractions that caused hydrolysis of Z-Ala-Ala-Leu-pNA, which is in agreement with the results of electrophoresis. The molecular forms of proteinases identified in the membrane may reflect various stages of biogenesis of the corresponding extracellular enzymes.

The investigation of the activity of extracellular hydrolytic enzymes and sporulation in the bacterium Bacillus intermedius 3-19 showed that the activity of ribonuclease is maximal in the glucose-containing growth medium, in which sporulation is suppressed. At the sporulation stages II-IV, the synthesis of phosphatase was not regulated by the factors that influence this synthesis in the phase of growth retardation. Caseinolytic activity exhibited two peaks. The first peak was observed when thiol-dependent proteinase began accumulating in the medium. The second peak corresponded to the late stages of sporulation, i.e., the stages of spore maturation and the autolysis of sporangium. The regulatory relationship between proteinase synthesis and sporulation and the possible role of extracellular phosphatases and proteinases in the sporulation are discussed.

The study of the accumulation pattern of extracellular proteins with chitinase activity in the parent Serratia marcescens strain Bú 211 (ATCC 9986) grown in the presence of mitomycin C and its mutant strain with the constitutive synthesis of chitinases grown in the absence of the inducer showed that chitinase activity appeared in the culture liquids of both strains at the end of the exponential phase (4 h of growth) and reached a maximum in the stationary phase (18-20 h of growth). The analysis of the culture liquids (12 h of growth) by denaturing electrophoresis in PAAG followed by the protein renaturation step revealed the presence of four extracellular proteins with chitinase activity and molecular masses of 21, 38, 52, and 58 kDa. © 2002 MAIK "Nauka/Interperiodica".

The biosynthesis of glutamyl endopeptidase from Bacillus intermedius 3-19 in recombinant strain of Bacillus subtilis has been investigated. The composition of culture medium, which yielded the maximum glutamyl endopeptidase production by B. subtilis strain, was developed, employing response surface methodology. The pathways of regulation of glutamyl endopeptidase synthesis in recombinant strain in general were found to be similar to those of other serine proteinases and of glutamyl endopeptidase in B. intermedius. Biosynthesis of glutamyl endopeptidase by recombinant strain was suppressed by easily metabolizable carbon sources. Ions of Ca2+(2mM), Mg2+ (1mM), and Co2+ (5mM) stimulated production of the proteinase by B. subtilis. In case of Co2+ ions strong stimulating effect (up to 400%) possibly was due to the release of the membrane-bound enzyme into the culture liquid, according to the mechanism described earlier for B. intermedius. The addition of Fe2+, Zn2+, and Cu2+ to the medium at concentrations of 1 to 10mM led to the gradual decrease in proteinase production by B. subtilis. This study has demonstrated a requirement by recombinant strain for excess carbon, nitrogen, and inorganic phosphate for active glutamyl endopeptidase production. In contrast with B. intermedius, for the maximum yield of endopeptidase by B. subtilis the presence in the culture medium of yeast extract at concentration of 2% and one of the organic substrates of proteinase - casein or gelatin (1%) was found to be necessary. Our study has revealed the changes in the pathways of secretion of glutamyl endopeptidase of B. intermedius by B. subtilis cells, expressing the gene for glutamyl endopeptidase from the plasmids: the part of the enzyme (2-5%) remained bound to the cell wall. © 2002 Elsevier Science Inc. All rights reserved.

The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of glutamyl endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of glutamyl endopeptidase. © 2002 MAIK "Nauka/Interperiodica".

The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of glutamyl endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of glutamyl endopeptidase.

The investigation of the activity of extracellular hydrolytic enzymes and sporulation in the bacterium Bacillus intermedius 3-19 showed that the activity of ribonuclease is maximal in the glucose-containing growth medium, in which sporulation is suppressed. At the sporulation stages II-IV, the synthesis of phosphatase was not regulated by the factors that influence this synthesis in the phase of growth retardation. Caseinolytic activity exhibited two peaks. The first peak was observed when thiol-dependent proteinase began accumulating in the medium. The second peak corresponded to the late stages of sporulation, i.e., the stages of spore maturation and the autolysis of sporangium. The regulatory relationship between proteinase synthesis and sporulation and the possible role of extracellular phosphatases and proteinases in the sporulation are discussed. © 2002 MAIK "Nauka/Interperiodica".

The study of the accumulation pattern of extracellular proteins with chitinase activity in the parent Serratia marcescens strain Bú 211 (ATCC 9986) grown in the presence of mitomycin C and its mutant strain with the constitutive synthesis of chitinases grown in the absence of the inducer showed that chitinase activity appeared in the culture liquids of both strains at the end of the exponential phase (4 h of growth) and reached a maximum in the stationary phase (18-20 h of growth). The analysis of the culture liquids (12 h of growth) by denaturing electrophoresis in PAAG followed by the protein renaturation step revealed the presence of four extracellular proteins with chitinase activity and molecular masses of 21, 38, 52, and 58 kDa.

Background: Proteinases are widely used in various fields of medicine, such as the treatment of burns, purulent wounds, or decubitus ulcers. On the basis of new microbial proteinases produced by nonpathogenic organisms, a new generation of medical preparations can be developed. Representatives of the Bacillus genera are nonpathogenic and are suitable for producing various proteases in large quantities. B. intermedius is shown to produce a set of alkaline proteases at the early and late stationary phase of growth. Material/Methods: The activity of alkaline proteinases was determined using synthetic chromogenous substrates Z-Glu-pNA and Z-Ala-Ala-Leu-pNA. To determine β-galactosidase activity, 2-nitro-β-D-galactopyranosid was used. Spores were calculated by phase-contrast microscopy. Results: During the late stages of sporulation B. intermedius 3-19 cells were shown to secrete two proteinases into the medium: glutamyl endopeptidase, with maximum activity at 40 hours of growth, and subtilisin, with maximum activity at 44 hours of growth. Evidence for the secretion of these enzymes into the medium was provided by measuring β-galactosidase activity. Conclusion: Our results show that proteinases from B. intermedius (glutamyl endopeptidase 2 and subtilisin 2) in the late stationary phase of growth are secreted enzymes. This suggests that these enzymes play a role in sporulation.

The effect of nutrients and growth conditions on the accumulation of glutamyl endopeptidase in the culture liquid of Bacillus intermedius 3-19 was studied. Glucose and other readily metabolizable carbon sources were found to suppress the production of the enzyme, whereas inorganic phosphate and ammonium cations enhanced it. Protein substrates, such as casein, gelatin, and hemoglobin, did not affect enzyme production. Some bivalent cations (Ca2+, Mg2+, Co2+) increased the production of glutamyl endopeptidase, but others (Zn2+, Fe2+, Cu2+) acted in the opposite way. The rate of enzyme accumulation in the culture liquid increased as the growth rate of the bacterium decreased, so that the maximum enzyme activity was observed in the stationary growth phase. Based on the results of this investigation, an optimal medium for the maximum production of glutamyl endopeptidase by B. intermedius 3-19 was elaborated. © 2000 MAIK "Nauka/Interperiodica".

Thiol-dependent serine proteinase and glutamylendopeptidase of Bacillus intermedius 3-19 being prevailing enzymes in the total pool of extracellular proteinases (95%) of this microorganism in catalytic active form were detected on the membrane of the cells. Production of these enzymes was maximum on the medium containing inorganic phosphate and gelatin and decreased 2-4-fold on the medium with glucose and lactate. The level of the activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of CoCl2 (2 mM) into the medium caused essential increase in extracellular glutamylendopeptidase activity and promoted the release of membrane-bound enzyme into cultural fluid. Proteolytic activity was detected in cytoplasm also. Proteinases localized in cytoplasm were shown to differ in properties from those secreted.

The activities of proteinases in the culture fluid and cellular fractions of Bacillus intermedius 3-19 grown under various conditions were studied. Thiol-dependent serine proteinase was the prevalent enzyme in the total pool of extracellular proteinases (70%); its catalytically active form was also detected in the cell membrane and, during active enzyme production, in the cell wall. Another enzyme, glutamyl endopeptidase (10% of the total pool), was detected in the cell membrane; it was also found in the cell wall and cytoplasm during active enzyme secretion into the growth medium. The production of these enzymes was maximal on medium containing inorganic phosphate and gelatin and decreased 2- to 4-fold on medium with glucose and lactate. The level of activity of extracellular enzymes correlated with that of corresponding membrane-bound proteins. The addition of CoCl2 (2 mM) into the medium caused an essential increase in extracellular glutamyl endopeptidase activity and promoted the release of the membrane-bound enzyme into the culture fluid. Proteolytic activity towards casein was also detected in the cytoplasm. The proteinases localized in the cytoplasm were shown to differ in their properties from those secreted. © 2000 MAIK "Nauka/Interperiodica".

The biosynthesis of glutamylendopeptidase from Bacillus intermedius strain 3-19 and localization of the enzyme in the bacterial cells was studied. The synthesis of the enzyme was suppressed by easily metabolizable carbon sources. Inorganic phosphate and NH+ 4 ions stimulated the production of glutamylendopeptidase. Complicated organic substrates such as casein, gelatine, and haemoglobin did not affect the biosynthesis of the enzyme. The divalent metallic ions Ca2+, Mg2+, Co2+ increased the production of glutamylendopeptidase while Zn2+, Cu2+, and Fe2+ reduced the biosynthesis of proteinase. The rate of synthesis of the enzyme increased when the rate of the bacterial growth decreased. The maximum enzyme activity in the culture fluid was determined at the stationary phase of growth. In the cells glutamylendopeptidase was bound to the cytoplasmic membrane, and the maximal enzyme activity was detected in the stationary growth phase. The results facilitated the development of a medium which yielded the maximum glutamylendopeptidase production by B. intermedius strain 3-19.

The growth of a mutant strain of Serratia marcescens with high chitinase activity and the biosynthesis of endochitinase by this strain were investigated. The study was carried out using semisynthetic culture medium without inducers and culture medium containing colloidal chitin as a sole nitrogen and carbon source, with and without mitomycin C. The mutant strain, unlike the native one, was shown to produce endochitinase and to secrete the enzyme into the medium during the growth on culture medium without the inducers, chitin and mitomycin C. During growth on the medium with chitin the mutant strain differed from the native one with a short lag-phase of growth, the early appearance of endochitinase in the culture liquid and a high level of endochitinase activity. The difference between the strains disappeared after the addition of mitomycin C, an inducer of the cell SOS-response, to the culture medium containing chitin. Specific endochitinase activity of S. marcescens mutant strain grown on various culture media had two maxima, namely at the beginning and at the end of the stationary phase. Mitomycin C increased the specific activity in a second peak of endochitinase activity during the growth of the mutant strain.

The extracellular chitinolytic complex of Serratia marcescens Bú 211 ATCC 9986 was shown to include three proteins with molecular masses of 58, 52, and 45 kDa (chitinases A, B, and C, respectively). Chitinases A and B were separated from chitinase C and purified from protein admixtures by chromatography on chitin. Chitinase A possessed two isoforms with pI values of 6.25 and 4.85-5.25. Chitinase B had only one isoform with a pI of 4.85-5.25 and appeared to be an endochitinase. In the absence of chitin, the biosynthesis of extracellular chitinases was induced by mitomycin C (MC), an inducer of the SOS-response in cells. In the presence of chitin in the cultivation medium, MC increased the chitinase activity. MC induced the synthesis of all three extracellular chitinases, but not of chitobiase, whose biosynthesis was induced by the substrate. © 1997 MAHK Hayka/Interperiodica Publishing.

The influence of actinomycin D on the synthesis of extracellular ribonucleases by "Bacillus intermedius" (binase), B.pumilus (RNAse Bp) and B.amyloliquefaciens (barnase) was studied comparatively. When added during the active synthesis of the enzymes actinomycin D stimulated the biosynthesis of binase and RNAse Bp and had no influence on the barnase biosynthesis. The response of the bacillary RNAse biosynthesis to the added actinomycin D correlated with the differences in the nucleotide sequences of the genes encoding the enzymes. The Escherichia coli SURE recombinant strains carrying the plasmids with the genes of binase, RNAse Bp and barnase under different regulatory sequences, as well as the E.coli MC4100 recombinant strains carrying the plasmids with the β-galactosidase gene under the promoters of the bacillary RNAse were isolated. However, the expression of the bacillary ribonuclease genes in the E.coli recombinant strains carrying the plasmids with the genes of the enzymes, as well as the expression of the β-galactosidase gene from the promotors of the bacillary RNAses was not stimulated by actinomycin D irrespective of the dose and addition time.

The gene for the extracellular ribonuclease of B. pumilus KMM62 (RNase Bp) has been cloned and sequenced. The structural gene for this enzyme is similar to those of the extracellular ribonucleases of B. intermedius 7P (binase) and B. amyloliquefaciens H2 (barnase), as are the regulatory regions of binase and RNase Bp. The regulatory region of the barnase gene, however, is quite different from the other two. In the promoter of the genes for binase and RNase Bp, but not in that for barnase, is a region similar to the Pho box of E. coli. We have established that inorganic phosphate suppresses the synthesis of the binase and RNase Bp, but does not effect the synthesis of barnase. © 1995.