Intracellular serine proteinase of Bacillus subtilis strain Marburg 168. Comparison with the homologous enzyme from Bacillus subtilis strain A-50.

Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN', produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.

Direct comparison of the subtilisin-like intracellular protease of Bacillus licheniformis with the homologous enzymes of Bacillus subtilis.

Intracellular serine proteases of Bacillus licheniformis and Bacillus subtilis are closely related.

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium. Comparison of the enzyme and other Bacillus subtilis serine proteases.

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by non-ionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.

[Effect of Bacillus subtilis A-50 "streptomycin" mutations on the formation of molecular forms of subtilisin, an extracellular alkaline proteinase]. / Vliianie "streptomitsinovykh" mutatsii Bacillus subtilis A-50 na obrazovanie molekuliarnykh form subtilizina--vnekletochnoi shchelochnoi proteinazy.

The patterns of subtilisin molecular forms of streptomycin-resistant (Strr) and streptomycin-dependent (Strd) mutants of Bacillus subtilis A-50, as well as the revertants of Strd to streptomycin-independence (Str1) were studied. Strr mutants had different quantitative pattern of the same subtilisin molecular forms as compared with the initial strain A-50 (the forms with Rf 0.08, 0.16 and 0.3). In comparison with the initial strain A-50, Strd mutants and Str1 revertants revealed three additional forms of the active enzyme with Rf 0.02, 0.5 and 0.7 and the molecular weights less than 35,000, 28,000 and 20,000 respectively. It was suggested that the rate and character of the enzyme secretion of the degree of its post-translational modifications might result in the different pattern of subtilisin molecular forms produced by these streptomycin mutants.

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.

Two related structural genes coding two homologous serine proteases in the Bacillus subtilis genome.

A Bacillus subtilis intracellular serine protease is homologous in sequence to an extracellular serine protease from the same strain, indicating the presence of two related structural genes in the Bacillus subtilis genome.

[Effects of mutations in Bacillus subtilis genome decreasing the protease activity on the formation of subtilisin molecular forms]. / Vliianie mutatsii Bacillus subtilis, snizhaiushchikh proteoliticheskuiu aktivnost; na obrazovanie mnozhestvennykh molekuliarnykh form subtilizina.

Multiple molecular forms of subtilisin--extracellular serine protease produced by the wild strain Bac. subtilis A-50 and its mutant strains with the protease activity decreased two-fold and more were studied. Six molecular forms of subtilisin were found on the whole when 33 mutant strains have been investigated under the experimental conditions. It is essential that both the wild and each of mutant strains under study produced not more than three out of these six forms. Three molecular forms of subtilisin from the mutant strains are similar to those found in the wild strain A-50, and have the molecular weight, of 27 000-30 000. Three other forms of subtilisin were revealed only in the mutant strains, and had the molecular weight of about 20 000. Apparently there is only one structural gene for subtilisin in Bac. subtilis genome. The appearence of multiple molecular forms of subtilisin may be due to the post-translational modifications (limited proteolysis) of the initial type of enzyme, i.e. pre-subtilisin. Probably, that certain mulations not affecting the structural gene can significantly change the expression of such gene by varying of the degree of product modifications.

[Comparative study of the multiplicity of exoenzymes produced by different strains of Bacillus subtilis]. / Sravnitel'noe izuchenie mnozhestvennosti ékzofermentov, produtsiruemykh razlichnymi mutantami Bacillus subtilis.

Molecular forms of two exoenzymes, subtilisin and alpha-amylase, produced by mutants of Bacillus subtilis were studied. The degree of the post-translational modification of subtilisin was less pronounced for P and M mutants than for R mutants. Some of the P and M mutants produced subtilisin having higher molecular weight and hydrophobicity as compared to the R mutants. This form of subtilisin may be the primary product of translation of the structural gene and therefore a precursor of subtilisin. Its appearance outside the cell may be due to the alteration in the cell surface structures in the P and M mutants and abnormal post-translational modification. The P and M mutants produced also differing exocellular proteins as compared to the R mutants, e.g. alpha-amylase forms with the higher isoelectric points.

Intracellular proteinase of Bacillus subtilis.

An intracellular serine proteinase was isolated from Bacillus subtilis, strain A-50. The molecular weight of the enzyme is 30000 +/- 1000, its amino acid composition is enriched in glutamic acid residues, the isoelectric point is 4.3, the N-terminal sequence Glu-Leu-Pro-Glu-Gly-Ile-Gln-Val-Ile-Lys-Ala-Pro-Glu-Leu-Xxx-Ala-Gln-Gly-Phe-Lys Gly-Ser-Asx-Ile-Lys-Ile-Ala-Val-Leu-Asx. The enzyme is structuraly homologous with secretory subtilisins.

[Multiple forms of Bacillus subtilis subtilisin and effects of mutations on the distribution of its molecular forms]. / Mnozhestvennost' subtilizinov Bacillus subtilis i vliianie mutatsii na raspredelenie molekuliarnykh form

Using polyacrylamide-gel electrophoresis, isoelectric focusing and gel-filtration it was demonstrated that the auxotrophic mutant strains of Bac. subtilis A-50 and their prototrophic revertant strains produce multiple molecular forms of subtilisin. Three of them are the same as the corresponding molecular forms of subtilisin from the wild strain A-50. In different mutant strains the relative amounts of the main three forms varies considerably resulting in the absence of certain forms in several strains. There is the additional minor form of subtilisin possessing high electrophoretic mobility in four prototrophic revertant strains and one Arg--auxotrophic strain of Bac. subtilis A-50. It would be reasonable to suppose that different molecular forms of subtilisin derive from the product of its single structural gene as a result of post-translational modifications (limited proteolysis). This enzyme and probably most, if not all secretory proteins may be synthesised as larger precursors and then specifically modified in the bacterial cell membranes. Thus, certain mutations, without affecting the structural gene of this secretory protein -- subtilisin -- have pronounced effects on this structural gene expression, varying the degree of its product modification and the amount of resulting secretory molecular forms of subtilisin.

[Temperature-dependent mutants of Bacillus subtilis A-50 with a diminished level of alkaline proteinase synthesis]. / Temperaturochuvstvitel'nye mutanty Bacillus subtilis A-50 so snizhennym urovnem sinteza shchelochnoi proteinazy

Temperature-sensitive proteolytically inactive mutants of Bacillus subtilis A-50 are obtained. The optimal ability to synthetise the enzyme both in the original strain and in mutants is expressed at 37 degrees C. The level of proteolytic activity of selected prot(ts) mutants depends both on the temperature of growth and on the temperature at which the activity of subtilisin is analysed. The optima of functioning ts-proteinases are shifted in mutants as compared with the original strain. The ability to form spores at 37 degrees C and 45 degrees C are considerably reduced as compared with this process at 28 degrees C. The results of disc electrophoresis in polyacrylamide gel indicate the presence of structural alteration of proteinases in the mutants obtained.