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A comparative study of DNA homology among 12 strains of thermophilic streptococci currently used as ferments in the dairy industry in different regions of the CIS and 16 strains of mesophilic lactococci obtained from different sources was performed by the method of optical reassociation in solution. These strains were found to form three groups with DNA homology generally not exceeding 30-35% between each other. These data indicate that strains commonly classified as Streptococcus thermophilus may belong to different taxa. At the same time, the DNA base composition was similar in all these strains (the G + C content was 38-40 mol %). Among 16 strains of mesophilic lactococci DNA homology was generally higher than 50%, i.e., all these strains indeed belong to the same species, Lactococcus lactis.

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Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.

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The growth of Bacillus subtilis FU-79 and its production of alpha-amylase (EC 3.2.1.1) were studied under the conditions of batch and continuous cultivation in a semisynthetic medium. The enzyme activity fell down abruptly upon a pulse addition of either glucose or yeast extract to the chemostat culture, and remained at a low level for the following ten generations. Apparently, a double limitation of the culture growth (viz., with residual glucose and with yeast extract components) is required for the activity of alpha-amylase to be high.

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lambda vector phages--pUC19 phasmid hybrids were constructed. The hybrids, phasmids lambda pMYF131 and lambda pSL51, were used as vector molecules for making genomic libraries. Vector phasmids exist as plasmids in vivo. They contain all the genes and specific sites necessary for lytic development, but the DNA molecules are not long enough to be packaged in lambda capsid. Elongation of the molecule due to insertion of a foreign DNA fragment renders phasmid all features of non-defective phage. Output of recombinants is up to 3 X 10(6) per 1 microgram of phasmid vector DNA. The fraction of non-recombinants in libraries is less than 1-0.1%. The capacity of the vectors is 19.6 and 22 kb for lambda pMYE131 and lambda pSL51 accordingly. It is possible to clone DNA fragments with blunt ends and various sticky-end fragments obtained by digestion with 14 prototype restrictases, into nine unique restriction sites of vector lambda pMYF131. The created vectors allowed to construct more than 30 representative genomic libraries of eukaryotic and prokaryotic organisms. 13 individual genes were identified within the libraries.

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Bacteriophage beta 45 of Corynebacterium diphtheriae was harvested. The extracted DNA of the bacteriophage was digested by the restriction endonuclease BamHI and inserted into the BamHI cleavage site of pUC19 vector plasmid. Plasmid pNVY5 containing a mutant gene crm45 of diphtheriae toxin in a 3.9 bpn fragment was isolated from the hybrid plasmids obtained. Cell free extracts of E. coli strain TG1 (pVNY5) contain the nontoxic protein crm45 possessing the specific enzymatic activity of diphtheriae toxin (ADP ribosylation on wheat elongation factor two). According to orientation of BamHI fragment in pNVY5 plasmid it is concluded that the crm45 gene is expressed using its own promoter.

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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.

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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.

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Vegetative cells and spores of the colonial morphological mutants of Bacillus subtilis A-50 were studied by electron microscopy. The ultrastructure of vegetative cells from both asporogenic colonial-morphological mutants and those which were capable of forming spores in the presence of high concentrations of nitrogen and carbon sources with a decreased activity and a modified spectrum of serine proteases differed from the parent strain by the presence of a microcapsule, the uneven thickness of a cell wall, and the absence of a distinct periplasmic space. Crystalline inclusions of a regular shape were detected in the sporeforming mutant in those cells which were devoid of spores. Spores of the mutant had additional layers.

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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.

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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.

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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.

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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.

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Spontaneous and NG- and ICR-191-induced variability of Bacillus subtilis A-50 with respect to the production of alkaline proteinase (subtilisine) is studied. It is found that ICR-191 induces greater variability as compared with NG under conditions of a low survival. Six mutants synthesizing proteinases showing a changed ability to form enzymes were obtained under the action of the same mutagens.

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