[The role of the promoter structure in the efficiency of the expression of guanylspecific ribonucleases from Bacillus intermedius and Bacillus pumilus].

Guanylspecific ribonucleases from B. intermedius (binase) and B.pumilus (RNase Bpu) are structural and functional homologues, and their biosynthesis is subjected to the same laws. At the same time, there are essential differences in the expression efficiency of binase and RNase Bpu genes. This was first suggested to be due to differences in nucleotide sequences of promoters of the genes. Therefore, we constructed plasmids changing each different nucleotide in binase promoter for corresponding one from RNase Bpu and vise versa. It was found that the difference in RNase Bpu and binase expression was due to the only nucleotide in RNase Bpu promoter.

[The role of the promoter and leader sequences of extracellular ribonuclease from Bacillus amyloliquefaciens in regulation of enzyme biosynthesis].

It is shown that in the medium rich with inorganic phosphate there is a stimulation of biosynthesis of ribonuclease from B. amyloliquefaciens (barnase) by actinomycin D, while biosynthesis of ribonucleases from B. intermedius (binase) and B. pumilus (KNase Bpu) in these conditions was suppressed. Features of biosynthesis of binase and RNase Bpu, directed by the barnase promoter, and also expression of chimeric gene of RNase Bpu with leader peptide of barnase were investigated. It was established that stimulation of synthesis of extracellular ribonuclease from B. amyloliquefaciens in the presence of actinomycin D was defined by structure of leader sequences.

[The Pho regulons of bacteria]. / Pho regulony bakterii.

Bacterial Pho regulons contain genes whose expression is regulated by a specific two-component signal transduction system. The products of these genes are involved in the transport and assimilation of inorganic phosphate. The paper summarizes data on the organization and function of Pho regulons in gram-negative and gram-positive bacteria, with particular emphasis on the Pho regulons of the best studied bacteria Escherichia coli and Bacillus subtilis.

[The biosynthesis of new secretory high-molecular-weight ribonucleases in Bacillus intermedius and Bacillus subtilis]. / Biosintez novykh vysokomolekuliarnykh sekretiruemykh ribonukleaz u Bacillus intermedius i Bacillus subtilis.

The investigation of new secretory ribonucleases, the Bacillus intermedius binase II expressed in the recombinant B. subtilis strain 3922 and the native RNase Bsn of B. subtilis, showed that they are synthesized in the growth retardation phase, when inorganic phosphate is exhausted in the medium. The biosynthesis of these ribonucleases was found to be suppressed by the presence of inorganic phosphate in the medium and activated by small amounts of the transcriptional inhibitor actinomycin D. The cultivation media of the producing strains were optimized for the maximum production of the enzymes.

[Regulation of extracellular phosphohydrolase biosynthesis in bacilli]. / Reguliatsiia biosinteza vnekletochnykh fosfogidrolaz u batsill.

Under phosphate-deficient conditions, B. intermedius, B. pumilus, and B. thuringiensis secrete phosphohydrolases, including phosphomono-, phosphodiesterases, and guanyl-specific ribonucleases which cleave RNA molecules to nucleoside-3'-phosphatases. The enzymes are synthesized by phosphate-starved vegetative cells, which is not associated with sporulation. Using B. subtilis strains with mutation in the regulatory protein genes phoP and phoR, it was shown that these proteins regulate expression of B. intermedius, B. pumilus, and B. thuringiensis ribonuclease genes in B. subtilis cells. Genes of heterologous RNAses were activated in recombinant B. subtilis strains simultaneously with its own PHO regulon genes. Presumably a regulatory system homologous to B. subtilis two-component PhoP-PhoR signal transduction system functions in other representatives of the Bacillus genus.

[Ribonuclease from Bacillus thuringiensis var. subtoxicus. Gene structure and biosynthesis regulation]. / Ribonucleaza iz Bacillus thuringiensis var. subtoxicus. Struktura gena i reguliatsiia biosinteza.

The gene for extracellular guanyl-specific ribonuclease of Bacillus thuringiensis var. subtoxicus (RNase Bth), a close homologue of the B. intermedius RNase (binase), was completely sequenced. Analysis of nucleotide sequences in the regions adjoining RNase genes revealed an identical organization of the chromosomal loci of RNase Bth and binase. Growth characteristics of the Bacillus thuringiensis var. subtoxicus strain and its synthesis of RNase were studied. It was shown that the exogenous inorganic phosphate inhibits the biosynthesis of RNase. At the same time, actinomycin D in low doses stimulates the enzyme synthesis. Comparative analysis of the influence of inorganic phosphate and actinomycin D on the biosynthesis of RNAse Bth and binase suggests a possibility of coincidence of regulatory pathways of synthesis of these enzymes.

[Expression of genes for guanyl-specific ribonucleases in Bacillus intermedius and Bacillus pumilus is regulated by the PhoP-PhoR two-component signal transduction system of PHO regulon of Bacillus subtilis]. / Ekspressiia genov guanilspetsifichnykh ribonukleaz Bacillus intermedius i Bacillus pumilus reguliruetsia dvukhkomponentnoi sistemoi transduktsii signala PHO regulona Bacillus subtilis PhoP-PhoR.

Promoters of the genes of guanyl-specific ribonucleases of Bacillus intermedius (binase) and Bacillus pumilus (RNase Bp) were found to contain sequences homologous to those recognizable by the regulatory protein PhoP in the promoters of the PHO regulon of B. subtilis, as well as regions partially homologous to the binding sites of another regulatory protein, PhoB, in the promoters of the PHO regulon of Escherichia coli. The role of the two-component regulatory systems PhoP-PhoR and PhoB-PhoR in the regulation of expression of the genes of binase and RNase Bp in recombinant strains of B. subtilis and E. coli was studied by using mutant strains. It was established that the expression of these genes in recombinant B. subtilis cells is stringently controlled by the PhoP-PhoR two-component regulatory system, whereas the expression of these genes in E. coli cells is not controlled by the regulatory proteins PhoB or PhoR. Presumably, regulatory systems of the response to phosphate starvation, analogous to the PHO regulon of B. subtilis, also function in other representatives of the genus Bacillus.

[Expression of secreted guanyl-specific ribonuclease genes from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells]. / Ekspressiia genov sekretiruemykh guanilspetsifichnykh ribonukleaz iz Bacillus intermedius i Bacillus pumilus v kletkakh Bacillus subtilis.

Plasmids with whole genes for ribonucleases from B. intermedius (binase) and B. pumilis (RNase Bp) assembled with the whole gene of barstar, a specific intracellular inhibitor, are constructed. The resultant plasmids pMZ55 and pMZ56 effectively express binase and RNase Bp genes in B. subtilis cells. A medium for maximum expression of RNase genes by recombinant strains is developed. The expression of binase and RNase Bp genes in B. subtilis cells is negatively regulated by exogenic inorganic phosphate.

[Biosynthesis of extracellular guanyl-specific ribonuclease from Bacillus circulans]. / Biosintez vnekletochnoi guanilspetsifichnoi ribonukleazy iz Bacillus circulans.

Biosynthesis of extracellular alkaline guanyl-specific RNase by Bacillus circulans (RNase Bci) was studied. Synthesis of the enzyme by the culture started in the late exponential phase and was inhibited by inorganic phosphate and glucose, in contrast to the biosynthesis of its structural and functional homologue, RNase Ba (barnase) of B. amyloliquefaciens. It is suggested that differences in the regulation of the biosynthesis of RNase Bci and Ba are related to different structures of their gene promoters.

[Effect of culture medium components on the accumulation of extracellular bacillary ribonucleases in culture fluid of recombinant strains of Escherichia coli]. / Vliianie komponentov pitatel'nykh sred na nakoplenie vnekletochnykh batsilliarnykh ribonukleas v kul'tural'noi zhidkosti rekombinantnykh shtammov Escherichia coli.

The effect of the concentrations of peptone, yeast extract, and inorganic phosphate on the expression of genes of extracellular ribonucleases from Bacillus intermedius 7P (binase) and Bacillus amyloliquefaciens H2 (barnase) was studied in Escherichia coli cells transformed with plasmids containing the structural genes of binase or barnase under the control of their own or synthetic regulatory region or the structural binase gene under the control of the regulatory regions of the genes of barnase or Bacillus pumilus RNase. Inorganic phosphate inhibited the expression of the binase gene under the control of its own regulatory region or the regulatory region of the RNase Bp gene. In all other cases, inorganic phosphate produced no effect on the synthesis of RNases by E. coli cells. This difference in the effects of phosphate may be due to the presence of a nucleotide sequence similar to the E. coli Pho box in the promoters of the binase and RNase Bp genes and the absence of this sequence in the barnase gene promoter. It was shown that high peptone and yeast extract concentrations in the cultivation medium are required for good growth of the recombinant E. coli strains and the biosynthesis of RNases.

[Biosynthesis of alkaline extracellular proteinases in Bacillus intermedius]. / Biosintez shchelochnoi vnekletochnoi proteinazy Bacillus intermedius.

The conditions for growth and serine proteinase biosynthesis were studied in the batch culture of Bacillus intermedius. The synthesis of the enzyme was found to be inhibited by glucose and by the amino acid mixture and activated by the addition of organic compounds-casein and gelatin-to the medium. Inorganic phosphate in the medium increased the enzyme yield. The enzyme activity was shown to be twofold higher upon the addition of corn extract (1.8%) in the medium than in the medium with meat peptone (1.7-2.2%) and inorganic phosphate (0.28-0.30 miligram).

[Biosynthetic regulation of extracellular ribonucleases in native strains of bacilli and in recombinant strains of Escherichia coli]. / Reguliatsiia biosinteza vnekleochnykh ribonukleaz i iskhodnykh batsilliarnykh i rekombinantnykh shtammakh Escherichia coli.

Regulation of the biosynthesis of extracellular ribonucleases of Bacillus amyloliquefaciens H2 (barnase), Bacillus intermedius 7P (binase), and Bacillus pumilus KMM 62 (RNase Bp) was studied in their native strains and recombinant Escherichia coli strains. Recombinant plasmids were obtained that contained genes encoding barnase, binase, and RNase Bp under the control of their own regulatory sequences (plasmids pMT415, pML5, and pML61), genes encoding barnase and binase under the control of the tac promoter and the phoA leader sequence (plasmids pMT416 and pML163), and the binase-encoding gene under the control of the regulatory sequences of the barnase and RNase Bp genes (plasmids pML53 and pML67, respectively). Inorganic phosphate (Pi) inhibited the biosynthesis of binase and RNase Bp in natural strains B. intermedius 7P and B. pumilus KMM 62 and recombinant E. coli strains when genes encoding these RNases were under the control of their own regulatory sequences. In contrast the biosynthesis of barnase-either in the natural B. amylolique-faciens strain or in recombinant E. coli strains-was not affected by Pi. Neither did inorganic phosphate produce any effect on the biosynthesis of binase when the binase-encoding gene was under the control of the barnase gene promoter (pML53). The leader sequences and promoters were found to be similar in the binase and RNase Bp genes and differed considerably from the leader sequence and promoter of the barnase gene. The promoter regions of the binase and RNase Bp genes, but not of the barnase gene, contained sequences that resembled the Pho box of the phosphate regulon from E. coli.

[Regulation of biosynthesis of intracellular enzymes in Bacillus intermedius 3S-19]. / Reguliatsiia biosinteza vnekletochnykh fermentov Bacillus intermedius 3S-19.

The addition of microelements (Co2+, Cu2+, Zn2+, and Fe2+) to a cultivation medium increased the activity of phosphomonoesterase but not of proteinase and ribonuclease. Glucose and inorganic phosphate (Pi) were the main factors that affected the direction and intensity of the biosynthesis of extracellular enzymes.

[Biosynthesis of extracellular ribonuclease by Bacillus pumilus]. / Biosintez vnekletochnoi ribonukleazy Bacillus pumilus.

Conditions for the biosynthesis of alkaline extracellular ribonuclease by Bacillus pumilus were studied and their comparison with that of the biosynthesis of ribonuclease by Bacillus intermedius was made. It was estimated that ribonuclease of Bacillus pumilus is synthesized post-exponentially, its synthesis is inhibited by inorganic phosphate and is stimulated by actinomycin D, as the synthesis of ribonuclease by Bacillus intermedius. At the same time essential differences in dynamic of enzyme formation, extent of phosphate inhibition and spores forming conditions of the comparing cultures were found.

[The effect of metal ions on the synthesis of extracellular enzymes by sporulating bacteria]. / Vliianie ionov metallov na sintez vnekletochnykh fermentov sporoobrazuiushchimi bakteriiami.

The action of metal ions which are present in nutritious medium on the synthesis of extracellular enzymes by sporulating bacteria is analysed. An important role of these ions in post-secretory modification of protein molecules and formation of functionally active molecules of enzyme is shown. The effect of metal ions on some cell envelope properties and extracellular enzyme secretion is under discussion.

[The isolation of highly purified bacillary ribonucleases from recombinant strains of Escherichia coli]. / Poluchenie vysokoochishchennykh batsilliarnykh ribonukleaz iz rekombinantnykh shtammov Escherichia coli.

Active and inactive highly purified extracellular alkaline ribonucleases (barnase) from recombinant Escherichia coli have been prepared according to the industrial technology of Bacillus intermedius ribonuclease purification. Electrophoretic parameters and ultraviolet spectra characterize these preparations as homogeneous proteins. Immunochemical test system of identification of inactive RNAse for its purification has been worked out.

[Antibiotic-resistant mutants of Bacillus intermedius--producers of a number of enzymes]. / Ustoichivye k antibiotikam mutanty Bacillus intermedius--produtsenty riada fermentov.

New antibiotic-resistant strains Bacillus intermedius--producers of phosphohydrolase and protease have been obtained and characterized. Strain S 19 is the more active producer of extracellular enzymes. Autotrophic mutants obtained on its basis possess reduced activity of phosphohydrolase.

[Effect of chloramphenicol and actinomycin D on extracellular alkaline RNAse biosynthesis by Bacillus intermedius]. / Vliianie khloramfenikola i aktinomitsina D na biosintez shchelochnoi vnekletochnoi RNKazy Bacillus intermedius.

By means of chloramphenicol it was found that biosynthesis of alkaline exocellular RNAase was repressed in Bacillus intermedius by inorganic phosphate. Actinomycin D at a low concentration stimulates RNAase biosynthesis in a medium with a minimal phosphorus concentration in model experiments with washed cells and in the batch culture. As a result, the activity of RNAase rises 2-4 times. The stimulating effect of actinomycin D decreases when phosphorus concentration in the medium is increased The effect of actinomycin D is maximal if the antibiotic is added to the medium when the specific growth rate of the bacterium falls down and the rate of RNAase biosynthesis rises.

[Optimization of Bacillus intermedius cultivation conditions to increase biosynthesis of alkaline extracellular RNAase]. / Optimizatsiia uslovii kul'tivirovaniia Bacillus intermedius dlia povysheniia biosinteza shchelochnoi vnekletochnoi RNKazy.

The effect of changes in the concentrations of peptone and glucose in the growth medium, the level of aeration and the original pH value on the biosynthesis of RNAase by Bacillus intermedius 7P was studied by means of a multifactor experiment. The optimal medium for the enzyme accumulation contains from 1.2 to 1.4% of glucose and from 2.8 to 3.1% of peptone, and is characterized by the original pH value of 8.45 to 8.7 and the aeration level of 1 : 7.5. The original pH value of the medium that is optimal for the enzyme biosynthesis is not optimal for the cultural growth. Under the optimized conditions of cultivation, the RNAase activity of Bac. intermedius 7P increases by 50 to 60%.

[Change in the normal microflora of the intestinal tract of rats in the process of their decontamination]. / Izmenenie normal'noi mikroflory kishechnogo trakta krys v protsesse ikh dekontaminatsii.

The authors studied a possibility of decontamination of animals by using antibiotics and a sterile diet, and also a combination of both methods. A reduction of the species composition of the intestinal microbial flora was noted in rats on sterile diet kept under conditions limiting the penetration of microbes into the organism from the external environment. Its greatest rarefaction -- practically to individual species -- was observed when the antibiotics were used in combination with a partial gnotobiotic isolation. In studying the action of antibiotics on the bacterial flora it is recommended to use the gnotobiotic isolation chamber to prevent the entrance of vulgar microbial flora from the surrounding environment.