[Antiviral activity of Bacillus intermedius RNAase in guinea-pigs and rabbits infected with outdoor rabies virus].

Single injection of Bacillus intermedius RNAase in a dose of 5 mg/kg could protect 40 and 50-70% of the outdoor rabies virus-preinfected guinea-pigs and rabbits, respectively. In the control group there were 100 and 75-100% deaths of the RNAase-untreated guinea-pigs and rabbits, respectively. Animal protection was observed only when RNAase was injected into the site of viral administration. The intramuscular injection of RNAase, other than the site of viral administration failed to protect the infected animals. The efficacy (75%) of RNAase injected into the rabbits was similar 1 and 24 hours after animal infection.

[Biosynthesis of Bacillus intermedius glutamyl endopeptidase in recombinant Bacillus subtilis cells during the stationary growth phase].

We studied the biosynthesis of Bacillus intermedius glutamyl endopeptidase in the recombinant Bacillus subtilis strain AJ73 delta58.21 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 growth retardation phase. Proteinase accumulation was activated by complex organic substrates (casein and gelatin). During 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.

[Secretion of bacterial ribonuclease].

The investigation of the effect of some components of the medium on the distribution of the secretory guanyl-specific ribonuclease of Bacillus intermedius (EC 3.1.4.23) among various cell fractions and culture liquid showed that the amount of this enzyme in the culture liquid does not depend on the concentration of calcium ions in the medium (within 1-5 mM). The study of the effect of the amino acid substitutions Trp34Asn and Trp70Asn in the ribonuclease molecule showed that the secretion of ribonuclease depends on the formation rate of its secondary structure. The amino acid substitution Trp34Asn completely inhibits ribonuclease secretion.

[The antiviral activity of RNAse Bacillus intermedius in experiments with mice preinfected with street rabies virus]. / Protivovirusnaia aktivnost' RNKazy Bacillus intermedius v éksperimentakh na myshakh linii CBA, predvaritel'no zarazhennykh ulichnym virusom beshenstva.

RNAse Bacillus intermedius, when administered once and according to 11 repeated experiments, protected the preliminarily infected CBA mice with street rabies virus (protection of 40-67%; p < 0.01-0.001). A reliable protection of Animals was registered only when RNAse was administered intramuscularly at the virus introduction spot; it was not effective, when the bacterial RNAse was injected in the brain, vein, under the skin or in muscles of a non-infected extremity. Neither did it produce any suppressive effect on the vaccinal antirabic immunity.

[The regulation of Bacillus intermedius glutamyl endopeptidase biosynthesis in the recombinant Bacillus subtilis strain during sporulation]. / Reguliatsiia biosinteza vnekletochnoi glutamiléndopeptidazy Bacillus intermedius v rekombinantnom shtamme Bacillus subtilis na stadii sporoobrazovaniia.

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 h 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.

[Membrane-bound forms of serine proteases of Bacillus intermedius]. / Membranno-sviazannye formy serinovykh proteinaz Bacillus intermedius.

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.

[Synthesis and secretion of Bacillus intermedius proteinases in the late stages of sporulation]. / Sintez i sekretsiia proteinaz Bacillus intermedius na pozdnikh stadiiakh sporoobrazovaniia.

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 beta-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.

[Hydrolytic enzymes and spore formation in Bacillus intermedius]. / Gidroliticheskie fermenty i sporoobrazovanie u Bacillus intermedius.

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.

[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.

[Effect of Bacillus intermedius ribonuclease on properties of the yeast Saccharomyces cerevisiae]. / Vliianie ribonukleazy Bacillus intermedius na svoistva drozhzhei Saccharomyces cerevisiae.

Effects of Bacillus intermedius ribonuclease on physiological, biochemical, and consumer properties of baker's yeast Saccharomyces cerevisiae were studied. This enzyme improved the yeast raising strength and increased the cell tolerance to various adverse factors. The antistress effect of RNase correlated with an earlier start of the stationary growth phase and increased trehalose pool.

[Dependence of the effect of RNAase from Bacillus intermedius on the growth of baker's yeast on the concentration of the exogenous enzyme]. / Zavisimost' vliianiia RNKazy Bacillus intermedius na rost pekarskikh drozhzhei ot kontsentratsii ékzogennogo fermenta.

Bacillus intermedius RNase added at a low concentration (0.001 microgram/ml) stimulated yeast growth, while a high RNase concentration (1500 micrograms/ml) was inhibitory to yeast growth. The inhibitory effect of RNase was transient and correlated with the increase in the trehalose pool of yeast cells. The number of unbudded cells in the yeast population tended to decrease under the action of low concentrations of bacillar RNase and to increase under the action of high concentrations of this enzyme.

[Effect of culture media components on accumulation of glutaminyl endopeptidase in culture liquid of Bacillus intermedius 3-19]. / Vliianie komponentov pitatel'noi sredy na nakoplenie glutamiléndopeptidazy v kul'tural'noi zhidkosti Bacillus intermedius 3-19.

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, while 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.

[The effect of carbon sources and mononucleotides on the production of extracellular alkaline phosphatase by Bacillus intermedius]. / Vliianie istochnikov ugleroda i mononukleotidov na produktsiiu vnekletochnoi shchelochnoi fosfatazy Bacillus intermedius.

The biosynthesis of extracellular alkaline phosphatase in the streptomycin-resistant strains Bacillus intermedius S3-19 and S7 in the presence in the medium of 5'-nucleoside monophosphates and different sources of carbon--glucose, sodium pyruvate, sodium lactate, or glycerol--was studied. It was established that, in the presence of mononucleotides, the content of extracellular alkaline phosphatase in both strains increased; the maximal effect was caused by 5'-AMP at a concentration of 20 micrograms/ml. In medium with a low orthophosphate content, where active biosynthesis of alkaline phosphatase occurred, 1% glucose and 0.5% pyruvate stimulated this process 2.5-4 times, and 2% sodium lactate and sodium pyruvate, on the contrary, inhibited it by 20-40%. Analysis of the dynamics of growth and accumulation of extracellular phosphatase in the presence of different sources of carbon in the medium gives evidence of an interrelationship between the biosynthesis of alkaline phosphatase and carbon metabolism in Bacillus intermedius.

[Localization of alkaline phosphatase in cells of Bacillus intermedius]. / Lokalizatsiia shchelochnoi fosfatazy v kletkakh Bacillus intermedius.

Alkaline phosphatase, an enzyme secreted by Bacillus intermedius S3-19 cells to the medium, was also detected in the cell wall, membrane, and cytoplasm. The relative content of alkaline phosphatase in these cell compartments depended on the culture age and cultivation medium. The vegetative growth of B. intermedius on 0.3% lactate was characterized by increased activity of extracellular and membrane-bound phosphatases. The increase in lactate concentration to 3% did not affect the activity of membrane-bound phosphatase but led to a decrease in the activity of the extracellular enzyme. Na2HPO4 at a concentration of 0.01% diminished the activity of membrane-bound and extracellular phosphatases. CoCl2 at a concentration of 0.1 mM released membrane-bound phosphatase into the medium. By the onset of sporulation, phosphatase was predominantly localized in the medium and in the cell wall. As is evident from zymograms, the multiple molecular forms of phosphatase varied depending on its cellular localization and growth phase.

[Medium for the production of Bacillus intermedius glutamyl endopeptidase by the recombinant Bacillus subtilis]. / Sreda dlia biosintheza glutamiléndopeptidazy Bacillus intermedius rekombinantnym shtammom Bacillus subtilis.

A nutrient medium was elaborated for the efficient production of glutamyl endopeptidase by the recombinant Bacillus subtilis strain AJ73 bearing the Bacillus intermedius 3-19 glutamyl endopeptidase gene within a multicopy plasmid. Optimal concentrations of the main nutrients, peptone and inorganic phosphate, were found using a multifactor approach. To provide for active growth and efficient glutamyl endopeptidase production, the cultivation medium of the recombinant strain should be enriched in phosphorus, organic and inorganic nitrogen sources, and yeast extract. Complex protein substrates, such as casein and gelatin, enhanced the biosynthesis of glutamyl endopeptidase. At the same time, easily metabolizable carbon sources suppressed it. The production of glutamyl endopeptidase was stimulated by the bivalent cations Ca2+, Mg2+, and Co2+.

[Localization of proteolytic enzymes in cells of Bacillus intermedius]. / Lokalizatsiia proteoliticheskikh fermentov v kletkakh Bacillus intermedius.

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. 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.

[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.

[Effect of the RNAase from Bacillus intermedius on growth and physiological characteristics of Escherichia coli]. / Issledavanie vliianiia RNKazy Bacillus intermedius na rost i fiziologicheskuiu kharakteristiku Esherichia coli.

The effect of the RNase from Bacillus intermedius on the growth of Escherichia coli was investigated. RNase added to growth medium enhanced the synthesis of DNA, RNA, and protein and stimulated cell division; the degree of stimulation depended on the enzyme concentration. A necessary condition for stimulation was the adsorption of the enzyme on the cell surface and its interaction with the cytoplasmic membrane, as demonstrated immunocytochemically. The adsorption of the enzyme was accompanied by a 43% decrease in the surface charge density. Other effects of RNase involved a 25% increase in the growth rate, a 38% biomass gain, and generation time shortening by 10 min. The stimulation of bacterial growth correlated with the stimulation of cellular respiration rate.

[Polydispersity of Serratia marcescens nuclease at optimal pH values]. / Polidispersnost' nukleazy Serratia marcescens pri optimal'nom znachenii pH.

Treatment with dimethyl suberimidate, a cross-linking bifunctional agent, showed that Sm1 and Sm2 nucleases of Serratia marcescens B10M1 are polydisperse in solution and consist of monomers and dimers at the level of pH optimal for the enzyme activity. The data suggest that nucleases from the strain B10M1 and any other strain are polydisperse at pH optimum if their amino acid sequences are identical.