RESUMEN
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.
Asunto(s)
Bacillus/genética , Endorribonucleasas/genética , Regulación Enzimológica de la Expresión Génica , Genes Bacterianos , Regiones Promotoras Genéticas , Ribonucleasa T1/genética , Bacillus/enzimología , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Escherichia coli/enzimología , Escherichia coli/genética , Plásmidos , Reacción en Cadena de la PolimerasaRESUMEN
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.
Asunto(s)
Regiones no Traducidas 5'/genética , Bacillus/enzimología , Regiones Promotoras Genéticas/fisiología , Ribonucleasas/genética , Bacillus/genética , Proteínas Bacterianas , Dactinomicina/farmacología , Regulación Enzimológica de la Expresión Génica , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Ribonucleasas/biosíntesisRESUMEN
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.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regulón , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Operón , Fosfatos/metabolismo , Regiones Promotoras Genéticas , Transducción de SeñalRESUMEN
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.
Asunto(s)
Bacillus subtilis/metabolismo , Bacillus/metabolismo , Ribonucleasas/biosíntesis , Bacillus/crecimiento & desarrollo , Bacillus subtilis/crecimiento & desarrollo , Medios de Cultivo , Dactinomicina/farmacología , Endorribonucleasas/biosíntesis , Fosfatos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Recombinantes/biosíntesis , Transformación GenéticaRESUMEN
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.
Asunto(s)
Bacillus/enzimología , Monoéster Fosfórico Hidrolasas/biosíntesis , Bacillus/genética , Secuencia de Bases , ADN Bacteriano , Genes Reguladores , Datos de Secuencia Molecular , Recombinación GenéticaRESUMEN
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.
Asunto(s)
Bacillus thuringiensis/enzimología , Ribonucleasas/biosíntesis , Ribonucleasas/genética , Secuencia de Aminoácidos , Bacillus thuringiensis/crecimiento & desarrollo , Secuencia de Bases , Dactinomicina/farmacología , Endorribonucleasas/genética , Datos de Secuencia Molecular , Fosfatos/farmacología , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Bacillus/genética , Proteínas Bacterianas/genética , Endorribonucleasas/genética , Regulón , Ribonucleasas/genética , Bacillus/enzimología , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Secuencia de Bases , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Transducción de SeñalRESUMEN
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.
Asunto(s)
Bacillus/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Ribonucleasa T1/genética , Secuencia de Aminoácidos , Bacillus/enzimología , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano , Datos de Secuencia Molecular , Plásmidos , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Bacillus/enzimología , Ribonucleasa T1/biosíntesis , Bacillus/genética , Bacillus/crecimiento & desarrollo , Secuencia de Bases , Medios de Cultivo , ADN Bacteriano , Glucosa/metabolismo , Datos de Secuencia Molecular , Ribonucleasa T1/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
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.
Asunto(s)
Endorribonucleasas/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Genes Bacterianos , Genes Reguladores , Ribonucleasas/genética , Bacillus , Proteínas Bacterianas , Medios de Cultivo , Endorribonucleasas/biosíntesis , Escherichia coli , Proteínas Recombinantes/biosíntesis , Ribonucleasas/biosíntesisRESUMEN
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).
Asunto(s)
Bacillus/enzimología , Endopeptidasas/biosíntesis , Aminoácidos/farmacología , Caseínas/metabolismo , Medios de Cultivo , Endopeptidasas/metabolismo , Gelatina/metabolismo , Glucosa/farmacología , Concentración de Iones de Hidrógeno , Peptonas/farmacología , Fosfatos/metabolismo , Extractos Vegetales/farmacología , Inhibidores de Proteasas/farmacología , Especificidad por Sustrato , Zea mays/químicaRESUMEN
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.
Asunto(s)
Bacillus/enzimología , Escherichia coli/genética , Ribonucleasas/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Bacteriano , Escherichia coli/enzimología , Datos de Secuencia Molecular , Fosfatos/metabolismo , Plásmidos , Regiones Promotoras Genéticas , Recombinación Genética , Regulón , Ribonucleasas/genéticaRESUMEN
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.
Asunto(s)
Bacillus/enzimología , Monoéster Fosfórico Hidrolasas/biosíntesis , Proteínas Quinasas/biosíntesis , Ribonucleasas/biosíntesis , Oligoelementos/farmacología , Cationes Bivalentes , Cobalto/farmacología , Cobre/farmacología , Medios de Cultivo , Activación Enzimática , Compuestos Ferrosos/farmacología , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Quinasas/metabolismo , Ribonucleasas/metabolismo , Zinc/farmacologíaRESUMEN
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.
Asunto(s)
Bacillus/enzimología , Ribonucleasas/biosíntesis , Dactinomicina/farmacología , Fosfatos/farmacologíaRESUMEN
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.
Asunto(s)
Bacterias/efectos de los fármacos , Bacterias/enzimología , Enzimas/biosíntesis , Enzimas/efectos de los fármacos , Metales/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Esporas Bacterianas/efectos de los fármacos , Esporas Bacterianas/enzimologíaRESUMEN
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.
Asunto(s)
Escherichia coli/enzimología , Recombinación Genética , Ribonucleasas/aislamiento & purificación , Animales , Bacillus/enzimología , Proteínas Bacterianas , Cromatografía DEAE-Celulosa , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Inmunización , Inmunodifusión , Plásmidos , Conejos , Ribonucleasas/análisis , Ribonucleasas/inmunología , Espectrofotometría UltravioletaRESUMEN
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.
Asunto(s)
Antibacterianos/antagonistas & inhibidores , Bacillus/enzimología , Mutación/efectos de los fármacos , Bacillus/efectos de los fármacos , Bacillus/efectos de la radiación , Farmacorresistencia Microbiana , Endopeptidasas/biosíntesis , Exorribonucleasas/biosíntesis , Mutación/efectos de la radiación , Monoéster Fosfórico Hidrolasas/biosíntesis , Rayos UltravioletaRESUMEN
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.
Asunto(s)
Bacillus/efectos de los fármacos , Cloranfenicol/farmacología , Dactinomicina/farmacología , Exorribonucleasas/biosíntesis , Ribonucleasas/biosíntesis , Bacillus/enzimología , Bacillus/crecimiento & desarrollo , Medios de Cultivo/metabolismo , Relación Dosis-Respuesta a Droga , Fosfatos/metabolismo , Factores de TiempoRESUMEN
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%.
Asunto(s)
Bacillus/enzimología , Ribonucleasas/biosíntesis , Bacillus/crecimiento & desarrollo , Glucosa/farmacología , Concentración de Iones de Hidrógeno , Peptonas/farmacologíaRESUMEN
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.