RESUMEN
Bacillus pumilus ribonuclease (binase) exhibits cytotoxic and oncolytic properties, while causing genotoxic effects at high concentrations. Mutants that have reduced catalytic activity and preserve the antitumor properties of the native enzyme could exert lower toxic side effects. Mutant binase forms with the Lys26Ala and His101Glu single substitutions were obtained by site-directed mutagenesis. A comparative analysis of Escherichia coli- and Bacillus subtilis-based expression systems demonstrated that the latter is better to use to produce the binase mutants. The binase mutants with reduced catalytic activity were isolated and purified to homogeneity by ion exchange chromatography; the maximum yield was 25 mg/L. Catalytic activities of the mutants toward natural RNA-substrates in comparison with those for native binase were estimated at 11% and 0.02%, respectively. Like native binase, the Lys26Ala mutant was found to be cytotoxic to the A549, BT-20, and HuTu 80 tumor cell lines, but did not substantially affect normal WI-38 cells. The His101Glu mutant did not show cytotoxicity.
RESUMEN
Mycoplasmas are incapable of de novo synthesis of nucleotides and must therefore secrete nucleases in order to replenish the pool of nucleic acid precursors. The nucleolytic activity of mycoplasmas is an important factor in their pathogenicity. Bacterial ribonucleases (RNases) may produce a broad spectrum of biological effects, including antiviral and antitumor activity. Mycoplasma RNases are therefore of interest. In the present work, capacity of Acholeplasma laidlawii and Mycoplasma hominis for RNase synthesis and secretion was studied. During the stationary growth phase, these organisms were found to synthesize Mg(2+)-dependent RNases, with their highest activity detected outside the cells. Localization of A. laidlawii RNases was determined: almost 90% of the RNase activity was found to be associated with the membrane vesicles. Bioinformational analysis revealed homology between the nucleotide sequences of 14 Bacillus subtilis genes encoding the products with RNase activity and the genes of the mycoplasmas under study. Amino acid sequences of 4 A. laidlawii proteins with ribonuclease activity and the Bsn RNase was also established.
Asunto(s)
Mycoplasma/metabolismo , Ribonucleasas/metabolismo , Bacillus subtilis/genética , Magnesio/metabolismo , Mycoplasma/crecimiento & desarrollo , Mycoplasma hominis/crecimiento & desarrollo , Mycoplasma hominis/metabolismo , Ribonucleasas/biosíntesis , Ribonucleasas/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoAsunto(s)
Bacillus/enzimología , Bacillus/genética , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , Ribonucleasas/biosíntesis , Sulfato de Amonio/metabolismo , Bacillus subtilis/metabolismo , Secuencia de Bases , Medios de Cultivo , Endorribonucleasas/biosíntesis , Endorribonucleasas/genética , Genes Bacterianos , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Ribonucleasas/genética , Alineación de SecuenciaRESUMEN
The role of the ResD-ResE two-component signal transduction system in regulation of the bacilli guanyl-specific ribonucleases genes expression was studied. The proteins with the homology to the Bacillus subtilis ResD and ResE regulatory proteins were found in all sequenced genomes of the Bacillus. Using the B. subtilis strains deficient in the genes for these proteins it was shown that the ResD-ResE signal transduction system positively regulates the expression of the genes for B. intermedius, B. pumilus, and B. thuringiensis ribonucleases in the B. subtilis host cell. The data obtained in this work indicate that regulatory system similar to the B. subtilis ResD-ResE two-component signal transduction system also functions in other representatives of the Bacillus genus.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Ribonucleasa T1/metabolismo , Factores de Transcripción/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Filogenia , Ribonucleasa T1/genética , Factores de Transcripción/genéticaRESUMEN
Guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus are actively secreted under phosphate starvation by recombinant strains of Bacillus subtilis with native regulatory systems and by strains defective in some proteins of the Spo0A phosphorylation pathway. The level of expression of ribonuclease genes has been shown to increase approximately sixfold in recombinant strains with mutation in the spo0A gene and threefold in the spo0A/abrB mutants, as compared with native strains. These results demonstrate that the Spo0A protein regulates the production of ribonucleases and thus acts as a repressor, while the AbrB protein is an activator of expression of the genes encoding ribonucleases from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells.
Asunto(s)
Bacillus subtilis/genética , Bacillus/enzimología , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Ribonucleasa T1/genética , Factores de Transcripción/genética , Genes BacterianosRESUMEN
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
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
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
Promoters of the genes for guanyl-specific ribonucleases, secreted by B. intermedius (binase) and B. pumilus (Rnase Bp) in phosphate deficient conditions, contain regions similar to appropriate consensus sequences in promoters of the PHO regulated genes of B. subtilis. A number of genes expressed in response to phosphate starvation in B. subtilis are regulated by the two component signal transduction system PhoP-PhoR. Expression of recombinant genes for binase and RNase Bp in B. subtilis strains with mutations in the regulatory protein genes of the PHO regulon was studied. Their expression is strongly regulated by the regulatory proteins of the B. subtilis PHO regulon.
Asunto(s)
Bacillus/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Ribonucleasa T1/genética , Transducción de Señal/genética , Bacillus/enzimología , Secuencia de Bases , Endorribonucleasas , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Regulón , Ribonucleasa T1/biosíntesis , Análisis de Secuencia de ADNRESUMEN
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 structure gene of extracellular alkaline ribonuclease Bacillus intermedius (binase) has been cloned in E. coli cells in composition of pMT 316 plasmid carrying the inhibitor gene (barstar of barnase--binase structure homologue. The possibility to use such vector has been proved during the barstar action on binase catalytic activity. Using biochemical immunochemical analysis the expression of binase gene in E. coli cells has been confirmed. The recombinant clone E. coli which contains both plasmids simultaneously--carrying gene for barster and for benase has been produced. The given vector is suggested to be used for cloning of inhibitor gene to obtain a viable producer of alkaline intracellular ribonuclease.
Asunto(s)
Bacillus/enzimología , Bacillus/genética , Clonación Molecular/métodos , Exorribonucleasas/genética , Regulación Bacteriana de la Expresión Génica/genética , Genes Bacterianos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Endorribonucleasas/análisis , Endorribonucleasas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Exorribonucleasas/análisis , Vectores Genéticos/genética , Plásmidos/genética , Ribonucleasas/análisis , Ribonucleasas/genéticaRESUMEN
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 UltravioletaAsunto(s)
Alérgenos/análisis , Ácaros/inmunología , Animales , Cobayas , Métodos , Ácaros/crecimiento & desarrollo , Polisacáridos/análisis , Proteínas/análisis , ConejosRESUMEN
Normal human serum antibodies to the protein complex more than 20 common (species-specific) STr. Pneumoniae antigens have been studied. In commercial lots of gamma globulin, manufactured during the last 8 years, antibodies to 9 pneumococcal antigens have been detected. The spectrum of antibodies and the intensity of reactions Str. pneumoniae has been found to be not inferior to Str. aureus and pyogenes (group A) and to considerably exceed Str. faecalis, Enterobacteriaceae, St. epidermidis, Str. salivarius, Str. viridans, N. perflava, Ps. aeruginosa, H. influenzae, B. bifidum.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Antígenos Bacterianos/inmunología , Streptococcus pneumoniae/inmunología , Humanos , Inmunodifusión , Especificidad de la Especie , gammaglobulinas/inmunologíaRESUMEN
The content and type of antibodies to the common antigens of enterobacteria, enterococci and bifidobacteria were studied in serum specimens obtained from 220 donors. Antibody titers to enterobacteria were almost twice as high as those to enterococci and 8 times higher than those in bifidobacteria. The final titer of the reaction with enterobacterial antigens in 90% of the donors was determined by IgG (7S); in respect of enterococci and bifidobacteria IgM played the leading role in 56% and 74% of the cases, respectively. In most cases the profile of immune responsiveness was characterized by marked individuality and comprised various titers and types of antibodies to the antigens of all three groups of bacteria. The results thus obtained are analyzed from the viewpoint of normal responsiveness to standard immunological stimuli; all individuals come in contact with such stimuli with an equal degree of probability.
Asunto(s)
Anticuerpos Antibacterianos , Antígenos Bacterianos , Enterococcus faecalis/inmunología , Lactobacillus/inmunología , Proteus/inmunologíaRESUMEN
The immunotyping of the intracellular protein complex in enterobacteria has allowed to reveal that the intracellular proteins of E. carotovora and Y. enterocolitica possess an antigenic profile characteristics of bacteria belonging to the family Enterobacteriaceae. E. carotovora are similar to the main group of enterobacteria (Escherichia, Shigella, Salmonella, Enterobacter, Klebsiella, Citrobacter, Arizona) in the degree of the antigenic homology of their intracellular protein complex. Y. enterocolitica considerably differ from other enterobacteria, forming an independent immunotype of the family Enterobacteriaceae.