ABSTRACT
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.
Subject(s)
Bacillus/enzymology , Ribonucleases/metabolism , Bacillus/genetics , Culture Media , Enzyme Activation , Escherichia coli/enzymology , Escherichia coli/genetics , Mutation , Recombinant Proteins/metabolism , Ribonucleases/geneticsABSTRACT
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.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Regulon , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Base Sequence , Escherichia coli/metabolism , Molecular Sequence Data , Operon , Phosphates/metabolism , Promoter Regions, Genetic , Signal TransductionABSTRACT
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.
Subject(s)
Bacillus/enzymology , Phosphoric Monoester Hydrolases/biosynthesis , Bacillus/genetics , Base Sequence , DNA, Bacterial , Genes, Regulator , Molecular Sequence Data , Recombination, GeneticABSTRACT
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.
Subject(s)
Bacillus/genetics , Bacterial Proteins/genetics , Endoribonucleases/genetics , Regulon , Ribonucleases/genetics , Bacillus/enzymology , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Base Sequence , Gene Expression Regulation, Enzymologic , Molecular Sequence Data , Promoter Regions, Genetic , Signal TransductionABSTRACT
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.
Subject(s)
Bacillus/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Ribonuclease T1/genetics , Amino Acid Sequence , Bacillus/enzymology , Bacterial Proteins/genetics , Base Sequence , DNA, Bacterial , Molecular Sequence Data , Plasmids , Species SpecificityABSTRACT
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.
Subject(s)
Bacillus/genetics , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Ribonuclease T1/genetics , Signal Transduction/genetics , Bacillus/enzymology , Base Sequence , Endoribonucleases , Molecular Sequence Data , Plasmids , Promoter Regions, Genetic , Regulon , Ribonuclease T1/biosynthesis , Sequence Analysis, DNAABSTRACT
Alkaline phosphatase (APase) was isolated from the culture liquid of the streptomycin-resistant strain of Bacillus intermedius S3-19 and purified as a homogeneous preparation by ion-exchange chromatography and FPLC. Electrophoresis and gel-filtration revealed that the active enzyme is a monomer with molecular weight of 46-47 kD. The enzyme possessed phosphomonoesterase and phosphodiesterase activities with maximal levels at pH 9.5 and 55 degreesC and was stable until 60 degreesC at pH 8.0-10.0. The isolated APase exhibits a broad specificity towards a wide variety of substrates. The effect of divalent metal ions and other reagents on its catalytic activities was studied. It was concluded that alkaline phosphatase of B. intermedius is similar to the secreted alkaline phosphatases from other Bacillus species in its physicochemical and catalytic properties.
