Phosphate regulation of biosynthesis of extracellular RNases of endospore-forming bacteria.

The gene for the extracellular ribonuclease of B. pumilus KMM62 (RNase Bp) has been cloned and sequenced. The structural gene for this enzyme is similar to those of the extracellular ribonucleases of B. intermedius 7P (binase) and B. amyloliquefaciens H2 (barnase), as are the regulatory regions of binase and RNase Bp. The regulatory region of the barnase gene, however, is quite different from the other two. In the promoter of the genes for binase and RNase Bp, but not in that for barnase, is a region similar to the Pho box of E. coli. We have established that inorganic phosphate suppresses the synthesis of the binase and RNase Bp, but does not effect the synthesis of barnase.

Contribution of arginine-82 and arginine-86 to catalysis of RNases from Bacillus intermedius (binase).

To elucidate the functional role of Arg82 and Arg86 in the enzyme activity of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant Arg86 Ala is 2.7 x 10(3) - 7.7 x 10(3) times less than that of the native enzyme. The decrease in activity is determined preferentially by the decrease in the molecular rate constant kcat with a relatively small change of enzyme-substrate affinity, characterized by Km. This is the expected result if Arg86 acts to lower the energy of a transition state of the reaction. The replacement of Arg82 by Ala causes a 5-19-fold activity decrease, depending on the substrate. We propose that this residue does not have a direct catalytic function in the molecular mechanism of the binase action and that the activity decrease of binase on the replacement of Arg82 by alanine is mediated by the effect of Arg82 on the pK of catalytic residues.

Mutational analysis of the active site of RNase of Bacillus intermedius (BINASE).

To elucidate the functional role of some residues in the active site of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant His101 Glu is 2.0-2.7% of that for the native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant kcat, with almost unchanged affinity of the enzyme for the substrate, characterized by KM. This is the expected result if His101 acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lys26 by Ala causes a reduction in the enzyme activity to 13-33%, depending on the substrate. The activity decreases are due to changes in both kcat and KM for poly(A) and poly(A) but in kcat alone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, barnase.

Expression of the genes for guanyl-specific ribonucleases from Bacillus intermedius and Bacillus pumilus is regulated by the two component signal transduction system PhoP-PhoR in B. subtilis.

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.

[Actinomycin D influence on biosynthesis of extracellular ribonucleases by sporulating bacteria]. / Vliyanie aktinomitsina D na biosintez vnekletochnykh ribonukleaz sporoobrazuyushchikh bakteril

The influence of actinomycin D on the synthesis of extracellular ribonucleases by "Bacillus intermedius" (binase), B.pumilus (RNAse Bp) and B.amyloliquefaciens (barnase) was studied comparatively. When added during the active synthesis of the enzymes actinomycin D stimulated the biosynthesis of binase and RNAse Bp and had no influence on the barnase biosynthesis. The response of the bacillary RNAse biosynthesis to the added actinomycin D correlated with the differences in the nucleotide sequences of the genes encoding the enzymes. The Escherichia coli SURE recombinant strains carrying the plasmids with the genes of binase, RNAse Bp and barnase under different regulatory sequences, as well as the E.coli MC4100 recombinant strains carrying the plasmids with the beta-galactosidase gene under the promoters of the bacillary RNAse were isolated. However, the expression of the bacillary ribonuclease genes in the E.coli recombinant strains carrying the plasmids with the genes of the enzymes, as well as the expression of the beta-galactosidase gene from the promotors of the bacillary RNAses was not stimulated by actinomycin D irrespective of the dose and addition time.

Novel extracellular ribonuclease from Bacillus intermedius--binase II: purification and some properties of the enzyme.

The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene. The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel. The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg. The molecular weight of binase II is 30 kD. The enzyme is activated by Mg2+ and virtually completely inhibited by EDTA. The pH optimum for the reaction of RNA hydrolysis is 8.5. The properties of the enzyme are close to those of RNase Bsn from B. subtilis. The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom.

A novel secreted ribonuclease from Bacillus intermedius: gene structure and regulatory control.

A second secreted ribonuclease, designated binase II, has been detected in Bacillus intermedius 7P, and its structural gene was cloned and sequenced. Unlike the well-known binase I, a 109-amino acid guanyl-specific enzyme, the 292-residue binase II is closely related to the B. subtilis nuclease Bsn, in structure and in its enzymatic properties. Binase II is also insensitive to inactivation by barstar, an inhibitor protein that is specific for guanyl-specific ribonucleases. While both B. intermedius enzymes are induced upon phosphate starvation, only the gene for binase I belongs to the pho regulon system and carries pho-box elements adjacent to its promoter sequence. The gene for binase II is similar to that for Bsn in lacking such elements. The birB gene coding for binase II appears to be located next to the 3'-end of a ferric ion transport operon, with which it convergently overlaps. This would allow attenuator control over binase II expression under conditions of starvation for ferric ions.