The Pho regulons of bacteria

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

The Pho regulons of bacteria

Bacterial Pho regulons contain genes whose products are involved in the transport and assimilation of inorganic phosphate. The expression of these genes is regulated by a specific two-component signal transduction system. 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. © 2002 MAIK "Nauka/Interperiodica"

Actinomycin D Influence on Biosynthesis of Extracellular Ribonucleases by Sporulating Bacteria

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 β-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 β-galactosidase gene from the promotors of the bacillary RNAses was not stimulated by actinomycin D irrespective of the dose and addition time

Production of high-molecular-weight ribonuclease Bsn from the recombinant strain of Bacillus subtilis

Background: Ribonucleases (RNases) can be used in both basic and clinical sciences, e.g. in research on developmental processes or on antiviral and antitumor therapy. RNases have great potential as therapeutic entities. On the basis of new ribonucleases new medications can be created. Bacilli synthesize two types of secretory ribonucleases, the well-studied low-molecular-weight ribonucleases and high-molecular-weight ribonucleases. Only two RNases of the second type have so far been described: RNase Bsn from B. subtilis and binase II from B. intermedius. Materials/Methods: The activity of ribonucleases was determined from the amount of the acid-soluble products of RNA hydrolysis. The cultivation media were optimized for maximum RNase production in terms of the experimental factorial design B2 using BIOPT software. Results: Our investigation of a novel secretory ribonuclease, the Bacillus subtilis RNase Bsn expressed in the recombinant B. subtilis strain 168, showed that it is synthesized in the growth retardation phase, when inorganic phosphate is exhausted in the medium. The biosynthesis of Bsn was found to be suppressed by inorganic phosphate in the medium and activated by small amounts of the transcriptional inhibitor actinomycin D. Conclusion: Our results show that the biosynthesis of the novel secretory ribonuclease Bsn in recombinant strain Bacillus subtilis 168 is subject to negative regulation by inorganic phosphate, and is activated by small doses of actinomycin D. The stimulating effect of this antibiotic is well pronounced during the active synthesis of ribonucleases, but insignificant when ribonuclease synthesis is inhibited by Pi

Expression of guanyl-specific ribonuclease genes of Bacillus intermedium and Bacillus pumilus is regulated by the PhoP-PhoB two-component signal-transduction system of the PHO regulon in recombinant Bacillus subtilis strains

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. © 1999 MAHK "Hayka/Interperiodica"

The biosynthesis of new secretory high-molecular-weight ribonucleases in Bacillus intermedius and Bacillus subtilis

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

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. © 1995

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

Effects of cultivation medium components on the accumulation of bacillar ribonucleases in the culture liquid of escherichia coli recombinant strains

-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

Effects of cultivation medium components on the accumulation of bacillar ribonucleases in the culture liquid of Escherichia coli recombinant strains

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. © 1996 MAHK Hayka/Interperiodica Publishing