The role of the promoter structure in the efficiency of the expression of guanylspecific ribonucleases from Bacillus intermedius and Bacillus pumilus

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.     

Ribonuclease from Bacillus thuringiensis var. subtoxicus. Gene structure and biosynthesis regulation

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.     

Directed evolution of barnase stability using proteolytic selection

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability.Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.     

Use of plasmid pTV1 in transposon mutagenesis and gene cloning in Bacillus amyloliquefaciens

The plasmid pTV1, constructed in Bacillus subtilis as a tool for insertional mutagenesis by the transposon Tn917, has been transferred to Bacillus amyloliquefaciens by transduction with the phage PBS1. Insertional mutants containing Tn917 were observed in the new host. Southern blot analysis of such mutants indicated no preference for insertion sites. The copy numbers of pTV1 in B. amyloliquefaciens and B. subtilis were found to be 1.4 and 14, respectively; the plasmid is less stable against loss in B. amyloliquefaciens. The overall transposition rate in B. amyloliquefaciens is nevertheless comparable to that in B. subtilis and large numbers of mutants are readily obtained. The yield of auxotrophs was about 0.7% of all mutants, but the preponderance of glutamate auxotrophs seen in B. subtilis was not observed. A number of auxotrophs were identified as to nutritional requirements and those tested were found to be stable. Mutants deficient in extracellular proteases, amylase, and ribonuclease (barnase) were also found and the inactivated barnase gene has been cloned in Escherichia coli. It seems likely, therefore, that any B. amyloliquefaciens gene for which there is a functional test could be cloned by this technique.     

Expression of Bacillus amyloliquefaciens extracellular ribonuclease (barnase) in Escherichia coli following an inactivating mutation

An inactivated gene for Bacillus amyloliquefaciens extracellular ribonuclease (barnase) has previously been cloned and sequenced following transposon mutagenesis. The intact gene could not be assembled in Escherichia coli and is presumed to be lethal. Therefore, we introduced specific mutations into the barnase gene to prevent its lethal effect. A Gln-73 mutant gene was stable in E. coli but only produced low amounts of barnase antigen. Mutants containing Asp, Gln or Arg, instead of His-102, at the active site were identified by immunological screening for barnase antigen. None of the mutant proteins with alterations at aa residue 102 possessed RNase activity. The level of barnase (Asp-102) was higher in E. coli than in B. subtilis but the protein was not processed to the correct size in E. coli. To obtain correct processing, the barnase (Asp-102) structural gene was fused to the E. coli alkaline phosphatase promoter and signal sequence (phoA). Cells containing this construct secreted correctly processed barnase (Asp-102) into the periplasmic space and culture supernatant at a level of 20 mg/l. Barnase (Asp-102) was purified and found to have an identical N-terminus and a thermal unfolding curve that was nearly identical to that of active barnase (His-102). The cloning and expression of barnase in E. coli will allow detailed analysis of barnase protein folding by molecular genetic approaches.     

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.     

Barnase and binase: twins with distinct fates

RNases are enzymes that cleave RNAs, resulting in remarkably diverse biological consequences. Many RNases are cytotoxic. In some cases, they attack selectively malignant cells triggering an apoptotic response. A number of eukaryotic and bacterial RNase-based strategies are being developed for use in anticancer and antiviral therapy. However, the physiological functions of these RNases are often poorly understood. This review focuses on the properties of the extracellular RNases from Bacillus amyloliquefaciens (barnase) and Bacillus intermedius (binase), the characteristics of their biosynthesis regulation and their physiological role, with an emphasis on the similarities and differences. Barnase and binase can be regarded as molecular twins according to their highly similar structure, physical-chemical and catalytic properties. Nevertheless, the 'life paths' of these enzymes are not the same, as their expression in bacteria is controlled by diverse signals. Binase is predominantly synthesized under phosphate starvation, whereas barnase production is strictly dependent on the multifunctional Spo0A regulator controlling sporulation, biofilm formation and cannibalism. Barnase and binase also have some distinctions in practical applications. Barnase was initially suggested to be useful in research and biotechnology as a tool for studying protein-protein interactions, for RNA elimination from biological samples, for affinity purification of RNase fusion proteins, for the development of cloning vectors and for sterility acquisition by transgenic plants. Binase, as later barnase, was tested for antiviral, antitumour and immunogenic effects. Both RNases have found their own niche in cancer research as a result of success in targeted delivery and selectivity towards tumour cells.     

Brownian dynamics simulation of the association of Bacillus amyloliquefaciens ribonuclease (barnase) and Bacillus intermedius ribonuclease (binase) with barstar

A comparative study of the association of two ribonucleases, barnase and binase, with the polypeptide inhibitor barstar has been performed by the Brownian dynamics simulation method. It was shown that the method adequately reproduced the dependence of the association rate on pH and ionic strength of solution and the influence of mutations of some ribonuclease amino acids. Two types of energetically favorable complexes of binase-barstar encounter were determined. In the type I complex, the amino acids of binase active center take part in the complex formation. In the second complex, the active center is free. It was supposed that the temporary binding of barstar into complex of type II is competitive relative to the inhibition reaction. This can partially explain the decrease in the rate of binase inhibition as compared with the corresponding reaction of barnase.     

A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells

Tumor-targeted vectors with controllable expression of therapeutic genes and specific antitumor antibodies are promising tools for the reduction of malignant tumors. Here we describe a new plasmid for the eukaryotic expression of an anti-HER2/neu mini-antibody-barnase fusion protein (4D5 scFv-barnase-His(5)) with an NH(2)-terminal leader peptide. The 4D5 scFv-barnase-His(5) gene was placed downstream of the tetracycline responsive-element minimal promoter in the vector using the Tet-Off gene-expression system. The Bacillus amyloliquefaciens ribonuclease barnase is toxic for the host cells. To overcome this problem, barstar gene under its own minimal cytomegalovirus promoter was used in designed vector. Barstar inhibits the background level of barnase in the cells in the presence of tetracycline in culture medium. The HEK 293T cells were transfected with the designed vector, and the 4D5 scFv-barnase-His(5) fusion protein was identified by anti-barnase antibodies in cell culture medium and after purification from cell lysates using metal-affinity chromatography. The overexpression of the anti-HER2/neu mini-antibody-barnase fusion protein decreased the intensity of fluorescence of HEK 293T cells co-transfected with the generated plasmid and a plasmid containing the gene of enhanced green fluorescent protein (pEGFP-N1), in comparison with the intensity of fluorescence of HEK 293T cells transfected with pEGFP-N1, in the absence of tetracycline in the medium. The effect of the 4D5 scFv-barnase-His(5) on EGFP fluorescence indicates that the introduced barnase functions as a ribonuclease inside the cells. The anti-HER2/neu mini-antibody could be used to deliver barnase to HER2/neu-positive cells and provide its penetration into the target cells, as HER2/neu is a ligand-internalizing receptor. This expression vector has potential applications to both gene and antibody therapies of cancer.     

Expression of secreted guanyl-specific ribonuclease genes from Bacillus intermedius and Bacillus pumilus in Bacillus subtilis cells

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.     

A comparative study of the interaction of Bacillus amyloliquefaciens ribonuclease (barnase) and Bacillus intermedius ribonuclease (binase) with barstar by brownian dynamics simulation

A comparative study of the interaction of two RNases (binase and barnase) with the polypeptide inhibitor barstar was performed by Brownian dynamics simulation. It was demonstrated that this method adequately reproduced the dependence of the association rate on the pH of solution as well as the effect of mutations at individual amino acid residues on the inhibition of barnase by barstar. Two types of energy-favorable binase-barstar encounter complexes were found. In type I complex, the amino acid residues of the binase active center are involved in formation of the complex; in type II complex, the active center remains free. It is suggested that temporary binding of free barstar into type II complex competes with the inhibition reaction. Presumably, this explains the decrease in the rate of binase inhibition by barstar as compared with the analogous reaction of barnase.     

Comparative Study of Thermostability and Structure of Close Homologues - Barnase and Binase

Abstract

Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase in the pH region 2–6 have been determined. The barnase heat denaturation (pH 2.85.5) proceeds according to the “all-or-none” principle. Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 °C at pH 5 to 7 °C at pH 3. Enthalpy values of barnase and binase denaturation coincide only at pH 4.5–5.5, but as far as pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase. The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp94by the His 18 residue, absent in binase. Secondary structures of both native and denaturated proteins also do not differ. Some differences in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moments distribution, have been found.     

Antibiosis by Bacillus amyloliquefaciens ribonuclease barnase expressed in Escherichia coli against symbiotic and endophytic nitrogen-fixing bacteria

A modified antibiosis assay was used to evaluate growth inhibition of symbiotic and endophytic bacteria by E. coli strains producing Bacillus amyloliquefaciens ribonuclease, barnase. Inhibition zones were only observed when the assays were performed in minimal medium agar. However, bacterial growth inhibition was not detected when using rich medium or susceptible strains expressing the ribonuclease inhibitor protein, barstar. Our results suggest that barnase may act as a broad range bacteriocin. The ecological significance of these results is discussed.     

Barnase toxin: a new chimeric toxin composed of pseudomonas exotoxin A and barnase

We have constructed a chimeric toxin composed of Pseudomonas exotoxin A (PE) and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase. The chimeric protein, termed PE-Bar, reacted with both anti-PE and anti-barnase antisera and had both ADP ribosylation and ribonuclease activities. The chimeric toxin was cytotoxic to the murine fibroblast cell line L929 and to a murine hybridoma resistant to PE. A mutant form of PE-Bar lacking ADP-ribosylating activity was still cytotoxic to L929 cells. Because treatment of cells prelabeld with [3H]uridine resulted in a decrease in their RNA content, we conclude that this cytotoxic effect was due to the ribonuclease activity of barnase molecules that had been translocated to the cytosol. It is now possible to construct chimeric toxins with two or more enzymatic activities that can be delivered to the cytosol of the target cells.     

Bacillus pumilus Ribonuclease Inhibits Migration of Human Duodenum Adenocarcinoma HuTu 80 Cells

Migration of cancer cells from the primary tumor site to nearby tissues is the starting point of the metastatic process. The invasive properties of cells are especially important for carcinomas, since tumor cells need to overcome the basement membrane and go beyond its boundaries to the underlying tissues. Substances that reduce the invasive ability of malignant cells are promising as antimetastatic agents. In the present work, the possibility of inhibiting the ability of different cancer cell lines to migrate under the influence of the Bacillus pumilus ribonuclease (binase) was analyzed using the scratch-wound assay. It was established that binase at non-toxic concentrations (10 µg/mL) reliably suppressed the migratory ability of HuTu 80 human duodenum adenocarcinoma cells incubated with RNase for 48‒72 h. The antimetastatic potential of binase is confirmed by molecular modeling data demonstrating the ability of binase to inhibit cellular metalloproteinases that determine the migration of tumor cells.     

Gel-based oligonucleotide microarray approach to analyze protein-ssDNA binding specificity

Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to single-stranded DNA (ssDNA). To demonstrate additional capabilities of this method, we analyzed the binding specificity of ribonuclease (RNase) binase from Bacillus intermedius (EC 3.1.27.3) to ssDNA using generic hexamer oligodeoxyribonucleotide microchip. Single-stranded octamer oligonucleotides were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5'-{N}N(1)N(2)N(3)N(4)N(5)N(6){N}-3' consisted of a fixed hexamer motif N(1)N(2)N(3)N(4)N(5)N(6) in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096 pads with a complete set of hexamer sequences. The affinity was determined by measuring dissociation of the RNase-ssDNA complexes with the temperature increasing from 0 degrees C to 50 degrees C in quasi-equilibrium conditions. RNase binase showed the highest sequence-specificity of binding to motifs 5'-NNG(A/T/C)GNN-3' with the order of preference: GAG > GTG > GCG. High specificity towards G(A/T/C)G triplets was also confirmed by measuring fluorescent anisotropy of complexes of binase with selected oligodeoxyribonucleotides in solution. The affinity of RNase binase to other 3-nt sequences was also ranked. These results demonstrate the applicability of the method and provide the ground for further investigations of nonenzymatic functions of RNases.     

Key role of barstar Cys-40 residue in the mechanism of heat denaturation of bacterial ribonuclease complexes with barstar

The mechanism by which barnase and binase are stabilized in their complexes with barstar and the role of the Cys-40 residue of barstar in that stabilization have been investigated by scanning microcalorimetry. Melting of ribonuclease complexes with barstar and its Cys-82-Ala mutant is described by two 2-state transitions. The lower-temperature one corresponds to barstar denaturation and the higher-temperature transition to ribonuclease melting. The barstar mutation Cys-40-Ala, which is within the principal barnase-binding region of barstar, simplifies the melting to a single 2-state transition. The presence of residue Cys-40 in barstar results in additional stabilization of ribonuclease in the complex.     

Barnase and barstar. Expression of its cloned inhibitor permits expression of a cloned ribonuclease

Barnase is the extracellular ribonuclease of Bacillus amyloliquefaciens and barstar its specific intracellular inhibitor. The gene for barstar has now been cloned and sequenced. When the wild-type gene for barnase is reconstructed from its previously cloned parts on the same plasmid as the barstar gene, the lethal effect of its expression is suppressed. A plasmid has been devised which directs the secretion of 100 mg per active barnase liter by Escherichia coli and another which provides large (500 to 1000 mg/l) yields of barstar. The structure of these plasmids and the derived 89 amino acid sequence of barstar are reported.