The role of the promoter and leader sequences of extracellular ribonuclease from Bacillus amyloliquefaciens in regulation of enzyme biosynthesis

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.     

Extracellular alkaline ribonuclease of Bacillus thuringiensis var. subtoxicus]

Intraspecific selection of Bacillus thuringiensis strains producing extracellular alkaline ribonucleases was carried out. Subtoxicus subspecies with increased expression of the enzyme was detected. A method was developed to isolate preparative amounts of homogeneous extracellular RNase of B. thuringiensis var. subtoxicus. The physico-chemical and catalytic properties of the enzyme was studied and compared with extracellular RNases of others Bacillus species. The conclusion about the structural and evolutional conservation of Bacillus extracellular RNases was drawn.     

Phagocytosis-stimulating mediators in insects

The ability of insect blood cells to ingest all kinds of synthetic particles and also a wide range of microorganisms in a very short time after injection has up to now been regarded as a phagocytic function without any humoral mediators. In a phagocytosis model with latex beads and nonhagocytosable cells of Bacillus thuringiensis subtoxicus, we are able to demonstrate the existence of lymphokine-like factors, which intervene in cellular defence reactions of insect. The following results were obtained: 1. Immediately after injection of latex beads, normally non-phagocytosable cells of Bacillus thuringiensis subtoxicus are phagocytosed. 2. Cell-free haemolymph of larvae of Galleria mellonella previously injected with latex beads, stimulates in new larvae phagocytosis of Bacillus thuringiensis subtoxicus after transfusion. 3. The fractionation of homogenates of latex-treated larvae on Sephadex G 50 shows two fractions which stimulate phagocytosis. We suppose that the appearance of these phagocytosis-stimulating factors is the result of a successful recognition of foreign material.     

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.     

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.     

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.     

Inorganic phosphate has a crucial effect on Cry3Aa delta-endotoxin production

AIMS: The study aimed at increasing Cry3Aa delta-endotoxin production by a local isolate of Bacillus thuringiensis (B.t. strain Mm2). To this end, different nutritional conditions were tested for their effects on Cry3Aa yields. METHODS AND RESULTS: Bacillus thuringiensis Mm2 was grown by shaking at 30 degrees C in different media. Samples were taken from the cultures at intervals and used for protein extraction. SDS-PAGE was performed for toxin analysis. Inclusion of inorganic phosphate (Pi) into the Difco's sporulation medium at an increased level of 200 mmol l-1 caused a fivefold increase (from 3 to 15.6 microg ml-1) in toxin production. Omission of FeSO4 from the medium decreased this yield by half. Resuspension experiments suggested catabolite repression of toxin biosynthesis by glucose. The inclusion of high Pi invariably increased toxin synthesis, even in the absence of sugars. CONCLUSIONS: Inorganic phosphate had the most striking effect on toxin biosynthesis. Iron effect was found to be unique to our isolate whereas Pi effect seemed to be common to the biosynthesis of Cry3Aa-type toxins. Stimulation of toxin synthesis by Pi did not seem to represent a relief from glucose repression. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thuringiensis is the most versatile biopesticide for use in pest management. Regarding cost-effectiveness of related fermentations, high Pi supplement drastically increases Coleoptera-specific toxin synthesis.     

Extracellular ribonuclease from Bacillus thuringiensis

The ability of the strain Bacillus thuringiensis var. subtoxicus to produce extracellular ribonuclease (ribonuclease Bt) was studied. It was found that the culture medium possesses a RNA-depolymerizing activity whose maximum is observed 4-5 hours after the beginning of the linear growth phase. A three-step chromatography of the culture extract on phosphocellulose resulted in a homogeneous enzyme with a molecular mass of 12000 Da. The enzyme showed the maximum activity towards RNA at pH 8.5, catalyzed the hydrolysis of polyribonucleotides and guanosine-2',3'-cyclophosphate. Hence, the enzyme can be related to base-nonspecific cyclizing ribonucleases showing the guanylic specificity towards nucleoside-2',3'-cyclophosphates.     

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms

AIMS: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them. METHODS AND RESULTS: Using 16S rRNA gene restriction fragment length polymorphisms generated by HindIII and EcoRI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensis serovars and five more strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis. All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia, at the 92.5% DNA relatedness rate. CONCLUSION: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.     

Exogenous <Emphasis Type="Italic">Bacillus pumilus</Emphasis> RNase (binase) suppresses the reproduction of reovirus serotype 1

The experimental study identified the antiviral activity of Bacillus pumilus RNase (binase) against the reovirus of serotype 1/strain Lang. For the first time, it has been found that 50 μg/mL of binase effectively reduced the hemagglutinin and cytocidal activity of reovirus in Vero cell line. The preincubation of the enzyme with reovirus before infection of the cells inhibited the viral replication. To determine the stagedependent effect of reovirus reproduction upon binase inhibition, the infected cells were treated with binase or RNase A at different phases of the infectious cycle. The treatment of virus-infected cells has revealed that both enzymes have a maximal antiviral effect on the reovirus propagation during early phases of the reovirus reproduction cycle, with binase being more effective than RNase A. It has been hypothesized that the combined action of the oncolytic reovirus and binase is promising for the elimination of tumor cells carrying mutated RAS gene.     

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.     

Effect of chloramphenicol and actinomycin D on extracellular alkaline RNAse biosynthesis by Bacillus intermedius

By means of chloramphenicol it was found that biosynthesis of alkaline exocellular RNAase was repressed in Bacillus intermedius by inorganic phosphate. Actinomycin D at a low concentration stimulates RNAase biosynthesis in a medium with a minimal phosphorus concentration in model experiments with washed cells and in the batch culture. As a result, the activity of RNAase rises 2-4 times. The stimulating effect of actinomycin D decreases when phosphorus concentration in the medium is increased The effect of actinomycin D is maximal if the antibiotic is added to the medium when the specific growth rate of the bacterium falls down and the rate of RNAase biosynthesis rises.     

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms

K.-B. JOUNG AND J.-C. CÔTÉ. 2001 .
Aims: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them.
Methods and Results: Using 16S rRNA gene restriction fragment length polymorphisms generated by Hin dIII and Eco RI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensi s serovars and five more strains, kurstaki HD-1, subtoxicus , dendrolimus , tenebrionis and sandiego , to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis . All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia , at the 92·5% DNA relatedness rate.
Conclusions: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains.
Significance and Impact of the Study: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.     

Site-specific restriction endonuclease BtcI from Bacillus thuringiensis var. canadensis

Efficiency of bacteriophage Tp4 plating on Bacillus thuringiensis var. canadensis H5 (Can) is decreased 10(7)-fold as compared with the efficiency of plating on Bacillus thuringiensis var. galleriae H5 (Gal). Bacteriophage Tp4 having propagated for one cycle in Can cells might be further grown in this strain without restriction. The sitespecific restriction endonuclease BtcI isolated from Bacillus thuringiensis var. canadensis recognises the same nucleotide sequence GATC in DNA as recognised by restriction endonuclease Sau3A.     

Spore Refractility in Variants of Bacillus cereus Treated with Actinomycin D

Refractility as indicated by light microscopy, electron microscopy of thin sections, and freeze fracture etching was increased and maintained in a cortexless mutant, A(-)1, of Bacillus cereus var. alesti by the addition during sporulation stage 4 of actinomycin D, which prevents the terminal lysis of spore core associated with sporulation in this organism. (45)Calcium uptake levels and dipicolinic acid (DPA) content were similarly maintained. The location of these components appears to be in the spore protoplast. In the parent A(-), treated with actinomycin D during stage 4, spore particles with similar morphology to the mutant, that is without a cortex and with the characteristics of refractility, were obtained. A major difference in sensitivity to actinomycin D between the processes of (45)Ca uptake and DPA synthesis was observed. Some heat resistance in A(-) made cortexless by actinomycin D could be observed. These studies indicate that the role of the cortex is not to produce the dehydrated refractile spore state but to maintain it.     

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.     

Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis

Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers. Recently, the aiiA gene (encoding an enzyme catalyzing the degradation of AHL) has been cloned from Bacillus sp. strain 240B1. During investigations in the course of the ongoing Bacillus thuringiensis subsp. morrisoni genome project, an aiiA homologue gene in the genome sequence was found. These results led to consideration of the possibility of the widespread existence of the gene in B. thuringiensis. aiiA homologue genes were found in 16 subspecies of B. thuringiensis, and their sequences were determined. Comparison of the Bacillus sp. strain 240B1 aiiA gene with the B. thuringiensis aiiA homologue genes showed high homologies of 89 to 95% and 90 to 96% in the nucleotide sequence and deduced amino acid sequence, respectively. Among the subspecies of B. thuringiensis having an aiiA gene, the subspecies aizawai, galleriae, kurstaki, kyushuensis, ostriniae, and subtoxicus were shown to degrade AHL. It was observed that recombinant Escherichia coli producing AiiA proteins also had AHL-degrading activity and could also attenuate the plant pathogenicity of Erwinia carotovora. These results indicate that insecticidal B. thuringiensis strains might have potential to compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity.