The site-specific endonucleases of bacteria in the genus Bacillus

Production regularities of site-specific endonucleases by aerobic spore-forming bacteria, separated from different ecological sources have been studied. It is shown that more than 1/3 of all the studied cultures produce site-specific endonucleases. A dependence of occurrence frequency of bacteria producers on the econiche of their separation has been noticed. The data on production of species-specific restrictases are obtained which can serve additional characteristics for differentiation of close species of Bacillus.     

Entomopathogenic bacteria Bacillus thuringiensis as producers of restriction endonucleases

A total of 115 collection strains of Bacillus thuringiensis, belonging to various subspecies, have been studied for the presence of DNA restriction-modification systems. Restriction endonucleases of 13 strains have been isolated and characterized. No considerable correlations between the taxonomic positions of the bacteria and the specificities of the endonucleases isolated have been detected. It is concluded that the enzymes with identical specificities are present in both the crystalliferous and acrystalliferous strains of the same subspecies.     

Chromosomal loci of genes controlling site-specific restriction endonucleases of Bacillus subtilis

Summary In Saccharomyces cerevisiae, diploid strains which are respiratory deficient (e.g., rho^–) or are homozygous for the mating-type locus (i.e., either a/a or /) are unable to sporulate. In order to induce sporulation in these nonsporulating strains, the technique of protoplast fusion mediated by polyethylene glycol was adopted. In this study, the products of protoplast fusion were induced to sporulate without reversion to normal cells.Protoplasts from a respiratory-deficient diploid strain were mixed with those from a respiratory-competent haploid one carrying mitochondrial drug resistance markers, treated with 30% polyethylene glycol-4000 and 25 mM CaCl₂, and incubated in 0.1 M potassium acetate containing 0.8 M sorbitol as an osmotic stabilizer. After two days' incubation, asci with three to eight spores were formed at a frequency of 1×10^–3 to 2×10^–4. Sporulation was also observed in products of fusion between an a/a diploid and haploid strains and between an / diploid and a haploid strains. The analysis of the genotypes of spores revealed that when fusion products were cultured under conditions for sporulation, karyogamy did not take place, diploid nuclei underwent meiosis, and both diploid and haploid nuclei were able to develop into spores.     

Chromosomal loci of genes controlling site-specific restriction endonucleases of Bacillus subtilis

Molecular Genetics and Genomics - We constructed transformants of B. subtilis 168 which acquired genes for site-specific restriction endonucleases. These endonucleases originated from various...     

Biochemical and genetic properties of site-specific restriction endonucleases in Bacillus globigii

Bacillus globigii contains two site-specific endonucleases, BPGLI AND BglI. A rapid technique for selection of mutants deficient in each of these enzymes was developed using sensitivity to infection by bacteriophage SP50 as an indication of the levels of enzyme. Mutants defective in BglI, BglII, and both BglI and BglII retained the wild-type modification phenotype. Genetic and biochemical studies have established that these enzymes are involved in restriction in vivo. Simplified purification procedures for BglI and BglII using these mutants are described.     

New restriction endonucleases from Acetobacter aceti and Bacillus aneurinolyticus.

Two restriction endonucleases with new sequence specificities have been isolated from Acetobacter aceti IFO 3281 and Bacillus aneurinolyticus IAM 1077 and named AatII and BanII, respectively. Based on analysis of the sequences around the restriction sites, the recognition sequences and cleavage sites of these endonucleases were deduced as below: (formula; see text)     

New producers of biologically active compounds--fungal strains of the genus Penicillium isolated from permafrost

Screening of producers of secondary metabolites was carried out among 25 fungal strains of Penicillium genus isolated from permafrost in Arctic and Antarctic regions and Kamchatka. Nearly 50% of the investigated strains synthesize biologically active substances of alkaloid nature: ergot alkaloids, diketopiperazines, and quinoline derivatives. A large group of the identified metabolites belongs to mycotoxins. A strain of Penicillium waksmanii was found producing epoxiagroclavine-I and quinocitrinins. The main physiological and biochemical characteristics of this producer were investigated.     

Two restriction endonucleases from Bacillus globiggi.

The sites of action of the restriction enzyme Bgl II on lambda DNA are mapped. This enzyme recognises the sequence 5' ...AGATCT...3' and makes staggered cuts producing sticky ends. In lambda DNA, the second A in this sequence is methylated about 50% of the time by a bacterial methylase absent in E. coli dam. In contrast to Bgl II, Bgl I makes many cuts in lambda DNA and produces 5' terminals which are not substrates for polynucleotide kinase.     

Type II restriction endonucleases from Bacillus sphaericus

Abstract The galactophilic lectin of the bacterium Pseudomonas aeruginosa (PA-I) was used for mitogenic stimulation of peripheral bloodlymphocytes from cancer-bearing patients and healthy subjects. This lectin, which preferentially stimulates sialidase-treated lymphocytes, was shown to be useful in the detection of an impairment in the mitogenic response of the patients' lymphocytes. Its efficiency was at least as that of the Phaseolus vulgaris lectin (PHA), which is widely used for the diagnosis and prognosis of deficient immunocompetence states.     

Two new site-specific endonucleases from Staphylococcus species strain D5

Staphylococcus species strain D5 containing two site-specific endonucleases, SspD5 I and SspD5 II, was found during screening of a bacterial strain collection from soil. These endonucleases were purified to functional homogeneity by sequential chromatography. Site-specific endonuclease SspD5 I recognizes sequence 5;-GGTGA(8N/8N) downward arrow-3; on DNA. Unlike Hph I, it cleaves DNA at a distance of 8 nucleotides from the recognized sequence on both chains producing blunt-end DNA fragments, while endonuclease Hph I cleaves DNA forming mononucleotide 3;-OH protruding ends. Thus, endonuclease SspD5 I is a new type II site-specific endonuclease and a neoschizomer of endonuclease Hph I. The advantage of this new endonuclease is that the blunt-end DNA products of this enzyme can be inserted without additional treatment into vector DNAs cleaved with endonucleases yielding DNA blunt-ends. Endonuclease SspD5 II recognizes site 5'-ATGCA T-3' and thus is an isoschizomer of endonuclease Nsi I. The molecular mass of SspD5 I is about 35 kD and that of SspD5 II is 40 kD. The enzymes exhibit maximal activity at 37 degrees C. The optimal buffer for the reaction is HRB (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, and 1 mM dithiothreitol).     

Detection of aquatic microorganisms from the Black Sea--producers of restriction endonucleases

300 clones of microorganisms isolated at different stations and from different depths in the Black Sea were screened for restriction endonucleases production. The production of restriction endonucleases was found in 17 clones screened. Three of them were identified to be Alteromonas haloplanktis B1. Restriction endonuclease AhaB1 is an isoshizomer of Sau961. An identified Alteromonas haloplanktis clone B8 produces AhaB8I restriction endonuclease the prototype to which is KpnI. Of the clones isolated three are Moraxella species B4 producing MapB4I restriction endonuclease analogous to BanI, three are Bacillus species producing BspB2I and one is Micrococcus lylae 113 producing Mly1131 analogue of NarI, six Moraxella species B6 produce MspB6I. The isolated producer strains may be used for isolation of above mentioned restriction endonucleases.     

2 site-specific endonucleases of the cyanobacterium Nostoc linckia]

Two restrictases Nli387/7 I and Nli387/7 II have been isolated from cyanobacterium Nostoc linckia using chromatography on phosphocellulose, "Mono Q" column, and heparin sepharose 4B. The preparations are described by the method of electrophoresis in polyacrylamide gel under denaturing conditions. Catalytic properties of restrictases are determined: optimal pH of the action--9.0--9.5, optimal concentration of Na+--5 mM, that of Mg2+--6 mM, optimal temperature--37 degrees C. The isolated enzymes are isoschizomers of restrictases avaI and AvaII. The point of cutting is determined for enzyme Nli387/7 I. It is shown that restrictase Nli387/7 I is a false isoschizomer Ava I.     

Reaction kinetics of some important site-specific endonucleases.

Reaction kinetics of the site-specific endonucleases BamHI, BgIII, C1aI, EcoRI, HpaII, PstI, SaII, SmaI, and XorII were investigated employing some frequently used substrates. Six of these enzymes could be analyzed under steady-state conditions. Kinetic data were obtained from progress curves applying an integrated Michaelis-Menten equation. KM ranged from 4 x 10(-9) M to 4 x 10(-11) M. Activities also spanned two orders of magnitude. In the case of C1aI the analysis of the pre-steady-state kinetics ("burst reaction") allowed the assessment of several rate constants. The rate-limiting step is the very slow dissociation of the enzyme-product complex (0.22 min(-1)). This complex is formed from the enzyme-bound nicked intermediate at a rate of 1.7 min(-1). The introduction of the first cut is again faster by a factor of about 6. SmaI and XorII resembled C1aI in their kinetics. The burst reaction can be used for the easy and unambiguous determination of molar concentrations of site-specific endonucleases in any preparation, which is free of non-specific DNases.     

Substrate specificity and properties of methyl-directed site-specific DNA endonucleases

Methyl-directed site-specific DNA endonucleases (MD endonucleases) are a small group of enzymes that specifically cleave only methylated DNA. The group includes N6-methyladenine- and 5-methylcytosine-directed enzymes. Although poorly understood, MD endonucleases are of interest for both basic research and application in biotechnology and epigenomics. The review for the first time summarizes the properties of MD endonucleases and considers their role in the bacterial cell and their possible uses in biotechnology and epigenomics.     

BbvII--a new site-specific endonuclease from Bacillus brevis 80

BbvII, a new site-specific endonuclease, has been isolated from Bacillus brevis 80 by gel-filtration and chromatography on heparin-Sepharose. The endonuclease recognizes a non-symmetrical sequence 5'-GTCTTC-3' in double-stranded DNA and cleaves DNA 3'-CAGAAG-5' in both strands outside the recognition sequence.     

Bvui: a site-specific endonuclease from Bacillus vulgatis

A site-specific endodeoxyribonuclease was partially purified from an extract of osmotically shocked spheroplasts of a Bacillus vulgatis strain; the enzyme has been designated BvuI and its activity was characterized. The locations of BvuI-generated cleavages on bacteriophage lambda and M13 derivatives mp7, mp8 and mp9, SV40 and PBR322 DNA molecules were determined. BvuI was shown to recognize the DNA sequence decreases 5'-G-Pu-G-C-Py-C-3' 3'-C-Py-C-G-Pu-G-5' increases and cleaves it at the positions indicated by arrows. Two identical BvuI recognition sites separated by fourteen nucleotide pairs were shown to occur within the tetracycline resistance gene of PBR322; BvuI should be useful for molecular cloning in that plasmid vector.     

Biosynthesis of fibrinolytic enzymes with different mechanisms of action by microorganisms of the genus Bacillus

The effect exerted by the composition of a growth medium on the biosynthesis of fibrinolytic enzymes was studied in Bacillus licheniformis 125, B. macerans P1 and Bacillus sp. Fibrinolytic enzymes with the plasmin and activator effects on fibrin were found to be formed predominantly depending on the specified conditions, e.g. in a chemically defined medium with potato broth.     

Search for producers of restriction endonucleases with a given specificity]

Six site-specific restriction endonucleases were isolated from Bacillus thuringiensis strains chosen of 58 strains ought purposefully for production of restriction enzymes. All six strains produce isoschisomers of Sau3A.     

Isolation, purification and properties of new restriction endonucleases from Bacillus badius and Bacillus lentus

We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689 respectively, by ammonium sulphate precipitation, phosphocellulose and heparin-sepharose column chromatography. SDS-PAGE protein profiles for BbaI and BleI showed denatured molecular weights of 52 and 48 kDa, respectively. BbaI hydrolyzed pUC18 DNA into 1900 and 700 bp, pBR322 DNA into two fragments of 2800 and 1500 bp and Φ×174 DNA into 3800 and 1600 bp. BleI hydrolyzed pUC18 DNA into 1800 and 800 bp, pBR322 DNA into two fragments of 2700 and 1600 bp and Φ×174 DNA into 3700 and 1700 bp. The effects of temperature, ionic strength, pH and Mg²⁺ ion concentrations were studied to demonstrate some biochemical properties of BbaI and BleI. Maximum activities of these enzymes were observed at 37 °C (pH 8.0) with 100 mM NaCl and 10 mM Mg²⁺ concentrations.