A simple and rapid method for screening bacteria for type II restriction endonucleases: enzymes in Aphanothece halophytica

A method is described which allows a large number of bacterial strains to be rapidly and easily screened for the presence of site-specific endonucleases. The method involves selective permeabilization of the bacterial cell and analysis of the exuded material. Type II restriction endonucleases from cyanobacteria and Gram-negative eubacteria have been detected and new enzymes have been found. The method should be widely applicable and easy to modify for use in genera other than those tested. Three-site-specific endonuclease activities, detected by this method in Aphanothece halophytica PCC 7412, were purified and their recognition and cleavage specificities were determined AhaI and AhaII recognise and cleave the same DNA sequences as CauII and AcyI respectively; the specificity of AhaIII (TTTAAA) has been reported previously (Whitehead and Brown, 1982, FEBS Letters 143:296–300).Abbreviations Brij-58 20 cetyl ether - Pu purine nucleoside - Py pyrimidine nucleoside     

Recognition sequence of a new methyl-specific restriction system from Clostridium acetobutylicum strain ABKn8

A new type II restriction endonuclease, named Cac8I was detected in Clostridium acetobutylicum strain ABKn8. Cac8I cleaved the hexanucleotide sequence [5'-GCN decreases NGC-3'] and generated blunt ends. Up to now no isoschizomer of Cac8I has been described [corrected].     

Restriction of DNA encoding selectable markers decreases the transformation efficiency of Helicobacter pylori

Helicobacter pylori populations recovered from the human stomach display extensive recombination and quasispecies development, and this suggests frequent exchange of DNA between different strains in vivo. In vitro, however, most H. pylori strains display restriction to the uptake of non-self DNA, as measured using selectable markers, regardless of their competency for transformation with self DNA. We have examined the effect of different selectable markers on double-crossover recombination efficiencies in three reference strains (1061, 26695 & SS1) and one clinical isolate (CHP1) of H. pylori. All strains were efficiently transformable to kanamycin or chloramphenicol resistance by using self-genomic DNA from isogenic mutants bearing the aphA3 or cat cassettes, respectively. However, strains 26695 and CHP1 showed a 3-5-log reduction in transformation efficiency by non-self recombinant DNA containing aphA3, when compared to cat. Strain 1061 readily accepted either cassette, and strain SS1 was poorly tolerant of any non-self DNA. Genome-wide random mutagenesis of these strains was only achievable with a selectable marker that allowed high transformation efficiency. Digestion of 32P-labelled cassettes by H. pylori lysates mirrored the transformation results and indicated that in some strains these cassettes are the targets of enzymatic restriction.     

Natural transformation of an engineered Helicobacter pylori strain deficient in type II restriction endonucleases

Restriction-modification (RM) systems are important for bacteria to limit foreign DNA invasion. The naturally competent bacterium Helicobacter pylori has highly diverse strain-specific type II systems. To evaluate the roles of strain-specific restriction in H. pylori natural transformation, a markerless type II restriction endonuclease-deficient (REd) mutant was constructed. We deleted the genes encoding all four active type II restriction endonucleases in H. pylori strain 26695 using sacB-mediated counterselection. Transformation by donor DNA with exogenous cassettes methylated by Escherichia coli was substantially (1.7 and 2.0 log(10) for cat and aphA, respectively) increased in the REd strain. There also was significantly increased transformation of the REd strain by donor DNA from other H. pylori strains, to an extent corresponding to their shared type II R-M system strain specificity with 26695. Comparison of the REd and wild-type strains indicates that restriction did not affect the length of DNA fragment integration during natural transformation. There also were no differentials in cell growth or susceptibility to DNA damage. In total, the data indicate that the type II REd mutant has enhanced competence with no loss of growth or repair facility compared to the wild type, facilitating H. pylori mutant construction and other genetic engineering.     

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.     

Engineering of restriction endonucleases: using methylation activity of the bifunctional endonuclease Eco57I to select the mutant with a novel sequence specificity

Type II restriction endonucleases (REs) are widely used tools in molecular biology, biotechnology and diagnostics. Efforts to generate new specificities by structure-guided design and random mutagenesis have been unsuccessful so far. We have developed a new procedure called the methylation activity-based selection (MABS) for generating REs with a new specificity. MABS uses a unique property of bifunctional type II REs to methylate DNA targets they recognize. The procedure includes three steps: (1) conversion of a bifunctional RE into a monofunctional DNA-modifying enzyme by cleavage center disruption; (2) mutagenesis and selection of mutants with altered DNA modification specificity based on their ability to protect predetermined DNA targets; (3) reconstitution of the cleavage center's wild-type structure. The efficiency of the MABS technique was demonstrated by altering the sequence specificity of the bifunctional RE Eco57I from 5'-CTGAAG to 5'-CTGRAG, and thus generating the mutant restriction endonuclease (and DNA methyltransferase) of a specificity not known before. This study provides evidence that MABS is a promising technique for generation of REs with new specificities.     

Molecular methods for typing of Helicobacter pylori and their applications

Microbial typing is a useful tool in clinical epidemiology for defining the source and route of infection, for studying the persistence and reinfection rates, clonal selection in the host and bacterial evolution. Phenotypic methods such as biotyping, serotyping and hemagglutinin typing have little discriminatory power compared to genotypic methods concerning the typing of Helicobacter pylori. Therefore great efforts have been made to establish useful molecular typing methods. In this context, the most frequently used genotypic methods are described based on our own experience and the literature: (1) restriction endonuclease analysis, (2) endonuclease analysis using pulsed-field gel electrophoresis, (3) ribotyping, (4) polymerase chain reaction (using either random primers or repetitive DNA sequence primers), and (5) polymerase chain reaction-restriction fragment length polymorphism analysis of e.g. the urease genes. Furthermore, reproducibility, discriminatory power, ease of performance and interpretation, cost and toxic procedures of each method are assessed. To date no direct comparison of all the molecular typing methods described has been performed in the same study with the same H. pylori strains. However, PCR analysis of the urease gene directly on suspensions of H. pylori or gastric biopsy material seems to be useful for routine use and applicable in specific epidemiological situations.     

Purification and properties of a site-specific restriction endonuclease AaaI from Acetobacter aceti subsp. aceti No. 1023

Abstract A type II restriction endonuclease, named Aaa I, was purified from Acetobacter aceti subsp. aceti No. 1023. The optimum pH and temperature were determined to be 8.5 and 37°C, respectively. The enzyme activity was stimulated by the addition of either NaCl or KCl and their optimum concentrations were 100 mM for both cations. Aaa I recognized the hexanucleotide sequence and cleaved it at the positions indicated by the arrows. Aaa I was an isoschizomer of Xma III from Xanthomonas malvacaerum and Eco 52I from Escherichia coli .     

BpaI and BpnI: novel type II restriction endonucleases from Bacillus pasteurii and Bacillus pantothenticus

Two novel type II restriction endonucleases, designated as BpaI and BpnI, were isolated from Bacillus pasteurii strain1761 and Bacillus pantothenticus strain1639, respectively. They were partially purified and SDS-PAGE indicated Mr values of 28 and 67 kDa for BpaI, 28 and 48 kDa for BpnI. The partially purified endonucleases hydrolyzed DNA into discrete fragments: pUC18 (2.6 kb for BpaI; 1.8 and 0.8 kb for BpnI), pBR322 (2.5 and 1.8 kb for BpaI; 2.6 and 1.7 kb for BpnI) and phix174 DNA (3.2 and 2.1 kb for BpaI; 4 and 1.3 kb for BpnI).     

A combined polymerase chain reaction and restriction endonuclease enzyme assay for discriminating between Campylobacter coli and Campylobacter jejuni

Abstract A combined polymerase chain reaction and restriction endonuclease (RE) enzyme assay was developed to discriminate between Campylobacter coli and Campylobacter jejuni . Amplimers of the FlaA gene obtained by PCR were digested with Alu I and Hin fI to distinguish C. coli from C. jejuni . With Alu I digestion C. jejuni -specific bands were observed at 110, 140 and 160 bp and C. coli -specific bands at 293 and 147 bp. C. jejuni -specific bands of 349 and 109 bp were found by Hin fI digestion but Hin fI did not digest the Fla A amplimer of C. coli . This combined technique is fast and easy to perform, and distinguishes the two campylobacters unequivocally.     

Differential inhibition of a restriction enzyme by quinoxaline antibiotics

The inhibition of cleavage by HpaI at two well-defined restriction sites in linearised ØX174-RF DNA by quinoxaline antibiotics has been investigated. Echinomycin, which displays a certain preference for binding to GC basepairs, inhibits cleavage at one site much more than the other, whereas triostin A, which displays less pronounced sequence-selectivity, inhibits both sites about equally. Other congeners inhibit reaction at the two sites with varying effectiveness. The results demonstrate the usefulness of studying inhibition of cleavage at specific sites by restriction enzymes as a means of exploring the specificity of DNA-ligand interactions.     

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.     

A simulation study on Nei and Li's model for estimating DNA divergence from restriction enzyme maps

Summary A simulation study has been conducted to check the accuracy of Nei and Li's (1979) formulas for the mean and variance of the proportion (S) of identical restriction sites between two DNA sequences and for estimating the mean and variance of the number () of base substitutions per nucleotide site between two DNA sequences. The results show that these formulas are quite accurate as long as the probability of S becoming zero is negligibly small. In addition to the simulation, approximate formulas have also been obtained for the probability for S to become zero at time t and for the contribution to S due to parallel mutation.     

Genetic relationship among isolates of Helicobacter pylori: evidence for the existence of a Helicobacter pylori species-complex

We investigated the population genetics of 23 isolates of H. pylori by allozyme electrophoresis using 16 enzyme loci. Isolates were obtained from adult patients of whom 48% were of Greek extraction. Eight patients (35%) had an active duodenal ulcer. Allelic variation per loci ranged from 2 to 11 alleles. Four major genetic clusters were apparent, having >75% fixed genetic differences. There was no distinct clustering (clonal structure) on the basis of the geographical origin of the persons from whom isolates were obtained, indicating that this bacterium has not recently jumped a species barrier into humans. Isolates associated with ulcer disease were not monophyletic, with isolates from ulcer patients being found in phylogenetically diverse branches of the dendogram derived from the data. Based on the genetic diversity of H. pylori isolates, we propose that isolates should be classified as belonging not to a single species but to a `Helicobacter pylori species-complex'.     

Purification and characterization of protein methylase II from Helicobacter pylori

Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS-PAGE. On non-denaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4x(78+29)=428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a K(m) value of 5.0x10(-6) M for S-adenosyl-L-methionine and a V(max) of 205 pmol methyl-(14)C transferred min(-1) mg(-1) protein.     

Isolation of restriction enzyme EcoVIII, an isoschizomer of HindIII, produced by Escherichia coli E1585-68

A restriction endonuclease designated EcoVIII, an isoschizomer of HindIII, was isolated from Escherichia coli E1585-68 and purified by dextran-polyethylene glycol (DPG) phase partition, ammonium sulfate precipitation, phospho- and DEAE-cellulose chromatography, and hydroxylapatite chromatography. The purified EcoVIII was stable during the purification procedure and its high specific activity required 10 mM Mg²⁺. Unlike HindIII, Eco VIII exhibited a high specific activity even at low pH (pH 6.3) and showed the highest activity at 48° C. Transformation of purified plasmid DNA from E. coli E1585-68 into K-12 indicated that the EcoVIII gene was carried on a multicopy 4.4-kb miniplasmid. EcoVIII seems to be preferable to HindIII for its production and use because of easier handling of producer cells and a wider range of activity.     

A novel synthetic peptide from a tomato defensin exhibits antibacterial activities against Helicobacter pylori

Defensins are a class of cysteine‐rich proteins, which exert broad spectrum antimicrobial activity. In this work, we used a bioinformatic approach to identify putative defensins in the tomato genome. Fifteen proteins had a mature peptide that includes the well‐conserved tetradisulfide array. We selected a representative member of the tomato defensin family; we chemically synthesized its γ‐motif and tested its antimicrobial activity. Here, we demonstrate that the synthetic peptide exhibits potent antibacterial activity against Gram‐positive bacteria, such as Staphylococcus aureus A170, Staphylococcus epidermidis, and Listeria monocytogenes, and Gram‐negative bacteria, including Salmonella enterica serovar Paratyphi, Escherichia coli, and Helicobacter pylori. In addition, the synthetic peptide shows minimal (<5%) hemolytic activity and absence of cytotoxic effects against THP‐1 cells. Finally, SolyC exerts an anti‐inflammatory activity in vitro, as it downregulates the level of the proinflammatory cytokines TNF‐α and IFN‐γ. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A new restriction endonuclease Eco31I recognizing a non-palindromic sequence

A restriction endonuclease with a novel site-specificity has been isolated from the Escherichia coli strain RFL31. The nucleotide sequences around a single Eco31I cut on pBR322 DNA and two cuts of λ DNA have been compared. A common ^5′GAGACC_3′CTCTGG sequence occurs near each cleavage site. Precise mapping of the cleavages in both DNA strands places the cuts five nucleotides to the left of the upper sequence and one nucleotide to the left of the lower sequence. This enabled us to deduce the following recognition and cleavage specificity of Eco31I: 5 ′ G G T C T C N ↓ 3 ′ C C A G A G N N N N N ↑