Mesophilic cyanobacteria producing thermophilic restriction endonucleases

When searching for the site-specific endonucleases in several strains of Phormidium we made the following observations. Among the 16 strains that originated from 15 species of Phormidium, 12 produced one or more restriction enzymes, of which two produced the highly thermophilic restriction endonucleases PtaI and PpaAII with their optimum activity at 65-80 degrees C, which is far above the lethal temperature for the host microorganism (40 degrees C). These two temperature-resistant enzymes are isoschizomers of known BspMII and TaqI endonucleases, respectively. The presence of the thermophilic TaqI isoschizomer does not seem to play any role in the mesophilic host microorganism, which does not even contain an active cognate methyltransferase. Among the remaining 10 strains, six produced isoschizomers of endonucleases which we first described in cyanobacteria, namely: PfaAII (NdeI), PinBII and PtaI (BspMII), PlaAII (RsalI), PpaAII, PpeI (ApaI). Two enzymes, PauAII (AhaIII) and PfaAII (NdeI), belong to a group of a very rarely occurring isoschizomers. Out of 21 cyanobacterial endonucleases investigated by us, four were active in a wide range of temperatures (from 15 to 60 degrees C) which also extended the optimal growth temperature of the hosts. We assume that our observation on the presence of temperature-resistant restriction enzymes in mesophilic hosts supports the idea of horizontal gene transfer. Restriction modification systems may be an excellent tool for investigation of that phenomenon.     

Endophytic Bacilli Producing Type II Restriction Endonucleases

Two of thirteen bacillar strains isolated from the inner tissues of cotton plants were found to produce type II restriction endonucleases. The investigation of the site specificity of these enzymes showed that they are AsuI and Eco31I isoschizomers.     

Substrate specificity of restriction endonucleases Eco471 and Eco521

Cleavage sites for Eco47I and Eco52I restriction endonucleases, which are isoschizomers of Ava II and Xma III, respectively, have been structurally elucidated.     

Endophytic bacilli--producers of type II restriction endonucleases

Two of thirteen bacillar strains isolated from the inner tissues of cotton plants were found to produce type II restriction endonucleases. The investigation of the site specificity of these enzymes showed that they are AsuI and Eco31I isoschizomers.     

Screening for restriction endonucleases in aerobic,thermophilic eubacteria

Summary A total of 216 Icelandic aerobic, heterotrophic, thermophiles belonging to three different genera were screened for type II restriction endonucleases. The frequency of positive strains was 44% for both Thermus and Bacillus but 63% for Rhodothermus. Approximately half of the enzymes from each group were characterised and a total of 14 different restriction enzymes were found. In all cases they were isoschizomers of known enzymes. Thermus contained 9 different types, Bacillus 6 and Rhodothermus had 3. This is the first time that isoschizomers of BspEI, BglI, EagI and EcoRV are found in Thermus and BstBI and EcoRV are found in Rhodothermus.     

Prevalence of CTGCAG recognizing restriction and modification systems in ruminal selenomonades

Analysis of restriction and modification activities in natural population of Selenomonas ruminantium have revealed the prevalence of CTGCAG (Pst I isoschizomers) recognizing restriction and/or modification systems in these bacteria. Pst I isoschizomeric restriction endonucleases were detected in 4 out of 15 strains tested. In one strain, the Pst I isoschizomeric restriction system was accompanied by another restriction and modification system recognizing GAATTC sequence (Eco RI isoschizomer). Four other strains contained CTGCAG specific methylases which lacked cognate endo-nuclease activities. Presence of identical restriction and modification systems in both of subspecies of S. ruminantium, as well as the occurrence of Pst I isoschizomers in various combinations, indicate the possibility of horizontal transfer of genes coding for these systems.     

Site-specific endonucleases LplI and AagI

New site-specific endonucleases LplI and AagI have been isolated from the Lactobacillus plantarum and Achromobacter agile cells, respectively. The enzymes' purification stages included treatment of cell-free extracts with polyethylenimine, fractionation in two-phase system by Albertsson's method, chromatography on blue Sepharose and DEAE-cellulose. The results of cleavage of a 5'-32P-labelled oligodeoxynucleotide duplex by restriction endonucleases LplI and AagI indicate that these enzymes recognize and cut the sequence AT decreases CGAT, being therefore true isoschizomers of the ClaI restriction endonuclease from Caryophanon latum. The L. plantarum strain has 400 fold endonuclease productivity as compared with the ClaI producent and is perspective for preparative isolation of LplI.     

A new endonuclease recognizing the deoxynucleotide sequence CCNNGG from the cyanobacterium Synechocystis 6701.

A new sequence-specific endonuclease from the cyanobacterium Synechocystis species PCC 6701 has been purified and characterized. This enzyme, SecI, is unique in recognizing the nucleotide sequence: 5' -CCNNGG-3' 3' -GGNNCC-5' and cleaves it at the position indicated by the symbol. Two other restriction endonucleases, SecII and SecIII, found in this organism are isoschizomers of MspI and MstII, respectively.     

A method for detecting new strains producing DNA modification and restriction enzymes--isoschizomers and isomethylomers of known restrictases and methylases

The paper describes a technique for the detection of new strains producing enzymes which mediate DNA modification and restriction, and isoschizomers and isomethylomers of the known restriction endonucleases and methylases. Three Bacillus subtilis strains whose DNA carries a BamH1 modification have been found. Two of these strains exert the restrictase activity with an R BamH1 specificity.     

Production of restriction endonucleases using multicopy Hsd plasmids occurring naturally in pathogenic Escherichia coli and Shigella boydii

A convenient procedure has been devised for detection of restriction endonucleases in the Escherichia coli-Shigella group. With this procedure, two restriction endonucleases, designated Sbo 13 and Eco T22, were found and later were identified as isoschizomers of NruI and AvaIII, respectively. These endonucleases were shown to be produced from small multicopy plasmids. They were isolated from nonpathogenic E. coli into which the plasmids had been introduced by transformation, and purified from contaminating nuclease activity. The yield was high, 1,000 units/g of wet cells for Sbo 13 and 500 units/g for Eco T22. Sbo 13 and Eco T22 should be preferable to NruI and AvaIII because of the high yield and ease in handling the producer cells.     

REBASE - restriction enzymes and methylases

REBASE is a comprehensive database of information about restriction enzymes and related proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, methylation sensitivity, crystal and sequence data. DNA methyltransferases, homing endonucleases, nicking enzymes, specificity subunits and control proteins are also included. Most recently, putative DNA methyltransferases and restriction enzymes, as predicted from analysis of genomic sequences, are also listed. The data is distributed via Email, ftp (ftp.neb.com), and the Web (http://rebase.neb.com).     

Substrate specificity of restriction endonuclease Eco781

The recognition sequence and cleavage point of restriction endonuclease Eco781 have been determined as 5'-GGCGCC-. There are several known enzymes recognizing the same sequence, although the prototype NarI and isoschizomers NdaI and NunII cleave the substrate to produce 5'-protruding ends, whereas cleavage with isoschizomer BbeI results in 3'-protruding ends. Therefore, restrictase Eco78I, generating flush ends, may be regarded as an enzyme with new specificity among the restriction endonucleases recognizing the 5'-GGCGCC-sequence.     

REBASE: restriction enzymes and methyltransferases

REBASE contains comprehensive information about restriction enzymes, DNA methyltransferases and related proteins such as nicking enzymes, specificity subunits and control proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, crystal and sequence data. Homing endonucleases are also included. REBASE contains the most complete and up-to-date information about the methylation sensitivity of restriction endonucleases. In addition, there is extensive information about the known and putative restriction-modification (R-M) systems in more than 100 sequenced bacterial and archaeal genomes. The data is available on the web (http://rebase.neb.com/rebase/rebase.html), through ftp (ftp.neb.com) and as monthly updates via email.     

Isolation and characterization of new restriction endonucleases from Vibrio parahaemolyticus: VpaK32I enzyme with the class-IIS heptanucleotide specificity, GCTCTTCN1/N4.

Six restriction endonucleases (ENases), classified into four different specificities, were found in a screen among 68 reference strains of Vibrio parahaemolyticus of human origin. Five of these ENases are isoschizomers of well-known ENases, while the remaining one, designated VpaK32I, is a novel and highly efficient class-IIS ENase with the hepatanucleotide recognition site, 5'-GCTCTTC(1/4)-3'.     

New distinctions between regions of centromeric heterochromatin in human chromosomes by treatments with the isoschizomers NdeII-Sau3AI

The isoschizomers NdeII-Sau3AI (decreases GATC) have been used to characterize heterochromatic regions in human chromosomes. The findings with NdeII are identical with those previously published with MboI, but the results with Sau3AI provide evidence for new distinctions of centromeric heterochromatin in chromosomes 5 and 6. The results are discussed in relation to the chromatin organization at these regions an the mechanisms of the action of restriction endonucleases.     

The search for strains producing new restriction endonucleases

The search for restrictases in 154 strains belonging to 104 species of 32 genera of microorganisms has been carried out by the method of rapid toluene assay. In 10 strains the activity of endonucleases specifically fragmenting the DNA of phage lambda in the presence of Mg2+ ions has been detected. Restrictases Pae I and Pae II formed by two Pseudomonas aeruginosa strains have been identified as the true isoschizomers of restriction endonucleases Sph I and Sma I respectively. The results of the screening of restrictase-producing strains indicate that the production of restrictases is widely spread among microorganisms of the genus Bacillus.     

Sequence-specific endonucleases in strains of Anabaena and Nostoc

The complements of restriction endonucleases of 12 strains of cyanobacteria were determined in cell-free extracts, and were compared with the complements of restriction activities assessed by measuring the relative efficiencies of plating of cyanophages on those cyanobacteria. The hosts which were susceptible to all of the phages contained endo R · AvaI and endo R · AvaII, and in several cases probably endo R · AvaIII, or isoschizomers of these enzymes. Three hosts which were lysed by only a subset (1 or 3) of the phages contained different restriction endonuclease. Anabaena sp. PCC 7120 showed apparent phenotypic restriction of phage An-22 grown in hosts with (isoschizomers of) AvaI, II and III, but no corresponding endonuclease has yet been detected in vitro. Nostoc sp. ATCC 29131 (PCC 6705) was found to contain a restriction enzyme, NspBII, with hitherot unknown specificity, C(A/C)GC(T/G)G.     

Isolation and characterization of a new poly(3-hydroxybutyrate)-degrading, denitrifying bacterium from activated sludge

One hundred and forty isolates of thermophilic bacteria from the genus Thermus were screened for the presence of restriction endonuclease activity. Thermostable isoschizomers of restriction endonucleases, such as AceIII, BbvI, BglI, BsePI, FnuDII, HgiAI, MaeII, MboI, MseI, PvuII, StuI, TaqI, Tsp4CI, TspEI, XhoI and XmaIII, were isolated. Two restriction enzymes, TatI and TauI, recognizing novel degenerate sequences 5'-W (downward arrow)GTACW-3' and 5'-GCSG (downward arrow)C-3' respectively were partially purified and the recognition and cleavage sites were determined.     

Screening for restriction endonucleases in methane-oxidizing bacteria.

51 methane-oxidizing bacteria strains such as Methylomonas methanica, M. rubra, Methylococcus capsulatus, M. thermophilus, M. luteus, M. ucrainicus, M. whittenburyi, Methylosinus trichosporium, M. sporium, Methylocystis parvus isolated from various ecological niches and geographical regions of the Ukraine and also the strains received from R. Whittenbury and Y. Heyer were screened for restriction endonucleases. Type II restriction endonucleases were detected in IMV B-3112 (= 12 b), IMV B-3027 (= 26), IMV B-3019 (= 9 c), IMV B-3017 (= 17 c), IMV B-3226 (= 26 v), IMV B-3033 (= Y), IMV B-3100 (= 100) and IMV B-3494 (= 1E494). The results obtained were indicative of relatively high frequency of restriction enzymes occurrence in methane-oxidizing bacteria. There were Kpn I (Asp 7181) restriction endonuclease isoschizomers in crude extracts of IMV B-3112, B-3017, B-3019, B-3027 isolated from fresh-water silt as well as in IMV B-3226 strain isolated from waste-water silt. Although these isolates had bee previously considered as untypical strains of M. ucrainicus, more detailed study of their properties allowed placing them with Methylovarius luteus (= Methylococcus luteus). IMV B-3494 strain was identified as Methylococcus capsulatus. Strain IMV B-3033 had earlier been allocated to Methylovarius whittenburyi (= Methylococcus whittenburyi). Specificity of restriction endonucleases of this strain was not tested. Therefore, for the first time restriction endonucleases were detected in methane-oxidizing bacteria. 8 strains (3 species) among 51 strains (13 species) were found to produce restriction endonucleases displaying three different types of specificity in the least. Producers of restriction endonucleases having Kpn I (Asp 7181) specificity were isolated from different water and silt samples of the Dnieper flood-land more than 20 years ago.     

REBASE--restriction enzymes and methylases

REBASE contains comprehensive information about restriction enzymes, DNA methylases and related proteins such as nicking enzymes, specificity subunits and control proteins. It contains published and unpublished references, recognition and cleavage sites, isoschizomers, commercial availability, methylation sensitivity, crystal data and sequence data. Homing endonucleases are also included. Most recently, extensive information about the methylation sensitivity of restriction enzymes has been added and a new feature contains complete analyses of the putative restriction systems in the sequenced bacterial and archaeal genomes. The data is distributed via email, ftp (ftp.neb.com) and the Web (http://rebase. neb.com).