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.     

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)     

Eight new restriction endonucleases fröm Herpetosiphon giganteus--divergent evolution in a family of enzymes.

Characterization of eight restriction endonucleases isolated from five strains of Herpetosiphon giganteus is described. HgiCI from strain Hpg9 recognizes and cleaves the degenerate sequence: GGPyPuCC, producing 5'-hexanucleotide protruding ends. Endonucleases HgiBI, HgiCII and HgiEI are isoschizomers of AvaII; HgiCIII and HgiDII are isoschizomers of SalI; and HgiDI and HgiGI are isoschizomers of AcyI. Based upon their closely related and in part overlapping recognition specificities a close evolutionary relationship is proposed for all known Hgi restriction endonucleases.     

Three restriction endonucleases from Anabaena flos-aquae

Three site-specific endonucleases, AflI, AflII and AflIII, have been partially purified from the cyanobacterium Anabaena flos-aquae CCAP 1403/13f. Their recognition and cleavage specificities have been determined to be: (formula; see text) AflII and AflIII are new specificities and may be useful in molecular cloning, as well as in the analysis of DNA. The distribution of type II restriction endonucleases in the cyanobacteria is briefly discussed.     

Restriction endonucleases from Herpetosiphon giganteus: an example of the evolution of DNA recognition specificity?

We describe the partial purification and characterisation of five Type II restriction endonucleases from two strains of Herpetosiphon giganteus. One of the activities, HgiJII, was the first enzyme found that cleaves DNA at the family of related sequences 5'-G-R-G-C-Y/C-3'. This enzyme may be related to the enzyme HgiAI from a different strain of the same species, and which cleaves at the sites 5'-G-W-G-C-W/C-3'. We have shown that DNAs from the strains producing HgiAI and HgiJII are resistant to both of these restriction endonucleases. The remaining four enzymes described here share recognition and cleavage specificities with other restriction endonucleases. The evolution of Type II restriction-modification systems and their role in vivo are discussed.     

Genetic polymorphism of the murine myogenic gene Myo-D1.

Polymorphism of the myogenic gene, Myo-D1, has been sought to examine genetic mechanisms which control skeletal muscle development. By Southern analysis, three restriction-fragment length polymorphisms (RFLPs) have been found in various mouse strains using the TaqI, SacI and BglII restriction endonucleases and a full-length cDNA Myo-D1 probe. Reference to the distribution of RFLPs in different mouse strains derived from Mus mus (M.m.) domesticus and M.m. musculus subspecies suggests that Myo-D1 rearrangements are subject to nonrandom association. The biological significance of RFLP of the Myo-D1 gene is yet to be determined.     

Ribotyping of Fusobacterium necrophorum strains isolated from bovine and ovine hepatic abscesses

A microbiological study was made of 100 strains of Fusobacterium necrophorum isolated from hepatic abscesses in bovine and ovine herds. Differences between the biological activity and ribotypes within the two F. necrophorum subspecies were studied. Conventional methods identified 89 isolates as F. necrophorum subsp. necrophorum and 11 as F. necrophorum subsp. funduliforme. For ribotyping, 50 strains (35 F.n. subsp. necrophorum, 11 F.n. subsp. funduliforme and 4 reference strains) were digested with restriction endonucleases (HindIII, EcoRI and BamHI) and examined after hybridization with digoxigenin-labelled cDNA probe transcribed from a 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzymes for ribotyping were EcoRI and BamHI. The presence or absence of two distinct band of 5 kb (EcoRI) and 10.5 kb (BamHI) differentiated the two subspecies. This technique also revealed genetic differences between isolates which could be used in the epidemiological study of clinical processes caused by F. necrophorum.     

Three new restriction endonucleases MaeI, MaeII and MaeIII from Methanococcus aeolicus

Three type II restriction endonucleases, MaeI, MaeII and MaeIII, with novel site specificities have been isolated and purified from the archaebacterium Methanococcus aeolicus PL-15/H. The recognition sequences of these enzymes are (formula: see text) with the sites of cleavage as indicated by the arrows. The sequences were confirmed by restriction and computer analyses on sequenced DNA's of plasmid pBR322, bacteriophages lambda and phi X174 and virus SV40.     

Bacteria associated with hazelnut (Corylus avellana L.) decline are of two groups: Pseudomonas avellanae and strains resembling P. syringae pv. syringae

A total of 118 fluorescent pseudomonads associated with hazelnut decline, which has been occurring for many years in different areas of northern Greece and Italy, were assessed by performing a repetitive PCR analysis with enterobacterial repetitive intergenic consensus, box element, and repetive extragenic palindromic primer sets, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein extracts, a carbon compound utilization analysis, and an analysis to determine the presence of the syrB gene. A subset of 53 strains was also characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA) by using nine restriction endonucleases. The virulence of 40 representative strains was assessed by using serial doses. The pathogenic specificities of the strains were also verified. ARDRA carried out with HinfI revealed two main groups of strains, groups A and B, which exhibited a level of similarity of 57%. The other eight restriction endonucleases used did not separate the strains. In addition, a cluster analysis performed by the unweighted pair group method using arithmetic averages after repetitive PCR and SDS-PAGE of protein extracts also revealed the same two groups. Furthermore, the differential utilization of some carbon compounds made it possible to differentiate the groups. Virulence assessment clearly indicated that the group A strains are very virulent, whereas the group B strains proved to be mildly virulent for hazelnut. Group A included the strains isolated in northern Greece and central Italy (i.e., the province of Viterbo); these strains do not have the syrB gene, are pathogenically restricted to Corylus avellana, and belong to Pseudomonas avellanae. Group B includes the other strains obtained from hazelnut cultivated in Piedmont, Campania, Latium, Sicily, and Sardinia. They represent a distinct taxon closely related to Pseudomonas syringae pv. syringae.     

鲫不同种系线粒体DNA物理图谱的构建

用１３种限制性内切酶对鲫鱼三个亚种共七个品系的线粒体ＤＮＡ进行分析,其中有９种酶在种系间或种系内产生限制性片断长度多态性,发现了１６个线粒体ＤＮＡ组合单倍型,通过双酶切分析,构建了１６个线粒体ＤＮＡ组合单倍型的１３种限制性内切酶的发点的物理图谱。     

BglII and MunI: what a difference a base makes

Restriction endonucleases are resilient to alterations in their DNA-binding specificities. Structures of the BglII and MunI endonucleases bound to their palindromic DNA sites, which differ by only their outer base pairs from the recognition sequences of BamHI and EcoRI, respectively, have recently been determined. A comparison of these complexes reveals surprising differences and similarities in structure, and provides a basis for understanding the immutability of restriction endonucleases.     

Mitochondrial DNA polymorphisms in the two subspecies of Drosophila sulfurigaster: relationship between geographic structure of population and nucleotide diversity.

Recent empirical and theoretical studies on mitochondrial DNA (mtDNA) variation in higher animals have suggested that the extent of mtDNA polymorphism is largely affected by spatial population subdivision. To examine this we studied mtDNA polymorphism in two subspecies of Drosophila sulfurigaster: D. s. albostrigata and D. s. bilimbata. Drosophila sulfurigaster albostrigata is mainly distributed on the mainland of Southeast Asia. In contrast, D. s. bilimbata forms discontinuous populations on many islands scattered in the Pacific Ocean. Because of the difference in their distribution patterns, the two subspecies are thought to be different in the extent of spatial population subdivision. mtDNA was isolated from greater than 50 isofemale strains for each subspecies and were analyzed by eight restriction endonucleases. Nucleotide diversity within a population was higher in D. s. albostrigata than in D. s. bilimbata. However, haplotype diversity was 1.6 times greater in D. s. bilimbata (0.85) than in D. s. albostrigata (0.53). The large difference in overall heterogeneity was attributed to the difference in interpopulational nucleotide diversity. For the two subspecies the proportion of interpopulational gene diversity in a subdivided population was calculated to be 0.54 in D. s. bilimbata and 0.40 in D. s. albostrigata. These observations indicate that spatial population subdivision is a major factor in determining mtDNA polymorphism in these subspecies. The extent of mtDNA divergence between the subspecies was very high. The average nucleotide divergence between them was 7.6%, which is almost the interspecific level reported for other Drosophila species. The cause of the high degree of mtDNA divergence is discussed.     

Bacteria Associated with Hazelnut (Corylus avellana L.) Decline Are of Two Groups: Pseudomonas avellanae and Strains Resembling P. syringae pv. syringae

A total of 118 fluorescent pseudomonads associated with hazelnut decline, which has been occurring for many years in different areas of northern Greece and Italy, were assessed by performing a repetitive PCR analysis with enterobacterial repetitive intergenic consensus, box element, and repetive extragenic palindromic primer sets, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein extracts, a carbon compound utilization analysis, and an analysis to determine the presence of the syrB gene. A subset of 53 strains was also characterized by amplified 16S ribosomal DNA restriction analysis (ARDRA) by using nine restriction endonucleases. The virulence of 40 representative strains was assessed by using serial doses. The pathogenic specificities of the strains were also verified. ARDRA carried out with HinfI revealed two main groups of strains, groups A and B, which exhibited a level of similarity of 57%. The other eight restriction endonucleases used did not separate the strains. In addition, a cluster analysis performed by the unweighted pair group method using arithmetic averages after repetitive PCR and SDS-PAGE of protein extracts also revealed the same two groups. Furthermore, the differential utilization of some carbon compounds made it possible to differentiate the groups. Virulence assessment clearly indicated that the group A strains are very virulent, whereas the group B strains proved to be mildly virulent for hazelnut. Group A included the strains isolated in northern Greece and central Italy (i.e., the province of Viterbo); these strains do not have the syrB gene, are pathogenically restricted to Corylus avellana, and belong to Pseudomonas avellanae. Group B includes the other strains obtained from hazelnut cultivated in Piedmont, Campania, Latium, Sicily, and Sardinia. They represent a distinct taxon closely related to Pseudomonas syringae pv. syringae.     

Different restriction and modification phenotypes in ruminal lactate-utilizing bacteria

Analysis of restriction and modification activities in lactate-utilizing bacteria belonging to the Megasphaera elsdenii and Mitsuokella multiacida species revealed the presence of GATC-specific, MboI isospecific, restriction-modification (R-M) systems in all strains tested. While restriction endonucleases isolated from M. elsdenii strains were found to be sensitive to Dam methylation, enzymes from M. multiacida cleaved DNA irrespective of Dam methylation. The comparison of type II R-M systems specificities in three closely related lactate-utilizing ruminal bacterial species indicated complete lack of restriction and/or modification enzymes previously characterized from Selenomonas ruminantium in tested M. elsdenii and M. multiacida strains. R-M systems are believed to represent the main defense tool against phage infection. Based on the results of our experiments it could be assumed that M. elsdenii and M. multiacida use the different strategy for bacteriophage protection compared to S. ruminantium.     

Diversity of DNA methylation pattern and total DNA restriction pattern in symbiotic Nostoc

Attempts were made to use total DNA restriction patterns and the response of purified DNA to treatment with restriction endonucleases to characterize several symbiotic Nostoc strains which had been isolated from different host plants cultivated in Italy. Among 27 restriction endonucleases tested, several did not cut any DNA and no significant variation in the susceptibility of the genomes to DNA restriction was seen among the strains. Therefore the Nostoc strains could not be separated into groups based on their different susceptibilities to the action of restriction endonucleases. However, in studies of total DNA restriction patterns, the restriction endonucleases BfrI and HpaI gave unique band patterns for each cyanobacterial isolate. Different profiles were even found in strains isolated from host plants belonging to the same species. The results do not support any definition of symbiotic Nostoc genomic groups or species and show that a tight specificity between the host plant and the cyanobacterium might not exist in the symbiotic associations involving Nostoc.     

Limitation of phage multiplication by microbes of the genus Bordetella

Possible causes limiting the multiplication of Bordetella phages or inducing their restriction, such as the influence of lysogenic immunity and the restriction-modification (R-M) system or the incompatibility of the receptor apparatus, have been studied. The limitation of the multiplication of phages by some B. bronchiseptica and B. pertussis strains has been shown to be due to the presence of the R-M system and lysogenic immunity. In five B. bronchiseptica strains and two B. pertussis strains site-specific endonucleases (restrictases) with Hind III specificity have been detected. One B. bronchiseptica strain without the R-M system has been detected. B. bronchiseptica strains producing site-specific endonucleases are practically nonpathogenic for humans, grow in common culture media and selectively produce only one restrictase, type Hind III, which guarantees from the admixture of other specific endonucleases. The B. parapertussis strains under study (altogether 100 strains) have not been found to limit the multiplication of Bordetella test phages. The absence of site-specific endonucleases has also been confirmed biochemically. These strains are recommended as indicator strains for the multiplication of Bordetella phages.     

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.     

Restriction endonuclease activities in the legionellae

Fifteen legionellae isolates were studied for the presence of restriction endonucleases. At least three different specificities were found in nine of these isolates. One particular activity was present in two groups of isolates isolated from widely separated geographic areas.     

Restriction endonucleases from various bacterial strains exhibit an ice-nucleating activity

Six strains containing site-specific endonucleases II were selected from a collection of 45 ice-nucleating bacterial strains isolated from rhizosphere of plants growing in various geographical regions. Endonucleases Pfl211I, Psp8I, and Psp23I were isolated and purified from two Pseudomonas sp. strains and a Pseudomonas fluorescens strain. Restriction endonucleases Pfl21I and Psp23I were shown to recognize and cleave the DNA nucleotide sequence 5'-CTGCA decrease G-3'. Endonuclease Psp8I recognized and cleaved the DNA nucleotide sequence 5'-G decrease GATCC-3'. These endonucleases were found to be true isoschizomers of PstI and BamHI, respectively.     

A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.