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.     

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.     

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.     

Isolation of plasmids present in thermophilic strains from hot springs in Jordan

The plasmid profile of two thermophilic bacterial strains isolated from recreation thermal springs in Jordan has been investigated. These strains are Streptococcus thermophilus and Bacillus sp1, which have been isolated from Zerka – Maeen and Himma hot springs respectively. Supercoiled and circular plasmid forms were detected, explaining the effect of DNA conformation on the mobility of the plasmid in the agarose gel electrophoresis. Two plasmids have been isolated and characterized by restriction endonucleases to facilitate their use as cloning vectors in thermophilic strains. The sizes of the plasmids were approximately 3 kb (from Streptococcus thermophilus) and 7 kb (from Bacillus spl). These plasmids were then digested with three different restriction enzymes (EcoRI, Bam HI, and HindIII), one of which was found to possess a single site for both plasmids, this enzyme is EcoRI.     

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     

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.     

Deoxyribonucleotide sequence divergence betweenStaphylococcus cohnii subspecies populations living on primate skin

Staphylococcus cohnii strains isolated from various primates could be separated into three distinct groups or subspecies on the basis of phenotypic characterization and DNA-DNA hybridization techniques. These included a human-specificS. cohnii subspecies (denoted here as subsp. 1), a widely distributed primateS. cohnii subspecies (subsp. 2), and a Ceboidea (New World monkey)-specificS. cohnii subspecies (subsp. 3). Divergence of the latter subspecies from the other two is great enough to place it in a near (separate)-species status.S. cohnii represents the third example of aStaphylococcus species where DNA divergence has been demonstrated between human and nonhuman primate-adapted populations. The data presented in this report continue to support the hypothesis that at least certain staphylococci have evolved together with their hosts by conjugate evolution.Paper No. 8555 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, N.C. 27650     

Restriction endonucleases from various bacterial strains displaying 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. EndonucleasesPft211I,Psp8I, andPsp23I were isolated and purified from twoPseudomonas sp. strains and aPseudomonas fluorescens strain. Restriction endonucleasesPfl2lI andPsp23I were shown to recognize and cleave the DNA nucleotide sequence 5′-CTGCA↓G-3′. Endonuclease Psp81 recognized and cleaved the DNA nucleotide sequence 5′-G↓GATCC-3′. These endonucleases were found to be true isoschizomers of PstI andBamHI, respectively.     

Sequence analysis of the yadA, inv, and ail genes and their expression in the main and nonmain Yersinia pestis subspecies and Yersinia pseudotuberculosis

The nucleotide sequences of the inv, yadA, and ail adhesin-invasin genes were analyzed in 24 strains of the main and nonmain Yersinia pestis subspecies, which were isolated from natural plague foci in Russia and neighbor countries, and ten Y. pseudotuberculosis strains. All of the five plague agent subspecies (main, caucasica, altaica, ulegeica, and hissarica) had the inv and yadA genes altered by insertion of the IS element and a single nucleotide deletion, respectively, as was earlier observed for the Y. pestis strains KIM and CO92. Consequently, the strains lacked functional activity of the Inv and YadA proteins. The ail gene of the main and ulegeica subspecies had a missense mutation, which replaced Val138 with Phe in the Ail protein. The strains of the caucasica subspecies had an AGT insertion in the ail gene, resulting in Ser148 insertion in the polypeptide chain. The changes in the ail sequence probably exerted no effect on ail expression, since the strains of all subspecies were resistant to blood serum complement.     

Correlation between polymerase chain reaction analysis of the histidine biosynthesis operon, randomly amplified polymorphic DNA analysis and phenotypic characterization of dairy Lactococcus isolates

A collection of 32 lactococcal strains isolated from raw milk in the Camembert RDO (registered designation of origin) area were phenotypically and genotypically characterized. As expected for environmental isolates, all strains had a Lactococcus lactis subsp. lactis phenotype. The strains were then genotypically identified by the randomly amplified polymorphic DNA (RAPD) technique, using reference strains of lactococci. Two major clusters were identified containing the two subspecies lactis and cremoris. The subspecies lactis cluster could be divided into five subgroups whereas there was a high coefficient of similarity between all strains in the subspecies cremoris cluster. This RAPD classification was then compared with that of a traditional PCR assay using L.lactis species-specific primers corresponding to part of the histidine biosynthesis operon. The two subspecies were differentiated by the size of the fragment amplified (about 200 bp longer for subspecies cremoris). Unlike preliminary phenotypic assignments, the results of PCR experiments corroborated the genotypic identification of the lactococcal strains by RAPD allowing the technique to be reconsidered on the basis of its taxonomic efficiency. Received: 14 May 1998 / Accepted: 3 September 1998     

Restriction and modification systems of ruminal bacteria

A high frequency of type II restriction endonuclease activities was detected inSelenomonas ruminantium but not in other rumen bacteria tested. Eight different restriction endonucleases were characterized in 17 strains coming from genetically homogeneous local population. Chromosomal DNA isolated fromS. ruminantium strains was found to be refractory to cleavage by various restriction enzymes, implying the presence of methylase activities additional to those required for protection against the cellular endonucleases. The presence of Dam methylation was detected inS. ruminantium strains as well as in several other species belonging to theSporomusa subbranch of low G+C Gram-positive bacteria (Megasphaera elsdenii, Mitsuokella multiacidus).     

Molecular analysis of organelle DNA of different subspecies of rice and the genomic stability of mtDNA in tissue cultured cells of rice

Summary Chloroplast (ct) and mitochondrial (mt) DNAs were isolated from two subspecies of rice (Oryza sativa), japonica (Calrose 76) and indica (PI353705) and compared by restriction endonuclease fragment pattern analysis. Similarly, PI353705 (A5) mtDNA was also compared with the mtDNA of its long term tissue cultured line, BL2. Variation in the ctDNA of the 2 subspecies was detected with two (AvaI and BglI) of the 11 restriction endonucleases tested, whereas their mtDNAs showed considerable variation when restricted by PstI, BamHI, HindIII and XhoI endonucleases. Thus, the chloroplast DNA was more highly conserved than the mtDNA in the subspecies comparisons. Only minor variation was observed between the restriction endonuclease patterns of the mtDNAs of BL2 and A5. Southern blots of mtDNA were hybridized with heterologous probes from maize and spinach organelle genes. Differences were found in the hybridization patterns of the two subspecies for six of the eight (mitochondrial and chloroplast) probes tested. Two of the seven (mitochondrial) probes (coxII and 26S rRNA) detected tissue culture generated variation in mtDNA. The relative values of restriction endonuclease and hybridization patterns for studying phylogenetic and genetic relationships in rice are discussed.Florida Agricultural Experiment Station Journal Series No. 8807. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable     

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.     

Restriction and modification of Halophage S45 inHalobacterium

A newly isolated group B1 bacteriophage, Halophage S45, is restricted and modified in vivo by strains ofHalobacterium. Three strain-specific activities have been observed. Two of these occur in the same bacterial strain and appear to be due to different kinds of enzymatic activities. One of the restriction specificities is shown to be associated with a strain-specific endonuclease active on unmodified halobacterial DNA.     

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.     

Evaluation of selected features of Staphylococcus cohnii enabling colonization of humans

Based on iron utilization, sensitivity to skin fatty acids, lipolytic and proteolytic activity the potential abilities ofStaphylococcus cohnii strains to colonize humans were evaluated. The investigation included 60 strains that belong to both subspecies,viz. S. cohnii ssp.cohnii andS. cohnii ssp.urealyticus. Strains were isolated from different sources of theIntensive Care Unit and from non-hospital environment. Most of the strains were multiple antibiotic-resistant. Strains of both subspecies revealed a relatively low iron requirement. These strains were capable of utilizing iron bound in oxo acids and from host iron-binding proteins.S. cohnii ssp.urealyticus were more effective in iron uptake thanS. cohnii ssp.cohnii. All investigated strains revealed sensitivity to skin fatty acids, butS. cohnii ssp.urealyticus strains were more resistant. Special features of strains of this subspecies promote colonization of humans.     

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.     

Rat kappa-chain allotypes

The presence of rat kappa-chain allotype specificities (RI-1a and b) has been studied in 13 subspecies of the seven native Australian species ofRattus. RI-la reactivity was not detected among these rats. On the other hand, extensive cross-reactivity was seen with RI-1b, some sera cross-reacting totally (R. leucopus cooktownensis), some not at all (R. colletti), and the remainder showing at least two distinct levels ofpartial cross-reactivity, confirming the existence of multiple specificities for Rl-lb. Three subspecies show polymorphism with respect to Rl-lb cross-reactivity (R. sordidus, R. colletti, andR. l. leucopus) and in one case (leucopus) breeding studies have indicated that there is allelic inheritance of this trait. The segregation of RI-1b reactivity has been studied in crosses and backcrosses made between species differing in their RI-1b reactivity, and the results are consistent with the existance of codominant alleles at a single locus. The fact that these species differ extensively in their karyotype opens the door to possible chromosomal localization of this and other genetic traits.     

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.