A second site-specific restriction endonuclease from Staphylococcus aureus.

A site-specific restriction endonuclease has been isolated from Staphylococcus aureus PS 96. This enzyme, Sau96 I, recognizes the DNA sequence 5'--G-G-N-C-C--3' and cleaves as indicated by the arrows. The enzyme 3'--C-C-N-G-G--5' cleaves adenovirus type 5 and lambda DNA many times, SV40 DNA 10 times and 0X174 RF DNA 2 times. Evidence is presented that the enzyme is involved in biological restriction-modification.     

Type II SsrI restriction endonuclease from Staphylococcus saprophyticus

The recognition sequence and cleavage site for restriction endonuclease SsrI have been determined, the latter being 5'-GTT decreases AAC-3'. The enzyme was isolated from Staphylococcus saprophyticus strain and may be used in DNA investigation instead of its isoshizomer HpaI.     

A type IV modification-dependent restriction enzyme SauUSI from Staphylococcus aureus subsp. aureus USA300

A gene encoding a putative DNA helicase from Staphylococcus aureus USA300 was cloned and expressed in Escherichia coli. The protein was purified to over 90% purity by chromatography. The purified enzyme, SauUSI, predominantly cleaves modified DNA containing 5mC and 5-hydroxymethylcytosine. Cleavage of 5mC-modified plasmids indicated that the sites S5mCNGS (S = C or G) are preferentially digested. The endonuclease activity requires the presence of adenosine triphosphate (ATP) or dATP whereas the non-hydrolyzable γ-S-ATP does not support activity. SauUSI activity was inhibited by ethylenediaminetetraacetic acid. It is most active in Mg⁺⁺ buffers. No companion methylase gene was found near the SauUSI restriction gene. The absence of a cognate methylase and cleavage of modified DNA indicate that SauUSI belongs to type IV restriction endonucleases, a group that includes EcoK McrBC and Mrr. SauUSI belongs to a family of highly similar homologs found in other sequenced S. aureus, S. epidermidis and S. carnosus genomes. More distant SauUSI orthologs can be found in over 150 sequenced bacterial/archaea genomes. Finally, we demonstrated the biological function of the type IV REase in restricting 5mC-modified plasmid DNA by transformation into clinical S. aureus strain SA564, and in restricting phage λ infection when the endonuclease is expressed in E. coli.     

Characterization of plasmids in aminocyclitol-resistant Staphylococcus aureus: electron microscopic and restriction endonuclease analysis

Plasmid species isolated from aminocyclitol-resistant Staphylococcus aureus have been analyzed by restriction endonuclease digestion and electron microscopy. These plasmids can be divided into two interrelated groups; intergroup variability is due to the gain or loss of defined DNA sequences. Plasmids pSJ1 and pSJ24 are related to staphylococcal penicillinase plasmid pI524 which was first described over 20 years ago. Both pSJ1 and pSJ24 differ from pI524 by the acquisition of 8 and 4 kbp, respectively, and encode additional resistance to the antibiotics erythromycin and kanamycin. The gain of these resistance determinants suggests that the evolution of staphylococcal resistance plasmids parallels that observed for plasmids of gram-negative bacteria and has serious implications for the spread of antibiotic resistance among the staphylococci.     

A new restriction endonuclease from Spirulina platensis.

Three restriction endonucleases, Sp1I, Sp1II and Sp1III have been purified partially from Spirulina platensis subspecies siamese and named. Sp1I cleaves bacteriophage lambda DNA at one site, phi X 174 RF DNA at two sites, but does not cleave pBR322 DNA. This enzyme recognizes the sequence 5'CGTACG3' 3'GCATCG5' and cuts the site indicated by the arrows. Sp1II is an isoschizomer of Tth111I and Sp1III is an isoschizomer of HaeIII.     

A new restriction endonuclease from Acetobacter pasteurianus.

A restriction endonuclease, ApaI, has been partially purified from Acetobacter pasteurianus. This enzyme cleaves bacteriophage lambda DNA and Simian virus 40 DNA at one site, adenovirus-2 DNA at more than nine sites, but it does not cleave phi X174 DNA nor plasmid pBR322 DNA. This enzyme recognizes the sequence (formula; see text) and cuts at the sites indicated by the arrows.     

A third restriction endonuclease from Xanthomonas malvacearum.

An additional sequence-specific endonuclease, XmaIII, has been partially purified from Xanthomonas malvacearum. XmaIII recognizes ten cleavage sites in adenovirus 2 DNA, two sites in bacteriophage lambda and no site in either simian virus 40 DNA or φX174 DNA. It recognizes the sequence

and cleaves at the sites indicated by the arrows. No other endonuclease with this particular nucleotide sequence specificity has been reported.     

A new restriction endonuclease from Streptomyces albus G.

A restriction endonuclease, SalI, has been partially purified from Streptomyces albus G. This enzyme cleaves adenovirus-2 DNA at three sites, bacteriophage λ DNA at two sites, but does not cleave simian virus 40 DNA or φX174 DNA. It recognizes the sequence

and cuts at the sites indicated by the arrows. An endonuclease (XamI) with similar specificity has also been isolated from Xanthomonas amaranthicola.     

Purification and characterization of SepII a new restriction endonuclease from Staphylococcus epidermidis

A Type II restriction enzyme SepII has been purified to apparent homogeneity from the gram-positive coccus, Staphylococcus epidermidis. The purification included an ammonium sulfate precipitation followed by Q-sepharose, heparin-sepharose and MonoQ column chromatography on an FPLC system. SDS-PAGE analysis showed a denatured molecular weight of 29 kDa. The effects of temperature, pH, NaCl, Mn(2+), Ca(2+), and Mg(2+) ion concentrations were studied to determine the optimal reaction conditions. The enzyme exhibits near maximal levels of activity between pH 8-10, at 10-20mM MgCl(2), 100-150 mM NaCl and 1mM DTT. The results also show that in NEB Buffer 3 the enzyme is active over a broad temperature range from 0 to 70 °C, and in the absence of DNA, enzyme thermostability is observed up to 50 °C for 20 min, while most of the original activity is conserved in 50% glycerol for weeks at room temperature. Single and double digestion in presence of commercial restriction enzymes of known DNA substrates (lambda, pBR322, pET21, pTrcHisB, pPB67) showed that the purified SepII recognized and cleaved the same site as EcoRV. Genomic DNA modification status was also determined.     

A site-specific endonuclease from Caulobacter crescentus CB-13: restriction endonuclease CcrI

A site-specific restriction endonuclease (CcrI) has been identified from Caulobacter crescentus CB-13. This enzyme has been purified to homogeneity and the cleavage patterns with various DNAs and sequence data show that CcrI recognizes the same sequence as the XhoI restriction endonuclease (5′-C^↓-T-C-G-A-G-3′). Ccr has an absolute requirement for magnesium ions with an optimum concentration of 4 mM. The enzyme is optimally active at pH 8.0 and is stable up to 70°C. CcrI has a molecular weight of 65300 and exists as a monomer in its native state. Most of the physical characteristics observed for CcrI were similar to those observed for XhoI. Kinetic studies on CcrI and XhoI suggest that the enzymes interact with λ DNA in the same manner; however, with ?X-174 R.F. DNA, CcrI has a greater affinity for the supercoiled molecule than XhoI.     

Two restriction and modification systems in Staphylococcus aureus NCTC8325.

The presence of two distinct host specificities in Staphylococcus aureus strain NCTC8325 was revealed by the isolation of restriction- and modification-deficient mutants. The two host specificity systems, designated S1 and S2, are both active on phage 80mualpha but are not additive in their restricting activity. Restriction-deficient, modification-proficient mutants were invariably affected in both restriction systems. The functional relationship between these two systems is discussed.     

SwaI, a unique restriction endonuclease from Staphylococcus warneri, which recognizes 5'-ATTTAAAT-3'.

A novel class-II restriction endonuclease designated SwaI was purified from Staphylococcus warneri. This enzyme cleaves adenovirus 2 DNA, SV40 DNA and M13mp7 at one site each, but does not cleave lambda, PhiX174, pBR322 or pBR328 DNA. SwaI recognizes the octanucleotide sequence 5'-ATTTAAAT-3', cleaving in the center of the recognition sequence creating blunt ended DNA fragments. SwaI was used to digest chromosomal DNA from various microorganisms and human cells.     

A thermostable, sequence-specific restriction endonuclease from Bacillus stearothermophilus: BstPI.

A restriction endonuclease, BstPI, was purified from a strain of B. stearothermophilus, and its cleavage specificity was determined. The enzyme cleaves at palindromic sites of the general structure: (Formula: see text) where N.N' can be any base pair. It produces phosphorylated 5'-termini which are single stranded over a length of 5 nucleotides. Ends generated by cleavage with BstPI can be rejoined by DNA ligase.     

NgoII, a restriction endonuclease from Neisseria gonorrhoeae.

EndoR . NgoII, a class II restriction endonuclease isolated from Neisseria gonorrhoeae, was purified to electrophoretic homogeneity. We were able to separate it from another restriction endonuclease of N. gonorrhoeae, NgoI, by phosphocellulose chromatography. NgoII is an isoschizomer of HaeIII, a restriction endonuclease of Haemophilus aegyptius, and was found to recognize the deoxyribonucleic acid nucleotide base sequence GGCC. NgoII was able to digest phage lambda deoxyribonucleic acid over a wide pH range, with optimal activity at pH 8.5. The enzyme has an absolute requirement for Mg2+; maximal enzyme activity was observed at 1 mM Mg2+. The active enzyme has a molecular weight of 65,000 and appears to be composed of six subunits of identical molecular weight (11,000). No methylase activity could be detected in the purified enzyme preparation.