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Isolation and characterization of a new restriction endonuclease, Sru30DI, from Selenomonas ruminantium
Gene, 158 (1995) 139-140 © 1995 Elsevier ScienceB.V. All rights reserved.0378-1119/95/$09.50
139
GENE 08811 Brief Notes
Isolation and characterization of a new restriction endonuclease, Sru3ODI, from Selenomonas ruminantium* (Type-II restriction endonuclease; StuI isoschizomer; Dcm sensitivity)
Peter Prista~, Ivan Vanat and Peter Javorsk~, Institute of Animal Physiology, Si'ovakAcademy of Sciences, 040 01 Kogice, SIovakia
Receivedby Z. Hradern~i:26 November 1994;Accepted:27 December 1994;Receivedat publishers: 30 January 1995
SUMMARY The restriction endonuclease (ENase) Sru3ODI, an isoschizomer of StuI, which recognizes the sequence 5'-AGG/ CCT-3', was purified from a natural isolate of Selenomonas ruminantium. The ENase was isolated from cell extracts using single-step purification by phosphocellulose column chromatography. Activity of Sru3ODI is inhibited by overlapping Dcm methylation. The ENase is extremely stable at 37°C and is active over a wide range of pH, temperature and salt concentrations.
Restriction endonucleases (ENases) play a crucial role in the control of phage propagation and gene spreading in natural environment, e.g., the digestive tracts of animals. A group of ruminal bacteria was shown to be a promissing source of ENases (Morrison et al., 1992a,b; Lee et al., 1992; Vanat et al., 1993a). From the ruminal selenomonades two ENases, SruI and Sru4DI, were isolated, both recognizing pure A + T sequences (Vanat et al., 1993b; Pristag et al., 1994). Because such ENases are rare, we looked for other ENase activities among natural isolates of Selenomonas ruminantium. In the S. ruminantium strain 30D a site-specific ENase was detected. As no unspecific nucleolytic activities were observed in S. ruminanti~m 30D cell extracts, Sru3ODI Correspondence to: Dr. P. Prista~, Institute of Animal Physiology, Slovak Academy of Sciences, Palackrho 12, 040 01 Ko~ice, Slovakia. Tel. (42-95) 622-8151; Fax (42-o5) 622-4491. e-mail: [email protected] *On request, the authors will supply detailed experimentalevidencefor the conclusionsreached in this BriefNote.
Abbreviations:bp, base pair(s); ENase, restriction endonuclease;kb, kilobase(s) or 1000bp; nt, nucleotide(s);S., Selenomonas. SSDI 0378-1119(95)00093-3
ENase was isolated from cell extracts using a one-step purification by phosphocellulose column chromatography. The yield of Sru3ODI was 40 000 units from 1 g (wet weight) of S. ruminantium 30D cells. The specificity of Sru3ODI was shown to be 5'-AGG/ CCT-3' (Fig. 1), thus Sru3ODI is a true isoschizomer of StuI (Shimotsu et al., 1980). Using plasmid pRES3 (see legend to Fig. 1) isolated from E. coli dcm ÷ and d c m strains it was shown that activity of Sru3ODI is inhibited by overlapping Dcm methylation. Sru30DI is rather insensitive to the reaction conditions, and for optimal activity it required 1-20 m M Mg 2÷ . Its activity was stimulated by the presence of monovalent cations at 20-250 mM. Without these cations and with excess of enzyme some 'star' activity was observed. Sru3ODI is active over a wide pH range (5.5-9.5). Although isolated from a mesophile microorganism with an optimal growth temperature of 39°C its activity remains constant at 37-57°C; however, Sru3ODI is rapidly denaturated at 67°C. Sru3ODI retains full activity for 1-6 h at 37°C, even in the absence of DNA. Under similar conditions StuI has lost its enzymatic activity after 2 h of incubation.
140 -
G
A
T
C
+
Ij
of E. coli and Dr. Gabriela Bukovsk~i (Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia) for the technical assistance provided during this work.
m
REFERENCES
Fig. 1. Sru3ODI cleavage site. Determination of the Sru3ODI cleavage position within its recognition sequence was carried out according to Brown and Smith (1980). Single-stranded DNA of pRES3 plasmid, a pBluescript SK + derivative containing a KpnI-EcoRI fragment of SV40 DNA (nt 294-1782) as insert, which carries the preliminary examined Sru3ODI site and the universal - 4 0 primer were used for standard dideoxy DNA sequencing reactions. The double-stranded DNA recovered from an additional sequencing reaction, lacking dideoxy terminators, was used as substrate for Sru3ODI digestion. The resulting cleavage products (lane - ) were separated on a 8 M urea-6% polyacrylamide sequencing gel alongside the sequencing ladder (lanes A, C, G and T); products filled-in with the Klenow fragment of DNA polymerase I are shown in lane +. The arrowhead shows the position of Sru3ODIgenerated band.
Therefore, Sru3ODI appears superior to StuI because of its high yield, remarkable stability and wide range of the reaction conditions.
ACKNOWLEDGEMENTS
We thank Dr. Nassal from the University of Heidelberg, Germany for providing us the dcm- strain
Brown, N.L. and Smith, M.: A general method for defining restriction enzyme cleavage and recognition sites. Methods Enzymol. 65 (1980) 391-404. Lee, S.F., Forsberg, C.W. and Gibbins, A.M.: Type II DNA restrictionmodification system and a endonuclease from the ruminal bacterium Fibrobacter succinogenes $85. J. Bacteriol. 174 (1992) 5275-5283. Morrison, M., Mackie, R.I. and White, B.A.: Partial characterization of a DNA endonuclease from Ruminococcus flavefaciensFD-1 and its inhibition by site-specific adenine methylation. Appl. Environ. Microbiol. 58 (1992a) 66-69. Morrison, M., Mackie, R.I. and White, B.A.: Partial purification and characterization of Ral8I, a class-IIS restriction endonuclease from Ruminococcus albus 8 which recognizes 5'-GGATC. Gene 111 (1992b) 105-108. Prista~, P., Vanat, I., God~iny, A. and Javorsk~, P.: Restriction endonucleases from Selenomonas ruminantium which recognize and cleave 5'-AT/TAAT-3'. Arch. Microbiol. 161 (1994) 439-441. Shimotsu, H., Takahashi, H. and Saito, H.: A new site-specific endonuclease StuI from Streptomyces tubercidicus. Gene 11 (1980) 219-225. Vanat, I., Prista~, P., Kutejov~, E., JOdov~t, J., God~iny, A. and Javorsk~, P.: SbvI restriction endonuclease from Streptococcus bovis. Lett. Appl. Microbiol. 17 (1993a) 297-299. Vanat, I., Prista~, P., Rybo~ov~t, E., God~ny, A. and Javorsk~, P.: SruI restriction endonuclease from Selenomonas ruminantium. FEMS Microbiol. Lett. 113 (1993b) 129 132.
139
GENE 08811 Brief Notes
Isolation and characterization of a new restriction endonuclease, Sru3ODI, from Selenomonas ruminantium* (Type-II restriction endonuclease; StuI isoschizomer; Dcm sensitivity)
Peter Prista~, Ivan Vanat and Peter Javorsk~, Institute of Animal Physiology, Si'ovakAcademy of Sciences, 040 01 Kogice, SIovakia
Receivedby Z. Hradern~i:26 November 1994;Accepted:27 December 1994;Receivedat publishers: 30 January 1995
SUMMARY The restriction endonuclease (ENase) Sru3ODI, an isoschizomer of StuI, which recognizes the sequence 5'-AGG/ CCT-3', was purified from a natural isolate of Selenomonas ruminantium. The ENase was isolated from cell extracts using single-step purification by phosphocellulose column chromatography. Activity of Sru3ODI is inhibited by overlapping Dcm methylation. The ENase is extremely stable at 37°C and is active over a wide range of pH, temperature and salt concentrations.
Restriction endonucleases (ENases) play a crucial role in the control of phage propagation and gene spreading in natural environment, e.g., the digestive tracts of animals. A group of ruminal bacteria was shown to be a promissing source of ENases (Morrison et al., 1992a,b; Lee et al., 1992; Vanat et al., 1993a). From the ruminal selenomonades two ENases, SruI and Sru4DI, were isolated, both recognizing pure A + T sequences (Vanat et al., 1993b; Pristag et al., 1994). Because such ENases are rare, we looked for other ENase activities among natural isolates of Selenomonas ruminantium. In the S. ruminantium strain 30D a site-specific ENase was detected. As no unspecific nucleolytic activities were observed in S. ruminanti~m 30D cell extracts, Sru3ODI Correspondence to: Dr. P. Prista~, Institute of Animal Physiology, Slovak Academy of Sciences, Palackrho 12, 040 01 Ko~ice, Slovakia. Tel. (42-95) 622-8151; Fax (42-o5) 622-4491. e-mail: [email protected] *On request, the authors will supply detailed experimentalevidencefor the conclusionsreached in this BriefNote.
Abbreviations:bp, base pair(s); ENase, restriction endonuclease;kb, kilobase(s) or 1000bp; nt, nucleotide(s);S., Selenomonas. SSDI 0378-1119(95)00093-3
ENase was isolated from cell extracts using a one-step purification by phosphocellulose column chromatography. The yield of Sru3ODI was 40 000 units from 1 g (wet weight) of S. ruminantium 30D cells. The specificity of Sru3ODI was shown to be 5'-AGG/ CCT-3' (Fig. 1), thus Sru3ODI is a true isoschizomer of StuI (Shimotsu et al., 1980). Using plasmid pRES3 (see legend to Fig. 1) isolated from E. coli dcm ÷ and d c m strains it was shown that activity of Sru3ODI is inhibited by overlapping Dcm methylation. Sru30DI is rather insensitive to the reaction conditions, and for optimal activity it required 1-20 m M Mg 2÷ . Its activity was stimulated by the presence of monovalent cations at 20-250 mM. Without these cations and with excess of enzyme some 'star' activity was observed. Sru3ODI is active over a wide pH range (5.5-9.5). Although isolated from a mesophile microorganism with an optimal growth temperature of 39°C its activity remains constant at 37-57°C; however, Sru3ODI is rapidly denaturated at 67°C. Sru3ODI retains full activity for 1-6 h at 37°C, even in the absence of DNA. Under similar conditions StuI has lost its enzymatic activity after 2 h of incubation.
140 -
G
A
T
C
+
Ij
of E. coli and Dr. Gabriela Bukovsk~i (Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia) for the technical assistance provided during this work.
m
REFERENCES
Fig. 1. Sru3ODI cleavage site. Determination of the Sru3ODI cleavage position within its recognition sequence was carried out according to Brown and Smith (1980). Single-stranded DNA of pRES3 plasmid, a pBluescript SK + derivative containing a KpnI-EcoRI fragment of SV40 DNA (nt 294-1782) as insert, which carries the preliminary examined Sru3ODI site and the universal - 4 0 primer were used for standard dideoxy DNA sequencing reactions. The double-stranded DNA recovered from an additional sequencing reaction, lacking dideoxy terminators, was used as substrate for Sru3ODI digestion. The resulting cleavage products (lane - ) were separated on a 8 M urea-6% polyacrylamide sequencing gel alongside the sequencing ladder (lanes A, C, G and T); products filled-in with the Klenow fragment of DNA polymerase I are shown in lane +. The arrowhead shows the position of Sru3ODIgenerated band.
Therefore, Sru3ODI appears superior to StuI because of its high yield, remarkable stability and wide range of the reaction conditions.
ACKNOWLEDGEMENTS
We thank Dr. Nassal from the University of Heidelberg, Germany for providing us the dcm- strain
Brown, N.L. and Smith, M.: A general method for defining restriction enzyme cleavage and recognition sites. Methods Enzymol. 65 (1980) 391-404. Lee, S.F., Forsberg, C.W. and Gibbins, A.M.: Type II DNA restrictionmodification system and a endonuclease from the ruminal bacterium Fibrobacter succinogenes $85. J. Bacteriol. 174 (1992) 5275-5283. Morrison, M., Mackie, R.I. and White, B.A.: Partial characterization of a DNA endonuclease from Ruminococcus flavefaciensFD-1 and its inhibition by site-specific adenine methylation. Appl. Environ. Microbiol. 58 (1992a) 66-69. Morrison, M., Mackie, R.I. and White, B.A.: Partial purification and characterization of Ral8I, a class-IIS restriction endonuclease from Ruminococcus albus 8 which recognizes 5'-GGATC. Gene 111 (1992b) 105-108. Prista~, P., Vanat, I., God~iny, A. and Javorsk~, P.: Restriction endonucleases from Selenomonas ruminantium which recognize and cleave 5'-AT/TAAT-3'. Arch. Microbiol. 161 (1994) 439-441. Shimotsu, H., Takahashi, H. and Saito, H.: A new site-specific endonuclease StuI from Streptomyces tubercidicus. Gene 11 (1980) 219-225. Vanat, I., Prista~, P., Kutejov~, E., JOdov~t, J., God~iny, A. and Javorsk~, P.: SbvI restriction endonuclease from Streptococcus bovis. Lett. Appl. Microbiol. 17 (1993a) 297-299. Vanat, I., Prista~, P., Rybo~ov~t, E., God~ny, A. and Javorsk~, P.: SruI restriction endonuclease from Selenomonas ruminantium. FEMS Microbiol. Lett. 113 (1993b) 129 132.