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. 1991 Dec 11;19(23):6457–6463. doi: 10.1093/nar/19.23.6457

Cloning, characterization and evolution of the BsuFI restriction endonuclease gene of Bacillus subtilis and purification of the enzyme.

W Kapfer 1, J Walter 1, T A Trautner 1
PMCID: PMC329197  PMID: 1721700

Abstract

The restriction endonuclease (R.BsuFI) of Bacillus subtilis recognizes the target DNA sequence 5' CCGG. The R.BsuFI gene was found in close proximity to the cognate M.BsuFI gene, which had previously been characterized (1). Cloning of the R.BsuFI gene in E.coli was only possible with the M.BsuFI Mtase gene present on a compatible plasmid. The cloned R.BsuFI gene was expressed in E. coli and restriction activity was observed in vivo and in vitro. The R.BsuFI gene consists of 1185 bp, coding for a protein of 395 amino acids with a calculated molecular weight of 45.6 kD. The R.BsuFI enzyme was purified to homogeneity following overexpression. It presumably works as a dimer and cleaves the 5' CCGG target sequence between the two cytosines to produce sticky ends with 5' CG overhangs, like the isoschizomers R.MspI and R.HpaII. The relatedness between R.BsuFI and R.MspI is reflected by significant similarities of the amino acid sequences of both enzymes. This is the first case where such similarities have been observed between isoschizomeric restriction endonucleases which belong to 5mC specific R/M systems. This observation suggests that R.BsuFI and R.MspI genes derive from a common ancestor. In spite of such functional and evolutionary relatedness, the R/M systems differ in the arrangement of their R and M genes. In the BsuFI system transcription of the two genes is convergent, whereas divergent transcription occurs in the MspI system.

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Selected References

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  1. Biswal N., Kleinschmidt A. K., Spatz H. C., Trautner T. A. Physical properties of the DNA of bacteriophage SP50. Mol Gen Genet. 1967;100(1):39–55. doi: 10.1007/BF00425774. [DOI] [PubMed] [Google Scholar]
  2. Bocklage H., Heeger K., Müller-Hill B. Cloning and characterization of the MboII restriction-modification system. Nucleic Acids Res. 1991 Mar 11;19(5):1007–1013. doi: 10.1093/nar/19.5.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Bron S., Murray K., Trautner T. A. Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclease from strain R. Mol Gen Genet. 1975 Dec 30;143(1):13–23. doi: 10.1007/BF00269416. [DOI] [PubMed] [Google Scholar]
  6. Brown N. L., Smith M. A general method for defining restriction enzyme cleavage and recognition sites. Methods Enzymol. 1980;65(1):391–404. doi: 10.1016/s0076-6879(80)65050-2. [DOI] [PubMed] [Google Scholar]
  7. Churchward G., Belin D., Nagamine Y. A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene. 1984 Nov;31(1-3):165–171. doi: 10.1016/0378-1119(84)90207-5. [DOI] [PubMed] [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
  10. Gryczan T. J., Contente S., Dubnau D. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J Bacteriol. 1978 Apr;134(1):318–329. doi: 10.1128/jb.134.1.318-329.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Howard K. A., Card C., Benner J. S., Callahan H. L., Maunus R., Silber K., Wilson G., Brooks J. E. Cloning the DdeI restriction-modification system using a two-step method. Nucleic Acids Res. 1986 Oct 24;14(20):7939–7951. doi: 10.1093/nar/14.20.7939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ikawa S., Shibata T., Ando T., Saito H. Genetic studies on site-specific endodeoxyribonucleases in Bacillus subtilis: multiple modification and restriction systems in transformants of Bacillus subtilis 168. Mol Gen Genet. 1980 Feb;177(3):359–368. doi: 10.1007/BF00271474. [DOI] [PubMed] [Google Scholar]
  13. Jentsch S. Restriction and modification in Bacillus subtilis: sequence specificities of restriction/modification systems BsuM, BsuE, and BsuF. J Bacteriol. 1983 Nov;156(2):800–808. doi: 10.1128/jb.156.2.800-808.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kessler C., Manta V. Specificity of restriction endonucleases and DNA modification methyltransferases a review (Edition 3). Gene. 1990 Aug 16;92(1-2):1–248. doi: 10.1016/0378-1119(90)90486-b. [DOI] [PubMed] [Google Scholar]
  15. Kiss A., Posfai G., Keller C. C., Venetianer P., Roberts R. J. Nucleotide sequence of the BsuRI restriction-modification system. Nucleic Acids Res. 1985 Sep 25;13(18):6403–6421. doi: 10.1093/nar/13.18.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Klimasauskas S., Timinskas A., Menkevicius S., Butkienè D., Butkus V., Janulaitis A. Sequence motifs characteristic of DNA[cytosine-N4]methyltransferases: similarity to adenine and cytosine-C5 DNA-methylases. Nucleic Acids Res. 1989 Dec 11;17(23):9823–9832. doi: 10.1093/nar/17.23.9823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. LURIA S. E., BURROUS J. W. Hybridization between Escherichia coli and Shigella. J Bacteriol. 1957 Oct;74(4):461–476. doi: 10.1128/jb.74.4.461-476.1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Larsen J. E., Gerdes K., Light J., Molin S. Low-copy-number plasmid-cloning vectors amplifiable by derepression of an inserted foreign promoter. Gene. 1984 Apr;28(1):45–54. doi: 10.1016/0378-1119(84)90086-6. [DOI] [PubMed] [Google Scholar]
  19. Lauster R. Evolution of type II DNA methyltransferases. A gene duplication model. J Mol Biol. 1989 Mar 20;206(2):313–321. doi: 10.1016/0022-2836(89)90481-6. [DOI] [PubMed] [Google Scholar]
  20. Lauster R., Trautner T. A., Noyer-Weidner M. Cytosine-specific type II DNA methyltransferases. A conserved enzyme core with variable target-recognizing domains. J Mol Biol. 1989 Mar 20;206(2):305–312. doi: 10.1016/0022-2836(89)90480-4. [DOI] [PubMed] [Google Scholar]
  21. Lin P. M., Lee C. H., Roberts R. J. Cloning and characterization of the genes encoding the MspI restriction modification system. Nucleic Acids Res. 1989 Apr 25;17(8):3001–3011. doi: 10.1093/nar/17.8.3001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Messing J., Vieira J. A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene. 1982 Oct;19(3):269–276. doi: 10.1016/0378-1119(82)90016-6. [DOI] [PubMed] [Google Scholar]
  23. Moran C. P., Jr, Lang N., LeGrice S. F., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339–346. doi: 10.1007/BF00729452. [DOI] [PubMed] [Google Scholar]
  24. Niaudet B., Goze A., Ehrlich S. D. Insertional mutagenesis in Bacillus subtilis: mechanism and use in gene cloning. Gene. 1982 Oct;19(3):277–284. doi: 10.1016/0378-1119(82)90017-8. [DOI] [PubMed] [Google Scholar]
  25. Pósfai J., Bhagwat A. S., Pósfai G., Roberts R. J. Predictive motifs derived from cytosine methyltransferases. Nucleic Acids Res. 1989 Apr 11;17(7):2421–2435. doi: 10.1093/nar/17.7.2421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rottländer E., Trautner T. A. Genetic and transfection studies with B, subtilis phage SP 50. I. Phage mutants with restricted growth on B. subtilis strain 168. Mol Gen Genet. 1970;108(1):47–60. doi: 10.1007/BF00343184. [DOI] [PubMed] [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Smith H. O., Annau T. M., Chandrasegaran S. Finding sequence motifs in groups of functionally related proteins. Proc Natl Acad Sci U S A. 1990 Jan;87(2):826–830. doi: 10.1073/pnas.87.2.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stephenson F. H., Ballard B. T., Boyer H. W., Rosenberg J. M., Greene P. J. Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases. Gene. 1989 Dec 21;85(1):1–13. doi: 10.1016/0378-1119(89)90458-7. [DOI] [PubMed] [Google Scholar]
  30. Szilák L., Venetianer P., Kiss A. Cloning and nucleotide sequence of the genes coding for the Sau96I restriction and modification enzymes. Nucleic Acids Res. 1990 Aug 25;18(16):4659–4664. doi: 10.1093/nar/18.16.4659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Trautner T. A., Pawlek B., Bron S., Anagnostopoulos C. Restriction and modification in B. subtilis. Biological aspects. Mol Gen Genet. 1974;131(3):181–191. doi: 10.1007/BF00267958. [DOI] [PubMed] [Google Scholar]
  32. Vosman B., Kooistra J., Olijve J., Venema G. Integration of vector-containing Bacillus subtilis chromosomal DNA by a Campbell-like mechanism. Mol Gen Genet. 1986 Sep;204(3):524–531. doi: 10.1007/BF00331035. [DOI] [PubMed] [Google Scholar]
  33. Walder R. Y., Langtimm C. J., Chatterjee R., Walder J. A. Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII. J Biol Chem. 1983 Jan 25;258(2):1235–1241. [PubMed] [Google Scholar]
  34. Walter J., Noyer-Weidner M., Trautner T. A. The amino acid sequence of the CCGG recognizing DNA methyltransferase M.BsuFI: implications for the analysis of sequence recognition by cytosine DNA methyltransferases. EMBO J. 1990 Apr;9(4):1007–1013. doi: 10.1002/j.1460-2075.1990.tb08203.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wilke K., Rauhut E., Noyer-Weidner M., Lauster R., Pawlek B., Behrens B., Trautner T. A. Sequential order of target-recognizing domains in multispecific DNA-methyltransferases. EMBO J. 1988 Aug;7(8):2601–2609. doi: 10.1002/j.1460-2075.1988.tb03110.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilson G. G. Organization of restriction-modification systems. Nucleic Acids Res. 1991 May 25;19(10):2539–2566. doi: 10.1093/nar/19.10.2539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wilson G. G. Type II restriction--modification systems. Trends Genet. 1988 Nov;4(11):314–318. doi: 10.1016/0168-9525(88)90109-6. [DOI] [PubMed] [Google Scholar]
  38. Wood W. B. Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J Mol Biol. 1966 Mar;16(1):118–133. doi: 10.1016/s0022-2836(66)80267-x. [DOI] [PubMed] [Google Scholar]
  39. Woodcock D. M., Crowther P. J., Doherty J., Jefferson S., DeCruz E., Noyer-Weidner M., Smith S. S., Michael M. Z., Graham M. W. Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res. 1989 May 11;17(9):3469–3478. doi: 10.1093/nar/17.9.3469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  41. Zahler S. A., Korman R. Z., Rosenthal R., Hemphill H. E. Bacillus subtilis bacteriophage SPbeta: localization of the prophage attachment site, and specialized transduction. J Bacteriol. 1977 Jan;129(1):556–558. doi: 10.1128/jb.129.1.556-558.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]

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