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. 1992 Aug;174(16):5275–5283. doi: 10.1128/jb.174.16.5275-5283.1992

Type II DNA restriction-modification system and an endonuclease from the ruminal bacterium Fibrobacter succinogenes S85.

S F Lee 1, C W Forsberg 1, A M Gibbins 1
PMCID: PMC206363  PMID: 1644754

Abstract

Fibrobacter succinogenes is an important cellulolytic bacterium found in the rumen and cecum of herbivores. Numerous attempts to introduce foreign DNA into F. succinogenes S85 have failed, suggesting the presence of genetic barriers in this organism. Results from this study clearly demonstrate that F. succinogenes S85 possesses a type II restriction endonuclease, FsuI, which recognizes the sequence 5'-GG(A/T)CC-3'. Analysis of the restriction products on sequencing gels showed that FsuI cleaves between the two deoxyguanosine residues, yielding a 3-base 5' protruding end. These data demonstrate that FsuI is an isoschizomer of AvaII. A methyltransferase activity has been identified in the cell extract of F. succinogenes S85. This activity modified DNA in vitro and protected the DNA from the restriction by FsuI and AvaII. DNA modified in vivo by a cloned methylase gene, which codes for M.Eco47II, also protected the DNA from restriction by FsuI, suggesting that FsuI is inhibited by methylation at one or both deoxycytosine residues of the recognition sequence. The methyltransferase activity in F. succinogenes S85 is likely modifying the same deoxycytosine residues, but the exact site(s) is unknown. A highly active DNase (DNase A) was also isolated from the cell extract of this organism. DNase A is an endonuclease which showed high activity on all forms of DNA (single stranded, double-stranded, linear, and circular) but no activity on RNA. In vitro, the DNase A hydrolyzed F. succinogenes S85 DNA extensively, indicating the lack of protection against hydrolysis by this enzyme. In the presence of Mg2+, DNA was hydrolyzed to fragments of 8 to 10 nucleotides in length. The presence of DNase A and the type II restriction-modification system of F. succinogenes S85 may be the barriers preventing the introduction of foreign DNA into this bacterium.

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Selected References

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  1. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Cavicchioli R., East P. D., Watson K. endAFS, a novel family E endoglucanase gene from Fibrobacter succinogenes AR1. J Bacteriol. 1991 May;173(10):3265–3268. doi: 10.1128/jb.173.10.3265-3268.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Elhai J., Wolk C. P. Conjugal transfer of DNA to cyanobacteria. Methods Enzymol. 1988;167:747–754. doi: 10.1016/0076-6879(88)67086-8. [DOI] [PubMed] [Google Scholar]
  4. Flint H. J., Thomson A. M. Deoxyribonuclease activity in rumen bacteria. Lett Appl Microbiol. 1990 Jul;11(1):18–21. doi: 10.1111/j.1472-765x.1990.tb00126.x. [DOI] [PubMed] [Google Scholar]
  5. Gilbert H. J., Hazlewood G. P. Genetic modification of fibre digestion. Proc Nutr Soc. 1991 Aug;50(2):173–186. doi: 10.1079/pns19910028. [DOI] [PubMed] [Google Scholar]
  6. Gong J. H., Lo R. Y., Forsberg C. W. Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85. Appl Environ Microbiol. 1989 Jan;55(1):132–136. doi: 10.1128/aem.55.1.132-136.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hu Y. J., Smith D. C., Cheng K. J., Foresberg C. W. Cloning of a xylanase gene from Fibrobacter succinogenes 135 and its expression in Escherichia coli. Can J Microbiol. 1991 Jul;37(7):554–561. doi: 10.1139/m91-093. [DOI] [PubMed] [Google Scholar]
  8. Hughes S. G., Murray K. The nucleotide sequences recognized by endonucleases AvaI and AvaII from Anabaena variabilis. Biochem J. 1980 Jan 1;185(1):65–75. doi: 10.1042/bj1850065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ikemura T., Dahlberg J. E. Small ribonucleic acids of Escherichia coli. I. Characterization by polyacrylamide gel electrophoresis and fingerprint analysis. J Biol Chem. 1973 Jul 25;248(14):5024–5032. [PubMed] [Google Scholar]
  10. Janulaitis A., Petrusyté M., Butkus V. Three sequence-specific endonucleases from Escherichia coli RFL47. FEBS Lett. 1983 Sep 19;161(2):213–216. doi: 10.1016/0014-5793(83)81010-2. [DOI] [PubMed] [Google Scholar]
  11. KUNITZ M. Crystalline desoxyribonuclease; isolation and general properties; spectrophotometric method for the measurement of desoxyribonuclease activity. J Gen Physiol. 1950 Mar;33(4):349–362. doi: 10.1085/jgp.33.4.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  13. LEHMAN I. R., ROUSSOS G. G., PRATT E. A. The deoxyribo-nucleases of Escherichia coli. III. Studies on the nature of the inhibition of endonuclease by ribonucleic acid. J Biol Chem. 1962 Mar;237:829–833. [PubMed] [Google Scholar]
  14. LEHMAN I. R., ROUSSOS G. G., PRATT E. A. The deoxyribonucleases of Escherichia coli. II. Purification and properties of a ribonucleic acid-inhibitable endonuclease. J Biol Chem. 1962 Mar;237:819–828. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lee S. F., Progulske-Fox A., Bleiweis A. S. Molecular cloning and expression of a Streptococcus mutans major surface protein antigen, P1 (I/II), in Escherichia coli. Infect Immun. 1988 Aug;56(8):2114–2119. doi: 10.1128/iai.56.8.2114-2119.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matte A., Forsberg C. W. Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85. Appl Environ Microbiol. 1992 Jan;58(1):157–168. doi: 10.1128/aem.58.1.157-168.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McGavin M. J., Forsberg C. W., Crosby B., Bell A. W., Dignard D., Thomas D. Y. Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3. J Bacteriol. 1989 Oct;171(10):5587–5595. doi: 10.1128/jb.171.10.5587-5595.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Morrison M., Mackie R. I., White B. A. Partial characterization of a DNA restriction endonuclease from Ruminococcus flavefaciens FD-1 and its inhibition by site-specific adenine methylation. Appl Environ Microbiol. 1992 Jan;58(1):66–69. doi: 10.1128/aem.58.1.66-69.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nelson M., McClelland M. Effect of site-specific methylation on DNA modification methyltransferases and restriction endonucleases. Nucleic Acids Res. 1989;17 (Suppl):r389–r415. doi: 10.1093/nar/17.suppl.r389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nelson M., McClelland M. Purification and assay of type II DNA methylases. Methods Enzymol. 1987;155:32–41. doi: 10.1016/0076-6879(87)55007-8. [DOI] [PubMed] [Google Scholar]
  22. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  23. Roberts R. J. Restriction enzymes and their isoschizomers. Nucleic Acids Res. 1990 Apr 25;18 (Suppl):2331–2365. doi: 10.1093/nar/18.suppl.2331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Russell J. B., Wilson D. B. Potential opportunities and problems for genetically altered rumen microorganisms. J Nutr. 1988 Feb;118(2):271–279. doi: 10.1093/jn/118.2.271. [DOI] [PubMed] [Google Scholar]
  25. SCOTT H. W., DEHORITY B. A. VITAMIN REQUIREMENTS OF SEVERAL CELLULOLYTIC RUMEN BACTERIA. J Bacteriol. 1965 May;89:1169–1175. doi: 10.1128/jb.89.5.1169-1175.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Sipat A., Taylor K. A., Lo R. Y., Forsberg C. W., Krell P. J. Molecular cloning of a xylanase gene from Bacteroides succinogenes and its expression in Escherichia coli. Appl Environ Microbiol. 1987 Mar;53(3):477–481. doi: 10.1128/aem.53.3.477-481.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  29. Teather R. M., Erfle J. D. DNA sequence of a Fibrobacter succinogenes mixed-linkage beta-glucanase (1,3-1,4-beta-D-glucan 4-glucanohydrolase) gene. J Bacteriol. 1990 Jul;172(7):3837–3841. doi: 10.1128/jb.172.7.3837-3841.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Worrall A. F., Connolly B. A. The chemical synthesis of a gene coding for bovine pancreatic DNase I and its cloning and expression in Escherichia coli. J Biol Chem. 1990 Dec 15;265(35):21889–21895. [PubMed] [Google Scholar]

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