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. 2004 Feb;166(2):661–668. doi: 10.1534/genetics.166.2.661

Developing a genetic system in Deinococcus radiodurans for analyzing mutations.

Mandy Kim 1, Erika Wolff 1, Tiffany Huang 1, Lilit Garibyan 1, Ashlee M Earl 1, John R Battista 1, Jeffrey H Miller 1
PMCID: PMC1470732  PMID: 15020457

Abstract

We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the beta-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif(r) system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif(r) in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

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Selected References

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  1. Albertini A. M., Hofer M., Calos M. P., Miller J. H. On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions. Cell. 1982 Jun;29(2):319–328. doi: 10.1016/0092-8674(82)90148-9. [DOI] [PubMed] [Google Scholar]
  2. Aubry-Damon H., Soussy C. J., Courvalin P. Characterization of mutations in the rpoB gene that confer rifampin resistance in Staphylococcus aureus. Antimicrob Agents Chemother. 1998 Oct;42(10):2590–2594. doi: 10.1128/aac.42.10.2590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aubry-Damon Hélène, Galimand Marc, Gerbaud Guy, Courvalin Patrice. rpoB mutation conferring rifampin resistance in Streptococcus pyogenes. Antimicrob Agents Chemother. 2002 May;46(5):1571–1573. doi: 10.1128/AAC.46.5.1571-1573.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Battista J. R. Against all odds: the survival strategies of Deinococcus radiodurans. Annu Rev Microbiol. 1997;51:203–224. doi: 10.1146/annurev.micro.51.1.203. [DOI] [PubMed] [Google Scholar]
  5. Cambau Emmanuelle, Bonnafous Pascale, Perani Evelyne, Sougakoff Wladimir, Ji Baohong, Jarlier Vincent. Molecular detection of rifampin and ofloxacin resistance for patients who experience relapse of multibacillary leprosy. Clin Infect Dis. 2001 Nov 21;34(1):39–45. doi: 10.1086/324623. [DOI] [PubMed] [Google Scholar]
  6. Campbell E. A., Korzheva N., Mustaev A., Murakami K., Nair S., Goldfarb A., Darst S. A. Structural mechanism for rifampicin inhibition of bacterial rna polymerase. Cell. 2001 Mar 23;104(6):901–912. doi: 10.1016/s0092-8674(01)00286-0. [DOI] [PubMed] [Google Scholar]
  7. Cupples C. G., Miller J. H. A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5345–5349. doi: 10.1073/pnas.86.14.5345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Farabaugh P. J., Schmeissner U., Hofer M., Miller J. H. Genetic studies of the lac repressor. VII. On the molecular nature of spontaneous hotspots in the lacI gene of Escherichia coli. J Mol Biol. 1978 Dec 25;126(4):847–857. doi: 10.1016/0022-2836(78)90023-2. [DOI] [PubMed] [Google Scholar]
  9. Garibyan Lilit, Huang Tiffany, Kim Mandy, Wolff Erika, Nguyen Anh, Nguyen Theresa, Diep Amy, Hu Kaibin, Iverson Ayuko, Yang Hanjing. Use of the rpoB gene to determine the specificity of base substitution mutations on the Escherichia coli chromosome. DNA Repair (Amst) 2003 May 13;2(5):593–608. doi: 10.1016/s1568-7864(03)00024-7. [DOI] [PubMed] [Google Scholar]
  10. Heep M., Odenbreit S., Beck D., Decker J., Prohaska E., Rieger U., Lehn N. Mutations at four distinct regions of the rpoB gene can reduce the susceptibility of Helicobacter pylori to rifamycins. Antimicrob Agents Chemother. 2000 Jun;44(6):1713–1715. doi: 10.1128/aac.44.6.1713-1715.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jin D. J., Gross C. A. Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J Mol Biol. 1988 Jul 5;202(1):45–58. doi: 10.1016/0022-2836(88)90517-7. [DOI] [PubMed] [Google Scholar]
  12. Karunakaran P., Davies J. Genetic antagonism and hypermutability in Mycobacterium smegmatis. J Bacteriol. 2000 Jun;182(12):3331–3335. doi: 10.1128/jb.182.12.3331-3335.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kerszman G. Induction of mutation to streptomycin resistance in Micrococcus radiodurans. Mutat Res. 1975 Apr;28(1):9–14. doi: 10.1016/0027-5107(75)90308-5. [DOI] [PubMed] [Google Scholar]
  14. Kim Mandy, Huang Tiffany, Miller Jeffrey H. Competition between MutY and mismatch repair at A x C mispairs In vivo. J Bacteriol. 2003 Aug;185(15):4626–4629. doi: 10.1128/JB.185.15.4626-4629.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klein J. L., Brown T. J., French G. L. Rifampin resistance in Mycobacterium kansasii is associated with rpoB mutations. Antimicrob Agents Chemother. 2001 Nov;45(11):3056–3058. doi: 10.1128/AAC.45.11.3056-3058.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lee Grace, Wolff Erika, Miller Jeffrey H. Mutagenicity of the cytidine analog zebularine in Escherichia coli. DNA Repair (Amst) 2004 Feb 3;3(2):155–161. doi: 10.1016/j.dnarep.2003.10.010. [DOI] [PubMed] [Google Scholar]
  17. Moseley B. E., Mattingly A. Repair of irradiation transforming deoxyribonucleic acid in wild type and a radiation-sensitive mutant of Micrococcus radiodurans. J Bacteriol. 1971 Mar;105(3):976–983. doi: 10.1128/jb.105.3.976-983.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nielsen K., Hindersson P., Hoiby N., Bangsborg J. M. Sequencing of the rpoB gene in Legionella pneumophila and characterization of mutations associated with rifampin resistance in the Legionellaceae. Antimicrob Agents Chemother. 2000 Oct;44(10):2679–2683. doi: 10.1128/aac.44.10.2679-2683.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ovchinnikov Y. A., Monastyrskaya G. S., Guriev S. O., Kalinina N. F., Sverdlov E. D., Gragerov A. I., Bass I. A., Kiver I. F., Moiseyeva E. P., Igumnov V. N. RNA polymerase rifampicin resistance mutations in Escherichia coli: sequence changes and dominance. Mol Gen Genet. 1983;190(2):344–348. doi: 10.1007/BF00330662. [DOI] [PubMed] [Google Scholar]
  20. Petersen-Mahrt Svend K., Harris Reuben S., Neuberger Michael S. AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature. 2002 Jul 4;418(6893):99–103. doi: 10.1038/nature00862. [DOI] [PubMed] [Google Scholar]
  21. Rangarajan S., Gudmundsson G., Qiu Z., Foster P. L., Goodman M. F. Escherichia coli DNA polymerase II catalyzes chromosomal and episomal DNA synthesis in vivo. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):946–951. doi: 10.1073/pnas.94.3.946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reynolds M. G. Compensatory evolution in rifampin-resistant Escherichia coli. Genetics. 2000 Dec;156(4):1471–1481. doi: 10.1093/genetics/156.4.1471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Severinov K., Soushko M., Goldfarb A., Nikiforov V. Rifampicin region revisited. New rifampicin-resistant and streptolydigin-resistant mutants in the beta subunit of Escherichia coli RNA polymerase. J Biol Chem. 1993 Jul 15;268(20):14820–14825. [PubMed] [Google Scholar]
  24. Stefanelli P., Fazio C., La Rosa G., Marianelli C., Muscillo M., Mastrantonio P. Rifampicin-resistant meningococci causing invasive disease: detection of point mutations in the rpoB gene and molecular characterization of the strains. J Antimicrob Chemother. 2001 Feb;47(2):219–222. doi: 10.1093/jac/47.2.219. [DOI] [PubMed] [Google Scholar]
  25. Sweet D. M., Moseley B. E. Accurate repair of ultraviolet-induced damage in Micrococcus radiodurans. Mutat Res. 1974 Jun;23(3):311–318. doi: 10.1016/0027-5107(74)90104-3. [DOI] [PubMed] [Google Scholar]
  26. Telenti A., Imboden P., Marchesi F., Lowrie D., Cole S., Colston M. J., Matter L., Schopfer K., Bodmer T. Detection of rifampicin-resistance mutations in Mycobacterium tuberculosis. Lancet. 1993 Mar 13;341(8846):647–650. doi: 10.1016/0140-6736(93)90417-f. [DOI] [PubMed] [Google Scholar]
  27. Tempest P. R., Moseley B. E. Lack of ultraviolet mutagenesis in radiation-resistant bacteria. Mutat Res. 1982 May-Jun;104(4-5):275–280. doi: 10.1016/0165-7992(82)90156-7. [DOI] [PubMed] [Google Scholar]
  28. Udupa K. S., O'Cain P. A., Mattimore V., Battista J. R. Novel ionizing radiation-sensitive mutants of Deinococcus radiodurans. J Bacteriol. 1994 Dec;176(24):7439–7446. doi: 10.1128/jb.176.24.7439-7446.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vogler Amy J., Busch Joseph D., Percy-Fine Stephanie, Tipton-Hunton Christine, Smith Kimothy L., Keim Paul. Molecular analysis of rifampin resistance in Bacillus anthracis and Bacillus cereus. Antimicrob Agents Chemother. 2002 Feb;46(2):511–513. doi: 10.1128/AAC.46.2.511-513.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

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