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. 2013 Nov 10;4(11):854–862. doi: 10.1007/s13238-013-3079-9

Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system

Guodong Luan 1,2, Zhen Cai 1,✉, Fuyu Gong 1,2, Hongjun Dong 1, Zhao Lin 1,2, Yanping Zhang 1, Yin Li 1,✉
PMCID: PMC4875452  PMID: 24214875

Abstract

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution. However, there have not been any similar systems developed for Clostridium, an important bacterial genus. Here we report a novel two-step strategy for developing controllable hypermutable cells of Clostridium acetobutylicum, an important and representative industrial strain. Firstly, the mutS/L operon essential for methyldirected mismatch repair (MMR) activity was inactivated from the genome of C. acetobutylicum to generate hypermutable cells with over 250-fold increased mutation rates. Secondly, a proofreading control system carrying an inducibly expressed mutS/L operon was constructed. The hypermutable cells and the proofreading control system were integrated to form a controllable hypermutable system SMBMutC, of which the mutation rates can be regulated by the concentration of anhydrotetracycline (aTc). Duplication of the miniPthl-tetR module of the proofreading control system further significantly expanded the regulatory space of the mutation rates, demonstrating hypermutable Clostridium cells with controllable mutation rates are generated. The developed C. acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress, indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.

Keywords: Clostridium acetobutylicum, mutation rates, hypermutable cells, artificial control

Contributor Information

Zhen Cai, Email: caiz@im.ac.cn.

Yin Li, Email: yli@im.ac.cn.

References

  1. Abe H, Fujita Y, Takaoka Y, Kurita E, Yano S, Tanaka N, Nakayama K. Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase delta. J Biosci Bioeng. 2009;108:199–204. doi: 10.1016/j.jbiosc.2009.03.019. [DOI] [PubMed] [Google Scholar]
  2. Andreesen JB, Andreesen H, Gottschalk G. Clostridia. 1989. Introduction to the physiology and biochemistry of the genus Clostridium; p. 36. [Google Scholar]
  3. Bao G, Wang R, Zhu Y, Dong H, Mao S, Zhang Y, Chen Z, Li Y, Ma Y. Complete genome sequence of Clostridium acetobutylicum DSM 1731, a solvent-producing strain with multireplicon genome architecture. J Bacteriol. 2011;193:5007–5008. doi: 10.1128/JB.05596-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartlett JG. Narrative review: The new epidemic of Clostridium difficile-associated enteric disease. Ann Intern Med. 2006;145:758–764. doi: 10.7326/0003-4819-145-10-200611210-00008. [DOI] [PubMed] [Google Scholar]
  5. Conrad TM, Joyce AR, Applebee MK, Barrett CL, Xie B, Gao Y, Palsson BO. Genome Biol. 2009. Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolution in lactate minimal media reveals flexible selection of adaptive mutations; p. 10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dong H, Zhang Y, Dai Z, Li Y. Engineering Clostridium strain to accept unmethylated DNA. PLoS One. 2010;5:e9038. doi: 10.1371/journal.pone.0009038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dong HJ, Tao WW, Zhang YP, Li Y. Development of an anhydrotetracycline-inducible gene expression system for solvent-producing Clostridium acetobutylicum: A useful tool for strain engineering. Metab Eng. 2012;14:59–67. doi: 10.1016/j.ymben.2011.10.004. [DOI] [PubMed] [Google Scholar]
  8. Echols H, Goodman MF. Fidelity mechanisms in DNA replication. Annu Rev Biochem. 1991;60:477–511. doi: 10.1146/annurev.bi.60.070191.002401. [DOI] [PubMed] [Google Scholar]
  9. Emlyn-Jones D, Price GD, Andrews TJ. Nitrogenregulated hypermutator strain of Synechococcus sp. for use in vivo artificial evolution. Appl Environ Microbiol. 2003;69:6427–6433. doi: 10.1128/AEM.69.11.6427-6433.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Endo A, Sasaki M, Maruyama A, Kurusu Y. Temperature adaptation of Bacillus subtilis by chromosomal groEL replacement. Biosci Biotechnol Biochem. 2006;70:2357–2362. doi: 10.1271/bbb.50689. [DOI] [PubMed] [Google Scholar]
  11. Gentile CF, Yu SC, Serrano SA, Gerrish PJ, Sniegowski PD. Competition between high- and higher-mutating strains of Escherichia coli. Biol Lett. 2011;7:422–424. doi: 10.1098/rsbl.2010.1036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Greener A, Callahan M, Jerpseth B. An efficient random mutagenesis technique using an E. coli mutator strain. Mol Biotechnol. 1997;7:189–195. doi: 10.1007/BF02761755. [DOI] [PubMed] [Google Scholar]
  13. Hastings PJ, Bull HJ, Klump JR, Rosenberg SM. Adaptive amplification: An inducible chromosomal instability mechanism. Cell. 2000;103:723–731. doi: 10.1016/S0092-8674(00)00176-8. [DOI] [PubMed] [Google Scholar]
  14. Hermann M, Fayolle F, Marchal R, Podvin L, Sebald M, Vandecasteele JP. Isolation and characterization of butanol-resistant mutants of Clostridium acetobutylicum. Appl Environ Microbiol. 1985;50:1238–1243. doi: 10.1128/aem.50.5.1238-1243.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hirsch A, Grinsted E. Methods for the growth and enumeration of anaerobic spore-formers from cheese, with observations on the effect of nisin. J Dairy Res. 1954;21:101–110. doi: 10.1017/S0022029900007196. [DOI] [Google Scholar]
  16. Horst JP, Wu TH, Marinus MG. Escherichia coli mutator genes. Trends Microbiol. 1999;7:29–36. doi: 10.1016/S0966-842X(98)01424-3. [DOI] [PubMed] [Google Scholar]
  17. Ishii K, Matsuda H, Iwasa Y, Sasaki A. Evolutionarily stable mutation rate in a periodically changing environment. Genetics. 1989;121:163–174. doi: 10.1093/genetics/121.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jones DT, Woods DR. Acetone-butanol fermentation revisited. Microbiol Rev. 1986;50:484–524. doi: 10.1128/mr.50.4.484-524.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kennedy CL, Krejany EO, Young LF, O’Connor JR, Awad MM, Boyd RL, Emmins JJ, Lyras D, Rood JI. The alpha-toxin of Clostridium septicum is essential for virulence. Mol Microbiol. 2005;57:1357–1366. doi: 10.1111/j.1365-2958.2005.04774.x. [DOI] [PubMed] [Google Scholar]
  20. Kimura M. On the evolutionary adjustment of spontaneous mutation rates. Genet Res. 1967;9:23–34. doi: 10.1017/S0016672300010284. [DOI] [Google Scholar]
  21. Kondrashov AS. Modifiers of mutation-selection balance: general approach and the evolution of mutation rates. Genet Res. 1995;66:53–69. doi: 10.1017/S001667230003439X. [DOI] [Google Scholar]
  22. Kunkel TA. DNA replication fidelity. J Biol Chem. 2004;279:16895–16898. doi: 10.1074/jbc.R400006200. [DOI] [PubMed] [Google Scholar]
  23. Kunkel TA, Bebenek R. DNA replication fidelity. Annu Rev Biochem. 2000;69:497–529. doi: 10.1146/annurev.biochem.69.1.497. [DOI] [PubMed] [Google Scholar]
  24. LeClerc JE, Li B, Payne WL, Cebula TA. High mutation frequencies among Escherichia coli and Salmonella pathogens. Science. 1996;274:1208–1211. doi: 10.1126/science.274.5290.1208. [DOI] [PubMed] [Google Scholar]
  25. Lewis TA, Goszczynski S, Crawford RL, Korus RA, Admassu W. Products of anaerobic 2,4,6-trinitrotoluene (TNT) transformation by Clostridium bifermentans. Appl Environ Microbiol. 1996;62:4669–4674. doi: 10.1128/aem.62.12.4669-4674.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Liu XB, Gu QY, Yu XB. Repetitive domestication to enhance butanol tolerance and production in Clostridium acetobutylicum through artificial simulation of bio-evolution. Bioresour Technol. 2013;130:638–643. doi: 10.1016/j.biortech.2012.12.121. [DOI] [PubMed] [Google Scholar]
  27. Loh E, Salk JJ, Loeb LA. Optimization of DNA polymerase mutation rates during bacterial evolution. Proc Natl Acad Sci U S A. 2010;107:1154–1159. doi: 10.1073/pnas.0912451107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Matic I, Taddei F, Radman M. Survival versus maintenance of genetic stability: a conflict of priorities during stress. Res Microbiol. 2004;155:337–341. doi: 10.1016/j.resmic.2004.01.010. [DOI] [PubMed] [Google Scholar]
  29. Mermelstein LD, Welker NE, Bennett GN, Papoutsakis ET. Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824. Nat Biotechnol. 1992;10:190–195. doi: 10.1038/nbt0292-190. [DOI] [PubMed] [Google Scholar]
  30. Modrich P, Lahue R. Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu Rev Biochem. 1996;65:101–133. doi: 10.1146/annurev.bi.65.070196.000533. [DOI] [PubMed] [Google Scholar]
  31. Morrison A, Johnson AL, Johnston LH, Sugino A. Pathway correcting DNA replication errors in Saccharomyces cerevisiae. EMBO J. 1993;12:1467–1473. doi: 10.1002/j.1460-2075.1993.tb05790.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Perron GG, Hall AR, Buckling A. Hypermutability and compensatory adaptation in antibiotic-resistant bacteria. Am Nat. 2010;176:303–311. doi: 10.1086/655217. [DOI] [PubMed] [Google Scholar]
  33. Perutka J, Wang W, Goerlitz D, Lambowitz AM. Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. J Mol Biol. 2004;336:421–439. doi: 10.1016/j.jmb.2003.12.009. [DOI] [PubMed] [Google Scholar]
  34. Rehner SA, Samuels GJ. Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycol Res. 1994;98:625–634. doi: 10.1016/S0953-7562(09)80409-7. [DOI] [Google Scholar]
  35. Sakaguchi G. Clostridium-Botulinum toxins. Pharmacol Therapeut. 1982;19:165–194. doi: 10.1016/0163-7258(82)90061-4. [DOI] [PubMed] [Google Scholar]
  36. Sambrook J, Russell DW. Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press; 2001. [Google Scholar]
  37. Sasaki M, Yonemura Y, Kurusu Y. Genetic analysis of Bacillus subtilis mutator genes. J Gen Appl Microbiol. 2000;46:183–187. doi: 10.2323/jgam.46.183. [DOI] [PubMed] [Google Scholar]
  38. Selifonova O, Valle F, Schellenberger V. Rapid evolution of novel traits in microorganisms. Appl Environ Microbiol. 2001;67:3645–3649. doi: 10.1128/AEM.67.8.3645-3649.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shao L, Hu S, Yang Y, Gu Y, Chen J, Yang Y, Jiang W, Yang S. Targeted gene disruption by use of a group II intron (targetron) vector in Clostridium acetobutylicum. Cell Res. 2007;17:963–965. doi: 10.1038/cr.2007.91. [DOI] [PubMed] [Google Scholar]
  40. Shaver AC, Sniegowski PD. Spontaneously arising mutL mutators in evolving Escherichia coli populations are the result of changes in repeat length. J Bacteriol. 2003;185:6076–6082. doi: 10.1128/JB.185.20.6076-6082.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shimoda C, Itadani A, Sugino A, Furusawa M. Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae. Genes Genet Syst. 2006;81:391–397. doi: 10.1266/ggs.81.391. [DOI] [PubMed] [Google Scholar]
  42. Sniegowski PD, Gerrish PJ, Lenski RE. Evolution of high mutation rates in experimental populations of E. coli. Nature. 1997;387:703–705. doi: 10.1038/42701. [DOI] [PubMed] [Google Scholar]
  43. Stephanopoulos G. Metabolic engineering by genome shuffling — Two reports on whole-genome shuffling demonstrate the application of combinatorial methods for phenotypic improvement in bacteria. Nat Biotechnol. 2002;20:666–668. doi: 10.1038/nbt0702-666. [DOI] [PubMed] [Google Scholar]
  44. Taddei F, Radman M, MaynardSmith J, Toupance B, Gouyon PH, Godelle B. Role of mutator alleles in adaptive evolution. Nature. 1997;387:700–702. doi: 10.1038/42696. [DOI] [PubMed] [Google Scholar]
  45. Tanabe K, Kondo T, Onodera Y, Furusawa M. A conspicuous adaptability to antibiotics in the Escherichia coli mutator strain, dnaQ49. Fems Microbiol Lett. 1999;176:191–196. doi: 10.1111/j.1574-6968.1999.tb13661.x. [DOI] [PubMed] [Google Scholar]
  46. Tenaillon O, Toupance B, Le Nagard H, Taddei F, Godelle B. Mutators, population size, adaptive landscape and the adaptation of asexual populations of bacteria. Genetics. 1999;152:485–493. doi: 10.1093/genetics/152.2.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yang HJ, Wolff E, Kim M, Diep A, Miller JH. Identification of mutator genes and mutational pathways in Escherichia coli using a multicopy cloning approach. Mol Microbiol. 2004;53:283–295. doi: 10.1111/j.1365-2958.2004.04125.x. [DOI] [PubMed] [Google Scholar]

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