Skip to main content
SpringerLink
Log in

Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus

  • Methods Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

Markerless gene-disruption technology is particularly useful for effective genetic analyses of Thermus thermophilus (T. thermophilus), which have a limited number of selectable markers. In an attempt to develop a novel system for the markerless disruption of genes in T. thermophilus, we applied a Cre/lox system to construct a triple gene disruptant. To achieve this, we constructed two genetic tools, a loxP–htk–loxP cassette and cre-expressing plasmid, pSH-Cre, for gene disruption and removal of the selectable marker by Cre-mediated recombination. We found that the Cre/lox system was compatible with the proliferation of the T. thermophilus HB27 strain at the lowest growth temperature (50 °C), and thus succeeded in establishing a triple gene disruptant, the (∆TTC1454::loxP, ∆TTC1535KpnI::loxP, ∆TTC1576::loxP) strain, without leaving behind a selectable marker. During the process of the sequential disruption of multiple genes, we observed the undesired deletion and inversion of the chromosomal region between multiple loxP sites that were induced by Cre-mediated recombination. Therefore, we examined the effects of a lox66–htk–lox71 cassette by exploiting the mutant lox sites, lox66 and lox71, instead of native loxP sites. We successfully constructed a (∆TTC1535::lox72, ∆TTC1537::lox72) double gene disruptant without inducing the undesired deletion of the 0.7-kbp region between the two directly oriented lox72 sites created by the Cre-mediated recombination of the lox66–htk–lox71 cassette. This is the first demonstration of a Cre/lox system being applicable to extreme thermophiles in a genetic manipulation. Our results indicate that this system is a powerful tool for multiple markerless gene disruption in T. thermophilus.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J 7:649–659

    Article  CAS  PubMed  Google Scholar 

  • Angelov A, Li H, Geissler A, Leis B, Liebl W (2013) Toxicity of indoxyl derivative accumulation in bacteria and its use as a new counterselection principle. Syst Appl Microbiol 36:585–592

    Article  CAS  PubMed  Google Scholar 

  • Aoki K, Itoh T (2007) Characterization of the ColE2-like replicon of plasmid pTT8 from Thermus thermophilus. Biochem Biophys Res Commun 353:1028–1033

    Article  CAS  PubMed  Google Scholar 

  • Averhoff B (2006) Genetic systems for Thermus. Methods Microbiol 35:279–308

    Article  CAS  Google Scholar 

  • Blas-Galindo E, Cava F, López-Viñas E, Mendieta J, Berenguer J (2007) Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus. Appl Environ Microbiol 73:5138–5145

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brouns SJJ, Wu H, Akerboom J, Turnbull AP, de Vos WM, van der Oost J (2005) Engineering a selectable marker for hyperthermophiles. J Biol Chem 280:11422–11431

    Article  CAS  PubMed  Google Scholar 

  • Buchholz F, Ringrose L, Angrand PO, Rossi F, Stewart AF (1996) Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination. Nucleic Acids Res 24:4256–4262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Buchholz F, Angrand PO, Stewart AF (1998) Improved properties of FLP recombinase evolved by cycling mutagenesis. Nat Biotechnol 16:657–662

    Article  CAS  PubMed  Google Scholar 

  • Carr JF, Danziger ME, Huang AL, Dahlberg AE, Gregory ST (2015) Engineering the genome of Thermus thermophilus using a counterselectable marker. J Bacteriol 197:1135–1144

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Coppoolse ER, de Vroomen MJ, Roelofs D, Smit J, van Gennip F, Hersmus BJM, Nijkamp HJJ, van Haaren MJJ (2003) Cre recombinase expression can result in phenotypic aberrations in plants. Plant Mol Biol 51:263–279

    Article  CAS  PubMed  Google Scholar 

  • Faraldo MM, de Pedro MA, Berenguer J (1992) Sequence of the S-layer gene of Thermus thermophilus HB8 and functionality of its promoter in Escherichia coli. J Bacteriol 174:7458–7462

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fujita A, Sato T, Koyama Y, Misumi Y (2015) A reporter gene system for the precise measurement of promoter activity in Thermus thermophilus HB27. Extremophiles 19:1193–1201

    Article  CAS  PubMed  Google Scholar 

  • Henne A, Brüggemann H, Raasch C, Wiezer A, Hartsch T, Liesegang H, Johann A, Lienard T, Gohl O, Martinez-Arias R, Jacobi C, Starkuviene V, Schlenczeck S, Dencker S, Huber R, Klenk HP, Kramer W, Merkl R, Gottschalk G, Fritz HJ (2004) The genome sequence of the extreme thermophile Thermus thermophilus. Nat Biotechnol 22:547–553

    Article  CAS  PubMed  Google Scholar 

  • Hiratsu K, Shiotani S, Makino K, Nunoshiba T (2013) Construction of a supF-based system for detection of mutations in the chromosomal DNA of Arabidopsis. Mol Genet Genom 288:707–715

    Article  CAS  Google Scholar 

  • Hoess RH, Abremski K (1985) Mechanism of strand cleavage and exchange in the Cre-lox site-specific recombination system. J Mol Biol 181:351–362

    Article  CAS  PubMed  Google Scholar 

  • Hoseki J, Yano T, Koyama Y, Kuramitsu S, Kagamiyama H (1999) Directed evolution of thermostable kanamycin-resistance gene: a convenient selection marker for Thermus thermophilus. J Biochem 126:951–956

    Article  CAS  PubMed  Google Scholar 

  • Kovács ÁT, van Hartskamp M, Kuipers OP, van Kranenburg R (2010) Genetic tool development for a new host for biotechnology, the thermotolerant bacterium Bacillus coagulans. Appl Environ Microbiol 76:4085–4088

    Article  PubMed  PubMed Central  Google Scholar 

  • Koyama Y, Hoshino T, Tomizuka N, Furukawa K (1986) Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp. J Bacteriol 166:338–340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lambert JM, Bongers RS, Kleerebezem M (2007) Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum. Appl Environ Microbiol 73:1126–1135

    Article  CAS  PubMed  Google Scholar 

  • Leibig M, Krismer B, Kolb M, Friede A, Götz F, Bertram R (2008) Marker removal in staphylococci via Cre recombinase and different lox sites. Appl Environ Microbiol 74:1316–1323

    Article  CAS  PubMed  Google Scholar 

  • Leis B, Angelov A, Li H, Liebl W (2014) Genetic analysis of lipolytic activities in Thermus thermophilus HB27. J Biotechnol 191:150–157

    Article  CAS  PubMed  Google Scholar 

  • Nagy A (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26:99–109

    Article  CAS  PubMed  Google Scholar 

  • Nakamura A, Takakura Y, Kobayashi H, Hoshino T (2005) In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus. J Biosci Bioeng 100:158–163

    Article  CAS  PubMed  Google Scholar 

  • Nakane S, Nakagawa N, Kuramitsu S, Masui R (2012) The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair. DNA Repair (Amst) 11:906–914

    Article  CAS  Google Scholar 

  • Ohta T, Tokishita S, Imazuka R, Mori I, Okamura J, Yamagata H (2006) β-Glucosidase as a reporter for the gene expression studies in Thermus thermophilus and constitutive expression of DNA repair genes. Mutagenesis 21:255–260

    Article  CAS  PubMed  Google Scholar 

  • Ohtani N, Tomita M, Itaya M (2010) An extreme thermophile, Thermus thermophilus, is a polyploid bacterium. J Bacteriol 192:5499–5505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Oshima T, Imahori K (1974) Description of Thermus thermophilus (Yoshida and Oshima) comb. nov., a nonsporulating thermophilic bacterium from a Japanese thermal spa. Int J Syst Bacteriol 24:102–112

    Article  CAS  Google Scholar 

  • Sakai T, Tokishita S, Mochizuki K, Motomiya A, Yamagata H, Ohta T (2008) Mutagenesis of uracil-DNA glycosylase deficient mutants of the extremely thermophilic eubacterium Thermus thermophilus. DNA Repair (Amst) 7:663–669

    Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Schmidt EE, Taylor DS, Prigge JR, Barnett S, Capecchi MR (2000) Illegitimate Cre-dependent chromosome rearrangements in transgenic mouse spermatids. Proc Natl Acad Sci USA 97:13702–13707

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Short JM, Fernandez JM, Sorge JA, Huse WD (1988) λ ZAP: a bacteriophage λ expression vector with in vivo excision properties. Nucleic Acids Res 16:7583–7600

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sternberg N, Hamilton D (1981) Bacteriophage P1 site-specific recombination: I. Recombination between loxP sites. J Mol Biol 150:467–486

    Article  CAS  PubMed  Google Scholar 

  • Suzuki N, Nonaka H, Tsuge Y, Inui M, Yukawa H (2005) New multiple-deletion method for the Corynebacterium glutamicum genome, using a mutant lox sequence. Appl Environ Microbiol 71:8472–8480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tamakoshi M, Yaoi T, Oshima T, Yamagishi A (1999) An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus. FEMS Microbiol Lett 173:431–437

    Article  CAS  PubMed  Google Scholar 

  • Wang L, Hoffmann J, Watzlawick H, Altenbuchner J (2016) Markerless gene deletion with cytosine deaminase in Thermus thermophilus strain HB27. Appl Environ Microbiol 82:1249–1255

    Article  CAS  PubMed Central  Google Scholar 

  • Yan X, Yu HJ, Hong Q, Li SP (2008) Cre/lox system and PCR-based genome engineering in Bacillus subtilis. Appl Environ Microbiol 74:5556–5562

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119

    Article  CAS  PubMed  Google Scholar 

  • Yokoyama S, Hirota H, Kigawa T, Yabuki T, Shirouzu M, Terada T, Ito Y, Matsuo Y, Kuroda Y, Nishimura Y, Kyogoku Y, Miki K, Masui R, Kuramitsu S (2000) Structural genomics projects in Japan. Nat Struct Mol Biol 7:943–945

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Prof. Akira Nakamura (University of Tsukuba) for kindly providing the pT8H5-Pslp plasmid. We also thank Prof. Toshihiro Ohta (Tokyo University of Pharmacy and Life Science) for kindly providing the pTAP60 plasmid and wild-type T. thermophilus HB27 strain.

Author information

Authors and Affiliations

  1. Department of Applied Chemistry, National Defense Academy, Hashirimizu 1-10-20, Yokosuka, Kanagawa, 239-8686, Japan

    Yoichiro Togawa & Keiichiro Hiratsu

  2. College of Liberal Arts, International Christian University, Osawa 3-10-2, Mitaka, Tokyo, 181-8585, Japan

    Tatsuo Nunoshiba

Authors
  1. Yoichiro Togawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tatsuo Nunoshiba
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Keiichiro Hiratsu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Keiichiro Hiratsu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

Communicated by S. Hohmann.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 1296 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Togawa, Y., Nunoshiba, T. & Hiratsu, K. Cre/lox-based multiple markerless gene disruption in the genome of the extreme thermophile Thermus thermophilus . Mol Genet Genomics 293, 277–291 (2018). https://doi.org/10.1007/s00438-017-1361-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-017-1361-x

Keywords

Search

Navigation