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Transfer of megaplasmid pKB1 from the rubber-degrading bacterium Gordonia westfalica strain Kb1 to related bacteria and its modification

  • Applied Genetics and Molecular Biotechnology
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Abstract

Because engineering of the 101.016-bp megaplasmid pKB1 of Gordonia westfalica Kb1 failed due to the absence of an effective transfer system, pKB1 was transferred by conjugation from G. westfalica Kb1 to a kanamycin-resistant mutant of Rhodococcus opacus PD630 at a frequency of about 6.2 × 10−8 events per recipient cell. Furthermore, pKB1 was transferred to G. polyisoprenivorans strains VH2 and Y2K and to Mycobacterium smegmatis by electroporation at frequencies of 5.5 × 103, 1.9 × 103, and 8.3 × 102 transformants per microgram plasmid DNA. The pKB1-encoded cadmium resistance gene cadA was used for selection in these experiments. Recombinant pKB1-containing G. polyisoprenivorans VH2 and M. smegmatis were then used to engineer pKB1. A kanamycin resistance cassette was inserted into the pKB1-encoded cadA gene, ligated to suicide plasmid pBBR1MCS-5, and the resulting plasmid was electroporated into plasmid-harboring strains. Homologous recombination between cadA on suicide plasmid and the respective sequence in pKB1 led to its integration into pKB1. Thus, two selection markers were accommodated in pKB1 to monitor plasmid transfer into Gordonia and related taxa for analysis of genes essential for rubber degradation and others. In this study, two transfer methods for large plasmids and strategies for engineering of pKB1 were successfully applied, thereby, extending the tool box for Gordonia.

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References

  • Arenskötter M, Baumeister D, Berekaa MM, Pötter G, Kroppenstedt RM, Linos A, Steinbüchel A (2001) Taxonomic characterization of two rubber-degrading bacteria belonging to the species Gordonia polyisoprenivorans and analysis of hypervariable regions of 16S rDNA sequences. FEMS Microbiol Lett 205:277–282

    Article  PubMed  Google Scholar 

  • Arenskötter M, Baumeister D, Kalscheuer R, Steinbüchel A (2003) Identification and application of plasmids suitable for transfer of foreign DNA to members of the genus Gordonia. Appl Environ Microbiol 69:4971–4974

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Arenskötter M, Bröker D, Steinbüchel A (2004) Biology of the metabolically diverse genus Gordonia. Appl Environ Microbiol 70:3195–3204

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Arenskötter M, Linos A, Schumann P, Kroppenstedt RM, Steinbüchel A (2005) Gordonia nitida Yoon et al. 2001 is a later synonym of Gordonia alkanivorans Kummer et al. 1999. Int J Syst Evol Microbiol 55:695–697

    Article  PubMed  Google Scholar 

  • Banh Q, Arenskötter M, Steinbüchel A (2005) Establishment of Tn5096-based transposon mutagenesis in Gordonia polyisoprenivorans. Appl Environ Microbiol 71:5077–5084

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bröker D, Arenskötter M, Legatzki A, Nies DH, Steinbüchel A (2004) Characterization of the 101.016-kbp megaplasmid pKB1 isolated from the rubber degrading bacterium Gordonia westfalica Kb1. J Bacteriol 186:212–225

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bullock WO, Fernandez JM, Stuart JM (1987) XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. Bio Techniques 5:376–379

    CAS  Google Scholar 

  • Byrd DR, Matson SW (1997) Nicking by transesterification: the reaction catalysed by a relaxase. Mol Microbiol 25:1011–1022

    Article  CAS  PubMed  Google Scholar 

  • Desomer J, Dhaese P, van Montagu M (1988) Conjugative transfer of cadmium resistance plasmids in Rhodococcus fascians strains. J Bacteriol 170:2401–2405

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dogan I, Pagilla KR, Webster DA, Stark BC (2006) Expression of Vitreoscilla hemoglobin in Gordonia amarae enhances biosurfactant production. J Ind Microbiol Biotechnol 33:693–700

    Article  CAS  PubMed  Google Scholar 

  • Friedrich B, Hogrefe C, Schlegel HG (1981) Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus. J Bacteriol 147:198–205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Galamba A, Soetaert K, Wang X-M, De Bruyn J, Jacobs P, Content J (2001) Disruption of adhC reveals a large duplication in the Mycobacterium smegmatis mc2155 genome. Microbiology 147:3281–3294

    Article  CAS  PubMed  Google Scholar 

  • Gilbert SC, Morton J, Buchanan S, Oldfield C, McRoberts A (1998) Isolation of a unique benzothiophene-desulphurizing bacterium, Gordona sp. strain 213E (NCIMB 40816), and characterization of desulphurization pathway. Microbiology (SGM) 144:2545–2553

    Article  CAS  Google Scholar 

  • Grohmann E, Muth G, Espinosa M (2003) Conjugative plasmid transfer in Gram-positive bacteria. Microbiol Mol Biol Rev 67:277–301

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guilhot C, Otal I, van Rompaey I, Martìn C, Gicquel B (1994) Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries. J Bacteriol 176:535–539

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hernandez-Perez G, Fayolle F, Vandecasteele J-P (2001) Biodegradation of ethyl t-butyl ether (ETBE), methyl t-butyl ether (MTBE) and t-amyl methyl ether (TAME) by Gordonia terrae. Appl Microbiol Biotechnol 55:117–121

    Article  CAS  PubMed  Google Scholar 

  • Hinds J, Mahenthiralingam E, Kempsell KE, Duncan K, Stokes RW, Parish T, Stoker NG (1999) Enhanced gene replacement in mycobacteria. Microbiology 145:519–527

    Article  CAS  PubMed  Google Scholar 

  • Husson RN, James BE, Young RA (1990) Gene replacement and expression of foreign DNA in mycobacteria. J Bacteriol 172:519–524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ibrahim EMA, Arenskötter M, Luftmann H, Steinbüchel A (2006) Identification of poly(cis-1,4-isoprene) degradation intermediates during growth of moderately thermophilic Actinomycetes on rubber and cloning of a functional lcp homologue from Nocardia farcinica strain E1. Appl Environ Microbiol 72:3375–3382

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kalscheuer R, Arenskötter M, Steinbüchel A (1999) Establishment of a gene transfer system for Rhodococcus opacus PD630 based on electroporation and its application for recombinant biosynthesis of poly(3-hydroxyalkanoic acids). Appl Microbiol Biotech 52:508–515

    Article  CAS  Google Scholar 

  • Kim SB, Brown R, Oldfield C, Gilbert SC, Goodfellow M (1999) Gordonia desulfuricans sp. nov., a benzothiophene-desulfurizing actinomycete. Int J Syst Bacteriol 49:1845–1851

    Article  CAS  PubMed  Google Scholar 

  • Kim SB, Brown R, Oldfield C, Gilbert SC, Iliarionov S, Goodfellow M (2000) Gordonia amicalis sp. nov., a dibenzothiophene–desulfurizing actinomycete. Int J Syst Bacteriol 50:2031–2036

    Article  CAS  Google Scholar 

  • Kotani T, Yamamoto T, Yurimoto H, Sakai Y, Kato N (2003) Propane monooxygenase and NAD+-dependent secondary alcohol dehydrogenase in propane metabolism by Gordonia sp. strain TY-5. J Bacteriol 185:7120–7128

    CAS  PubMed  Google Scholar 

  • Kotani T, Yurimoto H, Kato N, Sakai Y (2007) Novel acetone metabolism in a propane-utilizing bacterium, Gordonia sp. strain TY-5. J Bacteriol 189:886–893

    Article  CAS  PubMed  Google Scholar 

  • Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM (1995) Four new derivates of the broad host range cloning vector pBBR1MCS, carrying different antibiotic resistance cassettes. Gene 166:175–176

    Article  CAS  PubMed  Google Scholar 

  • Kummer C, Schumann P, Stackebrandt E (1999) Gordonia alkanivorans sp. nov., isolated from tar-contaminated soil. Int J Syst Bacteriol 49:1513–1522

    Article  CAS  PubMed  Google Scholar 

  • Linos A, Steinbüchel A (1998) Microbial degradation of natural and synthetic rubbers by novel bacteria belonging to the genus Gordona. Kautsch Gummi Kunstst 51:496–499

    CAS  Google Scholar 

  • Linos A, Steinbüchel A, Spröer C, Kroppenstedt RM (1999) Gordonia polyisoprenivorans sp. nov., a rubber degrading actinomycete isolated from automobile tire. Int J Syst Bacteriol 49:1785–1791

    Article  CAS  PubMed  Google Scholar 

  • Linos A, Berekaa MM, Steinbüchel A, Kim KK, Spröer C, Kroppenstedt RM (2002) Gordonia westfalica sp. nov., a novel rubber-degrading actinomycete. Int J Syst Evol Microbiol 52:1133–1139

    CAS  PubMed  Google Scholar 

  • Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV (2006) A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct 1:7–32

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mozo T, Hooykaas PJJ (1991) Electroporation of megaplasmids into Agrobacterium. Plant Mol Biol 16:917–918

    Article  CAS  PubMed  Google Scholar 

  • McFadden J (1996) Recombination in mycobacteria. Mol Microbiol 21:2056–211

    Article  Google Scholar 

  • Navarre WW, McClelland M, Libby SJ, Fang FC (2007) Silencing of xenogeneic DNA by H-NS—facilitation of lateral gene transfer in bacteria by a defense system that recognizes foreign DNA. Genes Dev 21:1456–1471

    Article  CAS  PubMed  Google Scholar 

  • Pansegrau W, Lanka E (1996) Enzymology of DNA transfer by conjugative mechanisms. Prog Nucleic Acid Res Mol Biol 54:197–251

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Schlegel HG, Kaltwasser H, Gottschalk G (1961) Ein Submersverfahren zur Kultur wasserstoffoxidierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38:209–222

    Article  CAS  PubMed  Google Scholar 

  • Sheng Y, Mancino V, Birren B (1995) Transformation of Escherichia coli with large DNA molecules by electroporation. Nucleic Acids Res 23:1990–1996

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Snapper SB, Melton RE, Mustafa S, Kieser T, Jacobs WR Jr (1990) Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4:1911–1919

    Article  CAS  PubMed  Google Scholar 

  • Wilson GG, Murray NE (1991) Restriction and modification systems. Annu Rev Genet 25:585–627

    Article  CAS  PubMed  Google Scholar 

  • Yang JC, Lessard PA, Sengupta N, Windsor SD, O’Brien XM, Bramucci M, Tomb J-F, Nagarajan V, Sinskey AJ (2007) TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12. Plasmid 57:55–70

    Article  CAS  PubMed  Google Scholar 

  • Veiga-Crespo P, Feijoo-Siota L, de Miguel T, Poza M, Villa TG (2006) Proposal of a method for the genetic transformation of Gordonia jacobae. J Appl Microbiol 100:608–614

    Article  CAS  PubMed  Google Scholar 

  • Yoon J-H, Lee JJ, Kang SS, Takeuchi M, Shin YK, Lee ST, Kang KH, Park YH (2000) Gordonia nitida sp. nov., a bacterium that degrades 3-ethylpyridine and 3-methylpyridine. Int J Syst Evol Microbiol 50:1203–1210

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgment

Financial support by a grant of the Deutsche Forschungsgemeinschaft (STE 386/10–1) is gratefully acknowledged.

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  1. Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, 48149, Münster, Germany

    Daniel Bröker, Matthias Arenskötter & Alexander Steinbüchel

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  1. Daniel Bröker
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  2. Matthias Arenskötter
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  3. Alexander Steinbüchel
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Correspondence to Alexander Steinbüchel.

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Bröker, D., Arenskötter, M. & Steinbüchel, A. Transfer of megaplasmid pKB1 from the rubber-degrading bacterium Gordonia westfalica strain Kb1 to related bacteria and its modification. Appl Microbiol Biotechnol 77, 1317–1327 (2008). https://doi.org/10.1007/s00253-007-1262-8

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  • DOI: https://doi.org/10.1007/s00253-007-1262-8

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