Skip to main content
SpringerLink
Log in

Structural and functional analysis of the killer element pPin1-3 from Pichia inositovora

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

Abstract

Strains of the yeast Pichia inositovora that carry the linear plasmids pPin1-1 (18 kb) and pPin1-3 (10 kb) display a killer activity towards Saccharomyces cerevisiae. Cloning and sequencing of the smaller plasmid, pPin1-3, revealed that it is 9683 bp long and has 154-bp terminal inverted repeats. Comparison of pPin1-3 with the only other completely sequenced killer plasmid, pGKL1 of Kluyveromyces lactis, revealed differences in genome organization. The Pichia element has four ORFs that account for 95% of the sequence. ORF1 is homologous to the putative immunity gene of the K. lactis system. A viral B-type DNA polymerase is encoded by ORF2. The predicted product of ORF3 displays similarities to the α- and β-subunits of the heterotrimeric K. lactis killer toxin, also known as zymocin. A cysteine-rich chitin-binding site and a chitinase signature, characteristic for the α-subunit of zymocin were identified in Orf3p. Chitin affinity chromatography and Western analysis confirmed the plasmid specific expression and secretion of a protein that cross-reacts with an antibody raised against the α-subunit of K. lactis zymocin. Disruption of the major chitin synthase-gene ( CHS3) renders S. cerevisiae resistant to the toxin, providing further evidence that chitin is the cellular receptor for the P. inositovora toxin. Orf4p of pPin1-3 displays only weak similarities to the γ-subunit of zymocin, which causes a G1 cell-cycle arrest in S. cerevisiae. However, disruption of the S. cerevisiae gene ELP3/TOT3, which encodes a histone-acetyltransferase that is essential for zymocin action, resulted in reduced sensitivity to the P. inositovora toxin also. Thus, despite obvious differences in genome organization and protein architecture, both killer systems very probably have similar modes of action.

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. 1A, B.
Fig. 2.
Fig. 3.
Fig. 4A, B.
Fig. 5.
Fig. 6A, B.

Similar content being viewed by others

References

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    PubMed  Google Scholar 

  • Butler AR, Porter M, Stark MJR (1991) Intracellular expression of Kluyveromyces lactis toxin γ subunit mimics treatment with exogenous toxin and distinguishes two classes of toxin-resistant mutant. Yeast 7:617–625

    CAS  PubMed  Google Scholar 

  • Frohloff F, Fichtner L, Jablonowski D, Breuning KD, Schaffrath R (2001) Saccharomyces cerevisiae elongator mutations confer resistance to the Kluyveromyces lactis zymocin. EMBO J 20:1993–2003

    CAS  PubMed  Google Scholar 

  • Fukuda K, Maebuchi M, Takata H, Gunge N (1997) The linear plasmid pDHL1 from Debaryomyces hansenii encodes a protein highly homologous to the pGKL1-plasmid DNA polymerase. Yeast 13:613–620

    Article  CAS  PubMed  Google Scholar 

  • Fukuhara H (1995) Linear DNA plasmids of yeasts. FEMS Microbiol Lett 131:1–9

    Article  CAS  PubMed  Google Scholar 

  • Gietz RD, Schiestl RH (1995) Transforming yeast with DNA. Methods Mol Cell Biol 5:255–269

    Google Scholar 

  • Gunge N (1995) Plasmid DNA and the killer phenomenon in Kluyveromyces. In: Kück U (ed) Genetics and biotechnology (The Mycota, vol 2). Springer-Verlag, Berlin, pp 189–209

  • Gunge N, Tamaru A, Ozawa F, Sakaguchi K (1981) Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character. J Bacteriol 145:382–390

    CAS  PubMed  Google Scholar 

  • Gunge N, Takahashi S, Fukuda K, Ohnishi T, Meinhardt F (1994) UV hypersensitivity of yeast linear plasmids. Curr Genet 26:369–373

    CAS  PubMed  Google Scholar 

  • Hayman GT, Bolen BL (1991) Linear DNA plasmids of Pichia inositovora are associated with a novel killer toxin activity. Curr Genet 19:389–393

    CAS  PubMed  Google Scholar 

  • Hishinuma F, Nakamura K, Hirai K, Nishizawa R, Gunge N, Maeda T (1984) Cloning and nucleotide sequence of the DNA killer plasmids from yeast. Nucleic Acids Res 12:7581–7597

    CAS  PubMed  Google Scholar 

  • Jablonowski D, Schaffrath R (2002) Saccharomyces cerevisiae RNA polymerase II is affected by Kluyveromyces lactis zymocin. J Biol Chem 277:26276–80

    Article  CAS  PubMed  Google Scholar 

  • Jablonowski D, Fichtner L, Martin VJ, Klassen R, Meinhardt F, Stark MJR, Schaffrath R (2001a) Saccharomyces cerevisiae cell wall chitin, the potential Kluyveromyces lactis zymocin receptor. Yeast 18:1285–1299

    CAS  PubMed  Google Scholar 

  • Jablonowski D, Frohloff F, Fichtner L, Stark MJR, Schaffrath R (2001b) Kluyveromyces lactis zymocin mode of action is linked to RNA polymerase II function via elongator. Mol Microbiol 42:1095–1105

    CAS  PubMed  Google Scholar 

  • Kaiser C, Michaelis S, Mitchell A (1994) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

  • Kämper J, Esser K, Gunge N, Meinhardt F (1991) Heterologous gene expression on the linear DNA killer plasmid from Kluyveromyces lactis. Curr Genet 19:109–118

    PubMed  Google Scholar 

  • Kishida M, Tokunaga M, Katayose Y, Yajima H, Kawamura-Watabe A, Hishinuma F (1996) Isolation and genetic characterization of pGKL killer-insensitive mutants ( iki) from Saccharomyces cerevisiae. Biosci Biotechnol Biochem 60:798–801

    CAS  PubMed  Google Scholar 

  • Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132

    CAS  PubMed  Google Scholar 

  • Klassen R, Meinhardt F (2002) Linear plasmids pWR1A and pWR1B of the yeast Wingea robertsiae are associated with a killer phenotype. Plasmid 48:142–148

    Article  CAS  PubMed  Google Scholar 

  • Klassen R, Tontsidou L, Larsen M, Meinhardt F (2001) Genome organization of the linear cytoplasmic element pPE1B from Pichia etchellsii. Yeast 18:953–961

    Article  CAS  PubMed  Google Scholar 

  • Klassen R, Jablonowski D, Schaffrath R, Meinhardt F (2002) Genome organization of the linear Pichia etchellsii plasmid pPE1A: evidence for expression of an extracellular chitin binding protein homologous to the α-subunit of the Kluyveromyces lactis killer toxin. Plasmid 47:224–233

    Article  CAS  PubMed  Google Scholar 

  • Knopf CW (1998) Evolution of viral DNA-dependent DNA polymerases. Virus Genes 16:47–58

    Article  CAS  PubMed  Google Scholar 

  • Larsen M, Meinhardt F (2000) Kluyveromyces lactis killer system: identification of a new gene encoded by pGKL2. Curr Genet 38:271–275

    Article  CAS  PubMed  Google Scholar 

  • Larsen M, Gunge N, Meinhardt F (1998) Kluyveromyces lactis killer plasmid pGKL2: evidence for a viral like capping enzyme encoded by ORF3. Plasmid 40:243–246

    Article  CAS  PubMed  Google Scholar 

  • Ligon JM, Bolen PL, Hill DS, Bothast RJ, Kurtzman CP (1989) Physical and biological characterization of linear DNA plasmids of the yeast Pichia inositovora. Plasmid 21:185–194

    CAS  PubMed  Google Scholar 

  • Meinhardt F, Schaffrath R (2001) Extranuclear inheritance: cytoplasmic linear double-stranded DNA killer elements of the dairy yeast Kluyveromyces lactis. Prog Bot 62:51–70

    CAS  Google Scholar 

  • Romanos M, Boyd A (1988) A transcriptional barrier to expression of cloned toxin genes of the linear plasmid k1 of Kluyveromyces lactis: evidence that native k1 has novel promoters. Nucleic Acids Res 16:7333–7350

    CAS  PubMed  Google Scholar 

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

    Google Scholar 

  • Schaffrath R, Stark MJR, Gunge N, Meinhardt F (1992) Kluyveromyces lactis killer system: ORF1 of pGKL2 has no function in immunity expression and is dispensable for killer plasmid replication and maintenance. Curr Genet 21:357–363

    CAS  PubMed  Google Scholar 

  • Schickel J, Helmig C, Meinhardt F (1996) Kluyveromyces lactis killer system. Analysis of cytoplasmic promoters of linear plasmids. Nucleic Acids Res 24:1879–1886

    Article  CAS  PubMed  Google Scholar 

  • Stark MJR, Boyd A (1986) The killer toxin of Kluyveromyces lactis: characterization of the toxin subunits and identification of the genes which encode them. EMBO J 5:1995–2002

    CAS  PubMed  Google Scholar 

  • Stark MJR, Boyd A, Mileham AJ, Romanos MA (1990) The plasmid encoded killer system of Kluyveromyces lactis: a review. Yeast 6:1–29

    CAS  Google Scholar 

  • Tiggemann M, Jeske S, Larsen M, Meinhardt F (2001) Kluyveromyces lactis cytoplasmic plasmid pGKL2: Heterologous expression of Orf3p and prove of guanylyltransferase and mRNA-triphosphatase activities. Yeast 18:815–825

    Article  CAS  PubMed  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–80

    PubMed  Google Scholar 

  • Tokunaga M, Wada N, Hishinuma F (1987) Expression and identification of immunity determinants on linear DNA killer plasmids pGKL1 and pGKL2 in Kluyveromyces lactis. Nucleic Acids Res 15:1031–46

    CAS  PubMed  Google Scholar 

  • Tokunaga M, Kawamura A., Hishinuma F (1989) Expression of pGKL killer 28 K subunit in Saccharomyces cerevisiae: identification of 28 K subunit as a killer protein. Nucleic Acids Res 17:3435–3446

    CAS  PubMed  Google Scholar 

  • Tommasino M, Ricci S, Galeotti C (1988) Genome organization of the killer plasmid pGKL2 from Kluyveromyces lactis. Nucleic Acids Res 16:5863–5978

    CAS  PubMed  Google Scholar 

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    CAS  PubMed  Google Scholar 

  • Wach A, Brachat A, Pohlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808

    CAS  PubMed  Google Scholar 

  • Wilson DW, Meacock PA (1988) Extranuclear gene expression in yeast: evidence for a plasmid encoded RNA-polymerase of unique structure. Nucleic Acids Res 16:8097–8112

    CAS  PubMed  Google Scholar 

  • Worsham PL, Bolen PL (1990) Killer toxin production in Pichia acaciae is associated with linear DNA plasmids. Curr Genet 18:77–80

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Thanks are due to Dr. M. J. R. Stark (University of Dundee, UK) and Dr. P. Philippsen (University of Basel, Switzerland) for providing antibodies, strain GS1731 and plasmid pFA6a

Author information

Authors and Affiliations

  1. Institut für Mikrobielle Molekularbiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstr. 3, 48149, Münster, Germany

    R. Klassen & F. Meinhardt

Authors
  1. R. Klassen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. F. Meinhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to F. Meinhardt.

Additional information

Communicated by C. P. Hollenberg

Rights and permissions

Reprints and permissions

About this article

Cite this article

Klassen, R., Meinhardt, F. Structural and functional analysis of the killer element pPin1-3 from Pichia inositovora . Mol Genet Genomics 270, 190–199 (2003). https://doi.org/10.1007/s00438-003-0920-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-003-0920-5

Keywords

Search

Navigation