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

Abstract

Pichia etchellsii CBS2011 (synonym Debaryomyces etchellsii) is a non-killer yeast harbouring two cryptic linear cytoplasmic DNA-elements, pPE1A (6.7 kb) and pPE1B (12.8 kb). Cloning and complete sequencing of pPE1A revealed a 6749-bp element with a remarkably high A + T content of 77.6%. The termini of pPE1A were found to consist of inversely orientated identical nucleotide repetitions of 178 bp, to which proteins are linked at the 5 ′-ends. It is only the second small, non-autonomous cytoplasmic yeast linear plasmid for which the complete nucleotide sequence is known. Five open reading frames (ORFs) were identified preceded by upstream conserved sequence motifs (UCS) characteristic for cytoplasmic promoters and perfectly matching the UCS consensus (ATNTGA). As none of the putative genes encodes a DNA-polymerase, pPE1A is the first yeast linear plasmid known that does not possess its own element-specific replication machinery. No function could be attributed to ORF1, 3, 4, and 5; the predicted ORF2 gene product is similar to chitin-binding proteins and chitinases, highest homologies were found to the precursor of the α- and β-subunits of the secreted Kluyveromyces lactis zymocin. Consistently, the Orf2p could be isolated from the culture fluid by chitin–Sepharose affinity chromatography and characterized by immuno-probing with an antibody specific for the K. lactis killer toxin α-subunit. Production of the protein was found to be plasmid-dependent. The sequence of pPE1A has been submitted to the EMBL data library, Accession No. AJ409097.