Isolation and sequence analysis of a gene from the linear DNA plasmid pPacl-2 of Pichia acaciae that shows similarity to a killer toxin gene of Kluyveromyces lactis.

The toxin-encoding linear plasmid systems found in Pichia acaciae and Kluyveromyces lactis yeasts appear to be quite similar, both in function and structural organization. By Southern hybridization, a linear plasmid of P. acaciae, pPacl-2, was found to hybridize to the second open reading frame (ORF2) of K. lactis plasmid pGKL1, known to encode the alpha and beta subunits of the K. lactis toxin. A 1.7 kbp segment of pPacl-2 DNA was cloned, sequenced and shown to contain four regions of strong homology to four similarly oriented regions of K. lactis ORF2. This 1.7 kbp fragment also contained an ORF of 1473 bp that could encode a protein of approximately 55.8 kDa. Like the alpha subunit gene of K. lactis ORF2, a very hydrophobic region occurs at the N-terminus, perhaps representing a signal sequence for transport out of the cell. Unlike K. lactis ORF2, however, the encoded polypeptide is much smaller and lacks a recognizable domain common to chitinases. The structure of a toxin that includes the translation product of this P. acaciae ORF would likely be quite different from that of the K. lactis toxin. Analysis of the upstream region of the P. acaciae ORF revealed an upstream conserved sequence identical to that found before ORFs 8 and 9 of pGKL2. A possible hairpin loop structure, as has been described for each of the four K. lactis pGKL1 ORFs, was found just upstream of the presumed start codon. The similarity of the promoter-like elements found in the linear plasmid genes of these diverse yeasts reinforces the idea of the existence of a unique, but highly conserved, expression system for these novel plasmids. The sequence has been deposited in the GenBank data library under Accession Number U02596.