The Studies On The Cold-active Killer Toxin From The Psychrotolerant Yeast Mrakia Frigida

Killer toxin is the exotoxin that many yeasts will produce to kill other yeasts.The killer strains themselves are immune to their own toxin, but remain susceptible tothe toxins secreted by other killer yeasts.The psychrotolerant yeast Mrakia frigida2E00797isolated from sea sediment inAntarctica was found to be able to produce killer toxin against the pathogenic yeast(Metschnikowia bicuspidata WCY) in crab. When the psychrotolerant yeast wasgrown in the medium with pH4.5and3.0%(wt/vol) NaCl and at15°C, it couldproduce the highest amount of killer toxin against the pathogenic yeast M. bicuspidataWCY. The crude killer toxin activity against the pathogenic yeast M. bicuspidataWCY was the highest when it grew at15°C in the assay medium with3.0%(wt/vol)NaCl and pH4.5. At temperatures higher than25°C, the killing activity produced byM. frigida2E00797was completely lost. The killer toxin produced by M. frigida2E00797could kill only M. bicuspidata WCY, Candida tropicalis and Candidaalbicans among all the fungal species and bacterial species tested in this study.The killer toxin was purified and characterized. The molecular weight of thepurified killer toxin was estimated to be55.6kDa and the purified killer toxin shared35.1%sequence homology with a protein kinase. The purified killer toxin’s optimaltemperature and pH for killing activity were15°C and4.5, respectively, and it wasstable in the temperature range from10to25°C at pH4.5. The toxin’s highest killingactivity was observed in the presence of3.0g/100ml NaCl. The purified killer toxinwas able to actively kill whole cells of M. bicuspidata but could not kill the protoplastof the sensitive yeast. Of the twelve yeast species tested in this study, the killer toxinwas able to kill C. tropicalis and C. albicans in addition to M. bicuspidata.After the whole sensitive cells were treated by the killer toxin, the invaginations ofthe cell wall occurred, the cell walls were destroyed, and integrity of the cell walldisappeared. The treated cells also can be stained by PI. However, the protoplasts of the cells can not be affected by the killer toxin. This again confirmed that the killertoxin produced by M. frigida2E00797acted on the cell wall of the sensitive yeastcells, causing defective integrity of the cell wall, damage of cell membrane and celldeath.