Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 31

The determinants of the killer factor are both cytoplasmic and nuclear. In S. cerevisiae, the killer phenomenon is associated with the presence of double‐stranded RNA particles, called virus‐like particles (VLP), in the cytoplasm. They are in the same category as noninfectious mycoviruses. There are two kinds of VLP: M and L. The M genome (1.3–1.9 kb) codes for the toxin (K) and for the immunity factor (R). The L genome (4.5 kb) codes for an RNA polymerase and the protein capsid that encapsulates the two genomes. Killer strains (K⁺R⁺) secrete the toxin and are immune to it. The sensitive cells (K⁻R⁻) do not possess M VLP, but most of them have L VLP. The two types of viral particles are necessary for the yeast cell to express the killer phenotype (K⁺R⁺), since the L mycovirus is necessary for the maintenance of the M type.

FIGURE 1.17 Identification of the K2 killer phenotype in S. cerevisiae. The presence of a halo around the two streaks of the killer strain is due to the death of the sensitive strain cultivated in the medium.

There are four types of killer activity in S. cerevisiae strains, corresponding to K1, K2, K28, and Klus toxins. They are, respectively, coded by M1, M2, M28, and Mlus VLPs with sizes of 1.8, 1.7, 2.1, and 2.3 kb. The K2 activity group is by far the most widespread in the S. cerevisiae strains encountered in wine (Rodríguez‐Cousiño et al., 2011). Neutral strains (K⁻R⁺) are insensitive to a given toxin without being capable of producing it. They possess M VLPs of normal size that code only for the immunity factor, but they either do not produce toxins or are inactive because of mutations affecting the M‐type RNA.

Many chromosomic genes are involved in the maintenance and replication of L and M RNA particles as well as in the maturation and transport of the toxin produced.

The K1 toxin is a small protein made up of two subunits (9 and 9.5 kDa); it is active and stable in a very narrow pH range (4.2–4.6). It is therefore inactive in grape must. The K2 toxin, a 16 kDa glycoprotein produced by homothallic strains of S. cerevisiae encountered in wine, is active between pH 2.8 and 4.8 with a maximum activity between 4.2 and 4.4. It is therefore active at the pH of grape must and wine.

K1 and K2 toxins attack sensitive cells by attaching themselves to a receptor located in the cell wall—a β‐1,6‐glucan. Two chromosomal genes, KRE1 and KRE2 (killer resistant), determine the possibility of this linkage. The KRE1 gene produces a cell wall glycoprotein that has β‐1,6‐glucan synthase activity. KRE1 mutants are resistant to K1 and K2 toxins because they are deficient in this enzyme and devoid of a β‐1,6‐glucan receptor. The KRE2 gene is also involved in the fixation of toxins to the cell wall receptor; KRE2 mutants are also resistant. The toxin linked to a glucan receptor is then transferred to a membrane receptor site by a mechanism requiring energy. Cells in the log phase are therefore more sensitive to the killer effect than cells in the stationary phase. When the plasma membrane of the sensitive cell is exposed to the toxin, it manifests serious functional alterations after a lag phase of about 40 minutes. Serious functional alterations include the interruption of the coupled transport of amino acids and protons, the acidification of the cell contents, and potassium and ATP leakage. The cell dies in two to three hours after contact with the toxin because of the above damage, due to the formation of pores in the plasma membrane.

The killer effect exerts itself exclusively on yeasts, and the pharmacological tests reported on killer toxins are negative.