Genome Features of a New Double-Stranded RNA Helper Virus (LBCbarr) from Wine Torulaspora delbrueckii Killer Strains

The killer phenotype of Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) is encoded in the genome of medium-size dsRNA viruses (V-M). Killer strains also contain a helper large size (4.6 kb) dsRNA virus (V-LA) which is required for maintenance and replication of V-M. Another large-size (4.6 kb) dsRNA virus (V-LBC), without known helper activity to date, may join V-LA and V-M in the same yeast. T. delbrueckii Kbarr1 killer strain contains the killer virus Mbarr1 in addition to two L viruses, TdV-LAbarr1 and TdV-LBCbarr1. In contrast, the T. delbrueckii Kbarr2 killer strain contains two M killer viruses (Mbarr1 and M1) and a LBC virus (TdV-LBCbarr2), which has helper capability to maintain both M viruses. The genomes of TdV-LBCbarr1 and TdV-LBCbarr2 were characterized by high-throughput sequencing (HTS). Both RNA genomes share sequence identity and similar organization with their ScV-LBC counterparts. They contain all conserved motifs required for translation, packaging, and replication of viral RNA. Their Gag-Pol amino-acid sequences also contain the features required for cap-snatching and RNA polymerase activity. However, some of these motifs and features are similar to those of LA viruses, which may explain that at least TdV-LBCbarr2 has a helper ability to maintain M killer viruses. Newly sequenced ScV-LBC genomes contained the same motifs and features previously found in LBC viruses, with the same genome location and secondary structure. Sequence comparison showed that LBC viruses belong to two clusters related to each species of yeast. No evidence for associated co-evolution of specific LBC with specific M virus was found. The presence of the same M1 virus in S. cerevisiae and T. delbrueckii raises the possibility of cross-species transmission of M viruses.

Genome Sequence of Saccharomyces cerevisiae Double-Stranded RNA Virus L-A-28

We cloned and sequenced the complete genome of the L-A-28 virus from the Saccharomyces cerevisiae K28 killer strain. This sequence completes the set of currently identified L-A helper viruses required for expression of double-stranded RNA-originated killer phenotypes in baking yeast.

Quantitative analysis of the killer activity of some oenological yeasts. Effect of some additives

<p style="text-align: justify;">The Killer factor was discovered in 1963. Since this time it has been widely studied and nowadays a lot is known about genetics of the factor, the biochemistry of the toxin and also about the way of action of the toxin on sensitive yeasts. The yeast strains are classified in three groups : killer strains, sensitive strains and neutral strains. The killer strain is able to kill sensitive strains while neutral strains are unable to kill any strain and remain unaffected by the toxin. Certainly this classification depends on the couple of strains (killer and sensitive) taken as references. It has been clearly established that the toxin acts on the sensitive cells by inducing important losses of ATP : due to holes created by the toxin in the cell membrane, ATP leaves the cell.</p><p style="text-align: justify;">In the field of Enology, it’s generally accepted that a killer yeast has more probabilities to have a good implantation in a non sterile medium (as the must is) than a neutral or a sensitive strain. Nevertheless, it is difficult to have a precise idea of the sensitivity of a strain as well as of its toxicity, as most of the methods are only able to give a qualitative information.</p><p style="text-align: justify;">In this paper, a new method of evaluation of the killer effect is presented. Also some results dealing with its application to the classification of some enological yeast strains are given and discussed.</p><p style="text-align: justify;">This method is based on the measurement of the ATP released by the sensitive strain under the action of the killer toxin. The criterion that we define is the initial rate of ATP release, that means the quantity of ATP lost in the two first hours by the sensitive cells in contact with the toxin. In a first step, it is shown that this method is reliable and also that it is in a good agreement with the method using the flow cytometry. ATP leak could be correlated with the amount of affected cells (dead and damaged cells). So, using this method, it becomes possible to classify easily yeast strains with respect to their sensitivity or toxicity. Several killer yeast strains were tested against a sensitive strain and different sensitive strains were submitted to the action of a killer toxin produced by a killer strain. It was shown that the losses of ATP were quite different according to the sensitivity of the strain. The initial rate of ATP release was found to be in a range of 0.00 to 0.12 micromole/L/h. The second part of the work deals with the study of the possible effect of some products used for winemaking on the efficiency of the killer effect. The products we have studied were bentonite, tannic acid and enological tannins. It is shown that these products may interact with the killer effect. So, bentonite, for example is able to inhibit completely the efficiency of the killer toxin, as soon as its concentration is about 10 grams/hL.</p>