Reduction of Sulfur Compounds through Genetic Improvement of Native Saccharomyces cerevisiae Useful for Organic and Sulfite-Free Wine

Sulfites and sulfides are produced by yeasts in different amounts depending on different factors, including growth medium and specific strain variability. In natural must, some strains can produce an excess of sulfur compounds that confer unpleasant smells, inhibit malolactic fermentation and lead to health concerns for consumers. In organic wines and in sulfite-free wines the necessity to limit or avoid the presence of sulfide and sulfite requires the use of selected yeast strains that are low producers of sulfur compounds, with good fermentative and aromatic aptitudes. In the present study, exploiting the sexual mass-mating spores’ recombination of a native Saccharomyces cerevisiae strain previously isolated from grape, three new S. cerevisiae strains were selected. They were characterized by low sulfide and sulfite production and favorable aromatic imprinting. This approach, that occurs spontaneously also in nature, allowed us to obtain new native S. cerevisiae strains with desired characteristics that could be proposed as new starters for organic and sulfite-free wine production, able to control sulfur compound production and to valorize specific wine types.

MAT heterozygosity and the second sterility barrier in the reproductive isolation of Saccharomyces species

The genetic analysis of large numbers of Saccharomyces cerevisiae × S. uvarum (“cevarum”) and S. kudriavzevii × S. uvarum (“kudvarum”) hybrids in our previous studies revealed that these species are isolated by a postzygotic double-sterility barrier. We proposed a model in which the first barrier is due to the abruption of the meiotic process by the failure of the chromosomes of the subgenomes to pair (and recombine) in meiosis and the second barrier is assumed to be the result of the suppression of mating by allospecific MAT heterozygosity. While the former is analogous to the major mechanism of postzygotic reproductive isolation in plants and animals, the latter seems to be Saccharomyces specific. To bolster the assumed involvement of MAT in the second sterility barrier, we produced synthetic alloploid two-species cevarum and kudvarum hybrids with homo- and heterothallic backgrounds as well as three-species S. cerevisiae × S. kudvarum × S. uvarum (“cekudvarum”) hybrids by mass-mating and examined their MAT loci using species- and cassette-specific primer pairs. We found that the allospecific MAT heterozygosity repressed MAT switching and mating in the hybrids and in the viable but sterile spores produced by the cevarum hybrids that had increased (allotetraploid) genomes. The loss of heterozygosity by meiotic malsegregation of MAT-carrying chromosomes in the latter hybrids broke down the sterility barrier. The resulting spores nullisomic for the S. uvarum chromosome produced vegetative cells capable of MAT switching and conjugation, opening the way for GARMe (Genome Autoreduction in Meiosis), the process that leads to chimeric genomes.

Invasive traits of veronicellid slugs in the Hawaiian Islands and temperature response suggesting possible range shifts under a changing climate

Understanding life history traits is important for assessing potential invasiveness, particularly in the context of the future spread of invasive species under climate change. A number of species of Veronicellidae have been introduced beyond their native ranges and have become invasive. Two of these species, Veronicella cubensis and Laevicaulis alte, are widespread in Hawaii, yet little is known of their life histories. This study of growth and reproduction and their relation to temperature in these two species was undertaken using laboratory stocks derived from individuals collected in Hawaii. More data were collected for V. cubensis than for L. alte because of difficulty maintaining the latter in the lab. Veronicella cubensis grew faster at 22 °C than at 27 °C. At 22 °C, the mean age at which V. cubensis first mated was 203 d, and the mean age when eggs were first laid was 226 d. Mating in V. cubensis lasted more than a day, and it took up to 4 d to lay an egg mass. Mating took less than a day in L. alte. No self-fertilization was recorded in V. cubensis, but a single L. alte individual maintained alone from hatching laid fertilized eggs. Sperm storage after a single mating in V. cubensis was estimated to last up to 6 months. In both species the time for eggs to hatch was shorter at 27 °C than at 22 °C. Hatchability was between 74 and 93%. Veronicella cubensis lived for at least 2 years and was estimated to produce at least 400 eggs over its life. Climate warming will probably lead to expansion of the ranges of V. cubensis and L. alte to higher elevations in Hawaii and elsewhere. Growth and reproduction will also be affected by a warming climate and therefore impact the success of these invasive species.

Generation of a Novel Saccharomyces cerevisiae Strain That Exhibits Strong Maltose Utilization and Hyperosmotic Resistance Using Nonrecombinant Techniques

ABSTRACT A yeast strain capable of leavening both unsugared and sweet bread dough efficiently would reduce the necessity of carrying out the expensive procedure of producing multiple baker's yeast strains. But issues involving the use of genetically modified foods have rendered the use of recombinant techniques for developing yeast strains controversial. Therefore, we used strong selection and screening systems in conjunction with traditional mass mating techniques to develop a strain of Saccharomyces cerevisiaethat efficiently leavens both types of dough.

Identification of Genes in the RosR Regulon ofRhizobium etli

ABSTRACT RosR is a determinant of nodulation competitiveness and cell surface characteristics of Rhizobium etli and has sequence similarity to a family of transcriptional repressors. To understand how RosR affects these phenotypes, we mutagenized a rosR mutant derivative of R. etli strain CE3 with a mini-Tn5 that contains a promoterless gusA gene at one end, which acts as a transcriptional reporter. Using a mass-mating technique, we introduced rosR into each mutant in trans and screened for mutants that expressed different levels of β-glucuronidase activity in the presence and absence ofrosR. A screen of 18,000 mutants identified 52 insertions in genes negatively regulated by RosR and 1 insertion in a gene positively regulated by RosR. Nucleotide sequence analysis of the regions flanking the insertions suggests that RosR regulates genes of diverse function, including those involved in polysaccharide production and in carbohydrate metabolism and those in a region containing sequence similarity to virC1 and virD3 fromAgrobacterium tumefaciens. Two of the mutants produced colonies with altered morphology and were more competitive in nodulation than was CE3ΔrosR, the rosRparent. One mutant that contained an insertion in a gene with similarity to exsH of Sinorhizobium melilotidid not nodulate the plant host Phaseolus vulgaris withoutrosR. These results indicate that RosR directly or indirectly influences expression of diverse genes in R. etli, some of which affect the cell surface and nodulation competitiveness.

Horizontal Transfer of Genetic Material amongSaccharomyces Yeasts

ABSTRACT The genus Saccharomyces consists of several species divided into the sensu stricto and the sensu lato groups. The genomes of these species differ in the number and organization of nuclear chromosomes and in the size and organization of mitochondrial DNA (mtDNA). In the present experiments we examined whether these yeasts can exchange DNA and thereby create novel combinations of genetic material. Several putative haploid, heterothallic yeast strains were isolated from different Saccharomyces species. All of these strains secreted an a- or α-like pheromone recognized by S. cerevisiae tester strains. When interspecific crosses were performed by mass mating between these strains, hybrid zygotes were often detected. In general, the less related the two parental species were, the fewer hybrids they gave. For some crosses, viable hybrids could be obtained by selection on minimal medium and their nuclear chromosomes and mtDNA were examined. Often the frequency of viable hybrids was very low. Sometimes putative hybrids could not be propagated at all. In the case of sensu stricto yeasts, stable viable hybrids were obtained. These contained both parental sets of chromosomes but mtDNA from only one parent. In the case of sensu lato hybrids, during genetic stabilization one set of the parental chromosomes was partially or completely lost and the stable mtDNA originated from the same parent as the majority of the nuclear chromosomes. Apparently, the interspecific hybrid genome was genetically more or less stable when the genetic material originated from phylogenetically relatively closely related parents; both sets of nuclear genetic material could be transmitted and preserved in the progeny. In the case of more distantly related parents, only one parental set, and perhaps some fragments of the other one, could be found in genetically stabilized hybrid lines. The results obtained indicate that Saccharomyces yeasts have a potential to exchange genetic material. If Saccharomyces isolates could mate freely in nature, horizontal transfer of genetic material could have occurred during the evolution of modern yeast species.

Stimulation of Oviposition and Heterosis in Interstrain Crosses of European Corn Borer (Lepidoptera: Pyralidae)

Hybrid matings of two Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) colonies (N and O), established in the laboratory a year apart, demonstrated positive heterosis in the F1 generation as indicated by increases in pupal weight (6%), number of egg masses per female (65%) and number of eggs per female (77%) as compared to intra-colony crosses in mass matings. In contrast, pupation date, an indicator of development time, was intermediate in the hybrids relative to the parental colonies. There was also a 37% increase in the numbers of eggs/female and a 45% increase in egg masses/female in the hybrid parental cross. We investigated this stimulation of oviposition in single female, reciprocal crosses. Oviposition stimulation was directional with the cross of N colony males x O colony females exhibiting significantly higher numbers of total eggs/female and egg masses/female on a daily basis. Colony O females in this cross laid more large egg masses than females in other crosses. Data from individual female crosses demonstrated that egg production in hybrid mass mating experiments was consistent with random mating of males and females from both colonies.