Chemical signaling and insect attraction is a conserved trait in yeasts.

Yeast volatiles attract insects, which apparently is of mutual benefit, for both yeasts and insects. However, it is unknown whether biosynthesis of metabolites that attract insects is a basic and general trait, or if it is specific for yeasts that live in close association with insects. Our goal was to study chemical insect attractants produced by yeasts that span more than 250 million years of evolutionary history and vastly differ in their metabolism and lifestyle. We bioassayed attraction of the vinegar fly Drosophila melanogaster to odors of phylogenetically and ecologically distinct yeasts grown under controlled conditions. Baker's yeast Saccharomyces cerevisiae, the insect-associated species Candida californica, Pichia kluyveri and Metschnikowia andauensis, wine yeast Dekkera bruxellensis, milk yeast Kluyveromyces lactis, the vertebrate pathogens Candida albicans and Candida glabrata, and oleophilic Yarrowia lipolytica were screened for fly attraction in a wind tunnel. Yeast headspace was chemically analyzed, and co-occurrence of insect attractants in yeasts and flowering plants was investigated through a database search. In yeasts with known genomes, we investigated the occurrence of genes involved in the synthesis of key aroma compounds. Flies were attracted to all nine yeasts studied. The behavioral response to baker's yeast was independent of its growth stage. In addition to Drosophila, we tested the basal hexapod Folsomia candida (Collembola) in a Y-tube assay to the most ancient yeast, Y. lipolytica, which proved that early yeast signals also function on clades older than neopteran insects. Behavioral and chemical data and a search for selected genes of volatile metabolites underline that biosynthesis of chemical signals is found throughout the yeast clade and has been conserved during the evolution of yeast lifestyles. Literature and database reviews corroborate that yeast signals mediate mutualistic interactions between insects and yeasts. Moreover, volatiles emitted by yeasts are commonly found also in flowers and attract many insect species. The collective evidence suggests that the release of volatile signals by yeasts is a widespread and phylogenetically ancient trait, and that insect-yeast communication evolved prior to the emergence of flowering plants. Co-occurrence of the same attractant signals in yeast and flowers suggests that yeast-insect communication may have contributed to the evolution of insect-mediated pollination in flowers.

Novel Centromeric Loci of the Wine and Beer Yeast Dekkera bruxellensis CEN1 and CEN2.

The wine and beer yeast Dekkera bruxellensis thrives in environments that are harsh and limiting, especially in concentrations with low oxygen and high ethanol. Its different strains' chromosomes greatly vary in number (karyotype). This study isolates two novel centromeric loci (CEN1 and CEN2), which support both the yeast's autonomous replication and the stable maintenance of plasmids. In the sequenced genome of the D. bruxellensis strain CBS 2499, CEN1 and CEN2 are each present in one copy. They differ from the known "point" CEN elements, and their biological activity is retained within ~900-1300 bp DNA segments. CEN1 and CEN2 have features of both "point" and "regional" centromeres: They contain conserved DNA elements, ARSs, short repeats, one tRNA gene, and transposon-like elements within less than 1 kb. Our discovery of a miniature inverted-repeat transposable element (MITE) next to CEN2 is the first report of such transposons in yeast. The transformants carrying circular plasmids with cloned CEN1 and CEN2 undergo a phenotypic switch: They form fluffy colonies and produce three times more biofilm. The introduction of extra copies of CEN1 and CEN2 promotes both genome rearrangements and ploidy shifts, with these effects mediated by homologous recombination (between circular plasmid and genome centromere copy) or by chromosome breakage when integrated. Also, the proximity of the MITE-like transposon to CEN2 could translocate CEN2 within the genome or cause chromosomal breaks, so promoting genome dynamics. With extra copies of CEN1 and CEN2, the yeast's enhanced capacities to rearrange its genome and to change its gene expression could increase its abilities for exploiting new and demanding niches.

Resistance to Dutch elm disease reduces presence of xylem endophytic fungi in Elms (Ulmus spp.).

Efforts to introduce pathogen resistance into landscape tree species by breeding may have unintended consequences for fungal diversity. To address this issue, we compared the frequency and diversity of endophytic fungi and defensive phenolic metabolites in elm (Ulmus spp.) trees with genotypes known to differ in resistance to Dutch elm disease. Our results indicate that resistant U. minor and U. pumila genotypes exhibit a lower frequency and diversity of fungal endophytes in the xylem than susceptible U. minor genotypes. However, resistant and susceptible genotypes showed a similar frequency and diversity of endophytes in the leaves and bark. The resistant and susceptible genotypes could be discriminated on the basis of the phenolic profile of the xylem, but not on basis of phenolics in the leaves or bark. As the Dutch elm disease pathogen develops within xylem tissues, the defensive chemistry of resistant elm genotypes thus appears to be one of the factors that may limit colonization by both the pathogen and endophytes. We discuss a potential trade-off between the benefits of breeding resistance into tree species, versus concomitant losses of fungal endophytes and the ecosystem services they provide.

Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana.

Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.7.1.21)-like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. The results obtained in the present study suggest a crucial role for the salvage of thymidine during early plant development.

"This is not an apple"-yeast mutualism in codling moth.

The larva of codling moth Cydia pomonella (Tortricidae, Lepidoptera) is known as the worm in the apple, mining the fruit for food. We here show that codling moth larvae are closely associated with yeasts of the genus Metschnikowia. Yeast is an essential part of the larval diet and further promotes larval survival by reducing the incidence of fungal infestations in the apple. Larval feeding, on the other hand, enables yeast proliferation on unripe fruit. Chemical, physiological and behavioral analyses demonstrate that codling moth senses and responds to yeast aroma. Female moths are attracted to fermenting yeast and lay more eggs on yeast-inoculated than on yeast-free apples. An olfactory response to yeast volatiles strongly suggests a contributing role of yeast in host finding, in addition to plant volatiles. Codling moth is a widely studied insect of worldwide economic importance, and it is noteworthy that its association with yeasts has gone unnoticed. Tripartite relationships between moths, plants, and microorganisms may, accordingly, be more widespread than previously thought. It, therefore, is important to study the impact of microorganisms on host plant ecology and their contribution to the signals that mediate host plant finding and recognition. A better comprehension of host volatile signatures also will facilitate further development of semiochemicals for sustainable insect control.

Parallel evolution of the make-accumulate-consume strategy in Saccharomyces and Dekkera yeasts.

Saccharomyces yeasts degrade sugars to two-carbon components, in particular ethanol, even in the presence of excess oxygen. This characteristic is called the Crabtree effect and is the background for the 'make-accumulate-consume' life strategy, which in natural habitats helps Saccharomyces yeasts to out-compete other microorganisms. A global promoter rewiring in the Saccharomyces cerevisiae lineage, which occurred around 100 mya, was one of the main molecular events providing the background for evolution of this strategy. Here we show that the Dekkera bruxellensis lineage, which separated from the Saccharomyces yeasts more than 200 mya, also efficiently makes, accumulates and consumes ethanol and acetic acid. Analysis of promoter sequences indicates that both lineages independently underwent a massive loss of a specific cis-regulatory element from dozens of genes associated with respiration, and we show that also in D. bruxellensis this promoter rewiring contributes to the observed Crabtree effect.

Candida albicans--a pre-whole genome duplication yeast--is predominantly aerobic and a poor ethanol producer.

Yeast species belonging to the lineage that underwent the whole genome duplication (WGD), and including Saccharomyces cerevisiae, can grow under anaerobiosis and accumulate ethanol in the presence of glucose and oxygen. The pre-WGD yeasts, which branched from the S. cerevisiae lineage just before the WGD event, including Kluyveromyces lactis, are more dependent on oxygen and do not accumulate large amounts of ethanol in the presence of excess oxygen. Yeasts that belong to the so-called 'lower branches' of the yeast phylogenetic tree and diverged from S. cerevisiae more than 200 million years ago have so far not been thoroughly investigated for their physiology and carbon metabolism. Here, we have studied several isolates of Candida albicans and Debaryomyces hansenii for their dependence on oxygen. Candida albicans grew very poorly at an oxygen concentration <1 p.p.m. and D. hansenii could not grow at all. In aerobic batch cultivations, C. albicans exhibited a predominantly aerobic metabolism, accumulating only small amounts of ethanol (0.01-0.09 g g(-1) glucose). Apparently, C. albicans and several other pre-WGD yeasts still exhibit the original traits of the yeast progenitor: poor accumulation of ethanol under aerobic conditions and strong dependence on the presence of oxygen.

Plant thymidine kinase 1: a novel efficient suicide gene for malignant glioma therapy.

The prognosis for malignant gliomas remains poor, and new treatments are urgently needed. Targeted suicide gene therapy exploits the enzymatic conversion of a prodrug, such as a nucleoside analog, into a cytotoxic compound. Although this therapeutic strategy has been considered a promising regimen for central nervous system (CNS) tumors, several obstacles have been encountered such as inefficient gene transfer to the tumor cells, limited prodrug penetration into the CNS, and inefficient enzymatic activity of the suicide gene. We report here the cloning and successful application of a novel thymidine kinase 1 (TK1) from the tomato plant, with favorable characteristics in vitro and in vivo. This enzyme (toTK1) is highly specific for the nucleoside analog prodrug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. An important feature of toTK1 is that it efficiently phosphorylates its substrate AZT not only to AZT monophosphate, but also to AZT diphosphate, with excellent kinetics. The efficiency of the toTK1/AZT system was confirmed when toTK1-transduced human glioblastoma (GBM) cells displayed a 500-fold increased sensitivity to AZT compared with wild-type cells. In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumor growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls. The novel toTK1/AZT suicide gene therapy system in combination with stem cell-mediated gene delivery promises new treatment of malignant gliomas.

Genome survey sequencing of the wine spoilage yeast Dekkera (Brettanomyces) bruxellensis.

The hemiascomycete yeast Dekkera bruxellensis, also known as Brettanomyces bruxellensis, is a major cause of wine spoilage worldwide. Wines infected with D. bruxellensis develop distinctive, unpleasant aromas due to volatile phenols produced by this species, which is highly ethanol tolerant and facultatively anaerobic. Despite its importance, however, D. bruxellensis has been poorly genetically characterized until now. We performed genome survey sequencing of a wine strain of D. bruxellensis to obtain 0.4x coverage of the genome. We identified approximately 3,000 genes, whose products averaged 49% amino acid identity to their Saccharomyces cerevisiae orthologs, with similar intron contents. Maximum likelihood phylogenetic analyses suggest that the relationship between D. bruxellensis, S. cerevisiae, and Candida albicans is close to a trichotomy. The estimated rate of chromosomal rearrangement in D. bruxellensis is slower than that calculated for C. albicans, while its rate of amino acid evolution is somewhat higher. The proteome of D. bruxellensis is enriched for transporters and genes involved in nitrogen and lipid metabolism, among other functions, which may reflect adaptations to its low-nutrient, high-ethanol niche. We also identified an adenyl deaminase gene that has high similarity to a gene in bacteria of the Burkholderia cepacia species complex and appears to be the result of horizontal gene transfer. These data provide a resource for further analyses of the population genetics and evolution of D. bruxellensis and of the genetic bases of its physiological capabilities.

How did Saccharomyces evolve to become a good brewer?

Brewing and wine production are among the oldest technologies and their products are almost indispensable in our lives. The central biological agents of beer and wine fermentation are yeasts belonging to the genus Saccharomyces, which can accumulate ethanol. Recent advances in comparative genomics and bioinformatics have made it possible to elucidate when and why yeasts produce ethanol in high concentrations, and how this remarkable trait originated and developed during their evolutionary history. Two research groups have shed light on the origin of the genes encoding alcohol dehydrogenase and the process of ethanol accumulation in Saccharomyces cerevisiae.