The Oleaginous Yeast Metschnikowia pulcherrima Displays Killer Activity against Avian-Derived Pathogenic Bacteria

Metschnikowia pulcherrima is a non-conventional yeast with potential to be used in biotechnological processes, especially those involving low-cost feedstock exploitation and biocontrol applications. The combination of traits that supports these industrial applications in M. pulcherrima also makes it an attractive option to study in the context of livestock health. In this study, we examined the specific interactions between M. pulcherrima and multiple avian pathogenic bacteria. We tested individual bacteria–yeast interactions and bacterial combinations in both solid and liquid media and in variable nutrient environments. Across multiple isolates of M. pulcherrima, we observed different levels of antimicrobial activity, varying from supporting the growth of competing bacteria through suppression and bacterial killing, and we found that these responses varied depending on the bacterial strains and media. We identified multiple molecular routes, including proteins produced by M. pulcherrima strains, that acted to control these microbial interactions. Furthermore, protein screening revealed that M. pulcherrima strains were induced to produce proteins specifically when exposed to bacterial strains, suggesting that fine-tuned mechanisms allow M. pulcherrima to function as a potential lynchpin in a microbial community.

Insight in the quorum sensing-driven lifestyle of the non-pathogenic Agrobacterium tumefaciens 6N2 and the interactions with the yeast Meyerozyma guilliermondii

Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the importance of the QS regulation in the bacteria-yeast interactions.

A Taphrina strain infecting the model plant Arabidopsis thaliana.

Yeasts are important plant-associated organisms that can modulate host immunity to either promote or prevent disease. Mechanisms of plant-yeast interactions, specifically of yeast perception by the plant innate immune system, remain unknown. Progress has been hindered by the scarcity of yeast species associated with the model plant Arabidopsis thaliana (Arabidopsis). We have previously isolated Taphrina strain M11 from wild Arabidopsis in the field. Taphrina are poorly studied dimorphic yeast-like fungi that are plant pathogens, often producing plant hormones and causing tumour-like leaf deformation symptoms on their hosts. Here we characterize the interaction of M11 with Arabidopsis. Infection of Arabidopsis with the birch pathogen T. betulina, used as a non-host control, shows early HR, enhanced ROS accumulation, and limitation of growth, demonstrating that Arabidopsis has immunity against non-adapted yeasts. M11 triggered limited cell death, an attenuated ROS response, and grew in planta, as well as subtle but clear leaf deformation symptoms, demonstrating it is pathogenic. Hormone responsive promoter-reporter analysis demonstrated activation of cytokinin signalling during infection. Mutant infection assays indicate jasmonate and ethylene were required for immunity against M11. Analysis of the Taphrina M11 genome was used to mine evidence for yeast specific PAMPs which may underlie host immune responses against yeast-like fungi.

Understanding Wine through Yeast Interactions

Wine is a product of microbial activities and microbe–microbe interactions. Yeasts are the principal microorganisms responsible for the evolution and fulfillment of alcoholic fermentation. Several species and strains coexist and interact with their environment and with each other during the fermentation course. Yeast–yeast interactions occur even from the early stages of fermentation, determining yeast community structure and dynamics during the process. Different types of microbial interactions (e.g., mutualism and commensalism or competition and amensalism) may exert positive or negative effects, respectively, on yeast populations. Interactions are intimately linked to yeast metabolic activities that influence the wine analytical profile and shape the wine character. In this context, much attention has been given during the last years to the interactions between Saccharomyces cerevisiae (SC) and non-Saccharomyces (NS) yeast species with respect to their metabolic contribution to wine quality. Yet, there is still a significant lack of knowledge on the interaction mechanisms modulating yeast behavior during mixed culture fermentation, while much less is known about the interactions between the various NS species or between SC and Saccharomyces non-cerevisiae (SNC) yeasts. There is still much to learn about their metabolic footprints and the genetic mechanisms that alter yeast community equilibrium in favor of one species or another. Gaining deeper insights on yeast interactions in the grape–wine ecosystem sets the grounds for understanding the rules underlying the function of the wine microbial system and provides means to better control and improve oenological practices.

BFG-PCA: tools and resources that expand the potential for binary protein interaction discovery

Barcode fusion genetics (BFG) utilizes deep sequencing to improve the throughput of protein-protein interaction (PPI) screening in pools. BFG has been implemented in Yeast two-hybrid (Y2H) screens (BFG-Y2H). While Y2H requires test protein pairs to localize in the nucleus for reporter reconstruction, Dihydrofolate Reductase Protein-Fragment Complementation Assay (DHFR-PCA) allows proteins to localize in broader subcellular contexts and proves to be largely orthogonal to Y2H. Here, we implemented BFG to DHFR-PCA (BFG-PCA). This plasmid-based system can leverage ORF collections across model organisms to perform comparative analysis, unlike the original DHFR-PCA that requires yeast genomic integration. The scalability and quality of BFG-PCA were demonstrated by screening human and yeast interactions of >11,000 protein pairs. BFG-PCA showed high-sensitivity and high-specificity for capturing known interactions for both species. BFG-Y2H and BFG-PCA capture distinct sets of PPIs, which can partially be explained based on the domain orientation of the reporter tags. BFG-PCA is a high-throughput protein interaction technology to interrogate binary PPIs that exploits clone collections from any species of interest, expanding the scope of PPI assays.

Toward application of biocontrol to inhibit wine spoilage yeasts: The use of statistical designs for screening and optimisation

Spoilage yeasts generate considerable economic losses in the wine industry, and although sulphur dioxide (SO2) is traditionally used for control, its use has become controversial because of its negative effects on health. Biocontrol has emerged as a partial alternative to SO2, and most research has focused on the selection of biocontrol yeasts and/or the mechanisms involved, while little research has been directed to the environmental conditions that make biocontrol effective for application. When there are two or more interacting yeasts, the physicochemical factors that affect their antagonism are many and therefore the application of biocontrol is complex. To reduce SO2, the present study aimed to elucidate biocontrol mechanisms of two yeast interactions and to establish optimal physicochemical conditions for biocontrol of the spoilage yeast during grape must fermentation. Through the use of statistical design, it was possible to find relevant physicochemical factors and optimise them. Wickerhamomyces anomalus “BWa156” developed an active supernatant against ZygoSaccharomyces rouxii “BZr6” while supernatant from Metschnikowia pulcherrima “BMp29” was ineffective. In mixed must fermentations, the first interaction (BWa156 vs. BZr6) showed fewer physicochemical factors impacting biocontrol compared to the second interaction (BMp29 vs. BZr6). However, the fewer factors of the first interaction had a stronger effect on the decline in the spoilage population. Validations showed that the optimal conditions for biocontrol with the first interaction could be predicted. Analysis of the results with BWa156 vs. BZr6 and BMp29 vs. BZr6 suggests that the first interaction is a competition that includes a killer toxin, while the second interaction involves competition for iron resources. Response surface methodology (RSM) allowed a reduction in the number of experiments and permitted to find the optimal biocontrol conditions (SO2: 0 mg mL-1; pH: 3.7; Reducing sugars: 23 °Brix) for the interaction between BWa156 and BZr6.

Viability-PCR Allows Monitoring Yeast Population Dynamics in Mixed Fermentations Including Viable but Non-Culturable Yeasts

The use of controlled mixed inocula of Saccharomyces cerevisiae and non-Saccharomyces yeasts is a common practice in winemaking, with Torulaspora delbrueckii, Lachancea thermotolerans and Metschnikowia pulcherrima being the most commonly used non-Saccharomyces species. Although S. cerevisiae is usually the dominant yeast at the end of mixed fermentations, some non-Saccharomyces species are also able to reach the late stages; such species may not grow in culture media, which is a status known as viable but non-culturable (VBNC). Thus, an accurate methodology to properly monitor viable yeast population dynamics during alcoholic fermentation is required to understand microbial interactions and the contribution of each species to the final product. Quantitative PCR (qPCR) has been found to be a good and sensitive method for determining the identity of the cell population, but it cannot distinguish the DNA from living and dead cells, which can overestimate the final population results. To address this shortcoming, viability dyes can be used to avoid the amplification and, therefore, the quantification of DNA from non-viable cells. In this study, we validated the use of PMAxx dye (an optimized version of propidium monoazide (PMA) dye) coupled with qPCR (PMAxx-qPCR), as a tool to monitor the viable population dynamics of the most common yeast species used in wine mixed fermentations (S. cerevisiae, T. delbrueckii, L. thermotolerans and M. pulcherrima), comparing the results with non-dyed qPCR and colony counting on differential medium. Our results showed that the PMAxx-qPCR assay used in this study is a reliable, specific and fast method for quantifying these four yeast species during the alcoholic fermentation process, being able to distinguish between living and dead yeast populations. Moreover, the entry into VBNC status was observed for the first time in L. thermotolerans and S. cerevisiae during alcoholic fermentation. Further studies are needed to unravel which compounds trigger this VBNC state during alcoholic fermentation in these species, which would help to better understand yeast interactions.

Yeast–Yeast Interactions: Mechanisms, Methodologies and Impact on Composition

During the winemaking process, alcoholic fermentation is carried out by a consortium of yeasts in which interactions occurs. The consequences of these interactions on the wine matrix have been widely described for several years with the aim of controlling the winemaking process as well as possible. In this review, we highlight the wide diversity of methodologies used to study these interactions, and their underlying mechanisms and consequences on the final wine composition and characteristics. The wide variety of matrix parameters, yeast couples, and culture conditions have led to contradictions between the results of the different studies considered. More recent aspects of modifications in the composition of the matrix are addressed through different approaches that have not been synthesized recently. Non-volatile and volatile metabolomics, as well as sensory analysis approaches are developed in this paper. The description of the matrix composition modification does not appear sufficient to explain interaction mechanisms, making it vital to take an integrated approach to draw definite conclusions on them.

Peer pressure: evolutionary responses to biotic pressures in wine yeasts

ABSTRACT In the macroscopic world, ecological interactions between multiple species of fauna and flora are recognised as major role-players in the evolution of any particular species. By comparison, research on ecological interactions as a driver of evolutionary adaptation in microbial ecosystems has been neglected. The evolutionary history of the budding yeast Saccharomyces cerevisiae has been extensively researched, providing an unmatched foundation for exploring adaptive evolution of microorganisms. However, in most studies, the habitat is only defined by physical and chemical parameters, and little attention is paid to the impact of cohabiting species. Such ecological interactions arguably provide a more relevant evolutionary framework. Within the genomic phylogenetic tree of S. cerevisiae strains, wine associated isolates form a distinct clade, also matched by phenotypic evidence. This domestication signature in genomes and phenomes suggests that the wine fermentation environment is of significant evolutionary relevance. Data also show that the microbiological composition of wine fermentation ecosystems is dominated by the same species globally, suggesting that these species have co-evolved within this ecosystem. This system therefore presents an excellent model for investigating the origins and mechanisms of interspecific yeast interactions. This review explores the role of biotic stress in the adaptive evolution of wine yeast.