The yeast Saccharomyces cerevisiae has evolved to dominate grape juice fermentation. A suite of cellular properties, rapid nutrient depletion, production of inhibitory compounds and the metabolic narrowing of the niche, all enable a minor resident of the initial population to dramatically increase its relative biomass in the ecosystem. This dominance of the grape juice environment is fueled by a rapid launch of glycolysis and energy generation mediated by transport of hexoses and an efficient coupling of transport and catabolism. Fermentation occurs in the presence of molecular oxygen as the choice between respiratory or fermentative growth is regulated by the availability of sugar a phenomenon known as glucose or catabolite repression. Induction of the [GAR + ] prion alters the expression of the major hexose transporter active under these conditions, Hxt3, reducing glycolytic capacity. Bacteria present in the grape juice ecosystem were able to induce the [GAR + ] prion in wine strains of S. cerevisiae. This induction reduced fermentation capacity but did not block it entirely. However, dominance factors such as the rapid depletion of amino acids and other nitrogen sources from the environment were impeded enabling greater access to these substrates for the bacteria. Bacteria associated with arrested commercial wine fermentations were able to induce the prion state, and yeast cells isolated from arrested commercial fermentations were found to be [GAR + ] thus confirming the ecological relevance of prion induction. Subsequent analyses demonstrated that the presence of environmental acetic acid could lead to [GAR + ] induction in yeast strains under certain conditions. The induction of the prion enabled yeast growth on non-preferred substrates, oxidation and reduction products of glucose and fructose, present as a consequence of bacterial energy production. In native ecosystems prion induction never exceeded roughly 50-60% of the population of yeast cells suggesting that the population retains the capacity for maximal fermentation. Thus, the bacterial induction of the [GAR + ] prion represents a novel environmental response: the query of the environment for the presence of competing organisms and the biological decision to temper glucose repression and dominance and enter a metabolic state enabling coexistence.

The formation of multivesicular endosomes (MVEs) mediates the turnover of numerous integral membrane proteins and has been implicated in the down-regulation of growth factor signaling, thereby exhibiting properties of a tumor suppressor. The endosomal sorting complex required for transport (ESCRT) machinery plays a key role in MVE biogenesis, enabling cargo selection and intralumenal vesicle (ILV) budding. However, the spatiotemporal pattern of endogenous ESCRT complex assembly and disassembly in mammalian cells remains poorly defined. By combining CRISPR/Cas9-mediated genome editing and live cell imaging using lattice light sheet microscopy (LLSM), we determined the native dynamics of both early-and late-acting ESCRT components at MVEs under multiple growth conditions. Specifically, our data indicate that ESCRT-0 accumulates quickly on endosomes, typically in less than 30 seconds, and its levels oscillate in a manner dependent on the downstream recruitment of ESCRT-I. Similarly, levels of the ESCRT-I complex also fluctuate on endosomes, but its average residency time is more than fivefold shorter compared with ESCRT-0. Vps4 accumulation is the most transient, however, suggesting that the completion of ILV formation occurs rapidly. Upon addition of epidermal growth factor (EGF), both ESCRT-I and Vps4 are retained at endosomes for dramatically extended periods of time, while ESCRT-0 dynamics are only modestly affected. Our findings are consistent with a model in which growth factor stimulation stabilizes late-acting components of the ESCRT machinery at endosomes to accelerate the rate of ILV biogenesis and attenuate signal transduction initiated by receptor activation. lattice light sheet microscopy | ESCRT microdomain | CRISPR/Cas9 | epidermal growth factor receptor | organelle

A novel method of yeast immobilization, called "biocapsules", has been developed in which cells of the yeast Saccharomyces cerevisiae become attached to the hyphae of the fungus Penicillium chrysogenum, remaining adhered following loss of viability of this fungus. Yeast immobilization facilitates higher cell densities than traditional fermentation methods, improves yield, and allows the reuse of the biocatalyst. Yeast cells may adhere to each other via specific cell-surface molecular interactions (flocculation) or may attach to surfaces (biofilm formation), and the roles of these two distinct attachment mechanisms in biocapsule formation is unknown. To elucidate the influence of biofilm formation versus flocculation on the yeast-fungus coimmobilization, a screening of selected strains from the Viticulture and Enology Department collection at the University of California, Davis, was carried out, and their ability to flocculate and form biofilm was quantified. Eighteen yeast strains capable of flocculation and biofilm formation were identified in this screening. Strains displaying differential capabilities in flocculation or biofilm formation and two control strains were further evaluated for their ability to specifically immobilize with P. chrysogenum. Seven strains exhibiting different patterns of flocculation and biofilm formation were identified. Biofilm-forming yeast strains displayed higher rates of immobilization with P. chrysogenum and formed more consistent biocapsules. In contrast, strains able to flocculate developed smaller, inconsistent biocapsules. Although the size and number of biocapsules formed varied by yeast strain, the total mass of biocapsules generated was similar for all strains. These results shed light on parameters that influence yeast-fungus coimmobilization, which may lead to an improvement of biocapsule consistency and advance the field of application for this new immobilization system.

Importance: Lactobacillus kunkeei, also known as the "ferocious lactobacilli", causes fermentation arrest during wine production 1. L. kunkeei co-evolved with honeybees and is an important probiotic for bee and hive health 2. In the bee ecosystem, L. kunkeei is one of a suite of lactic acid bacteria that protect the bee and hive from pathogens as well as aid in preservation of sugar-rich hive resources. The protection of sugar-rich resources probably uses similar mechanisms to those inhibiting yeast during grape juice fermentation.

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