Biology subjects

Howell, K.

Publications and source records attributed to Howell, K..

27 records · Page 2Linked to original sources

bioRxiv2021

Clarifying intercellular signalling in yeast: Saccharomyces cerevisiae does not undergo a quorum sensing-dependent switch to filamentous growth

Saccharomyces cerevisiae can alter its morphology to a filamentous form associated with unipolar budding in response to environmental stressors. Induction of filamentous growth is suggested under nitrogen deficiency in response to alcoholic signalling molecules through a quorum sensing mechanism. To investigate this claim, we analysed the budding pattern of S. cerevisiae cells over time under low nitrogen while concurrently measuring cell density and extracellular metabolite concentration. We found that the proportion of cells displaying unipolar budding increased between local cell densities of 4.8x106 and 5.3x107 cells/ml. However, the observed increase in unipolar budding could not be reproduced when cells were prepared at the critical cell density and in conditioned media. Removing the nutrient restriction by growth under high nitrogen conditions also resulted in an increase in unipolar budding between local cell densities of 5.2x106 and 8.2x107 cells/ml, but there were differences in metabolite concentration compared to the low nitrogen conditions. This suggests that neither cell density, metabolite concentration, nor nitrogen deficiency were necessary or sufficient to increase the proportion of unipolar budding cells. It is therefore unlikely that quorum sensing is the mechanism controlling the switch to filamentous growth in S. cerevisiae. Only a high concentration of the putative signalling molecule, 2-phenylethanol resulted in an increase in unipolar budding, but this concentration was not physiologically relevant. We suggest that the compound 2-phenylethanol acts through a toxicity mechanism, rather than quorum sensing, to induce filamentous growth. IMPORTANCEInvestigating dimorphism in the model organism Saccharomyces cerevisiae has been instrumental in understanding the signalling pathways that control hyphal growth and virulence in human pathogenic fungi. Quorum sensing was proposed to signal morphogenesis in S. cerevisiae populations. This mechanism requires the switch to filamentous growth to occur at a critical quorum sensing molecule concentration corresponding to a critical cell density. However, evidence for this mechanism is sparse and limited by the use of non-physiologically relevant concentrations of signalling metabolites. Our study designed a methodology to address this gap and may be applied to further studies of dimorphism in other types of yeasts. A significant implication of our findings is that morphogenesis does not occur in S. cerevisiae via a quorum sensing mechanism, and this important definition needs to be corrected. Mechanistic studies to understand dimorphism in yeasts, by considering metabolite concentrations, will further shed light onto this important cellular behaviour.

microbiology↗

bioRxiv2021

The post-translational modification landscape of commercial beers

Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.

biochemistry↗

bioRxiv2020

Diversity and dynamics of fungi during spontaneous fermentations and association with unique aroma profiles in wine

Microbial activity is an integral part of an agricultural ecosystem and influences the quality of agricultural commodities. Microbial ecology influences grapevine health and crop production, conversion of sugar to ethanol during fermentation, thus wine aroma and flavour. There are regionally differentiated microbial patterns in grapevines and must but how microbial patterns contribute to wine regional distinctiveness (terroir) at small scale (<100 km) is not well defined. Here we characterise fungal communities, yeast populations, and Saccharomyces cerevisiae populations during spontaneous fermentation using metagenomics and population genetics to investigate microbial distribution and fungal contributions to the resultant wine. We found differentiation of fungi, yeasts, and S. cerevisiae between geographic origins (estate/vineyard), with influences from the grape variety. Growth and dominance of S. cerevisiae during fermentation reshaped the fungal community and showed geographic structure at the strain level. Associations between fungal microbiota diversity and wine chemicals suggest that S. cerevisiae plays a primary role in determining wine aroma profiles at a sub-regional scale. The geographic distribution at scales of less than 12 km supports that differential microbial communities, including the dominant fermentative yeast S. cerevisiae can be distinct in a local setting. These findings provide further evidence for microbial contributions to wine terroir, and perspectives for sustainable agricultural practices to maintain microbial diversity and optimise fermentation function to craft beverage quality.

microbiology↗

bioRxiv2020

Quantitative data independent acquisition glycoproteomics of sparkling wine

Sparkling wine is an alcoholic beverage enjoyed around the world. The sensory properties of sparkling wine depend on a complex interplay between the chemical and biochemical components in the final product. Glycoproteins have been linked to positive and negative qualities in sparkling wine, but the glycosylation profiles of sparkling wine have not been previously investigated in detail. We analysed the glyco/proteome of sparkling wines using protein- and glycopeptide-centric approaches. We developed an automated workflow that created ion libraries to analyse Sequential Window Acquisition of all THeoretical mass spectra (SWATH) Data Independent Acquisition (DIA) mass spectrometry data based on glycopeptides identified by Byonic. We applied our workflow to three pairs of experimental sparkling wines to assess the effects of aging on lees and of different yeast strains used in the Liqueur de Tirage for secondary fermentation. We found that aging a cuvee on lees for 24 months compared to 8 months led to a dramatic decrease in overall protein abundance and an enrichment in large glycans at specific sites in some proteins. Secondary fermentation of a Riesling wine with Saccharomyces cerevisiae yeast strain Siha4 produced more yeast proteins and glycoproteins than with S. cerevisiae yeast strain DV10. The abundance and glycosylation profiles of grape glycoproteins were also different between grape varieties. This work represents the first in-depth study into protein- and peptide-specific glycosylation in sparkling wines and describes a quantitative glycoproteomic SWATH/DIA workflow that is broadly applicable to other sample types.

biochemistry↗

bioRxiv2020

Bacterial influence on the maintenance of symbiotic yeast through Drosophila metamorphosis

Interactions between microbial symbionts of metazoan hosts are emerging as key features of symbiotic systems. Little is known about the role of such interactions on the maintenance of symbiosis through hosts life cycle. We studied the influence of symbiotic bacteria on the maintenance of symbiotic yeast through metamorphosis of the fly Drosophila melanogaster. To this end we mimicked the development of larvae in natural fruit. In absence of bacteria yeast was never found in young adults. However, yeast could maintain through metamorphosis when larvae were inoculated with symbiotic bacteria isolated from D. melanogaster faeces. Furthermore, an Enterobacteriaceae favoured yeast transstadial maintenance. Because yeast is a critical symbiont of D. melanogaster flies, bacterial influence on host-yeast association may have consequences for the evolution of insect-yeast-bacteria tripartite symbiosis and their cooperation. Summary statementBacterial symbionts of Drosophila influence yeast maintenance through fly metamorphosis, a novel observation that may have consequences for the evolution of insect-yeast-bacteria interactions.

ecology↗

bioRxiv2020

Community succession of the grapevine fungal microbiome in the annual growth cycle

Microbial ecology is an integral component of wine production. From the vineyard to the winery, microbial activity influences grapevine health and productivity, conversion of sugar to ethanol during fermentation, wine aroma production, wine quality and distinctiveness. Fungi in the vineyard ecosystem are not well described. Here, we characterised the spatial and temporal dynamics of fungal communities associated with the grapevine (grapes, flowers, leaves, and roots) and soils over an annual growth cycle in two vineyards to investigate the influences of grape habitat, plant developmental stage (flowering, fruit set, veraison, and harvest), vineyards, and climatic conditions. Fungi were influenced by both the grapevine habitat and plant development stage. The core microbiome was prioritised over space and time, and the identified core members drove seasonal community succession. The development stage of veraison, where the grapes undergo a dramatic change in metabolism and start accumulating sugar, displayed a distinct shift in fungal communities. Co-occurrence networks showed strong correlations between the plant microbiome, the soil microbiome, and weather indices. Our study describes the complex ecological dynamics that occur in microbial assemblages over a growing season and highlight the importance of core community succession in vineyards. In addition to enriching our understanding of how plants and microbes interact, these findings may provide insights to craft wine regional distinctiveness and cope with global climate change.

microbiology↗

bioRxiv2019

Vineyard ecosystems are structured and distinguished by fungal communities impacting the flavour and quality of wine

The flavours of foods and beverages are formed by the agricultural environment where the plants are grown. In the case of wine, the location and environmental features of the vineyard site imprint the wine with distinctive aromas and flavours. Microbial growth and metabolism play an integral role in wine production from the vineyard to the winery, by influencing grapevine health, wine fermentation, and the flavour, aroma and quality of finished wines. The mechanism by which microbial distribution patterns drive wine metabolites is unclear and while flavour has been correlated with bacterial composition for red wines, bacterial activity provides a minor biochemical conversion in wine fermentation. Here, we collected samples across six distinct winegrowing areas in southern Australia to investigate regional distribution patterns of both fungi and bacteria and how this corresponds with wine aroma compounds. Results show that soil and must microbiota distinguish winegrowing regions and are related to wine chemical profiles. We found a strong relationship between microbial and wine metabolic profiles, and this relationship was maintained despite differing abiotic drivers (soil properties and weather/ climatic measures). Notably, fungal communities played the principal role in shaping wine aroma profiles and regional distinctiveness. We found that the soil microbiome is a potential source of grape- and must-associated fungi, and therefore the weather and soil conditions could influence the wine characteristics via shaping the soil fungal community compositions. Our study describes a comprehensive scenario of wine microbial biogeography in which microbial diversity responds to surrounding environments and ultimately sculpts wine aromatic characteristics. These findings provide perspectives for thoughtful human practices to optimise food and beverage flavour and composition through understanding of fungal activity and abundance.

microbiology↗

bioRxiv2019

Environmental specificity in Drosophila-bacteria symbiosis affects host developmental plasticity

Environmentally acquired microbial symbionts could contribute to host adaptation to local conditions like vertically transmitted symbionts do. This scenario necessitates symbionts to have different effects in different environments. We investigated this idea in Drosophila melanogaster, a species which communities of bacterial symbionts vary greatly among environments. We isolated four bacterial strains isolated from the feces of a D. melanogaster laboratory strain and tested their effects in two conditions: the ancestral environment (i.e. the laboratory medium) and a new environment (i.e. fresh fruit with live yeast). All bacterial effects on larval and adult traits differed among environments, ranging from very beneficial to marginally deleterious. The joint analysis of larval development speed and adult size further shows bacteria affected developmental plasticity more than resource acquisition. This effect was largely driven by the contrasted effects of the bacteria in each environment. Our study illustrates that understanding D. melanogaster symbiotic interactions in the wild will necessitate working in ecologically realistic conditions. Besides, context-dependent effects of symbionts, and their influence on host developmental plasticity, shed light on how environmentally acquired symbionts may contribute to host evolution.

evolutionary biology↗

bioRxiv2019

Environmental specificity and evolution in Drosophila-bacteria symbiosis

Environmentally acquired microbial symbionts could contribute to host adaptation to local adaptation like vertically transmitted symbionts do. This scenario necessitates symbionts to have different effects in different environments. In Drosophila melanogaster, communities of extracellular bacterial symbionts vary largely among environments, which could be due to variable effects on phenotype. We investigated this idea with four bacterial strains isolated from the feces of a D. melanogaster lab strain, and tested their effects in two environments: the environment of origin (i.e. the laboratory medium) and a new one (i.e. fresh fruit with live yeast). All bacterial effects on larval and adult traits differed among environments, ranging from very beneficial to marginally deleterious. The joint analysis of larval development speed and adult size further suggests bacteria would affect developmental plasticity more than resource acquisition in males. The context-dependent effects of bacteria we observed, and its underlying mechanisms, sheds light on how environmentally acquired symbionts may contribute to host evolution.

evolutionary biology↗