Biology subjects

Verstrepen, K. J.

Publications and source records attributed to Verstrepen, K. J..

5 recordsLinked to original sources

bioRxiv2024

Genes involved in protein folding and chromatin organization buffer genetic variation

Mutations are not always phenotypically active or show different effects in different individuals. While the mechanisms underlying this variable relationship between mutations and phenotypes are largely elusive, some specific genes may influence the phenotypic effects of cryptic variation. We employ the toolbox of Saccharomyces cerevisiae to perform a genome-wide screen aimed at identifying these so-called genetic buffer genes. Measuring the fitness of 1.8 million mutated strains identified a small set of evolutionary conserved buffer genes involved in protein folding and chromatin organization, including GIM3, SSA2, HOG1 and FKH2. Deletion of these genes increases the fitness effect of de novo mutations as well as standing genetic variation, with some mutations becoming adaptive. Moreover, losing a buffer gene results in a decline of standing genetic variation. Together, these results reveal a set of conserved genes that influence the phenotypic outcome of mutations and play a role in complex genetics and incomplete penetrance.

evolutionary biology↗

bioRxiv2022

Development of a tractable model system to mimic wood-ageing of beer on a lab scale

Wood-ageing of conventionally fermented beers is gaining increased attention in the production of sour beers with a noteworthy balance between sourness, wood aroma and flavour complexity. Besides the extraction of wood-derived compounds into the beer, wood-aged sours owe their layered flavour profile to the activity of a variety of wild microorganisms that reside in the barrels or that emerge from the brewing or maturation environment. However, until now wood-ageing of craft beers largely remains a process of trial and error that often generates unexpected or undesirable results. Therefore, to better understand the process and develop control strategies to improve the consistency, predictability and overall quality of the resulting beer, more insight is needed into the interactions between the wood, the microorganisms and the maturing beer. Nevertheless, as studying these interactions on an industrial scale is highly challenging, the objective of this study was to develop a reproducible and easy-to-manipulate experimentally tractable system that can be used to study wood-ageing of beer on a lab scale. Barrel-ageing was mimicked in a 0.5 liter glass jar filled with beer and closed off by a wooden disk. Furthermore, the system was equipped with a synthetic community composed of four bacterial species (Acetobacter malorum, Gluconobacter oxydans, Lactobacillus brevis and Pediococcus damnosus) and four fungal species (Brettanomyces bruxellensis, Candida friedrichii, Pichia membranifaciens and Saccharomyces cerevisiae) that represented key microbes previously identified in wood-ageing experiments with 225-liter barrels. In order to test the hypothesis that the barrel-ageing process of beer can be replicated in the simplified in-vitro system, the system was subjected to 60 days of ageing and microbial community dynamics and beer chemistry were compared with a 38-week industrial barrel-ageing experiment using the same beer. Beer samples were collected at regular time points and subjected to both qPCR assays targeting the eight selected species and chemical analysis. Results revealed that in vitro ageing showed similar trends in the temporal dynamics of the microbial populations and beer chemistry as those observed during 38-weeks of barrel-ageing in 225-liter barrels. Furthermore, results were found to be highly reproducible. Altogether, the in-vitro system was found to be a robust and reproducible system that has great potential to perform more in-depth research about the intricate interactions between microbes, wood and maturing beer and to develop control strategies to improve the consistency, predictability and overall quality of the resulting beer.

microbiology↗

bioRxiv2022

Beer ethanol and iso-α-acid level affect microbial community establishment and beer chemistry throughout wood maturation of beer

Sour beers produced by barrel-aging of conventionally fermented beers are becoming increasingly popular. However, as the intricate interactions between the wood, the microbes and the beer are still unclear, wood maturation often leads to inconsistent end products with undesired sensory properties. Previous research on industrial barrel-aging of beer suggests that beer parameters like the ethanol content and bitterness play an important role in the microbial community composition and beer chemistry, but their exact impact still remains to be investigated. In this study, an experimentally tractable lab-scale system based on an in-vitro community of four key bacteria (Acetobacter malorum, Gluconobacter oxydans, Lactobacillus brevis and Pediococcus damnosus) and four key yeasts (Brettanomyces bruxellensis, Candida friedrichii, Pichia membranifaciens and Saccharomyces cerevisiae) that are consistently associated with barrel-aging of beer, was used to test the hypotheses that beer ethanol and bitterness impact microbial community composition and beer chemistry. Experiments were performed using different levels of ethanol (5.2 v/v%, 8 v/v% and 11 v/v%) and bitterness (13 ppm, 35 ppm and 170 ppm iso--acids), and beers were matured for 60 days. Samples were taken after 0, 10, 20, 30 and 60 days to monitor population densities and beer chemistry. Results revealed that all treatments and the maturation time significantly affected the microbial community composition and beer chemistry. More specifically, the ethanol treatments obstructed growth of L. brevis and G. oxydans and delayed fungal growth. The iso--acid treatments hindered growth of L. brevis and stimulated growth of P. membranifaciens, while the other strains remained unaffected. Beer chemistry was found to be affected by higher ethanol levels, which led to an increased extraction of wood-derived compounds. Furthermore, the distinct microbial communities also induced changes in the chemical composition of the beer samples, leading to concentration differences in beer- and wood-derived compounds like 4-ethyl guaiacol, 4-ethyl phenol, cis-oak lactone, vanillin, furfural and 5-methyl furfural. Altogether, our results indicate that wood-aging of beer is affected by biotic and abiotic parameters, influencing the quality of the final product. Additionally, this work provides a new, cost-effective approach to study the production of barrel-aged beers based on a simplified microbial community model.

microbiology↗

bioRxiv2021

Permissive aggregative group formation favors coexistence in yeast

In Saccharomyces cerevisiae, the FLO1 gene encodes flocculins that lead to formation of multicellular flocs, that offer protection to the constituent cells. Flo1p was found to preferentially bind to fellow cooperators compared to defectors lacking FLO1 expression, resulting in enrichment of cooperators within the flocs. Given this dual function in cooperation and kin recognition, FLO1 has been termed a green beard gene. Because of the heterophilic nature of Flo1p binding however, we hypothesize that kin recognition is permissive and depends on the relative stability of FLO1+/flo1- versus FLO1+/FLO1+ bonds, which itself can be dependent on environmental conditions and intrinsic cell properties. We combine single cell measurements of adhesion strengths, individual cell-based simulations of cluster formation and evolution, and in vitro flocculation experiments to study the impact of relative bond stability on defector exclusion as well as benefit and stability of cooperation. We hereto vary the relative bond stability by changing the shear flow rate and the inherent bond strength. We identify a marked trade-off between both aspects of the green beard mechanism, with reduced relative bond stability leading to increased kin recognition, but at the expense of decreased cluster sizes and benefit of cooperation. Most notably, we show that the selection of FLO1 cooperators is negative-frequency dependent, which we directly attribute to the permissive character of the Flo1p bond. Taking into account the costs associated to FLO1 expression, this asymmetric selection results in a broad range of ecological conditions where coexistence between cooperators and defectors is stable. Although the kin recognition aspect of the FLO1 green beard gene is thus limited and condition dependent, the negative-frequency dependency of selection can conserve the diversity of flocculent and non-flocculent phenotypes ensuring flexibility towards variable selective pressures.

evolutionary biology↗

bioRxiv2019

Isolation of persisters enabled by ss-lactam-induced filamentation reveals their single-cell awakening characteristics

When exposed to lethal doses of antibiotics, bacterial populations are most often not completely eradicated. A small number of phenotypic variants, defined as persisters, are refractory to antibiotics and survive treatment. Despite their involvement in relapsing infections caused by major pathogens, processes determining phenotypic switches from and to the persister state largely remain elusive. This is mainly due to the low frequency of persisters in a population and the lack of reliable persistence markers, both hampering studies of persistence at the single-cell level. Problematically, existing methods to enrich for persisters result in samples with very low persister densities and/or a too high abundance of other cell types. Here we present a novel and highly effective persister isolation method involving cephalexin, an antibiotic that induces extensive filamentation of susceptible cells. We show that antibiotic-tolerant cells can easily be separated by size after a short cephalexin treatment, and that the isolated cells are genuine persisters. We used our isolation method to monitor persister outgrowth at the single-cell level in a microfluidic device, thereby conclusively demonstrating that awakening is a stochastic phenomenon. We anticipate that our novel approach can have far-reaching consequences in the persistence field, by allowing single-cell studies at a much higher throughput than previously reported.

microbiology↗