A glimpse at an early stage of microbe domestication revealed in the variable genome of Torulaspora delbrueckii, an emergent industrial yeast

The yeast Torulaspora delbrueckii is gaining importance for biotechnology due to its ability to increase wine sensorial complexity and for enhancing pre-frozen bread dough leavening. However, little is known about its population structure, variation in gene content, or possible domestication routes. Here, we address these issues and update the delimitation of T. delbrueckii along five major clades. Among the three European clades, a basal lineage is associated with the wild arboreal niche, while the two other lineages are linked with anthropic environments, one to wine fermentations and the other to diverse sources including dairy products and bread dough (Mix- Anthropic clade). Using 62 genomes we identified 5629 genes in the pangenome of T. delbrueckii and 270 genes in the cloud genome. A pangenome tree analysis showed that wine strains have a genome composition more similar to European wild arboreal strains than to those of the Mix Anthropic clade, in contradiction with the phylogenetic analysis. An association of gene content and ecology gave further support to the hypothesis that the Mix - Anthropic clade has the most specialized genome content and indicated that some of the exclusive genes were implicated in galactose and maltose utilization. More detailed analyses traced the acquisition of a cluster of GAL genes in strains associated with dairy products and the expansion and functional diversification of MAL genes in strains isolated from bread dough. Contrary to S. cerevisiae, domestication in T. delbrueckii is not primed by alcoholic fermentation and appears to be a recent event.

Unique Brewing-Relevant Properties of a Strain of Saccharomyces jurei Isolated From Ash (Fraxinus excelsior)

The successful application of Saccharomyces eubayanus and Saccharomyces paradoxus in brewery fermentations has highlighted the potential of wild Saccharomyes yeasts for brewing, and prompted investigation into the application potential of other members of the genus. Here, we evaluate, for the first time, the brewing potential of Saccharomyces jurei. The newly isolated strain from an ash tree (Fraxinus excelsior) in Upper Bavaria, Germany, close to the river Isar, was used to ferment a 12°P wort at 15°C. Performance was compared directly with that of a reference lager strain (TUM 34/70) and the S. eubayanus type strain. Both wild yeast rapidly depleted simple sugars and thereafter exhibited a lag phase before maltose utilization. This phase lasted for 4 and 10 days for S. eubayanus and S. jurei, respectively. S. eubayanus utilized fully the available maltose but, consistent with previous reports, did not use maltotriose. S. jurei, in contrast, utilized approximately 50% of the maltotriose available, making this the first report of maltotriose utilization in a wild Saccharomyces species. Maltotriose use was directly related to alcohol yield with 5.5, 4.9, and 4.5% ABV produced by Saccharomyces pastorianus, S. jurei, and S. eubayanus. Beers also differed with respect to aroma volatiles, with a high level (0.4 mg/L) of the apple/aniseed aroma ethyl hexanoate in S. jurei beers, while S. eubayanus beers had a high level of phenylethanol (100 mg/L). A trained panel rated all beers as being of high quality, but noted clear differences. A phenolic spice/clove note was prominent in S. jurei beer. This was less pronounced in the S. eubayanus beers, despite analytical levels of 4-vinylguaiacol being similar. Tropical fruit notes were pronounced in S. jurei beers, possibly resulting from the high level of ethyl hexanoate. Herein, we present results from the first intentional application of S. jurei as a yeast for beer fermentation (at the time of submission) and compare its fermentation performance to other species of the genus. Results indicate considerable potential for S. jurei application in brewing, with clear advantages compared to other wild Saccharomyces species.

Unique brewing-relevant properties of a strain of Saccharomyces jurei isolated from ash (Fraxinus excelsior)

The successful application of Saccharomyces eubayanus and Saccharomyces paradoxus in brewery fermentations has highlighted the potential of wild yeast for brewing, and prompted investigation into the application potential of other members of the genus. Here, we evaluate, for the first time, the brewing potential of Saccharomyces jurei. The newly isolated strain from an ash tree (Fraxinus excelsior) in Upper Bavaria, Germany, close to the river Isar, was used to ferment a 12°P wort at 15°C. Performance was compared directly with that of a reference lager strain (TUM 34/70) and the S. eubayanus type strain. Both wild yeast rapidly depleted simple sugars and thereafter exhibited a lag phase before maltose utilization. This phase lasted for 4 and 10 days for S. eubayanus and S. jurei, respectively. S. eubayanus utilized fully the available maltose but, consistent with previous reports, did not use maltotriose. S. jurei, in contrast, utilized approx. 50% of the maltotriose available, making this the first report of maltotriose utilization in a wild Saccharomyces species. Maltotriose use was directly related to alcohol yield with 5.5, 4.9, and 4.5 % ABV produced by S. pastorianus, S. jurei and S. eubayanus. Beers also differed with respect to aroma volatiles, with a high level (0.4 mg/L) of the apple/aniseed aroma ethyl hexanoate in S. jurei beers, while S. eubayanus beers had a high level of phenylethanol (100 mg/L). A trained panel rated all beers as being of high quality, but noted clear differences. A phenolic spice/clove note was prominent in S. jurei beer. This was less pronounced in the S. eubayanus beers, despite analytical levels of 4-vinylguaiacol being similar. Tropical fruit notes were pronounced in S. jurei beers, possibly resulting from the high level of ethyl hexanoate. Herein, we present the successful results of the first intentional application of S. jurei as a yeast for beer fermentation known to us and compare its fermentation performance to other species of the genus. Results indicate considerable potential for S. jurei application in brewing, with clear advantages compared to other wild Saccharomyces species.

Evidence for two main domestication trajectories in Saccharomyces cerevisiae linked to distinct bread-making processes

SummaryDespite bread being one of the most historically and culturally important fermented products, its history and influence on the evolution of associated microbial species remains largely unknown. The first evidence of leavened bread dates to the second millenium BCE in Egypt and since, the art of bread-making developed and spread worldwide. Nowadays, leavened bread is made either by using a pure commercial culture of the yeast Saccharomyces cerevisiae or by propagating a sourdough, which is a mix of flour and water spontaneously fermented by yeast and bacteria. We studied the domestication of S. cerevisiae populations originating from industry and sourdough and tested whether these different bread-making processes led to population divergence. We found that the origin of S. cerevisiae bakery strains is polyphyletic with 67 % of strains clustering in two main clades: most commercial strains were tetraploid and clustered with strains having diverse origins, including beer. By contrast, most sourdough strains were diploids and found in a second clade of strains having mosaic genomes and diverse origins including fruits, or clinical and wild environments. When compared to the others, sourdough strains harboured in average a higher copy number of genes involved in maltose utilization, a common sugar produced from dough starch. Overall, a high level of gene flow from multiple contributors was detected. Phenotyping of bakery and non bakery strains further showed that sourdough and industrial bakery populations have undergone human selection for rapidly starting fermentations and for high CO2 production. Interestingly, sourdough strains also showed a better adaptation to a sourdough mimicking environment, suggesting that natural selection occurred as well. In summary, our results revealed that the domestication of bakery yeast populations has been accompanied by dispersion, hybridization and divergent selection through industrial and artisanal bakery processes. In addition, they unveiled for the first time a case of fungus domestication where species divergence occurred through autotetraploidisation.

Proteomic Analyses of Intracellular Salmonella enterica Serovar Typhimurium Reveal Extensive Bacterial Adaptations to Infected Host Epithelial Cells

Salmonellaspecies can gain access into nonphagocytic cells, where the bacterium proliferates in a unique membrane-bounded compartment. In order to reveal bacterial adaptations to their intracellular niche, here we conducted the first comprehensive proteomic survey ofSalmonellaisolated from infected epithelial cells. Among ∼3,300 identified bacterial proteins, we found that about 100 proteins were significantly altered at the onset ofSalmonellaintracellular replication. In addition to substantially increased iron-uptake capacities, bacterial high-affinity manganese and zinc transporters were also upregulated, suggesting an overall limitation of metal ions in host epithelial cells. We also found thatSalmonellainduced multiple phosphate utilization pathways. Furthermore, our data suggested upregulation of the two-component PhoPQ system as well as of many downstream virulence factors under its regulation. Our survey also revealed that intracellularSalmonellahas increased needs for certain amino acids and biotin. In contrast,Salmonelladownregulated glycerol and maltose utilization as well as chemotaxis pathways.

Inactivation of the phosphoglucomutase gene pgm in Corynebacterium glutamicum affects cell shape and glycogen metabolism

In Corynebacterium glutamicum formation of glc-1-P (α-glucose-1-phosphate) from glc-6-P (glucose-6-phosphate) by α-Pgm (phosphoglucomutase) is supposed to be crucial for synthesis of glycogen and the cell wall precursors trehalose and rhamnose. Furthermore, Pgm is probably necessary for glycogen degradation and maltose utilization as glucan phosphorylases of both pathways form glc-1-P. We here show that C. glutamicum possesses at least two Pgm isoenzymes, the cg2800 (pgm) encoded enzyme contributing most to total Pgm activity. By inactivation of pgm we created C. glutamicum IMpgm showing only about 12% Pgm activity when compared to the parental strain. We characterized both strains during cultivation with either glucose or maltose as substrate and observed that (i) the glc-1-P content in the WT (wild-type) and the mutant remained constant independent of the carbon source used, (ii) the glycogen levels in the pgm mutant were lower during growth on glucose and higher during growth on maltose, and (iii) the morphology of the mutant was altered with maltose as a substrate. We conclude that C. glutamicum employs glycogen as carbon capacitor to perform glc-1-P homeostasis in the exponential growth phase and is therefore able to counteract limited Pgm activity for both anabolic and catabolic metabolic pathways.

Obacunone Represses Salmonella Pathogenicity Islands 1 and 2 in anenvZ-Dependent Fashion

ABSTRACTObacunone belongs to a class of unique triterpenoids called limonoids, present inCitrusspecies. Previous studies from our laboratory suggested that obacunone possesses antivirulence activity and demonstrates inhibition of cell-cell signaling inVibrio harveyiandEscherichia coliO157:H7. The present work sought to determine the effect of obacunone on the food-borne pathogenSalmonella entericaserovar Typhimurium LT2 by using a cDNA microarray. Transcriptomic studies indicated that obacunone repressesSalmonellapathogenicity island 1 (SPI1), the maltose transporter, and the hydrogenase operon. Furthermore, phenotypic data for the Caco-2 infection assay and maltose utilization were in agreement with microarray data suggesting repression of SPI1 and maltose transport. Further studies demonstrated that repression of SPI1 was plausibly mediated throughhilA. Additionally, obacunone seems to repress SPI2 under SPI2-inducing conditions as well as in Caco-2 infection models. Furthermore, obacunone seems to represshilAin an EnvZ-dependent fashion. Altogether, the results of the study seems to suggest that obacunone exerts an antivirulence effect onS.Typhimurium and may serve as a lead compound for development of antivirulence strategies forS.Typhimurium.