Adaptation of Anaerobic Digestion Microbial Communities to High Ammonium Levels: Insights from Strain-Resolved Metagenomics.

Ammonia release from proteinaceous feedstocks represents the main inhibitor of the anaerobic digestion (AD) process, which can result in a decreased biomethane yield or even complete failure of the process. The present study focused on the adaptation of mesophilic AD communities to a stepwise increase in the concentration of ammonium chloride in synthetic medium with casein used as the carbon source. An adaptation process occurring over more than 20 months allowed batch reactors to reach up to 20 g of NH4+ N/L without collapsing in acidification nor ceasing methane production. To decipher the microbial dynamics occurring during the adaptation and determine the genes mostly exposed to selective pressure, a combination of biochemical and metagenomics analyses was performed, reconstructing the strains of key species and tracking them over time. Subsequently, the adaptive metabolic mechanisms were delineated by following the single nucleotide variants (SNVs) characterizing the strains and prioritizing the associated genes according to their function. An in-depth exploration of the archaeon Methanoculleus bourgensis vb3066 and the putative syntrophic acetate-oxidizing bacteria Acetomicrobium sp. ma133 identified positively selected SNVs on genes involved in stress adaptation. The intraspecies diversity with multiple coexisting strains in a temporal succession pattern allows us to detect the presence of an additional level of diversity within the microbial community beyond the species level.

Decoding Microbial Responses to Ammonia Shock Loads in Biogas Reactors through Metagenomics and Metatranscriptomics.

The presence of elevated ammonia levels is widely recognized as a significant contributor to process inhibition in biogas production, posing a common challenge for biogas plant operators. The present study employed a combination of biochemical, genome-centric metagenomic and metatranscriptomic data to investigate the response of the biogas microbiome to two shock loads induced by single pulses of elevated ammonia concentrations (i.e., 1.5 g NH4+/LR and 5 g NH4+/LR). The analysis revealed a microbial community of high complexity consisting of 364 Metagenome Assembled Genomes (MAGs). The hydrogenotrophic pathway was the primary route for methane production during the entire experiment, confirming its efficiency even at high ammonia concentrations. Additionally, metatranscriptomic analysis uncovered a metabolic shift in the methanogens Methanothrix sp. MA6 and Methanosarcina flavescens MX5, which switched their metabolism from the acetoclastic to the CO2 reduction route during the second shock. Furthermore, multiple genes associated with mechanisms for maintaining osmotic balance in the cell were upregulated, emphasizing the critical role of osmoprotection in the rapid response to the presence of ammonia. Finally, this study offers insights into the transcriptional response of an anaerobic digestion community, specifically focusing on the mechanisms involved in recovering from ammonia-induced stress.

Magnetite Alters the Metabolic Interaction between Methanogens and Sulfate-Reducing Bacteria.

It is known that the presence of sulfate decreases the methane yield in the anaerobic digestion systems. Sulfate-reducing bacteria can convert sulfate to hydrogen sulfide competing with methanogens for substrates such as H2 and acetate. The present work aims to elucidate the microbial interactions in biogas production and assess the effectiveness of electron-conductive materials in restoring methane production after exposure to high sulfate concentrations. The addition of magnetite led to a higher methane content in the biogas and a sharp decrease in the level of hydrogen sulfide, indicating its beneficial effects. Furthermore, the rate of volatile fatty acid consumption increased, especially for butyrate, propionate, and acetate. Genome-centric metagenomics was performed to explore the main microbial interactions. The interaction between methanogens and sulfate-reducing bacteria was found to be both competitive and cooperative, depending on the methanogenic class. Microbial species assigned to the Methanosarcina genus increased in relative abundance after magnetite addition together with the butyrate oxidizing syntrophic partners, in particular belonging to the Syntrophomonas genus. Additionally, Ruminococcus sp. DTU98 and other species assigned to the Chloroflexi phylum were positively correlated to the presence of sulfate-reducing bacteria, suggesting DIET-based interactions. In conclusion, this study provides new insights into the application of magnetite to enhance the anaerobic digestion performance by removing hydrogen sulfide, fostering DIET-based syntrophic microbial interactions, and unraveling the intricate interplay of competitive and cooperative interactions between methanogens and sulfate-reducing bacteria, influenced by the specific methanogenic group.

How multiple farming conditions correlate with the composition of the raw cow's milk lactic microbiome.

Questionnaires on farming conditions were retrieved from 2129 dairy farms and clustered, resulting in 106 representative raw cow's milk samples analysed in winter and summer. Substantiating the efficiency of our survey, some farming conditions affected the milk physicochemical composition. Culturing identified several species of lactic acid bacteria (LAB) per milk, whose number increased through 16S ribosomal RNA (rRNA) gene sequencing and shotgun metagenome analyses. Season, indoor versus outdoor housing, cow numbers, milk substitutes, ratio cattle/rest area, house care system during lactation, and urea and medium-chain fatty acids correlated with the overall microbiome composition and the LAB diversity within it. Shotgun metagenome detected variations in gene numbers and uniqueness per milk. LAB functional pathways differed among milk samples. Focusing on amino acid metabolisms and matching the retrieved annotated genes versus non-starter lactic acid bacteria (NSLAB) references from KEGG and corresponding to those identified, all samples had the same gene spectrum for each pathway. Conversely, gene redundancy varied among samples and agreed with NSLAB diversity. Milk samples with higher numbers of NSLAB species harboured higher number of copies per pathway, which would enable steady-state towards perturbations. Some farming conditions, which affected the microbiome richness, also correlated with the NSLAB composition and functionality.

Genome-Centric Metatranscriptomics Analysis Reveals the Role of Hydrochar in Anaerobic Digestion of Waste Activated Sludge.

Anaerobic digestion (AD) of waste activated sludge (WAS) has been widely used, while it poses problems including low methane yield and production rate. Hydrochar is produced by hydrothermal liquefaction of biomass; however, little is known about the role of hydrochar in promoting AD of WAS. The present study showed that hydrochar increased the methane production rate by 30.8% and yield by 31.4% of hydrothermal pretreated dewatered WAS. Hydrochar increased the methane production rate and yield by enhancing the acidification and methanogenesis processes. Genomic-centric metatranscriptomics were used to identify the metabolic activities and transcriptomic response of individual metagenome-assembled genomes that were enriched by hydrochar. Although Methanosarcina sp. FDU0106 had been shown unable to used H2, it had the complete pathway for the reduction of CO2 to methane. Syntrophomonas sp. FDU0164 expressed genes for extracellular electron transfer via electrically pili, suggesting that Syntrophomonas sp. FDU0164 and Methanosarcina sp. FDU0106 were exchanging electrons via direct interspecies electron transfer. The expression of pili was decreased, indicating that hydrochar could replace its roles. Additionally, Firmicutes sp. FDU0048, Proteiniphilum sp. FDU0082, and Aminobacterium mobile FDU0089 were related to the degradation of organics, which could be related to the enhanced methane yield.

Large-scale sequencing and comparative analysis of oenological Saccharomyces cerevisiae strains supported by nanopore refinement of key genomes.

Saccharomyces cerevisiae has long been part of human activities related to the production of food and wine. The industrial demand for fermented beverages with well-defined and stable characteristics boosted the isolation and selection of strains conferring a distinctive aroma profile to the final product. To uncover variants characterizing oenological strains, the sequencing of 65 new S. cerevisiae isolates, and the comparison with other 503 publicly available genomes were performed. A hybrid approach based on short Illumina and long Oxford Nanopore reads allowed the in-depth investigation of eleven genomes and the identification of putative laterally transferred regions and structural variants. A comparative analysis between clusters of strains belonging to different datasets allowed the identification of novel relevant genetic features including single nucleotide polymorphisms, insertions and structural variants. Detection of oenological single nucleotide variants shed light on the existence of different levels of modulation for the mevalonate pathway relevant for the biosynthesis of aromatic compounds.

Revealing metabolic mechanisms of interaction in the anaerobic digestion microbiome by flux balance analysis.

Anaerobic digestion is a key biological process for renewable energy, yet the mechanistic knowledge on its hidden microbial dynamics is still limited. The present work charted the interaction network in the anaerobic digestion microbiome via the full characterization of pairwise interactions and the associated metabolite exchanges. To this goal, a novel collection of 836 genome-scale metabolic models was built to represent the functional capabilities of bacteria and archaea species derived from genome-centric metagenomics. Dominant microbes were shown to prefer mutualistic, parasitic and commensalistic interactions over neutralism, amensalism and competition, and are more likely to behave as metabolite importers and profiteers of the coexistence. Additionally, external hydrogen injection positively influences microbiome dynamics by promoting commensalism over amensalism. Finally, exchanges of glucogenic amino acids were shown to overcome auxotrophies caused by an incomplete tricarboxylic acid cycle. Our novel strategy predicted the most favourable growth conditions for the microbes, overall suggesting strategies to increasing the biogas production efficiency. In principle, this approach could also be applied to microbial populations of biomedical importance, such as the gut microbiome, to allow a broad inspection of the microbial interplays.

The impact of CUP1 gene copy-number and XVI-VIII/XV-XVI translocations on copper and sulfite tolerance in vineyard Saccharomyces cerevisiae strain populations.

In wine production, sulfites are widely used as antimicrobials and antioxidants, whereas copper is associated with fungicides and wine fining treatments. Therefore, wine yeasts are constantly exposed to these agents. Copper tolerance is related to the copy number of the CUP1 gene, encoding for a metallothionein involved in copper detoxification. In wine yeasts, sulfite resistance mainly depends on the presence of the translocation t(XVI;VIII) in the promoter region of the SSU1 gene. This gene encodes for a plasma membrane sulfite pump involved in sulfite metabolism and detoxification. Recently, a new translocation, t(XVI;VIII), was identified. In this work, 253 Saccharomyces cerevisiae strains, representing three vineyard populations from two different continents, were analyzed, along with 20 industrial starters. Copper and sulfites tolerance as well as distribution of CUP1 gene copy-number, t(XVI;VIII)and t(XVI;XV) of SSU1 gene were studied to evaluate the impact of these genomic variations on population phenotypes. The CUP1 gene copy-number was found to be highly variable, ranging from zero to 79 per strain. Moreover it differently impacted the copper tolerance in the populations of the two continents. The diffusion of t(XVI;VIII) and, for the first time, t(XVI;XV) was determined in the three vineyard populations. The correlation between the presence of the translocation and strain sulfite tolerance levels was significant only for the t(XVI;VIII).

Insights into Ammonia Adaptation and Methanogenic Precursor Oxidation by Genome-Centric Analysis.

Ammonia released from the degradation of protein and/or urea usually leads to suboptimal anaerobic digestion (AD) when N-rich organic waste is used. However, the insights behind the differential ammonia tolerance of anaerobic microbiomes remain an enigma. In this study, the cultivation in synthetic medium with different carbon sources (acetate, methanol, formate, and H2/CO2) shaped a common initial inoculum into four unique ammonia-tolerant syntrophic populations. Specifically, various levels of ammonia tolerance were observed: consortia fed with methanol and H2/CO2 could grow at ammonia levels up to 7.25 g NH+-N/L, whereas the other two groups (formate and acetate) only thrived at 5.25 and 4.25 g NH+-N/L, respectively. Metabolic reconstruction highlighted that this divergent microbiome might be achieved by complementary metabolisms to maximize biomethane recovery from carbon sources, thus indicating the importance of the syntrophic community in the AD of N-rich substrates. Besides, sodium/proton antiporter operon, osmoprotectant/K+ regulator, and osmoprotectant synthesis operon may function as the main drivers of adaptation to the ammonia stress. Moreover, energy from the substrate-level phosphorylation and multiple energy-converting hydrogenases (e.g., Ech and Eha) could aid methanogens to balance the energy request for anabolic activities and contribute to thriving when exposed to high ammonia levels.

Interplay between gut microbiota and p66Shc affects obesity-associated insulin resistance.

The 66 kDa isoform of the mammalian Shc gene promotes adipogenesis, and p66Shc-/- mice accumulate less body weight than wild-type (WT) mice. As the metabolic consequences of the leaner phenotype of p66Shc-/- mice is debated, we hypothesized that gut microbiota may be involved. We confirmed that p66Shc-/- mice gained less weight than WT mice when on a high-fat diet (HFD), but they were not protected from insulin resistance and glucose intolerance. p66Shc deletion significantly modified the composition of gut microbiota and their modification after an HFD. This was associated with changes in gene expression of Il-1b and regenerating islet-derived protein 3 γ ( Reg3g) in the gut and in systemic trimethylamine N-oxide and branched chain amino acid levels, despite there being no difference in intestinal structure and permeability. Depleting gut microbiota at the end of HFD rendered both strains more glucose tolerant but improved insulin sensitivity only in p66Shc-/- mice. Microbiota-depleted WT mice cohoused with microbiota-competent p66Shc-/- mice became significantly more insulin resistant than WT mice cohoused with WT mice, despite no difference in weight gain. These findings reconcile previous inconsistent observations on the metabolic phenotype of p66Shc-/- mice and illustrate the complex microbiome-host-genotype interplay under metabolic stress.-Ciciliot, S., Albiero, M., Campanaro, S., Poncina, N., Tedesco, S., Scattolini, V., Dalla Costa, F., Cignarella, A., Vettore, M., Di Gangi, I. M., Bogialli, S., Avogaro, A., Fadini, G. P. Interplay between gut microbiota and p66Shc affects obesity-associated insulin resistance.

Spatial Distribution and Diverse Metabolic Functions of Lignocellulose-Degrading Uncultured Bacteria as Revealed by Genome-Centric Metagenomics.

The mechanisms by which specific anaerobic microorganisms remain firmly attached to lignocellulosic material, allowing them to efficiently decompose organic matter, have yet to be elucidated. To circumvent this issue, microbiomes collected from anaerobic digesters treating pig manure and meadow grass were fractionated to separate the planktonic microbes from those adhered to lignocellulosic substrate. Assembly of shotgun reads, followed by a binning process, recovered 151 population genomes, 80 out of which were completely new and were not previously deposited in any database. Genome coverage allowed the identification of microbial spatial distribution in the engineered ecosystem. Moreover, a composite bioinformatic analysis using multiple databases for functional annotation revealed that uncultured members of the Bacteroidetes and Firmicutes follow diverse metabolic strategies for polysaccharide degradation. The structure of cellulosome in Firmicutes species can differ depending on the number and functional roles of carbohydrate-binding modules. In contrast, members of the Bacteroidetes are able to adhere to and degrade lignocellulose due to the presence of multiple carbohydrate-binding family 6 modules in beta-xylosidase and endoglucanase proteins or S-layer homology modules in unknown proteins. This study combines the concept of variability in spatial distribution with genome-centric metagenomics, allowing a functional and taxonomical exploration of the biogas microbiome.IMPORTANCE This work contributes new knowledge about lignocellulose degradation in engineered ecosystems. Specifically, the combination of the spatial distribution of uncultured microbes with genome-centric metagenomics provides novel insights into the metabolic properties of planktonic and firmly attached to plant biomass bacteria. Moreover, the knowledge obtained in this study enabled us to understand the diverse metabolic strategies for polysaccharide degradation in different species of Bacteroidetes and Clostridiales Even though structural elements of cellulosome were restricted to Clostridiales species, our study identified a putative mechanism in Bacteroidetes species for biomass decomposition, which is based on a gene cluster responsible for cellulose degradation, disaccharide cleavage to glucose, and transport to cytoplasm.

Whole genome comparison of two Starmerella bacillaris strains with other wine yeasts uncovers genes involved in modulating important winemaking traits.

Starmerella bacillaris is an osmotolerant yeast with interesting winemaking traits such as low-ethanol and high-glycerol production, previously considered as wine spoilage and recently proposed to improve the sensory quality of wine. This is the first work performing a whole-genome analysis of the variants identified by comparing two S. bacillaris strains (PAS13 and FRI751). Additionally, an extensive search for orthologous genes against Saccharomyces and non-Saccharomyces yeasts produced a detailed reconstruction of the pan-genome for yeast species used in winemaking. Starmerella bacillaris PAS13 was able to produce 36% more glycerol than S. bacillaris FRI751 without increasing ethanol level over 5% (v/v). Orthologous genes revealed new insights in the response to osmotic stress determined by the mitogen-activated protein kinase (MAPK) from S. bacillaris strains. The comparison between the two S. bacillaris genomes revealed 33 771 high-quality variants that were ranked considering their predicted impact on gene functions. Furthermore, analysis of structural variations in the genome revealed five translocations. The absence of some transcriptional factors involved in the regulation of GPD (glycerol-3-phosphate dehydrogenase), like the protein kinases YpK1p and YpK2p, and the identification of a tandem duplication increasing the GPP1 (glycerol-3-phosphate phosphatase) gene copy number suggest a remarkably different regulation of the glycerol pathway for S. bacillaris in comparison to S. cerevisiae.

Microbial community changes in methanogenic granules during the transition from mesophilic to thermophilic conditions.

Upflow anaerobic sludge blanket (UASB) reactor is one of the most applied technologies for various high-strength wastewater treatments. The present study analysed the microbial community changes in UASB granules during the transition from mesophilic to thermophilic conditions. Dynamicity of microbial community in granules was analysed using high-throughput sequencing of 16S ribosomal RNA gene amplicons, and the results showed that the temperature strictly determines the diversity of the microbial consortium. It was demonstrated that most of the microbes which were present in the initial mesophilic community were not found in the granules after the transition to thermophilic conditions. More specifically, only members from family Anaerolinaceae managed to tolerate the temperature change and contributed in maintaining the physical integrity of granular structure. On the contrary, new hydrolytic and fermentative bacteria were quickly replacing the old members in the community. A direct result from this abrupt change in the microbial diversity was the accumulation of volatile fatty acids and the concomitant pH drop in the reactor inhibiting the overall anaerobic digestion process. Nevertheless, by maintaining deliberately the pH levels at values higher than 6.5, a methanogen belonging to Methanoculleus genus emerged in the community enhancing the methane production.

Genome comparison and physiological characterization of eight Streptococcus thermophilus strains isolated from Italian dairy products.

Eight Streptococcus thermophilus strains of dairy origin isolated in Italy were chosen to investigate autochthonous bacterial diversity in this important technological species. In the present study a comparative analysis of all the 17 S. thermophilus genomes publicly available was performed to identify the core and the variable genes, which vary among strains from 196 to 265. Additionally, correlation between the isolation site and the genetic distance was investigated at genomic level. Results highlight that the phylogenetic reconstruction differs from the geographical strain distribution. Moreover, strain M17PTZA496 has a genome of 2.15 Mbp, notably larger than that of the others, determined by lateral gene transfer (including phage-mediated incorporation) and duplication events. Important technological characters, such as growth kinetics, bacteriocin production, acidification kinetics and surface adhesion capability were studied in all the Italian strains. Results indicate a wide range of variability in adhesion properties that significantly clustered strains into four groups. Genomic differences among strains in relation to these characters were identified but a clear correlation between genotype and phenotype was not always found since most of the genomic modifications arise from single nucleotide polymorphisms. This research represents a step forward in the identification of strains-specific functions in Streptococcus thermophilus and it has also the potential to provide valuable information to predict strain specific behaviors in industrial processes.

Characterization of the planktonic microbiome in upflow anaerobic sludge blanket reactors during adaptation of mesophilic methanogenic granules to thermophilic operational conditions.

Upflow anaerobic sludge blanket (UASB) technology refers to reactor technology where granules, i.e. self-immobilised microbial associations, are the biological catalysts involved in the anaerobic digestion process. During the start-up period, UASB reactors operate at relatively long HRT and therefore the liquid phase of the reactor becomes a favourable environment for microbial growth. The current study aimed to elucidate the dynamicity of the suspended microbial community in UASB reactors, during the transition from mesophilic to thermophilic conditions. High throughput 16S rRNA amplicon sequencing was used to characterize the taxonomic composition of the microbiome. The results showed that the microbial community was mainly composed by hydrolytic and fermentative bacteria. Results revealed relevant shifts in the microbial community composition, which is mainly determined by the operational conditions and the reactor performance. Finally, shared OTUs between the microbial consortia of the suspended and the granular sludge showed that planktonic microbiota is significantly influencing the granule microbial community composition.

A novel archaeal species belonging to Methanoculleus genus identified via de-novo assembly and metagenomic binning process in biogas reactors.

Recently, a first comprehensive catalogue of microbial genomes populating biogas reactors treating manure and agro-industrial residues was determined by sequencing samples collected from 22 biogas reactors including laboratory and full scale. Among the archaeal community, one of the most abundant methanogens belongs to Methanoculleus genus and for this reason it was provisionally named Methanoculleus sp. DTU006. Its full length 16S rRNA sequence is 97% similar to Methanoculleus marisnigri JR1 and to Methanoculleus palmolei DSM 4273. Despite the high similarity of the 16S gene sequence, Average Nucleotide Identity calculation (ANI) calculated on all protein encoding genes indicated that the two most similar species, Methanoculleus bourgensis MS2T and Methanoculleus sp. MAB1, are divergent enough to define Methanoculleus sp. DTU006 as new archaeal species. Its genome (2.15 Mbp) has an estimated completeness around 93%. Analysis of the metabolic pathways using KEGG confirmed that it is a hydrogenotrophic methanogen and therefore it is proposed the Candidatus status by naming it as "Candidatus Methanoculleus thermohydrogenotrophicum".

Untangling the Effect of Fatty Acid Addition at Species Level Revealed Different Transcriptional Responses of the Biogas Microbial Community Members.

In the present study, RNA-sequencing was used to elucidate the change of anaerobic digestion metatranscriptome after long chain fatty acids (oleate) exposure. To explore the general transcriptional behavior of the microbiome, the analysis was first performed on shotgun reads without considering a reference metagenome. As a second step, RNA reads were aligned on the genes encoded by the microbial community, revealing the expression of more than 51 000 different transcripts. The present study is the first research which was able to dissect the transcriptional behavior at a single species level by considering the 106 microbial genomes previously identified. The exploration of the metabolic pathways confirmed the importance of Syntrophomonas species in fatty acids degradation, and also highlighted the presence of protective mechanisms toward the long chain fatty acid effects in bacteria belonging to Clostridiales, Rykenellaceae, and in species of the genera Halothermothrix and Anaerobaculum. Additionally, an interesting transcriptional activation of the chemotaxis genes was evidenced in seven species belonging to Clostridia, Halothermothrix, and Tepidanaerobacter. Surprisingly, methanogens revealed a very versatile behavior different from each other, even among similar species of the Methanoculleus genus, while a strong increase of the expression level in Methanosarcina sp. was evidenced after oleate addition.

Different mechanisms of resistance modulate sulfite tolerance in wine yeasts.

From a technological point of view, yeast resistance to sulfite is of great interest and represents an important technological character for winemaking. Several mechanisms are involved, and strain-dependent strategies to obtain SO2 resistance can deeply influence wine quality, although this choice is less relevant in determining the technological performance of the strain during fermentation. In this study, to better understand the strain-specific mechanisms of resistance, 11 Saccharomyces cerevisiae strains, whose genomes have been previously sequenced, were selected. Their attitude towards sulfites, in terms of resistance and production, was evaluated, and RNA-sequencing of four selected strains was performed during fermentation process in synthetic grape must in the presence of SO2. Results demonstrated that at molecular level, the physical effect of SO2 triggered multiple stress responses in the cell and high tolerance to general enological stressing condition increased SO2 resistance. Adaptation mechanism due to high basal gene expression level rather than specific gene induction in the presence of sulfite seemed to be responsible in modulating strain resistance. This mechanism involved higher basal gene expression level of specific cell wall proteins, enzymes for lipid biosynthesis, and enzymes directly involved in SO2 assimilation pathway and efflux.

Transcriptome structure variability in Saccharomyces cerevisiae strains determined with a newly developed assembly software.

BACKGROUND: RNA-seq studies have an important role for both large-scale analysis of gene expression and for transcriptome reconstruction. However, the lack of software specifically developed for the analysis of the transcriptome structure in lower eukaryotes, has so far limited the comparative studies among different species and strains. RESULTS: In order to fill this gap, an innovative software called ORA (Overlapped Reads Assembler) was developed. This software allows a simple and reliable analysis of the transcriptome structure in organisms with a low number of introns. It can also determine the size and the position of the untranslated regions (UTR) and of polycistronic transcripts. As a case study, we analyzed the transcriptional landscape of six S. cerevisiae strains in two different key steps of the fermentation process. This comparative analysis revealed differences in the UTR regions of transcripts. By extending the transcriptome analysis to yeast species belonging to the Saccharomyces genus, it was possible to examine the conservation level of unknown non-coding RNAs and their putative functional role. CONCLUSIONS: By comparing the results obtained using ORA with previous studies and with the transcriptome structure determined with other software, it was proven that ORA has a remarkable reliability. The results obtained from the training set made it possible to detect the presence of transcripts with variable UTRs between S. cerevisiae strains. Finally, we propose a regulatory role for some non-coding transcripts conserved within the Saccharomyces genus and localized in the antisense strand to genes involved in meiosis and cell wall biosynthesis.

The impact of genomic variability on gene expression in environmental Saccharomyces cerevisiae strains.

Environmental Saccharomyces cerevisiae strains are crucially important, as they represent the large pool from which domesticated industrial yeasts have been selected, and vineyard strains can be considered the genetic reservoir from which industrial wine strains with strong fermentative behaviour are selected. Four vineyard strains with different fermentation performances were chosen from a large collection of strains isolated from Italian vineyards. Their genomes were sequenced to identify how genetic variations influence gene expression during fermentation and to clarify the evolutionary relationship between vineyard isolates and industrial wine strains. RNA sequencing was performed on the four vineyard strains, as well as on the industrial wine yeast strain EC1118 and on the laboratory strain S288c, at two stages of fermentation. We showed that there was a large gene cluster with variable promoter regions modifying gene expression in the strains. Our results indicate that it is the evolvability of the yeast promoter regions, rather than structural variations or strain-specific genes, that is the main cause of the differences in gene expression. This promoter variability, determined by variable tandem repeats and a high number of single-nucleotide polymorphisms together with 49 differentially expressed transcription factors, explained the strong phenotypic differences in the strains.