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Background Carbon fixation through biological methanation has emerged as a promising technology to produce renewable energy in the context of the circular economy. The anaerobic digestion microbiome is the fundamental biological system operating biogas upgrading and is paramount in power-to-gas conversion. Carbon dioxide (CO2) methanation is frequently performed by microbiota attached to solid supports generating biofilms. Despite the apparent simplicity of the microbial community involved in biogas upgrading, the dynamics behind most of the interspecies interaction remain obscure. To understand the role of the microbial species in CO2 fixation, the biofilm generated during the biogas upgrading process has been selected as a case study. The present work investigates via genome-centric metagenomics, based on a hybrid Nanopore-Illumina approach the biofilm developed on the diffusion devices of four ex situ biogas upgrading reactors. Moreover, genome-guided metabolic reconstruction and flux balance analysis were used to propose a biological role for the dominant microbes. Results The combined microbiome was composed of 59 species, with five being dominant (> 70% of total abundance); the metagenome-assembled genomes representing these species were refined to reach a high level of completeness. Genome-guided metabolic analysis appointed Firmicutes sp. GSMM966 as the main responsible for biofilm formation. Additionally, species interactions were investigated considering their co-occurrence in 134 samples, and in terms of metabolic exchanges through flux balance simulation in a simplified medium. Some of the most abundant species (e.g., Limnochordia sp. GSMM975) were widespread (~ 67% of tested experiments), while others (e.g., Methanothermobacter wolfeii GSMM957) had a scattered distribution. Genome-scale metabolic models of the microbial community were built with boundary conditions taken from the biochemical data and showed the presence of a flexible interaction network mainly based on hydrogen and carbon dioxide uptake and formate exchange. Conclusions Our work investigated the interplay between five dominant species within the biofilm and showed their importance in a large spectrum of anaerobic biogas reactor samples. Flux balance analysis provided a deeper insight into the potential syntrophic interaction between species, especially Limnochordia sp. GSMM975 and Methanothermobacter wolfeii GSMM957. Finally, it suggested species interactions to be based on formate and amino acids exchanges.

BackgroundAntarctica is the coldest and driest continent on Earth, characterized by polyextreme environmental conditions, where species adapted to grow and thrive at low values of temperature, low nutrient concentration and relative humidity and high UV radiation form complex networks of interactions. Microbial communities growing in these harsh environments can form biofilms that help the associated species to survive in such harsh conditions. The knowledge on biofilms microbial community in the literature is extensive, however, most studies are focused on the dominant species and are not able to disclose the whole functional complexity and entire metabolic potential. MethodsTo overcome these limitations, the present study used genome-centric metagenomics and metatranscriptomics to analyze two biofilm samples collected in Deception Island, Maritime Antarctica. Species abundance and their functional activity were correlated with environmental parameters. Differential gene expression analysis based on the negative binomial distribution of the DeSeq2 package was used to compare the abundance of metagenome-assembled genome (MAG) in the biofilms (BR and BG) and the gene expression of the metatranscriptomic analysis (p-value 0.05).ResultsThe results showed a complex microbiome represented by 180 MAGs, highly divergent according to temperature, pH, and position along a transect of Whalers Bay sediments. Metabolic evaluations allowed to predict polyphenols and chitin as primary substrate feeding. The potential metabolic interactions were investigated using metabolic flux balance analysis and revealed that purple bacteria are the taxonomic group with the highest number of correlations with other bacteria. This behavior is well represented by the MAG WB_595 belonging to Alphaproteobacteria. ConclusionsDue to predicted mixothrophic behavior of Alaproteobacteria_WB_595 MAG, it may represent a crucial member of the microbiome, and it has the potential to support the heterotrophic species in the biofilms. Metatranscriptomics revealed that the chaperone system and proteins counteracting reactive oxygen species and toxic compounds have a key role in the maintenance of bacterial cell homeostasis in sediments of volcanic origin.

Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route.

Questionnaires on farming conditions were retrieved from 2,129 dairy farms and clustered, resulting in 106 representative raw cow`s milk samples analyzed 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 rRNA gene sequencing and shotgun metagenome analyses. Season, indoor vs. 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 vs. 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 harbored 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. This article is protected by copyright. All rights reserved.

The Euganean Thermal District has been known since Roman times for the therapeutic properties of peloids, obtained from natural clays that have undergone a traditional maturation process. This leads to the growth of a green microbial biofilm with Cyanobacteria and the target species Phormidium sp. ETS-05 as fundamental components for their ability to synthetize anti-inflammatory molecules. Currently, in-depth studies on the microbiota colonizing Euganean peloids, as in general on peloids utilized worldwide, are missing. This is the first characterization of the microbial community of Euganean thermal muds, also investigating the effects of environmental factors on its composition. We analysed 53 muds from 29 sites (Spas) using a polyphasic approach, finding a stable microbiota peculiar to the area. Differences among mud samples mainly depended on two parameters: water temperature and shading of mud maturation plants. In the range 37&ndash, 47 &deg, C and in the case of irradiance attenuation due to the presence of protective roofs, a statistically significant higher mud Chl a content was detected. Moreover, in these conditions, a characteristic microbial and Cyanobacteria population composition dominated by Phormidium sp. ETS-05 was observed. We also obtained the complete genome sequence of this target species using a mixed sequencing approach based on Illumina and Nanopore sequencing.

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).

Methanogenesis, a biological process mediated by complex microbial communities, has attracted great attention due to its contribution to global warming and potential in biotechnological applications. The current study unveiled the core microbial methanogenic metabolisms in anaerobic vessel ecosystems by applying combined genome-centric metagenomics and metatranscriptomics. Here, we demonstrate that an enriched natural system, fueled only with acetate, could support a bacteria-dominated microbiota employing a multi-trophic methanogenic process. Moreover, significant changes, in terms of microbial structure and function, were recorded after the system was supplemented with additional H2. Methanosarcina thermophila, the predominant methanogen prior to H2 addition, simultaneously performed acetoclastic, hydrogenotrophic, and methylotrophic methanogenesis. The methanogenic pattern changed after the addition of H2, which immediately stimulated Methanomicrobia-activity and was followed by a slow enrichment of Methanobacteria members. Interestingly, the essential genes involved in the Wood-Ljungdahl pathway were not expressed in bacterial members. The high expression of a glycine cleavage system indicated the activation of alternative metabolic pathways for acetate metabolism, which were reconstructed in the most abundant bacterial genomes. Moreover, as evidenced by predicted auxotrophies, we propose that specific microbes of the community were forming symbiotic relationships, thus reducing the biosynthetic burden of individual members. These results provide new information that will facilitate future microbial ecology studies of interspecies competition and symbiosis in methanogenic niches.

Non-conventional yeasts are increasingly applied in fermented beverage industry to obtain distinctive products with improved quality. Among these yeasts, Lachancea thermotolerans has multiple features of industrial relevance, especially the production of l(+)-lactic acid (LA), useful for the biological acidification of wine and beer. Since few information is available on this peculiar activity, the current study aimed to explore the physiological and genetic variability among L. thermotolerans strains. From a strain collection, mostly isolated from wine, a huge phenotypic diversity was acknowledged and allowed the selection of a high (SOL13) and a low (COLC27) LA producer. Comparative whole-genome sequencing of these two selected strains and the type strain CBS 6340T showed a high similarity in terms of gene content and functional annotation. Notwithstanding, target gene-based analysis revealed variations between high and low producers in the key gene sequences related to LA accumulation. More in-depth investigation of the core promoters and expression analysis of the genes ldh, encoding lactate dehydrogenase, indicated the transcriptional regulation may be the principal cause behind phenotypic differences. These findings highlighted the usefulness of whole-genome sequencing coupled with expression analysis. They provided crucial genetic insights for a deeper investigation of the intraspecific variability in LA production pathway.

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.

Lactobacillus helveticus belongs to the large group of lactic acid bacteria (LAB), which are the major players in the fermentation of a wide range of foods. LAB are also present in the human gut, which has often been exploited as a reservoir of potential novel probiotic strains, but several parameters need to be assessed before establishing their safety and potential use for human consumption. In the present study, six L. helveticus strains isolated from natural whey cultures were analyzed for their phenotype and genotype in exopolysaccharide (EPS) production, low pH and bile salt tolerance, bile salt hydrolase (BSH) activity, and antibiotic resistance profile. In addition, a comparative genomic investigation was performed between the six newly sequenced strains and the 51 publicly available genomes of L. helveticus to define the pangenome structure. The results indicate that the newly sequenced strain UC1267 and the deposited strain DSM 20075 can be considered good candidates for gut-adapted strains due to their ability to survive in the presence of 0.2% glycocholic acid (GCA) and 1% taurocholic and taurodeoxycholic acid (TDCA). Moreover, these strains had the highest bile salt deconjugation activity among the tested L. helveticus strains. Considering the safety profile, none of these strains presented antibiotic resistance phenotypically and/or at the genome level. The pangenome analysis revealed genes specific to the new isolates, such as enzymes related to folate biosynthesis in strains UC1266 and UC1267 and an integrated phage in strain UC1035. Finally, the presence of maltose-degrading enzymes and multiple copies of 6-phospho-β-glucosidase genes in our strains indicates the capability to metabolize sugars other than lactose, which is related solely to dairy niches.

Here the draft genome sequence of Lactobacillus paracasei strain DTA83, isolated from stools of healthy infants in Rio de Janeiro (Brazil), is reported. The 2.8-Mb genome possesses 2825 protein-coding sequences distributed on 330 SEED subsystems. This strain belongs to a set of potentially probiotic Lactobacillus spp. strains used to study genetic factors related to antibiotic resistance after stress conditions, such as simulated gastrointestinal conditions. The complete genome data have been deposited in GenBank under the accession number QRBH00000000 , https://www.ncbi.nlm.nih.gov/nuccore/ QRBH00000000 .

Background The expansion of renewable energy produced by windmills and photovoltaic panels has generated a considerable electricity surplus, which can be utilized in water electrolysis systems for hydrogen production. The resulting hydrogen can then be funneled to anaerobic digesters for biogas upgrading (biomethanation) purposes (power-to-methane) or to produce high value-added compounds such as short-chain fatty acids (power-to-chemicals). Genome-centric metagenomics and metatranscriptomic analyses were performed to better understand the metabolic dynamics associated with H2 injection in two different configurations of anaerobic digesters treating acidic wastes, specifically cheese manufacturing byproducts. These approaches revealed the key-genes involved in methanation and carbon fixation pathways at species level. Results The biogas upgrading process in the single-stage configuration increased the CH4 content by 7%. The dominant methanogenic species responsible for the upregulation of the hydrogenotrophic pathway in this reactor was Methanothermobacter wolfeii UC0008. In the two-stage configuration, H2 injection induced an upregulation of CO2 fixation pathways producing short-chain fatty acids, mainly acetate and butyrate. In this configuration, the abundant species Anaerobaculum hydrogeniformans UC0046 and Defluviitoga tunisiensis UC0050 primarily upregulated genes related to electron transport chains, suggesting putative syntrophisms with hydrogen scavenger microbes. Interestingly, Tepidanaerobacter acetatoxydans UC0018 did not act as an acetate-oxidizer in either reactor configurations, and instead regulated pathways involved in acetate production and uptake. A putative syntrophic association between Coprothermobacter proteolyticus UC0011 and M. wolfeii UC0008 was proposed in the two-stage reactor. In order to support the transcriptomic findings regarding the hydrogen utilization routes, an advanced bioconversion model was adapted for the simulation of the single- and two-stage reactor setups. Conclusions This is the first study investigating biogas reactor metatranscriptome dynamics following hydrogen injection for biomethanation and carbon fixation to short-chain fatty acids purposes. The same microbes showed different patterns of metabolic regulation in the two reactor configurations. It was observed an effect of the specialized acidogenic reactor on the overall microbial consortium composition and activity in the two-stage digester. There were also suggested the main species responsible for methanation, short-chain fatty acids production, and electron transport chain mechanisms, in both reactor configurations. Electronic supplementary material The online version of this article (10.1186/s40168-018-0583-4) contains supplementary material, which is available to authorized users.

Extended use of antibiotics in dairy farming for therapeutic and prophylactic reasons, but also the higher prevalence of antibiotic resistant bacteria (ARB) in the farm environment raised the concern of consuming raw cow's milk and its derived products. The aim of this study was to predict by shotgun metagenomic analyses the presence of antibiotic resistance genes (ARGs) mainly correlated with Gram-negative bacteria in antibiotic residue free raw cow's milk derived exclusively from healthy animal from South Tyrol (Northern Italy), chosen as a model system. Assessment of shotgun metagenomic data of reconstructed scaffolds, revealed the existence of Pseudomonas spp. as the most abundant Gram-negative species in the raw cow's milk samples bearing ARGs. Besides, ARGs also linked to lactic acid bacteria such as Lactococcus sp. and Lactobacillus sp. ARGs correlated to microbiome found in milk samples conferred resistance towards aminoglycoside-streptothricin, beta-lactamase, macrolide, tetracycline, carbapenem, cephalosporin, penam, peptide, penem, fluoroquinolone, chloramphenicol and elfamycin antibiotics. Further bioinformatic processing included de-novo reassembly of all metagenomic sequences from all milk samples in one, to reconstruct metagenome assembled genomes (MAGs), which were further used to investigate mobile genetic elements (MGE). Analyses of the reconstructed MAGs showed that, MAG 9 (Pseudomonas sp1.) contained the oriT gene (origin of transfer gene) needed for transferring virulent factors. Although the presence of Pseudomonas is common in raw cow's milk, pasteurization treatment reduces their survivability. Nevertheless, attention should be paid on Pseudomonas spp. due to their intrinsic resistance to antibiotics and their capability of transferring virulent factors to other bacteria.

TRAM domain proteins present in Archaea and Bacteria have a β-barrel shape with anti-parallel β-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins.

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.

Biogas upgrading via carbon dioxide hydrogenation is an emerging technology for electrofuel production. The biomethanation efficiency is strongly dependent on a balanced microbial consortium, whose high- resolution characterization along with their functional potential and interactions are pivotal for process optimization. The present work is the first genome-centric metagenomic study on mesophilic and thermophilic biogas upgrading reactors aiming to define the metabolic profile of more than 200 uncultivated microbes involved in hydrogen assisted methanogenesis. The outcomes from predictive functional analyses were correlated with microbial abundance variations to clarify the effect of process parameters on the community. The operational temperature significantly influenced the microbial richness of the reactors, while the H2 addition distinctively alternated the abundance of the taxa. Two different Methanoculleus species (one mesophilic and one thermophilic) were identified as the main responsible ones for methane metabolism. Finally, it was demonstrated that the addition of H2 exerted a selective pressure on the concerted or syntrophic interactions of specific microbes functionally related to carbon fixation, propionate and butanoate metabolisms. Novel bacteria were identified as candidate syntrophic acetate oxidizers (e.g., Tepidanaerobacter sp. DTU063), while the addition of H2 favored the proliferation of potential homoacetogens (e.g., Clostridia sp. DTU183). Population genomes encoding genes of Wood-Ljungdahl pathway were mainly thermophilic, while propionate degraders were mostly identified at mesophilic conditions. Finally, putative syntrophic interactions were identified between microbes that have either versatile metabolic abilities or are obligate/facultative syntrophs.

Bovine mastitis remains the main cause of economic losses for dairy farmers. Mammary pathogenic Escherichia coli (MPEC) is related to an acute mastitis and its treatment is still based on the use of antibiotics. In the era of antimicrobial resistance (AMR), bacterial viruses (bacteriophages) present as an efcient treatment or prophylactic option. However, this makes it essential that its genetic structure, stability and interaction with the host immune system be thoroughly characterized. The present study analyzed a novel, broad host-range anti-mastitis agent, the T4virus vB_EcoM-UFV13 in genomic terms, and its activity against a MPEC strain in an experimental E. coli-induced mastitis mouse model. 4,975 Single Nucleotide Polymorphisms (SNPs) were assigned between vB_EcoM-UFV13 and E. coli phage T4 genomes with high impact on coding sequences (CDS) (37.60%) for virion proteins. Phylogenetic trees and genome analysis supported a recent infection mix between vB_EcoM-UFV13 and Shigella phage Shf2. After a viral stability evaluation (e.g pH and temperature), intramammary administration (MOI 10) resulted in a 10-fold reduction in bacterial load. Furthermore, pro-infammatory cytokines, such as IL-6 and TNF-α, were observed after viral treatment. This work brings the whole characterization and immune response to vB_EcoM-UFV13, a biocontrol candidate for bovine mastitis. A mastite bovina continua sendo a principal causa de perdas econômicas para os produtores de leite. Patogênico mamário A Escherichia coli (MPEC) está relacionada a uma mastite aguda e seu tratamento ainda se baseia no uso de antibióticos. Na era da resistência antimicrobiana (RAM), os vírus bacterianos (bacteriófagos) apresentam-se como tratamento eficaz ou opção profilática. No entanto, isso torna essencial que sua estrutura genética, estabilidade e interação com o sistema imunológico do hospedeiro sejam completamente caracterizadas. O presente estudo analisaram um novo agente antimastite de ampla gama de hospedeiros, o T4virus vB_EcoM-UFV13 em termos genômicos, e sua atividade contra uma cepa MPEC em um modelo experimental de camundongo com mastite induzida por E. coli. 4.975 Polimorfismos de nucleotídeo único (SNPs) foram atribuídos entre vB_EcoM-UFV13 e fago T4 de E. coli genomas com alto impacto nas seqüências de codificação (CDS) (37,60%) para proteínas virion. Árvores filogenéticas e análise do genoma apoiou uma mistura de infecção recente entre vB_EcoM-UFV13 e fago Shigella Shf2. Após uma avaliação da estabilidade viral (por exemplo, pH e temperatura), administração intramamária (MOI 10) resultou em uma redução de 10 vezes na carga bacteriana. Além disso, citocinas pró-inflamatórias, como IL-6 e TNF-α, foram observados após o tratamento viral. Este trabalho traz toda a caracterização e imunidade resposta ao vB_EcoM-UFV13, um candidato a biocontrole para mastite bovina.

Four full-scale anaerobic digesters with a long history of stable operation were characterized in terms of active microbiome and metagenome. Isotopic fractionation of biogas demonstrated that acetotrophy was rather prevalent in reactors operated at 6 gTAN L−1, suggesting that syntrophic acetate oxidizing bacteria (SAOB) played a significant role in the latter. These results were generally coherent with the observed active bacterial and archaeal communities but no known SAOB were observed. Metagenome descriptions yielded 73 assembled population genomes, of which only 7 could be assigned at the species level. Gene annotation and association to relevant metabolic pathways indicated that the phyla Chloroflexi and Bacteroidales might encompass new, currently undescribed, SAOB/formate producing species that would metabolize acetate via the glycine cleavage system. The predominant hydrogenotrophic counterpart at a high ammonia content belonged to the genus Methanoculleus, which could also grow on acetate to a certain extent.

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.

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 MS2 T 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”.

Appl. Microbiol. Biotechnol.ISI Document Delivery No.: AF0VXTimes Cited: 0Cited Reference Count: 53Treu, Laura Campanaro, Stefano Nadai, Chiara Toniolo, Chiara Nardi, Tiziana Giacomini, Alessio Valle, Giorgio Blondin, Bruno Corich, VivianaUniversity of Padua [CPDA082991]; Provincia di TrevisoRNA-seq was performed at the CRIBI Biotechnology Centre (University of Padua) and we acknowledge the support of Michela D'Angelo in processing our samples. Fermentation curves and enological analysis were performed at the CIRVE laboratories of Conegliano (Treviso, Italy) and we acknowledge the support of Barbara Bovo and Milena Carlot. This work was supported by the University of Padua [CPDA082991] and by the Provincia di Treviso providing PhD fellowship. We express thanks to Georgine Faulkner of the Department of Biology (University of Padua) for her assistance in the manuscript revision.SpringerNew york; We used RNA-sequencing (RNA-seq) to analyze the expression profile of four vineyard strains of Saccharomyces cerevisiae having different fermentation performances. The expression profiles obtained in two steps of the fermentation process were compared with those obtained for the industrial wine strain EC1118 and for the laboratory strain S288c. The two strains with low fermentation efficiency, namely, S288c and the vineyard strain R103, exhibited markedly different expression profiles when compared to the other four strains. We also found that the vineyard strains P283 and P301 are characterized by a high expression of the transcription factor Met32p in the first step of the fermentation. Met32p, in coordination with the Hap4p transcription factor, determined the over-expression of the genes involved in the respiration processes, in the response to oxidative stress and in the sulfur amino acids biosynthesis. These combined actions are likely to increase the level of antioxidants whose protective effect could contribute to improve the fermentation process. Gene expression and phenotypic data revealed that the vineyard strain P301 has low nitrogen utilization in comparison to the other wine strains, combined with high fermentation efficiency. Analysis of the genes involved in fermentation stress response revealed a lower expression in strains characterized by low fermentation efficiency, particularly in the first fermentation phase. These findings evidenced the high variability of transcriptional profiles among different wine yeast strains and clarify their connection with complex phenotypic traits, such as the fermentation efficiency and the nitrogen sources utilization.

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.

Summary 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.

The process stability of biogas plants is often deteriorated by the accumulation of Long Chain Fatty Acids (LCFA). The microbial community shifts due to LCFA disturbances have been poorly understood as the molecular techniques used were not able to identify the genome characteristics of uncultured microorganisms and additionally, the presence of limited number of reference genomes in public databases prevented the comprehension of specific functional roles characterizing these microorganisms. The present study is the first research which deciphers by means of high throughput shotgun sequencing the dynamics of the microbial community during an inhibitory shock load induced by single pulses of unsaturated LCFA at two different concentrations (i.e. 2 g/L-reactor and 3 g/L-reactor). The metagenomic analysis showed that only the microbes associated with LCFA degradation could encode proteins related to “chemotaxis” and “flagellar assembly”, which promoted the ability to move towards the LCFA sources so as to degrade them. Moreover, the syntrophic interactions found between Syntrophomonas sp. together with Methanosarcina sp. were possibly assigned to the menaquinone-electron transfer. Finally, it was proven that a previously exposed to LCFA inoculum is more efficient in the degradation process of LCFA due to the specialization of the microbial consortium.

Vitis vinifera L. cv. Corvina grape forms the basis for the production of unique wines, such as Amarone, whose distinctive sensory features are strongly linked to the post-harvest grape withering process. Indeed, this process increases sugar concentration and changes must characteristics. While microorganisms involved in must fermentation have been widely investigated, few data are available on the microbiota of withered grapes. Thus, in this paper, a whole metagenome sequencing (WMS) approach was used to analyse the microbial consortium associated with Corvina berries at the end of the withering process performed in two different conditions ("traditional withering," TW or "accelerated withering," AW), and to unveil whether changes of drying parameters could have an impact on microbial diversity. Samples of healthy undamaged berries were collected and washed, to recover microorganisms from the surface and avoid contamination with grapevine genetic material. Isolated DNA was sequenced and the data obtained were analyzed with several bioinformatics methods. The eukaryotic community was mainly composed by members of the phylum Ascomycota, including Eurotiomycetes, Sordariomycetes, and Dothideomycetes. Moreover, the distribution of the genera Aspergillus and Penicillium (class Eurotiomycetes) varied between the withered berry samples. Instead, Botryotinia, Saccharomyces, and other wine technologically useful microorganisms were relatively scarce in both samples. For prokaryotes, 25 phyla were identified, nine of which were common to both conditions. Environmental bacteria belonging to the class Gammaproteobacteria were dominant and, in particular, the TW sample was characterized by members of the family Pseudomonadaceae, while members of the family Enterobacteriaceae dominated the AW sample, in addition to Sphyngobacteria and Clostridia. Finally, the binning procedure discovered 15 putative genomes which dominated the microbial community of the two samples, and included representatives of genera Erwinia, Pantoea, Pseudomonas, Clostridium, Paenibacillus, and of orders Lactobacillales and Actinomycetales. These results provide insights into the microbial consortium of Corvina withered berries and reveal relevant variations attributable to post-harvest withering conditions, underling how WMS could open novel perspectives in the knowledge and management of the withering process of Corvina, with an impact on the winemaking of important Italian wines.

RNA polymerase in Archaea is composed of 11 or 12 subunits - 9 or 10 that form the core, and a heterodimer formed from subunits E and F that associates with the core and can interact with general transcription factors and facilitate transcription. While the ability of the heterodimer to bind RNA has been demonstrated, it has not been determined whether it can recognize specific RNA targets. In this study we used a recombinant archaeal MbRpoE/F to capture cellular mRNA in vitro and a microarray to determine which transcripts it specifically binds. Only transcripts for 117 genes (4% of the total) representing 48 regions of the genome were bound by MbRpoE/F. The transcripts represented important genes in a number of functional classes: methanogenesis, cofactor biosynthesis, nucleotide metabolism, transcription, translation, import/export. The arrangement and characteristics (e.g. codon and amino acid usage) of genes relative to the putative origin of replication indicate that MbRpoE/F preferentially binds to mRNA of genes whose expression may be important for cellular fitness. We also compared the biophysical properties of RpoE/F from M. burtonii and Methanocaldococcus jannaschii, demonstrating a 50°C difference in their apparent melting temperatures. By using MbRpoE/F to capture and characterize cellular RNA we have identified a previously unknown functional property of the MbRpoE/F heterodimer.

Methanococcoides burtonii is a member of the Archaea that was isolated from Ace Lake in Antarctica and is a valuable model for studying cold adaptation. Low temperature transcriptional regulation of global gene expression, and the arrangement of transcriptional units in cold-adapted archaea has not been studied. We developed a microarray for determining which genes are expressed in operons, and which are differentially expressed at low (4°C) or high (23°C) temperature. Approximately 55% of genes were found to be arranged in operons that range in length from 2 to 23 genes, and mRNA abundance tended to increase with operon length. Analysing microarray data previously obtained by others for Halobacterium salinarum revealed a similar correlation between operon length and mRNA abundance, suggesting that operons may play a similar role more broadly in the Archaea. More than 500 genes were differentially expressed at levels up to ? 24-fold. A notable feature was the upregulation of genes involved in maintaining RNA in a state suitable for translation in the cold. Comparison between microarray experiments and results previously obtained using proteomics indicates that transcriptional regulation (rather than translation) is primarily responsible for controlling gene expression in M. burtonii. In addition, certain genes (e.g. involved in ribosome structure and methanogenesis) appear to be regulated post-transcriptionally. This is one of few experimental studies describing the genome-wide distribution and regulation of operons in archaea.

Sulfur dioxide is extensively used during industrial fermentations and contributes to determine the harsh conditions of winemaking together with low pH, high sugar content and increasing ethanol concentration. Therefore the presence of sulfite has to be considered in yeast gene expression studies to properly understand yeast behavior in technological environments such as winemaking. A reliable expression pattern can be obtained only using an appropriate reference gene set that is constitutively expressed regardless of perturbations linked to the experimental conditions. In this work we tested 15 candidate reference genes suitable for analysis of gene expression during must fermentation in the presence of sulfite. New reference genes were selected from a genome-wide expression experiment, obtained by RNA sequencing of four Saccharomyces cerevisiae wine strains grown in enological conditions. Their performance was compared to that of the most common genes used in previous studies. The most popular software based on different statistical approaches (geNorm, NormFinder and BestKeeper) were chosen to evaluate expression stability of the candidate reference genes. Validation was obtained using other wine strains by comparing normalized gene expression data with transcriptome quantification both in the presence and absence of sulfite. Among 15 reference genes tested ALG9, FBA1, UBC6 and PFK1 appeared to be the most reliable while ENO1, PMA1, DED1 and FAS2 were the worst. The most popular reference gene ACT1, widely used for S. cerevisiae gene expression studies, showed a stability level markedly lower than those of our selected reference genes. Finally, as the expression of the new reference gene set remained constant over the entire fermentation process, irrespective of the perturbation due to sulfite addition, our results can be considered also when no sulfite is added to the must.

Foam formation can lead to total failure of digestion process in biogas plants. In the present study, possible correlation between foaming and the presence of specific microorganisms in biogas reactors was elucidated. The microbial ecology of continuous fed digesters overloaded with proteins, lipids and carbohydrates before and after foaming incidents was characterized using 16S rRNA gene sequencing. Moreover, the microbial diversity between the liquid and foaming layer was assessed. A number of genera that are known to produce biosurfactants, contain mycolic acid in their cell wall, or decrease the surface tension of the media, increased their relative abundance after foam formation. Finally, a microorganism similar to widely known foaming bacteria (Nocardia and Desulfotomaculum) was found to increase its relative abundance in all reactors once foam was observed, regardless of the used substrate. These findings suggest that foaming and specific microorganisms might have direct association which requires to be further investigated.

Here, we report the draft genome assembly of Lactobacillus fabifermentans strain T30PCM01 isolated from grape marc. Its genome is the largest (3.58 Mbp) among Lactobacillus species and reveals an enormous potential for carbohydrate utilization and transcriptional regulation.

Here is presented the whole-genome sequences of Streptococcus thermophilus strains MTH17CL396 and M17PTZA496, isolated from fontina protected designation of origin (PDO) cheese in the Valle d'Aosta Region (Italy). S. thermophilus is a lactic acid bacterium widely present in dairy products, and these are the first publicly available genome sequences of S. thermophilus strains isolated from cheese.