Phenotypic, genotypic, and resistome of mesophilic spore-forming bacteria isolated from pasteurized liquid whole egg.

The production of whole-liquid eggs is of significant economic and nutritional importance. This study aimed to assess the phenotypic and genotypic diversity of mesophilic aerobic spore-forming bacteria (n = 200) isolated from pasteurized whole liquid egg and liquid egg yolk. The majority of the isolates were identified as belonging to the genera Bacillus (86 %), followed by Brevibacillus (10 %) and Lysinibacillus (4 %). For the phenotypic characterization, isolates were subjected to various heat shocks, with the most significant reductions observed at 80 °C/30 min and 90 °C/10 min for isolates recovered from raw materials. On the other hand, the decrease was similar for isolates recovered from raw material and final product at 100 °C/5 min and 110 °C/5 min. Genotypic genes related to heat resistance (cdnL, spoVAD, dacB, clpC, dnaK, and yitF/Tn1546) were examined for genotypic characterization. The dnaK gene showed a positive correlation with the highest thermal condition tested (110 °C/5 min), while 100 °C/5 min had the highest number of positively correlated genes (clpC, cdnL, yitF/Tn1546, and spoVAD). Whole Genome Sequencing of four strains revealed genes related to sporulation, structure formation, initiation and regulation, stress response, and DNA repair in vegetative cells. The findings of this study indicate that these mesophilic aerobic spore-forming bacteria may adopt several strategies to persist through the process and reach the final product. As the inactivation of these microorganisms during egg processing is challenging, preventing raw materials contamination and their establishment in processing premises must be reinforced.

Comparative genomics reveals probable adaptations for xylose use in Thermoanaerobacterium saccharolyticum.

Second-generation ethanol, a promising biofuel for reducing greenhouse gas emissions, faces challenges due to the inefficient metabolism of xylose, a pentose sugar. Overcoming this hurdle requires exploration of genes, pathways, and organisms capable of fermenting xylose. Thermoanaerobacterium saccharolyticum is an organism capable of naturally fermenting compounds of industrial interest, such as xylose, and understanding evolutionary adaptations may help to bring novel genes and information that can be used for industrial yeast, increasing production of current bio-platforms. This study presents a deep evolutionary study of members of the firmicutes clade, focusing on adaptations in Thermoanaerobacterium saccharolyticum that may be related to overall fermentation metabolism, especially for xylose fermentation. One highlight is the finding of positive selection on a xylose-binding protein of the xylFGH operon, close to the annotated sugar binding site, with this protein already being found to be expressed in xylose fermenting conditions in a previous study. Results from this study can serve as basis for searching for candidate genes to use in industrial strains or to improve Thermoanaerobacterium saccharolyticum as a new microbial cell factory, which may help to solve current problems found in the biofuels' industry.

Assessment of cecal microbiota modulation from piglet dietary supplementation with copper.

BACKGROUND: Swine production expanded in the last decades. Efforts have been made to improve meat production and to understand its relationship to pig gut microbiota. Copper (Cu) is a usual supplement to growth performance in animal production. Here, two performance studies were conducted to investigate the effects of three different sources of Cu on the microbiota of piglets. A total of 256 weaned piglets were randomly allocated into 4 treatments (10 replicates per treatment of 4 piglets per pen in Trial 1 and 8 replicates of 3 piglets per pen in Trial 2). Treatments included a control group (fed 10 mg/kg of Cu from CuSO4), a group fed at 160 mg/kg of Copper (II) sulfate (CuSO4) or tri-basic copper chloride (TBCC), and a group fed with Cu methionine hydroxy analogue chelated (Cu-MHAC) at 150, 80, and 50 mg/kg in Phases 1 (24-35 d), 2 (36-49 d), and 3 (50-70 d), respectively. At 70 d, the cecum luminal contents from one pig per pen were collected and polled for 16 S rRNA sequencing (V3/V4 regions). Parameters were analyzed in a completely randomized block design, in which each experiment was considered as a block. RESULTS: A total of 1337 Operational Taxonomic Units (OTUs) were identified. Dominance and Simpson ecological metrics were statistically different between control and treated groups (P < 0.10) showing that different Cu sources altered the gut microbiota composition with the proliferation of some bacteria that improve gut health. A high abundance of Prevotella was observed in all treatments while other genera were enriched and differentially modulated, according to the Cu source and dosage. The supplementation with Cu-MHAC can modify a group of bacteria involved in feed efficiency (FE) and short chain fatty acids (SCFA) production (Clostridium XIVa, Desulfovibrio, and Megasphera). These bacteria are also important players in the activation of ghrelin and growth hormones that were previously reported to correlate with Cu-MHAC supplementation. CONCLUSIONS: These results indicated that some genera seem to be directly affected by the Cu source offered to the animals. TBCC and Cu-MHAC (even in low doses) can promote healthy modifications in the gut bacterial composition, being a promising source of supplementation for piglets.

Wide distribution of D-xylose dehydrogenase in yeasts reveals a new element in the D-xylose metabolism for bioethanol production.

D-xylose utilization by yeasts is an essential feature for improving second-generation ethanol production. However, industrial yeast strains are incapable of consuming D-xylose. Previous analyzes of D-xylose-consuming or fermenting yeast species reveal that the genomic features associated with this phenotype are complex and still not fully understood. Here we present a previously neglected yeast enzyme related to D-xylose metabolism, D-xylose dehydrogenase (XylDH), which is found in at least 105 yeast genomes. By analyzing the XylDH gene family, we brought evidence of gene evolution marked by purifying selection on codons and positive selection evidence in D-xylose-consuming and fermenting species, suggesting the importance of XylDH for D-xylose-related phenotypes in yeasts. Furthermore, although we found no putative metabolic pathway for XylDH in yeast genomes, namely the absence of three bacterial known pathways for this enzyme, we also provide its expression profile on D-xylose media following D-xylose reductase for two yeasts with publicly available transcriptomes. Based on these results, we suggest that XylDH plays an important role in D-xylose usage by yeasts, likely being involved in a cofactor regeneration system by reducing cofactor imbalance in the D-xylose reductase pathway.

Plant microbiomes harbor potential to promote nutrient turnover in impoverished substrates of a Brazilian biodiversity hotspot.

The substrates of the Brazilian campos rupestres, a grassland ecosystem, have extremely low concentrations of phosphorus and nitrogen, imposing restrictions to plant growth. Despite that, this ecosystem harbors almost 15% of the Brazilian plant diversity, raising the question of how plants acquire nutrients in such a harsh environment. Here, we set out to uncover the taxonomic profile, the compositional and functional differences and similarities, and the nutrient turnover potential of microbial communities associated with two plant species of the campos rupestres-dominant family Velloziaceae that grow over distinct substrates (soil and rock). Using amplicon sequencing data, we show that, despite the pronounced composition differentiation, the plant-associated soil and rock communities share a core of highly efficient colonizers that tend to be highly abundant and is enriched in 21 bacterial families. Functional investigation of metagenomes and 522 metagenome-assembled genomes revealed that the microorganisms found associated to plant roots are enriched in genes involved in organic compound intake, and phosphorus and nitrogen turnover. We show that potential for phosphorus transport, mineralization, and solubilization are mostly found within bacterial families of the shared microbiome, such as Xanthobacteraceae and Bryobacteraceae. We also detected the full repertoire of nitrogen cycle-related genes and discovered a lineage of Isosphaeraceae that acquired nitrogen-fixing potential via horizontal gene transfer and might be also involved in nitrification via a metabolic handoff association with Binataceae. We highlight that plant-associated microbial populations in the campos rupestres harbor a genetic repertoire with potential to increase nutrient availability and that the microbiomes of biodiversity hotspots can reveal novel mechanisms of nutrient turnover.

Phylogenomics and gene selection in Aspergillus welwitschiae: Possible implications in the pathogenicity in Agave sisalana.

Aspergillus welwitschiae causes bole rot disease in sisal (Agave sisalana and related species) which affects the production of natural fibers in Brazil, the main worldwide producer of sisal fibers. This fungus is a saprotroph with a broad host range. Previous research established A. welwitschiae as the only causative agent of bole rot in the field, but little is known about the evolution of this species and its strains. In this work, we performed a comparative genomics analysis of 40 Aspergillus strains. We show the conflicting molecular identity of this species, with one sisal-infecting strain sharing its last common ancestor with Aspergillus niger, having diverged only 833 thousand years ago. Furthermore, our analysis of positive selection reveals sites under selection in genes coding for siderophore transporters, Sodium­calcium exchangers, and Phosphatidylethanolamine-binding proteins (PEBPs). Herein, we discuss the possible impacts of these gene functions on the pathogenicity in sisal.

Functional genomics analysis of a phyllospheric Pseudomonas spp with potential for biological control against coffee rust.

BACKGROUND: Pseudomonas spp. promotes plant growth and colonizes a wide range of environments. During the annotation of a Coffea arabica ESTs database, we detected a considerable number of contaminant Pseudomonas sequences, specially associated with leaves. The genome of a Pseudomonas isolated from coffee leaves was sequenced to investigate in silico information that could offer insights about bacterial adaptation to coffee phyllosphere. In parallel, several experiments were performed to confirm certain physiological characteristics that could be associated with phyllospheric behavior. Finally, in vivo and in vitro experiments were carried out to verify whether this isolate could serve as a biocontrol agent against coffee rust and how the isolate could act against the infection.  RESULTS: The isolate showed several genes that are associated with resistance to environmental stresses, such as genes encoding heat/cold shock proteins, antioxidant enzymes, carbon starvation proteins, proteins that control osmotic balance and biofilm formation. There was an increase of exopolysaccharides synthesis in response to osmotic stress, which may protect cells from dessication on phyllosphere. Metabolic pathways for degradation and incorporation into citrate cycle of phenolic compounds present in coffee were found, and experimentally confirmed. In addition, MN1F was found to be highly tolerant to caffeine. The experiments of biocontrol against coffee leaf rust showed that the isolate can control the progress of the disease, most likely through competition for resources. CONCLUSION: Genomic analysis and experimental data suggest that there are adaptations of this Pseudomonas to live in association with coffee leaves and to act as a biocontrol agent.

Genetic mapping of a bioethanol yeast strain reveals new targets for hydroxymethylfurfural- and thermotolerance.

Current technology that enables bioethanol production from agricultural biomass imposes harsh conditions for Saccharomyces cerevisiae's metabolism. In this work, the genetic architecture of industrial bioethanol yeast strain SA-1 was evaluated. SA-1 segregant FMY097 was previously described as highly aldehyde resistant and here also as thermotolerant: two important traits for the second-generation industry. A Quantitative Trait Loci (QTL) mapping of 5-hydroxymethylfurfural (HMF) -resistant segregants of hybrid FMY097/BY4742 disclosed a region in chromosome II bearing alleles with uncommon non-synonymous (NS) single nucleotide polymorphisms (SNPs) in FMY097: MIX23, PKC1, SEA4, and SRO77. Allele swap to susceptible laboratory strain BY4742 revealed that SEA4FMY097 enhances robustness towards HMF, but the industrial fitness could not be fully recovered. The genetic network arising from the causative genes in the QTL window suggests that intracellular signaling TOR (Target of Rapamycin) and CWI (Cell Wall Integrity) pathways are regulators of this phenotype in FMY097. Because the QTL mapping did not result in one major allelic contribution to the evaluated trait, a background effect in FMY097's HMF resistance is expected. Quantification of NADPH - cofactor implied in endogenous aldehyde detoxification reactions - supports the former hypothesis, given its high availability in FMY097. Regarding thermotolerance, SEA4FMY097 grants BY4742 ability to grow in temperatures as high as 38 ºC in liquid, while allele PKC1FMY097 allows growth up to 40 ºC in solid medium. Both SEA4FMY097 and PKC1FMY097 encode rare NS SNPs, not found in other > 1013S. cerevisiae. Altogether, these findings point towards crucial membrane and stress mediators for yeast robustness.

Genomic and physiological evaluation of two root associated Pseudomonas from Coffea arabica.

Many Pseudomonas species promote plant growth and colonize a wide range of environments. The annotation of a Coffea arabica ESTs database revealed a considerable number of Pseudomonas sequences. To evaluate the genomic and physiology of Pseudomonas that inhabit coffee plants, fluorescent Pseudomonas from C. arabica root environment were isolated. Two of them had their genomes sequenced; one from rhizospheric soil, named as MNR3A, and one from internal part of the root, named as EMN2. In parallel, we performed biochemical and physiological experiments to confirm genomic analyses results. Interestingly, EMN2 has achromobactin and aerobactin siderophore receptors, but does not have the genes responsible for the production of these siderophores, suggesting an interesting bacterial competition strategy. The two bacterial isolates were able to degrade and catabolize plant phenolic compounds for their own benefit. Surprisingly, MNR3A and EMN2 do not contain caffeine methylases that are responsible for the catabolism of caffeine. In fact, bench experiments confirm that the bacteria did not metabolize caffeine, but were resistant and chemically attracted to it. Furthermore, both bacteria, most especially MNR3A, were able to increase growth of lettuce plants. Our results indicate MNR3A as a potential plant growth promoting bacteria.

Oxidative cleavage of polysaccharides by a termite-derived superoxide dismutase boosts the degradation of biomass by glycoside hydrolases.

Wood-feeding termites effectively degrade plant biomass through enzymatic degradation. Despite their high efficiencies, however, individual glycoside hydrolases isolated from termites and their symbionts exhibit anomalously low effectiveness in lignocellulose degradation, suggesting hereto unknown enzymatic activities in their digestome. Herein, we demonstrate that an ancient redox-active enzyme encoded by the lower termite Coptotermes gestroi, a Cu/Zn superoxide dismutase (CgSOD-1), plays a previously unknown role in plant biomass degradation. We show that CgSOD-1 transcripts and peptides are up-regulated in response to an increased level of lignocellulose recalcitrance and that CgSOD-1 localizes in the lumen of the fore- and midguts of C. gestroi together with termite main cellulase, CgEG-1-GH9. CgSOD-1 boosts the saccharification of polysaccharides by CgEG-1-GH9. We show that the boosting effect of CgSOD-1 involves an oxidative mechanism of action in which CgSOD-1 generates reactive oxygen species that subsequently cleave the polysaccharide. SOD-type enzymes constitute a new addition to the growing family of oxidases, ones which are up-regulated when exposed to recalcitrant polysaccharides, and that are used by Nature for biomass degradation.

Fungal communities represent the majority of root-specific transcripts in the transcriptomes of Agave plants grown in semiarid regions.

Agave plants present drought resistance mechanisms, commercial applications, and potential for bioenergy production. Currently, Agave species are used to produce alcoholic beverages and sisal fibers in semi-arid regions, mainly in Mexico and Brazil. Because of their high productivities, low lignin content, and high shoot-to-root ratio, agaves show potential as biomass feedstock to bioenergy production in marginal areas. Plants host many microorganisms and understanding their metabolism can inform biotechnological purposes. Here, we identify and characterize fungal transcripts found in three fiber-producing agave cultivars (Agave fourcroydes, A. sisalana, and hybrid 11648). We used leaf, stem, and root samples collected from the agave germplasm bank located in the state of Paraiba, in the Brazilian semiarid region, which has faced irregular precipitation periods. We used data from a de novo assembled transcriptome assembly (all tissues together). Regardless of the cultivar, around 10% of the transcripts mapped to fungi. Surprisingly, most root-specific transcripts were fungal (58%); of these around 64% were identified as Ascomycota and 28% as Basidiomycota in the three communities. Transcripts that code for heat shock proteins (HSPs) and enzymes involved in transport across the membrane in Ascomycota and Basidiomycota, abounded in libraries generated from the three cultivars. Indeed, among the most expressed transcripts, many were annotated as HSPs, which appear involved in abiotic stress resistance. Most HSPs expressed by Ascomycota are small HSPs, highly related to dealing with temperature stresses. Also, some KEGG pathways suggest interaction with the roots, related to transport to outside the cell, such as exosome (present in the three Ascomycota communities) and membrane trafficking, which were further investigated. We also found chitinases among secreted CAZymes, that can be related to pathogen control. We anticipate that our results can provide a starting point to the study of the potential uses of agaves' fungi as biotechnological tools.

Machine learning and comparative genomics approaches for the discovery of xylose transporters in yeast.

BACKGROUND: The need to mitigate and substitute the use of fossil fuels as the main energy matrix has led to the study and development of biofuels as an alternative. Second-generation (2G) ethanol arises as one biofuel with great potential, due to not only maintaining food security, but also as a product from economically interesting crops such as energy-cane. One of the main challenges of 2G ethanol is the inefficient uptake of pentose sugars by industrial yeast Saccharomyces cerevisiae, the main organism used for ethanol production. Understanding the main drivers for xylose assimilation and identify novel and efficient transporters is a key step to make the 2G process economically viable. RESULTS: By implementing a strategy of searching for present motifs that may be responsible for xylose transport and past adaptations of sugar transporters in xylose fermenting species, we obtained a classifying model which was successfully used to select four different candidate transporters for evaluation in the S. cerevisiae hxt-null strain, EBY.VW4000, harbouring the xylose consumption pathway. Yeast cells expressing the transporters SpX, SpH and SpG showed a superior uptake performance in xylose compared to traditional literature control Gxf1. CONCLUSIONS: Modelling xylose transport with the small data available for yeast and bacteria proved a challenge that was overcome through different statistical strategies. Through this strategy, we present four novel xylose transporters which expands the repertoire of candidates targeting yeast genetic engineering for industrial fermentation. The repeated use of the model for characterizing new transporters will be useful both into finding the best candidates for industrial utilization and to increase the model's predictive capabilities.

Internode elongation in energy cane shows remarkable clues on lignocellulosic biomass biosynthesis in Saccharum hybrids.

Energy cane is a dedicated crop to high biomass production and selected during Saccharum breeding programs to fit specific industrial needs for 2G bioethanol production. Internode elongation is one of the most important characteristics in Saccharum hybrids due to its relationship with crop yield. In this study, we selected the third internode elongation of the energy cane. To characterize this process, we divided the internode into five sections and performed a detailed transcriptome analysis (RNA-Seq) and cell wall characterization. The histological analyses revealed a remarkable gradient that spans from cell division and protoxylem lignification to the internode maturation and complete vascular bundle lignification. RNA-Seq analysis revealed more than 11,000 differentially expressed genes between the sections internal. Gene ontology analyzes showed enriched categories in each section, as well as the most expressed genes in each section, presented different biological processes. We found that the internode elongation and division zones have a large number of unique genes. Evaluated the specific profile of genes related to primary and secondary cell wall formation, cellulose synthesis, hemicellulose, lignin, and growth-related genes. For each section these genes presented different profiles along the internode in elongation in energy cane. The results of this study provide an overview of the regulation of gene expression of an internode elongation in energy cane. Gene expression analysis revealed promising candidates for transcriptional regulation of energy cane lignification and evidence key genes for the regulation of internode development, which can serve as a basis for understanding the molecular regulatory mechanisms that support the growth and development of plants in the Saccahrum complex.

Brazilian industrial yeasts show high fermentative performance in high solids content for corn ethanol process.

An imminent change in the world energy matrix makes it necessary to increase the production of renewable fuels. The United States and Brazil are the world's largest producers, but their production methods are very different, using different raw materials, ground corn and sugarcane juice, respectively. In recent years, strong investments have been made to expand the use of corn in Brazilian ethanol production. The combination of the sugar cane and corn ethanol industries has generated innovations in the sector, such as the "flex" mills, which are traditional sugar cane mills adapted to produce corn ethanol in the sugar cane off-season. Brazil has a portfolio of robust industrial yeasts for sugarcane ethanol production, naturally evolved and selected over the past 50 years. In this work, we analyze for the first time the performance of Brazilian industrial strains (BG-1, CAT-1, PE-2 and SA-1, widely used in sugarcane ethanol production) in corn ethanol production using different stress conditions. Ethanol Red yeast, traditionally used in corn ethanol plants around the world, was used as a control. In terms of tolerance to temperature (35 °C), strains BG-1 and SA-1 stood out. In fermentations with high solids concentration (35%), strain BG-1 reached ethanol contents higher than 19% w/v and had a productivity gain of 5.8% compared to fermentation at 30%. This was the first time that these industrial strains were evaluated using the high solids concentration of 35% and the results point to ways to improve the corn ethanol production process.

Yeast Double Transporter Gene Deletion Library for Identification of Xenobiotic Carriers in Low or High Throughput.

The routes of uptake and efflux should be considered when developing new drugs so that they can effectively address their intracellular targets. As a general rule, drugs appear to enter cells via protein carriers that normally carry nutrients or metabolites. A previously developed pipeline that searched for drug transporters using Saccharomyces cerevisiae mutants carrying single-gene deletions identified import routes for most compounds tested. However, due to the redundancy of transporter functions, we propose that this methodology can be improved by utilizing double mutant strains in both low- and high-throughput screens. We constructed a library of over 14,000 strains harboring double deletions of genes encoding 122 nonessential plasma membrane transporters and performed low- and high-throughput screens identifying possible drug import routes for 23 compounds. In addition, the high-throughput assay enabled the identification of putative efflux routes for 21 compounds. Focusing on azole antifungals, we were able to identify the involvement of the myo-inositol transporter, Itr1p, in the uptake of these molecules and to confirm the role of Pdr5p in their export. IMPORTANCE Our library of double transporter deletion strains is a powerful tool for rapid identification of potential drug import and export routes, which can aid in determining the chemical groups necessary for transport via specific carriers. This information may be translated into a better design of drugs for optimal absorption by target tissues and the development of drugs whose utility is less likely to be compromised by the selection of resistant mutants.

QTL mapping of a Brazilian bioethanol strain links the cell wall protein-encoding gene GAS1 to low pH tolerance in S. cerevisiae.

BACKGROUND: Saccharomyces cerevisiae is largely applied in many biotechnological processes, from traditional food and beverage industries to modern biofuel and biochemicals factories. During the fermentation process, yeast cells are usually challenged in different harsh conditions, which often impact productivity. Regarding bioethanol production, cell exposure to acidic environments is related to productivity loss on both first- and second-generation ethanol. In this scenario, indigenous strains traditionally used in fermentation stand out as a source of complex genetic architecture, mainly due to their highly robust background-including low pH tolerance. RESULTS: In this work, we pioneer the use of QTL mapping to uncover the genetic basis that confers to the industrial strain Pedra-2 (PE-2) acidic tolerance during growth at low pH. First, we developed a fluorescence-based high-throughput approach to collect a large number of haploid cells using flow cytometry. Then, we were able to apply a bulk segregant analysis to solve the genetic basis of low pH resistance in PE-2, which uncovered a region in chromosome X as the major QTL associated with the evaluated phenotype. A reciprocal hemizygosity analysis revealed the allele GAS1, encoding a ß-1,3-glucanosyltransferase, as the casual variant in this region. The GAS1 sequence alignment of distinct S. cerevisiae strains pointed out a non-synonymous mutation (A631G) prevalence in wild-type isolates, which is absent in laboratory strains. We further showcase that GAS1 allele swap between PE-2 and a low pH-susceptible strain can improve cell viability on the latter of up to 12% after a sulfuric acid wash process. CONCLUSION: This work revealed GAS1 as one of the main causative genes associated with tolerance to growth at low pH in PE-2. We also showcase how GAS1PE-2 can improve acid resistance of a susceptible strain, suggesting that these findings can be a powerful foundation for the development of more robust and acid-tolerant strains. Our results collectively show the importance of tailored industrial isolated strains in discovering the genetic architecture of relevant traits and its implications over productivity.

Metabolite profiles of energy cane and sugarcane reveal different strategies during the axillary bud outgrowth.

Commercial cultivation of sugarcane is usually carried out by planting culm segments (sett) carrying buds in their internodes. However, this is an inefficient practice due to high sprouting irregularity. In this work, we inspect the first stages of the physiological preparation of the culm for sprouting, trying to identify compounds that actively participate in this process. We compared, during the first 48 h, the metabolic profile of sugarcane against energy cane, a cultivar known to have higher sprouting speed and consistency. In fact, during this short period it was possible to observe that energy cane already had a higher physiological activity than sugarcane, with significant changes in the catabolism of amino acids, increased levels of reducing sugars, lipids and metabolic activity in the phenylpropanoid pathway. On the other hand, sugarcane samples had just begun their activity during this same period, with an increase in the level of glutamate as the most significant change, which may be linked to the strategy of these cultivars to develop their roots before leaves, opposite of what is seen for energy cane. These results contribute to the development of strategies for increasing the efficiency of sprouting in sugarcane.

The Sisal Virome: Uncovering the Viral Diversity of Agave Varieties Reveals New and Organ-Specific Viruses.

Sisal is a common name for different plant varieties in the genus Agave (especially Agave sisalana) used for high-quality natural leaf fiber extraction. Despite the economic value of these plants, we still lack information about the diversity of viruses (virome) in non-tequilana species from the genus Agave. In this work, by associating RNA and DNA deep sequencing we were able to identify 25 putative viral species infecting A. sisalana, A. fourcroydes, and Agave hybrid 11648, including one strain of Cowpea Mild Mottle Virus (CPMMV) and 24 elements likely representing new viruses. Phylogenetic analysis indicated they belong to at least six viral families: Alphaflexiviridae, Betaflexiviridae, Botourmiaviridae, Closteroviridae, Partitiviridae, Virgaviridae, and three distinct unclassified groups. We observed higher viral taxa richness in roots when compared to leaves and stems. Furthermore, leaves and stems are very similar diversity-wise, with a lower number of taxa and dominance of a single viral species. Finally, approximately 50% of the identified viruses were found in all Agave organs investigated, which suggests that they likely produce a systemic infection. This is the first metatranscriptomics study focused on viral identification in species from the genus Agave. Despite having analyzed symptomless individuals, we identified several viruses supposedly infecting Agave species, including organ-specific and systemic species. Surprisingly, some of these putative viruses are probably infecting microorganisms composing the plant microbiota. Altogether, our results reinforce the importance of unbiased strategies for the identification and monitoring of viruses in plant species, including those with asymptomatic phenotypes.

Understanding the differences in 2G ethanol fermentative scales through omics data integration.

In this work, we evaluated the fermentative performance and metabolism modifications of a second generation (2G) industrial yeast by comparing an industrial condition during laboratory and industrial scale fermentations. Fermentations were done using industrial lignocellulosic hydrolysate and a synthetic medium containing inhibitors and analyses were carried out through transcriptomics and proteomics of these experimental conditions. We found that fermentation profiles were very similar, but there was an increase in xylose consumption rate during fermentations using synthetic medium when compared to lignocellulosic hydrolysate, likely due to the presence of unknown growth inhibitors contained in the hydrolysate. We also evaluated the bacterial community composition of the industrial fermentation setting and found that the presence of homofermentative and heterofermentative bacteria did not significantly change the performance of yeast fermentation. In parallel, temporal differentially expressed genes (tDEG) showed differences in gene expression profiles between compared conditions, including heat shocks and the presence of up-regulated genes from the TCA cycle during anaerobic xylose fermentation. Thus, we indicate HMF as a possible electron acceptor in this rapid respiratory process performed by yeast, in addition to demonstrating the importance of culture medium for the performance of yeast within industrial fermentation processes, highlighting the uniquenesses according to scales.

Adaptive evolution of Moniliophthora PR-1 proteins towards its pathogenic lifestyle.

BACKGROUND: Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches' broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. RESULTS: Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host-pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. CONCLUSIONS: Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.