Metagenomic approach to identify genes encoding for glycoside hydrolases in composting samples

Metagenomics involves the study of the genomic DNA from a set of microorganisms present in a particular environmental sample. This approach has emerged as a promising culture-independent technique to explore the diversity and function of microbiomes, allowing the discovery of novel biochemical compounds, namely enzymes with high potential for industrial applications. Composting habitats are characterized by a high microbial diversity, and represent a suitable source of robust enzymes able to convert the recalcitrant structure of lignocellulose, such as cellulases, endohemicellulases, oligosaccharide-degrading enzymes, and debranching enzymes. In fact, several lignocellulose-degrading enzymes have been successfully identified in composting samples following metagenomic approaches. The efficient handling, processing, and analysis of the large metagenomic datasets generated by next-generation sequencing platforms can be achieved using advanced bioinformatics pipelines. In this work, composting samples were collected from three Portuguese composting units, which handle different types of wastes. The metagenomic DNA was extracted from the composting samples, the three composting metagenomes were analyzed by shotgun sequencing and a comparative analysis was performed between our samples and composting samples selected from the literature to evaluate the potential of these environments for lignocellulosic biomass conversion. The metagenomic sequencing data from all samples were processed using appropriate bioinformatics tools and the functional annotation of genes encoding glycoside hydrolases was carried out using the CAZy database. Our bioinformatics pipeline revealed that all samples were enriched in cellulases, endoglucanases, and -glucosidases, which confirms the richness of composting habitats, regardless of waste compositions, in lignocellulose-degrading enzymes. As these compost samples were collected in the thermophilic phase, the identified enzymes may harbor interesting features for industrial purposes, including catalytic activity under high temperatures.info:eu-repo/semantics/publishedVersio

Functional screening for the detection of β-glucosidase activity in a metagenomic library obtained from a compost sample

There is an increasing need to find novel and robust biocatalysts with promising features that compete with those currently available on the market. Composting is an extreme habitat of high microbiological diversity that represents a suitable source of lignocellulose-degrading enzymes, such as cellulases, hemicellulases and ligninases, properly active under harsh conditions. These enzymes can convert the recalcitrant structure of lignocellulose into valuable bioproducts with great biotechnological potential. -Glucosidases are glycoside hydrolases responsible for degrading cellulose, namely in the disruption of the final glycosidic bonds of short-chain oligosaccharides to obtain glucose. Metagenomics is an emerging cultureindependent technique that has proven effective in identifying new biocatalysts with better catalytic activity through the analysis of DNA extracted from a vast number of environments. The metagenomic analysis is divided into two main technologies: sequence- and functionalbased approach. Function-based screening aims to discover and identify new genes capable of producing biocompounds/biomolecules with new or improved functions. This screening is based on the detection and isolation of clones with a positive response to the desired phenotype when activity-based techniques are applied. In this study, high-molecular-weight DNA extracted from a compost sample was used to construct a fosmid metagenomic library. This library was evaluated through a functional screening to identify clones that expressed cellulase activity, specifically -glucosidase activity. The enzymatic activity was unravelled using esculin as substrate through the formation of a brown halo as a positive response (Figure 1). The functional screening was performed in 96-well microplates and the detection of - glucosidase activity was evaluated at different temperature (25-60 °C) and pH (4.5-9.5) conditions. It was possible to identify clones with the enzymatic activity of -glucosidase in almost all tested conditions, except at 60 °C. The best conditions for clone growth occur in a longer initial incubation time (3 days, 37 °C). On the other hand, the lower pH and incubation temperature favoured a faster detection of -glucosidase activity.The study received financial support from the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the Project LIGNOZYMES (POCI-01-0145-FEDER-029773).info:eu-repo/semantics/publishedVersio

Mining bioactive molecules from extreme environments using sequence-based metagenomics

The unique features of extreme habitats, particularly the high temperature and salinity, make them promising reservoirs for the bioprospection and discovery of novel and interesting bioactive molecules. In fact, the microorganisms inhabiting this type of environments have adopted survival strategies to thrive under hostile conditions, synthesizing several biochemical compounds valuable for many biotechnological applications. Culture-independent techniques, namely metagenomics, have emerged as powerful tools to access and explore the genetic and metabolic diversity of the microbial communities present in extreme and complex ecosystems. Two different metagenomic approaches can be applied, namely, sequence- and function-based metagenomics. The sequence-based studies allow finding new gene sequences that reveal similarities with the annotated genomes available in the databases. In this study, thermophilic composting samples were collected from two Portuguese composting units (Terra Fértil and Lipor), which handle different types of wastes. The metagenomic DNA was extracted from the composting samples and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to characterize the taxonomic and functional profiles of the metagenomes using both reads and metagenome-assembled genomes (MAGs) as input. Their microbiome was dominated by Bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria and Balneolia stood out for their higher abundance. These data confirm the richness of the composting habitats in bacterial strains known to survive and play an important role in harsh conditions, namely extreme saline environments. Furthermore, the combined analysis of functional annotation against KEGG and COG databases revealed a clear dominance of the contigs associated with the metabolism of carbohydrates, energy, amino acids and xenobiotics biodegradation and metabolism. This work represents the first comparative study on the taxonomic and functional profiles of Portuguese composting samples.info:eu-repo/semantics/publishedVersio

Image processing tool for the detection and quantification of xylanase activity in a metagenomic study

The vast diversity of unexplored microbial communities inhabiting the planet drives the continuous screening for promising biocatalysts. Until recently, the strategies to find new microorganisms and their enzymes were mainly focused on laboratory studies of pure microbial cultures. However, a great amount of environmental microorganisms cannot be cultivated under laboratorial conditions [1]. Metagenomics has emerged as an innovative approach to explore these uncultivable microorganisms through the analysis of DNA extracted from environmental samples [2]. It is considered a powerful tool for the discovery of novel biocatalysts and two different approaches have been proposed. Sequence-based studies recognize candidate genes but do not provide direct conclusions about the functionality of the encoded enzymes. On the other hand, the function-based approach allows the identification of new enzymes and also leads to preliminary information about their activities and physicochemical parameters. Indeed, function-based screenings have been successfully used in different environments to find genes encoding lignocellulose-degrading enzymes, such as xylanases [2]. These enzymes are considered important catalysts in the biological decomposition of lignocellulosic residues. In this study, a fosmid library previously prepared in Escherichia coli with genomic DNA extracted from a compost sample collected in a national composting unit (Lipor) [3] was evaluated through a functional screening. To assess the xylanase activity of all the clones, a fast and simple chromogenic screening test using AZCL-xylan was performed in 96-well microplates at room temperature. Afterwards, the positive clones were selected and incubated at different temperatures (25, 37, 45 and 60 ºC) with the same substrate in Petri plates, for three days, to identify the most fast and promising clones. The presence of blue color was assumed as positive responses correlated with areas size. Area boundaries were extracted automatically by analyzing color images of the samples using MATLABs in-house functions. At 60 ºC, no positive clones were detected. Two positive clones simultaneously exhibited enzymatic activity under 25, 37 and 45 ºC. In general, 37 ºC proved to be the most suitable temperature for the detection of xylanase activity. The method herein reported can be further optimized for the automatic detection of different enzymatic activities in high throughput screenings.The study received financial support from Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the project LIGNOZYMES-Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01-0145-FEDER-029773). J.S. and A.M.A.C. acknowledge their research grants UMINHO/BIM/2020/28 and UMINHO/BPD/37/2018, respectively, under the scope of the project LIGNOZYMES. The authors also acknowledge the Portuguese composting unit Lipor for kindly supplying the compost sample.info:eu-repo/semantics/publishedVersio

Vibrio coralliilyticus infection triggers a behavioural response and perturbs nutritional exchange and tissue integrity in a symbiotic coral

Under homoeostatic conditions, the relationship between the coralPocillopora damicornisandVibrio coralliilyticusiscommensal. An increase in temperature, or in the abundance ofV. coralliilyticus, can turn this association pathogenic,causing tissue lysis, expulsion of the corals’symbiotic algae (genusSymbiodinium), and eventually coral death. Using acombination of microfluidics,fluorescence microscopy, stable isotopes, electron microscopy and NanoSIMS isotopicimaging, we provide insights into the onset and progression ofV.coralliilyticusinfection in the daytime and at night, atthe tissue and (sub-)cellular level. The objective of our study was to connect the macro-scale behavioural response ofthe coral to the micro-scale nutritional interactions that occur between the host and its symbiont. In the daytime, polypsenhanced their mucus production, and actively spewed pathogens.Vibrioinfection primarily resulted in the formationof tissue lesions in the coenosarc. NanoSIMS analysis revealed infection reduced13C-assimilation inSymbiodinium, butincreased13C-assimilation in the host. In the night incubations, no mucus spewing was observed, and a mucusfilm wasformed on the coral surface.Vibrioinoculation and infection at night showed reduced13C-turnover inSymbiodinium, but didnot impact host13C-turnover. Our results show that both the nutritional interactions that occur between the two symbioticpartners and the behavioural response of the host organism play key roles in determining the progression and severity ofhost-pathogen interactions. More generally, our approach provides a new means of studying interactions (ranging frombehavioural to metabolic scales) between partners involved in complex holobiont systems, under both homoeostatic andpathogenic conditions

Tuberculosis is associated with expansion of a motile, permissive and immunomodulatory CD16(+) monocyte population via the IL-10/STAT3 axis

The human CD14+ monocyte compartment is composed by two subsets based on CD16 expression. We previously reported that this compartment is perturbed in tuberculosis (TB) patients, as reflected by the expansion of CD16+ monocytes along with disease severity. Whether this unbalance is beneficial or detrimental to host defense remains to be elucidated. Here in the context of active TB, we demonstrate that human monocytes are predisposed to differentiate towards an anti-inflammatory (M2-like) macrophage activation program characterized by theCD16+CD163+MerTK+pSTAT3+ phenotype and functional properties such as enhanced protease-dependent motility, pathogen permissivity and immunomodulation. This process is dependent on STAT3 activation, and loss-of-function experiments point towards a detrimental role in host defense against TB. Importantly, we provide a critical correlation between the abundance of the CD16+CD163+MerTK+pSTAT3+ cells and the progression of the disease either at the local level in a non-human primate tuberculous granuloma context, or at the systemic level through the detection of the soluble form of CD163 in human sera. Collectively, this study argues for the pathogenic role of the CD16+CD163+MerTK+pSTAT3+ monocyte-to-macrophage differentiation program and its potential as a target for TB therapy,and promotes the detection of circulating CD163 as a potential biomarker for disease progression and monitoringof treatment efficacy.Fil: Lastrucci, Claire. Centre National de la Recherche Scientifique; FranciaFil: Bénard, Alan. Centre National de la Recherche Scientifique; FranciaFil: Balboa, Luciana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Pingris, Karine. Centre National de la Recherche Scientifique; FranciaFil: Souriant, Shanti. Centre National de la Recherche Scientifique; FranciaFil: Poincloux, Renaud. Centre National de la Recherche Scientifique; FranciaFil: Al Saati, Talal. Inserm; FranciaFil: Rasolofo, Voahangy. Pasteur Institute in Antananarivo; MadagascarFil: González Montaner, Pablo. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; ArgentinaFil: Inwentarz, Sandra. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; ArgentinaFil: Moraña, Eduardo José. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas ; ArgentinaFil: Kondova, Ivanela. Biomedical Primate Research Centre; Países BajosFil: Verreck, Franck A. W.. Biomedical Primate Research Centre; Países BajosFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Neyrolles, Olivier. Centre National de la Recherche Scientifique; FranciaFil: Maridonneau Parini, Isabel. Centre National de la Recherche Scientifique; FranciaFil: Lugo Villarino, Geanncarlo. Centre National de la Recherche Scientifique; FranciaFil: Cougoule, Celine. Centre National de la Recherche Scientifique; Franci

Hematopoietic cell kinase (Hck) isoforms and phagocyte duties – From signaling and actin reorganization to migration and phagocytosis

International audienc

Regulation of photoprotection gene expression in Chlamydomonas by a putative E3 ubiquitin ligase complex and a homolog of CONSTANS.

Photosynthetic organisms use nonphotochemical quenching (NPQ) mechanisms to dissipate excess absorbed light energy and protect themselves from photooxidation. In the model green alga Chlamydomonas reinhardtii, the capacity for rapidly reversible NPQ (qE) is induced by high light, blue light, and UV light via increased expression of LHCSR and PSBS genes that are necessary for qE. Here, we used a forward genetics approach to identify SPA1 and CUL4, components of a putative green algal E3 ubiquitin ligase complex, as critical factors in a signaling pathway that controls light-regulated expression of the LHCSR and PSBS genes in C. reinhardtii The spa1 and cul4 mutants accumulate increased levels of LHCSR1 and PSBS proteins in high light, and unlike the wild type, they express LHCSR1 and exhibit qE capacity even when grown in low light. The spa1-1 mutation resulted in constitutively high expression of LHCSR and PSBS RNAs in both low light and high light. The qE and gene expression phenotypes of spa1-1 are blocked by mutation of CrCO, a B-box Zn-finger transcription factor that is a homolog of CONSTANS, which controls flowering time in plants. CONSTANS-like cis-regulatory sequences were identified proximal to the qE genes, consistent with CrCO acting as a direct activator of qE gene expression. We conclude that SPA1 and CUL4 are components of a conserved E3 ubiquitin ligase that acts upstream of CrCO, whose regulatory function is wired differently in C. reinhardtii to control qE capacity via cis-regulatory CrCO-binding sites at key photoprotection genes