Mapping Meiotic DNA Breaks: Two Fully-Automated Pipelines to Analyze Single-Strand DNA Sequencing Data, hotSSDS and hotSSDS-extra.

Molecular approaches are required to detect DNA double-strand break (DSB) events and to map and quantify them at high resolution. One of the most popular molecular methods in the field of meiotic recombination is the ChIP-SSDS (Chromatin immuno-precipitation and single-strand DNA sequencing). Here, we present two fully-automated Nextflow-based pipelines to analyze the sequencing data generated by this method. The first one identifies highly reproducible DSB sites, while the second provides a characterization of recovered DSB sites, including the description of the hotspot distribution and intensity along the genome and the overlap with specific regions such as gene features or known DSB hotspots. Finally, we discuss limitations/advantages and key points to consider when applying this method to specific genotypes or unconventional species.

Transgenerational exposure to ocean acidification impacts the hepatic transcriptome of European sea bass (Dicentrarchus labrax).

Physiological effects of ocean acidification associated with elevated CO2 concentrations in seawater is the subject of numerous studies in teleost fish. While the short time within-generation impact of ocean acidification (OA) on acid-base exchange and energy metabolism is relatively well described, the effects associated with transgenerational exposure to OA are much less known. Yet, the impacts of OA can vary in time with the potential for acclimation or adaptation of a species. Previous studies in our lab demonstrated that transgenerational exposure to OA had extensive effects on the transcriptome of the olfactory epithelium of European sea bass (Dicentrarchus labrax), especially on genes related to ion balance, energy metabolism, immune system, synaptic plasticity, neuron excitability and wiring. In the present study, we complete the previous work by investigating the effect of transgenerational exposure to OA on the hepatic transcriptome of European sea bass. Differential gene expression analysis was performed by RNAseq technology on RNA extracted from the liver of two groups of 18 months F2 juveniles that had been exposed since spawning to the same AO conditions as their parents (F1) to either actual pH or end-of-century predicted pH levels (IPCC RCP8.5), respectively. Here we show that transgenerational exposure to OA significantly impacts the expression of 236 hepatic transcripts including genes mainly involved in inflammatory/immune responses but also in carbohydrate metabolism and cellular homeostasis. Even if this transcriptomic impact is relatively limited compared to what was shown in the olfactory system, this work confirmed that fish transgenerationally exposed to OA exhibit molecular regulation of processes related to metabolism and inflammation. Also, our data expand the up-regulation of a key gene involved in different physiological pathways including calcium homeostasis (i.e. pthr1), which we already observed in the olfactory epithelium, to the liver. Even if our experimental design does not allow to discriminate direct within F2 generation effects from transgenerational plasticity, these results offer the perspective of more functional analyses to determine the potential physiological impact of OA exposure on fish physiology with ecological relevance.

Tracing key genes associated with the Pinctada margaritifera albino phenotype from juvenile to cultured pearl harvest stages using multiple whole transcriptome sequencing.

BACKGROUND: Albino mutations are commonly observed in the animal kingdom, including in bivalves. In the black-lipped pearl oyster Pinctada margaritifera, albino specimens are characterized by total or partial absence of colouration resulting in typical white shell phenotype expression. The relationship of shell colour with resulting cultured pearl colour is of great economic interest in P. margaritifera, on which a pearl industry is based. Hence, the albino phenotype provides a useful way to examine the molecular mechanisms underlying pigmentation. RESULTS: Whole transcriptome RNA-sequencing analysis comparing albino and black wild-type phenotypes at three stages over the culture cycle of P. margaritifera revealed a total of 1606, 798 and 187 differentially expressed genes in whole juvenile, adult mantle and pearl sac tissue, respectively. These genes were found to be involved in five main molecular pathways, tightly linked to known pigmentation pathways: melanogenesis, calcium signalling pathway, Notch signalling pathway, pigment transport and biomineralization. Additionally, significant phenotype-associated SNPs were selected (N = 159), including two located in the Pif biomineralization gene, which codes for nacre formation. Interestingly, significantly different transcript splicing was detected between juvenile (N = 1366) and adult mantle tissue (N = 313) in, e.g., the tyrosinase Tyr-1 gene, which showed more complex regulation in mantle, and the Notch1 encoding gene, which was upregulated in albino juveniles. CONCLUSION: This multiple RNA-seq approach provided new knowledge about genes associated with the P. margaritifera albino phenotype, highlighting: 1) new molecular pathways, such as the Notch signalling pathway in pigmentation, 2) associated SNP markers with biomineraliszation gene of interest like Pif for marker-assisted selection and prevention of inbreeding, and 3) alternative gene splicing for melanin biosynthesis implicating tyrosinase.

Supplementation of live yeast based feed additive in early life promotes rumen microbial colonization and fibrolytic potential in lambs.

Rumen microbiota is of paramount importance for ruminant digestion efficiency as the microbial fermentations supply the host animal with essential sources of energy and nitrogen. Early separation of newborns from the dam and distribution of artificial milk (Artificial Milking System or AMS) could impair rumen microbial colonization, which would not only affect rumen function but also have possible negative effects on hindgut homeostasis, and impact animal health and performance. In this study, we monitored microbial communities in the rumen and the feces of 16 lambs separated from their dams from 12 h of age and artificially fed with milk replacer and starter feed from d8, in absence or presence of a combination of the live yeast Saccharomyces cerevisiae CNCM I-1077 and selected yeast metabolites. Microbial groups and targeted bacterial species were quantified by qPCR and microbial diversity and composition were assessed by 16S rDNA amplicon sequencing in samples collected from birth to 2 months of age. The fibrolytic potential of the rumen microbiota was analyzed with a DNA microarray targeting genes coding for 8 glycoside hydrolase (GH) families. In Control lambs, poor establishment of fibrolytic communities was observed. Microbial composition shifted as the lambs aged. The live yeast supplement induced significant changes in relative abundances of a few bacterial OTUs across time in the rumen samples, among which some involved in crucial rumen function, and favored establishment of Trichostomatia and Neocallimastigaceae eukaryotic families. The supplemented lambs also harbored greater abundances in Fibrobacter succinogenes after weaning. Microarray data indicated that key cellulase and hemicellulase encoding-genes were present from early age in the rumen and that in the Supplemented lambs, a greater proportion of hemicellulase genes was present. Moreover, a higher proportion of GH genes from ciliate protozoa and fungi was found in the rumen of those animals. This yeast combination improved microbial colonization in the maturing rumen, with a potentially more specialized ecosystem towards efficient fiber degradation, which suggests a possible positive impact on lamb gut development and digestive efficiency.

Procyanidin-Cell Wall Interactions within Apple Matrices Decrease the Metabolization of Procyanidins by the Human Gut Microbiota and the Anti-Inflammatory Effect of the Resulting Microbial Metabolome In Vitro.

B-type oligomeric procyanidins in apples constitute an important source of polyphenols in the human diet. Their role in health is not known, although it is suggested that they generate beneficial bioactive compounds upon metabolization by the gut microbiota. During apple processing, procyanidins interact with cell-wall polysaccharides and form stable complexes. These interactions need to be taken into consideration in order to better assess the biological effects of fruit constituents. Our objectives were to evaluate the impact of these interactions on the microbial metabolization of cell walls and procyanidins, and to investigate the potential anti-inflammatory activity of the resulting metabolome, in addition to analyzing the taxonomical changes which the microbiota undergo. In vitro fermentation of three model apple matrices with microbiota from 4 healthy donors showed that the binding of procyanidins to cell-wall polysaccharides, whether covalently or non-covalently, substantially reduced procyanidin degradation. Although cell wall-unbound procyanidins negatively affected carbohydrate fermentation, they generated more hydroxyphenylvaleric acid than bound procyanidins, and increased the abundance of Adlercreutzia and Gordonibacter genera. The best results in terms of production of anti-inflammatory bioactive metabolites were observed from the apple matrix with no bonds between procyanidins and cell wall polysaccharides, although the matrix with non-covalent bonds was not far behind.

Transcriptomic analysis reveals specific metabolic pathways of enterohemorrhagic Escherichia coli O157:H7 in bovine digestive contents.

BACKGROUND: The cattle gastrointestinal tract (GIT) is the main enterohemorrhagic Escherichia coli (EHEC) reservoir. In order to identify nutrients required for the survival or multiplication of EHEC in the bovine GIT, we compared the transcriptomes of the EHEC O157:H7 reference strain EDL933 cultured in vitro in bovine digestive contents (DCs) (rumen, small intestine and rectum) using RNA-sequencing. RESULTS: Gene expression profiles showed that EHEC EDL933 activated common but also specific metabolic pathways to survive in the different bovine DCs. Mucus-derived carbohydrates seem important in EHEC nutrition in posterior DCs (small intestine and rectum) but not in rumen content. Additional carbohydrates (xylose, ribose, mannitol, galactitol) as well as gluconeogenic substrates (aspartate, serine, glycerol) would also be used by EHEC as carbon and/or nitrogen sources all along the bovine GIT including the rumen. However, xylose, GalNac, ribose and fucose transport and/or assimilation encoding genes were over-expressed during incubation in rectum content compared with rumen and intestine contents, and genes coding for maltose transport were only induced in rectum. This suggests a role for these carbohydrates in the colonization of the cattle rectum, considered as the major site for EHEC multiplication. In contrast, the transcription of the genes associated with the assimilation of ethanolamine, an important nitrogen source for EHEC, was poorly induced in EHEC growing in rectum content, suggesting that ethanolamine is mainly assimilated in the cattle rumen and small intestine. Respiratory flexibility would also be required for EHEC survival because of the redundancy of dehydrogenases and reductases simultaneously induced in the bovine DCs, probably in response to the availability of electron donors and acceptors. CONCLUSION: EHEC EDL933 showed a high flexibility in the activation of genes involved in respiratory pathways and assimilation of carbon and nitrogen sources, most of them from animal origin. This may allow the bacterium to adapt and survive in the various bovine GIT compartments. Obtaining a better understanding of EHEC physiology in bovine GIT is a key step to ultimately propose strategies to limit EHEC carriage and shedding by cattle.

Factors Involved in the Persistence of a Shiga Toxin-Producing Escherichia coli O157:H7 Strain in Bovine Feces and Gastro-Intestinal Content.

Healthy cattle are the primary reservoir for O157:H7 Shiga toxin-producing E. coli responsible for human food-borne infections. Because farm environment acts as a source of cattle contamination, it is important to better understand the factors controlling the persistence of E. coli O157:H7 outside the bovine gut. The E. coli O157:H7 strain MC2, identified as a persistent strain in French farms, possessed the characteristics required to cause human infections and genetic markers associated with clinical O157:H7 isolates. Therefore, the capacity of E. coli MC2 to survive during its transit through the bovine gastro-intestinal tract (GIT) and to respond to stresses potentially encountered in extra-intestinal environments was analyzed. E. coli MC2 survived in rumen fluids, grew in the content of posterior digestive compartments and survived in bovine feces at 15°C predicting a successful transit of the bacteria along the bovine GIT and its persistence outside the bovine intestine. E. coli MC2 possessed the genetic information encoding 14 adherence systems including adhesins with properties related to colonization of the bovine intestine (F9 fimbriae, EhaA and EspP autotransporters, HCP pilus, FdeC adhesin) reflecting the capacity of the bacteria to colonize different segments of the bovine GIT. E. coli MC2 was also a strong biofilm producer when incubated in fecal samples at low temperature and had a greater ability to form biofilms than the bovine commensal E. coli strain BG1. Furthermore, in contrast to BG1, E. coli MC2 responded to temperature stresses by inducing the genes cspA and htrA during its survival in bovine feces at 15°C. E. coli MC2 also activated genes that are part of the GhoT/GhoS, HicA/HicB and EcnB/EcnA toxin/antitoxin systems involved in the response of E. coli to nutrient starvation and chemical stresses. In summary, the large number of colonization factors known to bind to intestinal epithelium and to biotic or abiotic surfaces, the capacity to produce biofilms and to activate stress fitness genes in bovine feces could explain the persistence of E. coli MC2 in the farm environment.

Draft Genome Sequence of Enterohemorrhagic Escherichia coli O157:H7 Strain MC2 Isolated from Cattle in France.

Enterohemorrhagic Escherichia coli (EHEC) with serotype O157:H7 is a major foodborne pathogen. Here, we report the draft genome sequence of EHEC O157:H7 strain MC2 isolated from cattle in France. The assembly contains 5,400,376 bp that encoded 5,914 predicted genes (5,805 protein-encoding genes and 109 RNA genes).

Draft genome sequence and characterization of commensal Escherichia coli strain BG1 isolated from bovine gastro-intestinal tract.

Escherichia coli is the most abundant facultative anaerobic bacteria in the gastro-intestinal tract of mammals but can be responsible for intestinal infection due to acquisition of virulence factors. Genomes of pathogenic E. coli strains are widely described whereas those of bovine commensal E. coli strains are very scarce. Here, we report the genome sequence, annotation, and features of the commensal E. coli BG1 isolated from the gastro-intestinal tract of cattle. Whole genome sequencing analysis showed that BG1 has a chromosome of 4,782,107 bp coding for 4465 proteins and 97 RNAs. E. coli BG1 belonged to the serotype O159:H21, was classified in the phylogroup B1 and possessed the genetic information encoding "virulence factors" such as adherence systems, iron acquisition and flagella synthesis. A total of 12 adherence systems were detected reflecting the potential ability of BG1 to colonize different segments of the bovine gastro-intestinal tract. E. coli BG1 is unable to assimilate ethanolamine that confers a nutritional advantage to some pathogenic E. coli in the bovine gastro-intestinal tract. Genome analysis revealed the presence of i) 34 amino acids change due to non-synonymous SNPs among the genes encoding ethanolamine transport and assimilation, and ii) an additional predicted alpha helix inserted in cobalamin adenosyltransferase, a key enzyme required for ethanolamine assimilation. These modifications could explain the incapacity of BG1 to use ethanolamine. The BG1 genome can now be used as a reference (control strain) for subsequent evolution and comparative studies.