An Insight into Functional Metagenomics: A High-Throughput Approach to Decipher Food-Microbiota-Host Interactions in the Human Gut.

Our understanding of the symbiotic relationship between the microbiota and its host has constantly evolved since our understanding that the "self" was not only defined by our genetic patrimony but also by the genomes of bugs living in us. The first culture-based methods highlighted the important functions of the microbiota. However, these methods had strong limitations and did not allow for a full understanding of the complex relationships that occur at the interface between the microbiota and the host. The recent development of metagenomic approaches has been a groundbreaking step towards this understanding. Its use has provided new insights and perspectives. In the present chapter, we will describe the advances of functional metagenomics to decipher food-microbiota and host-microbiota interactions. This powerful high-throughput approach allows for the assessment of the microbiota as a whole (including non-cultured bacteria) and enabled the discovery of new signaling pathways and functions involved in the crosstalk between food, the gut microbiota and its host. We will present the pipeline and highlight the most important studies that helped to develop the field. To conclude, we will emphasize the most recent developments and hot topics in functional metagenomics.

A High-Content Microscopy Screening Identifies New Genes Involved in Cell Width Control in Bacillus subtilis.

How cells control their shape and size is a fundamental question of biology. In most bacteria, cell shape is imposed by the peptidoglycan (PG) polymeric meshwork that surrounds the cell. Thus, bacterial cell morphogenesis results from the coordinated action of the proteins assembling and degrading the PG shell. Remarkably, during steady-state growth, most bacteria maintain a defined shape along generations, suggesting that error-proof mechanisms tightly control the process. In the rod-shaped model for the Gram-positive bacterium Bacillus subtilis, the average cell length varies as a function of the growth rate, but the cell diameter remains constant throughout the cell cycle and across growth conditions. Here, in an attempt to shed light on the cellular circuits controlling bacterial cell width, we developed a screen to identify genetic determinants of cell width in B. subtilis. Using high-content screening (HCS) fluorescence microscopy and semiautomated measurement of single-cell dimensions, we screened a library of ∼4,000 single knockout mutants. We identified 13 mutations significantly altering cell diameter, in genes that belong to several functional groups. In particular, our results indicate that metabolism plays a major role in cell width control in B. subtilis. IMPORTANCE Bacterial shape is primarily dictated by the external cell wall, a vital structure that, as such, is the target of countless antibiotics. Our understanding of how bacteria synthesize and maintain this structure is therefore a cardinal question for both basic and applied research. Bacteria usually multiply from generation to generation while maintaining their progenies with rigorously identical shapes. This implies that the bacterial cells constantly monitor and maintain a set of parameters to ensure this perpetuation. Here, our study uses a large-scale microscopy approach to identify at the whole-genome level, in a model bacterium, the genes involved in the control of one of the most tightly controlled cellular parameters, the cell width.

Prediction of the intestinal resistome by a three-dimensional structure-based method.

The intestinal microbiota is considered to be a major reservoir of antibiotic resistance determinants (ARDs) that could potentially be transferred to bacterial pathogens via mobile genetic elements. Yet, this assumption is poorly supported by empirical evidence due to the distant homologies between known ARDs (mostly from culturable bacteria) and ARDs from the intestinal microbiota. Consequently, an accurate census of intestinal ARDs (that is, the intestinal resistome) has not yet been fully determined. For this purpose, we developed and validated an annotation method (called pairwise comparative modelling) on the basis of a three-dimensional structure (homology comparative modelling), leading to the prediction of 6,095 ARDs in a catalogue of 3.9 million proteins from the human intestinal microbiota. We found that the majority of predicted ARDs (pdARDs) were distantly related to known ARDs (mean amino acid identity 29.8%) and found little evidence supporting their transfer between species. According to the composition of their resistome, we were able to cluster subjects from the MetaHIT cohort (n = 663) into six resistotypes that were connected to the previously described enterotypes. Finally, we found that the relative abundance of pdARDs was positively associated with gene richness, but not when subjects were exposed to antibiotics. Altogether, our results indicate that the majority of intestinal microbiota ARDs can be considered intrinsic to the dominant commensal microbiota and that these genes are rarely shared with bacterial pathogens.

Genetic variability of the activity of bidirectional promoters: a pilot study in bovine muscle.

Bidirectional promoters are regulatory regions co-regulating the expression of two neighbouring genes organized in a head-to-head orientation. In recent years, these regulatory regions have been studied in many organisms; however, no investigation to date has been done to analyse the genetic variation of the activity of this type of promoter regions. In our study, we conducted an investigation to first identify bidirectional promoters sharing genes expressed in bovine Longissimus thoracis and then to find genetic variants affecting the activity of some of these bidirectional promoters. Combining bovine gene information and expression data obtained using RNA-Seq, we identified 120 putative bidirectional promoters active in bovine muscle. We experimentally validated in vitro 16 of these bidirectional promoters. Finally, using gene expression and whole-genome genotyping data, we explored the variability of the activity in muscle of the identified bidirectional promoters and discovered genetic variants affecting their activity. We found that the expression level of 77 genes is correlated with the activity of 12 bidirectional promoters. We also identified 57 single nucleotide polymorphisms associated with the activity of 5 bidirectional promoters. To our knowledge, our study is the first analysis in any species of the genetic variability of the activity of bidirectional promoters.

Construction and validation of a novel dual reporter vector for studying mammalian bidirectional promoters.

Regulation of gene expression plays important role in cellular functions. With the development of sequencing techniques, more and more genomes are available and genome-wide analyses of genomic structures that may affect gene expression regulation are now possible. Analyses of several genomes have found a class of regulatory regions that contain elements that initiate transcription of two different genes positioned with a head-to-head arrangement in two opposite directions. These regulatory regions are known as bidirectional promoters. Although bidirectional promoters have been known for years, recent genome-scale studies have shown that the regulation of the expression of up to 10% of the genes are controlled by bidirectional promoters. These findings are based mostly on computational work and only a limited number of putative bidirectional promoters have been experimentally validated. Developing methods to study bidirectional promoters will allow researchers to understand how these regions are regulated and the roles that divergent transcription plays in the expression of genes. Here, we have developed a novel dual-fluorescence reporter gene vector to study the transcriptional output of mammalian bidirectional promoters. We demonstrate that this vector is capable of expressing reporter genes under the control of bidirectional promoters, using the known human OSGEP/APEX bidirectional promoter.

Bovine TWINKLE and mitochondrial ribosomal protein L43 genes are regulated by an evolutionary conserved bidirectional promoter.

TWINKLE is a mitochondrial DNA helicase playing an important role in mitochondrial DNA replication. In human, mutations in this gene cause progressive external ophtalmoplegia and mitochondrial DNA depletion syndrome-7. TWINKLE is well conserved among multicellular eukaryotes and is believed to be a key regulator of mitochondrial DNA copy number in mammals. Despite its involvement in several diseases and its important function in mitochondrial DNA metabolism, nothing is known about the regulation of the expression of TWINKLE. We have analysed the 5'-flanking genomic region of the bovine TWINKLE gene and found it was localised adjacent to the MRPL43 gene in a head-to-head orientation, suggesting that both genes are regulated by a shared bidirectional promoter. The bovine 75-bp long intergenic region shows substantial homology across different species and contains several conserved putative transcription factor binding sites. A TATA box, however, was lacking. Using a dual fluorescent reporter system and transient transfection assays, we have analysed the bovine intergenic region between TWINKLE and MRPL43. This small genomic fragment showed a bidirectional promoter activity. As the TWINKLE/MRPL43 bidirectional promoter tested was highly conserved, it is likely that the results we obtained here in cattle may be extended to the other species.

EGR1 and EGR2 involvement in vertebrate tendon differentiation.

The molecules involved in vertebrate tendon formation during development remain largely unknown. To date, only two DNA-binding proteins have been identified as being involved in vertebrate tendon formation, the basic helix-loop-helix transcription factor Scleraxis and, recently, the Mohawk homeobox gene. We investigated the involvement of the early growth response transcription factors Egr1 and Egr2 in vertebrate tendon formation. We established that Egr1 and Egr2 expression in tendon cells was correlated with the increase of collagen expression during tendon cell differentiation in embryonic limbs. Vertebrate tendon differentiation relies on a muscle-derived FGF (fibroblast growth factor) signal. FGF4 was able to activate the expression of Egr genes and that of the tendon-associated collagens in chick limbs. Egr gene misexpression experiments using the chick model allowed us to establish that either Egr gene has the ability to induce de novo expression of the reference tendon marker scleraxis, the main tendon collagen Col1a1, and other tendon-associated collagens Col3a1, Col5a1, Col12a1, and Col14a1. Mouse mutants for Egr1 or Egr2 displayed reduced amounts of Col1a1 transcripts and a decrease in the number of collagen fibrils in embryonic tendons. Moreover, EGR1 and EGR2 trans-activated the mouse Col1a1 proximal promoter and were recruited to the tendon regulatory regions of this promoter. These results identify EGRs as novel DNA-binding proteins involved in vertebrate tendon differentiation by regulating type I collagen production.

Scleraxis and NFATc regulate the expression of the pro-alpha1(I) collagen gene in tendon fibroblasts.

The combinatorial action of separate cis-acting elements controls the cell-specific expression of type I collagen genes. In particular, we have shown that two short elements located between -3.2 and -2.3 kb and named TSE1 and TSE2 are needed for expression of the mouse COL1a1 gene in tendon fibroblasts. In this study, we analyzed the trans-acting factors binding to TSE1 and TSE2. Gel shift experiments showed that scleraxis (SCX), which is a basic helix-loop-helix transcription factor that is expressed selectively in tendon fibroblasts, binds TSE2, preferentially as a SCX/E47 heterodimer. In transfection experiments, overexpression of SCX and E47 strongly enhanced the activity of reporter constructs harboring either four copies of TSE2 cloned upstream of the COL1a1 minimal promoter or a 3.2-kb segment of the COL1a1 proximal promoter. Analysis of TSE1 showed that it contains a consensus binding site for NFATc transcription factors. This led us to show that the NFATc4 gene is expressed in tendons of developing mouse limbs and in TT-D6 cells, a cell line that has characteristics of tendon fibroblasts. In gel shift assays, TSE1 bound NFATc proteins present in nuclear extracts from TT-D6 cells. In transfection experiments, overexpression of NFATc transactivated a reporter construct harboring four copies of TSE1 cloned upstream of the COL1a1 minimal promoter. By contrast, inhibition of the nuclear translocation of NFATc proteins in TT-D6 cells strongly inhibited the expression of the COL1a1 gene. Taken together, these results suggest that SCX and NFATc4 cooperate to activate the COL1a1 gene specifically in tendon fibroblasts.