Biology subjects

Forquet, R.

Publications and source records attributed to Forquet, R..

8 recordsLinked to original sources

bioRxiv2023

Quantitative spatial analysis of bacterial transcriptome and chromosome structural data with GRATIOSA: application to twin-supercoiled domain distribution

While classical models of transcriptional regulation focus on transcription factors binding at promoters, gene expression is also influenced by chromosome organization. Understanding this spatial regulation strongly benefits from integrated and quantitative spatial analyses of genome-scale data such as RNA-Seq and ChIP-Seq. We introduce Genome Regulation Analysis Tool Incorporating Organization and Spatial Architecture (GRATIOSA), a Python package making such combined analyses more automatic, systematic and reproducible. While current software focuses on initial analysis steps (read mapping and counting), GRAsTIOSA proposes an integrated framework for subsequent analyses, providing a broad range of spatially-resolved quantitative data comparisons and representations. As an example, we quantitatively assess the validity and extension of the twin-supercoiled domain model in Escherichia coli genome-wide transcription, using recent topoisomerase ChIP-Seq data. We show that topoisomerases are locally recruited by the 40% most highly expressed transcription units, with a magnitude correlating with the expression level. The recruitment of topoisomerase I extends to around 10 kb upstream, whereas DNA gyrase is recruited at least 30 kb downstream of transcription units. This organization is the primary determinant of topoisomerase I recruitment, whereas gyrase binding is additionally modulated at larger 100-200 kb length-scale. Further analyses of spatial regulation will be facilitated by GRATIOSA.

bioinformatics↗

bioRxiv2022

Quantitative contribution of the spacer length in the supercoiling-sensitivity of bacterial promoters

DNA supercoiling acts as a global transcriptional regulator in bacteria, but the promoter sequence or structural determinants controlling its effect remain unclear. It was previously proposed to modulate the torsional angle between the -10 and -35 hexamers, and thereby regulate the formation of the closed-complex depending on the length of the "spacer" between them. Here, we develop a ther-modynamic model of this notion based on DNA elasticity, providing quantitative and parameter-free predictions on the relative activation of promoters containing a short vs long spacer when the DNA supercoiling level is varied. The model is tested through an analysis of in vitro and in vivo expression assays of mutant promoters with variable spacer lengths, confirming its accuracy for spacers ranging from 15 to 19 nucleotides, except those of 16 nucleotides where other regulatory mechanisms likely overcome the effect of this specific step. An analysis at the whole-genome scale in E. coli then demonstrates a significant effect of the spacer length on the genomic expression after transient or inheritable superhelical variations, validating the models predictions. Altogether, this study shows that the torsional constraints associated to promoter binding by RNA Polymerase underpin a basal and global regulatory mechanism independent of specific transcription factors.

bioinformatics↗

bioRxiv2021

Protein interaction network analysis reveals growth conditions-specific crosstalk between chromosomal DNA replication and other cellular processes in E. coli

E. coli and many other bacterial species can alter their cell cycle according to nutrient availability. Under optimal conditions bacteria grow and divide very fast but they slow down the cell cycle when conditions deteriorate. This adaptability is underlined by mechanisms coordinating cell growth with duplication of genetic material and cell division. Several mechanisms regulating DNA replication process in E. coli have been described with biochemical details so far. Nevertheless we still dont fully understand the source of remarkable precision that allows bacterial cells to coordinate their growth and chromosome replication. To shed light on regulation of E. coli DNA replication at systemic level, we used affinity purification coupled with mass spectrometry (AP-MS) to characterize protein-protein interactions (PPIs) formed by key E. coli replication proteins, under disparate bacterial growth conditions and phases. We present the resulting dynamic replication protein interaction network (PIN) and highlight links between DNA replication and several cellular processes, like outer membrane synthesis, RNA degradation and modification or starvation response. ImportanceDNA replication is a vital process, ensuring propagation of genetic material to progeny cells. Despite decades of studies we still dont fully understand how bacteria coordinate chromosomal DNA duplication with cell growth and cell division under optimal and stressful conditions. At molecular level, regulation of processes, including DNA replication, is often executed through direct protein-protein interactions (PPIs). In this work we present PPIs formed by the key E. coli replication proteins under three different bacterial growth conditions. We show novel PPIs with confirmed impact on chromosomal DNA replication. Our results provide also alternative explanations of genetic interactions uncovered before by others for E.coli replication machinery.

microbiology↗

bioRxiv2021

What is a supercoiling-sensitive gene? Insights from topoisomerase I inhibition in the Gram-negative bacterium Dickeya dadantii

DNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of these enzymes with antibiotics leads to global supercoiling modifications and subsequent changes in global gene expression. In previous studies, genes responding to DNA relaxation induced by gyrase inhibition were categorised as "supercoiling-sensitive". Here, we studied the opposite variation of DNA supercoiling in the phytopathogen Dickeya dadantii using the non-marketed antibiotic seconeolitsine. We showed that the drug is active against topoisomerase I from this species, and analysed the first transcriptomic response of a Gram-negative bacterium to topoisomerase I inhibition. We find that the responding genes essentially differ from those observed after DNA relaxation, and further depend on the growth phase. We characterised these genes at the functional level, and also detected distinct patterns in terms of expression level, spatial and orientational organisation along the chromosome. Altogether, these results highlight that the supercoiling-sensitivity is a complex feature, which depends on the action of specific topoisomerases, on the physiological conditions, and on their genomic context. Based on previous in vitro expression data of several promoters, we propose a qualitative model of SC-dependent regulation that accounts for many of the contrasting transcriptomic features observed after gyrase or topoisomerase I inhibition.

genomics↗

bioRxiv2020

Bacterial promoter opening underpins ubiquitous transcriptional regulation by DNA supercoiling

DNA supercoiling acts as a global transcriptional regulator, which contributes to the rapid transcriptional response of bacteria to many environmental changes. Although a large fraction of promoters from distant species respond to superhelical variations, the sequence or structural determinants of this behaviour remain elusive. Here, we propose the sequence of the "discriminator" element located downstream of the -10 hexamer to play an important role in this response, by modulating the facility of open-complex formation during transcription initiation. We develop a quantitative model of this regulatory mechanism relying on known parameters of DNA thermodynamics, and show that its predictions quantitatively match the in vitro and in vivo supercoiling response of stable RNA promoters previously measured, as well as the in vivo response of selected mRNA promoters with mutated discriminator sequences. We then test the universality of this mechanism by statistical analysis of promoter sequences in transcriptomes of phylogenetically distant bacteria under conditions of supercoiling variations, (1) by gyrase inhibitors, (2) by environmental stresses, (3) inherited in the longest-running evolution experiment. In all cases, we identify a robust and significant sequence signature in the discriminator region, suggesting that promoter opening underpins an ubiquitous regulatory mechanism in the prokaryotic kingdom, based on the fundamental mechanical properties of DNA.

microbiology↗

bioRxiv2020

Mapping the complex transcriptional landscape of the phytopathogenic bacterium Dickeya dadantii

Dickeya dadantii is a phytopathogenic bacterium that causes soft rot in a wide range of plant hosts worldwide, and a model organism for studying virulence gene regulation. The present study provides a comprehensive and annotated transcriptomic map of D. dadantii obtained by a computational method combining five independent transcriptomic datasets: (i) paired-end RNA-seq data for a precise re-construction of the RNA landscape; (ii) DNA microarray data providing transcriptional responses to a broad variety of environmental conditions; (iii) long-read Nanopore native RNA-seq data for isoform-level transcriptome validation and determination of transcription termination sites; (iv)dRNA-seq data forthe precise mapping of transcription start sites; (v) in planta DNA microarray data for a comparison of gene expression profiles between in vitro experiments and the early stages of plant infection. Our results show that transcription units sometimes coincide with predicted operons but are generally longer, most of them comprising internal promoters and terminators that generate alternative transcripts of variable gene composition. We characterise the occurrence of transcriptional read-through at terminators, which might play a basal regulation role and explain the extent of transcription beyond the scale of operons. We finally highlight the presence of noncontiguous operons and excludons in the D. dadantii genome, novel genomic arrangements that might contribute to the basal coordination of transcription. The highlighted transcriptional organisation may allow D. dadantii to finely adjust its gene expression programme for a rapid adaptation to fast changing environments. IMPORTANCEThis is the first transcriptomic map of a Dickeya species. It may therefore significantly contribute to further progress in the field of phytopathogenicity. It is also one of the first reported applications of long-read Nanopore native RNA-seq in prokaryotes. Our findings yield insights into basal rules of coordination of transcription that might be valid for other bacteria, and may raise interest in the field of microbiology in general. In particular, we demonstrate that gene expression is coordinated at the scale of transcription units rather than operons, which are larger functional genomic units capable of generating transcripts with variable gene composition for a fine-tuning of gene expression in response to environmental changes. In line with recent studies, our findings indicate that the canonical operon model is insufficient to explain the complexity of bacterial transcriptomes.

microbiology↗

bioRxiv2020

The nucleoid-associated protein IHF acts as a domainin protein coordinating the bacterial virulence traits with global transcription

Bacterial pathogenic growth requires a swift coordination of pathogenicity functions with various kinds of environmental stresses encountered in the course of host infection. Among the factors critical for bacterial adaptation are changes of DNA topology and binding effects of nucleoid-associated proteins transducing the environmental signals to the chromosome and coordinating the global transcriptional response to stress. In this study we use the model phytopathogen Dickeya dadantii to analyse the organisation of transcription by the nucleoid-associated protein IHF. We determine both phenotypic effects of ihfA mutation on D. dadantii virulence and the transcriptional response under various conditions of growth. For the first time in enterobacteria, we examine the transcriptome of an IHF-depleted mutant under conditions of DNA relaxation, revealing a subtle interplay between IHF and DNA topology. We show that this mutation reorganises the genomic expression by altering the distribution of DNA supercoils along the chromosome at different length scales, thus affecting many virulence genes involved in both symptomatic and asymptomatic phases of infection, including those required for pectin catabolism. Altogether, we propose that IHF is a "domainin" protein, the inactivation of which impairs the coordination of chromosomal stress-response domains harbouring various virulence traits, thus abrogating the pathogenicity of D. dadantii.

microbiology↗

bioRxiv2019

Bacterial genome architecture shapes global transcriptional regulation by DNA supercoiling

DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.

microbiology↗