Biology subjects

Nasser, W.

Publications and source records attributed to Nasser, W..

15 recordsLinked to original sources

bioRxiv2024

Fis family members synergistically control the virulence of Legionella pneumophila

Legionella pneumophila virulence is controlled in a growth phase-dependent manner by a complex regulatory network involving several two-component systems, small regulatory RNAs and the translational CsrA regulator. Here, we address the additional role of Nucleoid-associated proteins (NAP) regulators in this network, by investigating the regulatory functions of the three Fis paralogs (Fis1, Fis2, Fis3), a unique feature among bacteria, in the infection cycle of L. pneumophila. Specifically, we show that deletion of fis1 has a major impact on L. pneumophila virulence, and that deletion of fis2 enhances the intensity of this phenotype. Consistently, RNA-seq analysis and reporter gene fusions demonstrate the predominant role of Fis1 in the regulation of many virulence-related genes, including those involved in the flagellum, pili biosynthesis, and Dot/Icm type 4 secretion machinery, as well as several genes encoding Dot/Icm effectors. Both Fis1 and Fis2 bind to AT-rich motifs upstream their target genes, but Fis1 with higher affinity than Fis2. Importantly, Fis1 and Fis2 would be capable of forming heterodimers that could bind with variable affinity to this AT-rich motif. It is also important to note that the three Fis proteins are not produced at the same time and in the same amounts. We therefore hypothesize that the duplication of fis genes in L. pneumophila is not simply a back-up system to compensate for potentially deleterious mutations in a fis gene, but rather a means to fine-tune the expression of targeted genes, particularly virulence genes. IMPORTANCEAppropriate control of virulence gene expression is crucial to the success of bacterial infection. Nucleoid-associated protein regulators, including Fis proteins, have been shown to participate in the virulence of several human pathogens. The importance of our discovery lies in the fact that L. pneumophila possesses three non-homologous Fis proteins instead of just one. We demonstrate that Fis1 and Fis2 are not functional duplicates of each other. On the contrary, Fis1 and Fis2 are synthesized neither simultaneously nor in equal amounts during the bacterial growth phase, and they cooperate to regulate virulence gene expression by targeting similar AT-rich motifs, albeit with distinct affinity, and by being capable of forming heterodimers. Taken together, our data suggest that the high conservation of fis gene duplication results from the need for fine-tuned control of Legionella virulence in response to its different environmental and human hosts, rather than from functional redundancy to circumvent deleterious fis mutations.

microbiology↗

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

Soft limbal niche maintains stem cell compartmentalization and function through YAP

Stem cells (SCs) decision to self-renew or differentiate largely depends on the external control of their niche. However, the complex mechanisms that underlie this crosstalk are poorly understood. To address this question, we focused on the corneal epithelial SC model in which the SC niche, known as the limbus, is spatially segregated from the differentiation compartment. We report that the unique biomechanical property of the limbus supports the nuclear localization and function of Yes-associated protein (YAP), a putative mediator of the mechanotransduction pathway. Perturbation of tissue stiffness or YAP activity affects SC function as well as tissue integrity under homeostasis and significantly inhibited the regeneration of the SC population following SC depletion. In vitro experiments revealed that substrates with the rigidity of the corneal differentiation compartment inhibit YAP localization and induce differentiation, a mechanism that is mediated by the TGF{beta}-SMAD2/3 pathway. Taken together, these results indicate that SC sense biomechanical niche signals and that manipulation of mechano-sensory machinery or its downstream biochemical output may bear fruits in SC expansion for regenerative therapy. HighlightsO_LIYAP is essential for limbal SC function, regeneration, and dedifferentiation C_LIO_LILox over-expression stiffens the limbal niche, affects SC phenotype and corneal integrity C_LIO_LICorneal rigidity represses YAP and stemness in a SMAD2/3-dependent manner C_LIO_LIManipulation of mechanosensory or TGF-{beta} pathway influences limbal SC expansion in vitro C_LI

cell biology↗

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↗

bioRxiv2021

Carbon catabolite repression in pectin digestion by phytopathogen Dickeya dadantii

The catabolism of pectin from the plant cell walls plays a crucial role in the virulence of the phytopathogen Dickeya dadantii. In particular, the timely expression of pel genes encoding major pectate lyases is essential to circumvent the plant defense systems and induce a massive pectinolytic activity during the maceration phase. While previous studies identified the role of a positive feedback loop specific to the pectin degradation pathway, here we show that the pel expression pattern is controlled by a metabolic switch between glucose and pectin. We develop a dynamical and quantitative regulatory model of this process integrating the two main regulators CRP and KdgR related to these two sources of carbon, and reproducing the concentration profiles of the associated metabolites, cAMP and KDG respectively, quantified using a new HPLC method. The model involves only 5 adjustable parameters, and recapitulates the dynamics of these metabolic pathways during bacterial growth together with the regulatory events occurring at the promoters of two major pel genes, pelE and pelD. It highlights their activity as an instance of carbon catabolite repression occurring at the transcriptional regulatory level, and directly related to the virulence of D. dadantii. The model also shows that quantitative differences in the binding properties of common regulators at these two promoters resulted in a qualitative different role of pelD and pelE in the metabolic switch, and also likely in conditions of infection, explaining their evolutionary conservation as separate genes in this species.

systems biology↗

bioRxiv2021

RNA chaperones Hfq and ProQ play a key role in the virulence of the plant pathogenic bacterium Dickeya dadantii

Dickeya dadantii is an important pathogenic bacterium that infects a number of crops including potato and chicory. While extensive works have been carried out on the control of the transcription of its genes encoding the main virulence functions, little information is available on the post-transcriptional regulation of these functions. We investigated the involvement of the RNA chaperones Hfq and ProQ in the production of the main D. dadantii virulence functions. Phenotypic assays on the hfq and proQ mutants showed that inactivation of hfq resulted in a growth defect, a modified capacity for biofilm formation and strongly reduced motility, and in the production of degradative extracellular enzymes (proteases, cellulase and pectate lyases). Accordingly, the hfq mutant failed to cause soft rot on chicory leaves. The proQ mutant had reduced resistance to osmotic stress, reduced extracellular pectate lyase activity compared to the wild-type strain, and reduced virulence on chicory leaves. Most of the phenotypes of the hfq and proQ mutants were related to the low amounts of mRNA of the corresponding virulence factors. Complementation of the double mutant hfq-proQ by each individual protein and cross-complementation of each chaperone suggested that they might exert their effects via partially overlapping but different sets of targets. Overall, it clearly appeared that the two Hfq and ProQ RNA chaperones are important regulators of pathogenicity in D. dadantii. This underscores that virulence genes are regulated post transcriptionally by non-coding RNAs.

microbiology↗

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

Development and validation of an HPLC method to quantify 2-Keto-3-deoxy-gluconate (KDG), a major metabolite in pectin and alginate degradation pathways

A rapid and sensitive High Performance Liquid Chromatography (HPLC) method with photometric and fluorescence detection is developed for routine analysis of 2-Keto-3-deoxy-gluconate (KDG), a catabolite product of pectin and alginate. These polysaccharides are primary-based compounds for biofuel production and for generation of high-value-added products. HPLC is performed, after derivatization of the 2-oxo-acid groups of the metabolite with o-phenylenediamine (oPD), using a linear gradient of trifluoroacetic acid and acetonitrile. Quantification is accomplished with an internal standard method. The gradient is optimized to distinguish KDG from its close structural analogues such as 5-keto-4-deoxyuronate (DKI) and 2,5-diketo-3-deoxygluconate (DKII). The proposed method is simple, highly sensitive and accurate for time course analysis of pectin or alginate degradation. HighlightsA fluorescent based-HPLC method report the quantification of KDG, a metabolite originating from alginate and from pectin degradation pathways, using derivatization with o-phenylenediamine (oPD)

biochemistry↗

bioRxiv2020

Capturing limbal epithelial stem cell population dynamics, signature and their niche

Stem cells (SCs) are traditionally viewed as rare, slow-cycling cells that follow deterministic rules dictating their self-renewal or differentiation. It was several decades ago, when limbal epithelial SCs (LSCs) that regenerate the corneal epithelium were one of the first sporadic, quiescent SCs ever discovered. However, LSC dynamics, heterogeneity and genetic signature are largely unknown. Moreover, recent accumulating evidence strongly suggested that epithelial SCs are actually abundant, frequently dividing cells that display stochastic behavior.In this work, we performed an in-depth analysis of the murine limbal epithelium by single-cell RNA sequencing and quantitative lineage tracing. The generated data provided an atlas of cell states of the corneal epithelial lineage, and particularly, revealed the co-existence of two novel LSC populations that reside in separate and well-defined sub-compartments. In the “outer” limbus, we identified a primitive widespread population of quiescent LSCs (qLSCs) that uniformly express Krt15/Gpha2/Ifitm3/Cd63 proteins, while the “inner” limbus host prevalent active LSCs (aLSCs) co-expressing Krt15-GFP/Atf3/Mt1-2/Socs3. Analysis of LSC population dynamics suggests that while qLSCs and aLSCs possess different proliferation rates, they both follow similar stochastic rules that dictate their self-renewal and differentiation. Finally, T cells were distributed in close proximity to qLSCs. Indeed, their absence or inhibition resulted in the loss of quiescence and delayed wound healing. Taken together, we propose that divergent regenerative strategies are tailored to properly support tissue-specific physiological constraints. The present study suggests that in the case of the cornea, quiescent epithelial SCs are abundant, follow stochastic rules and neutral drift dynamics.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

DNA sequence-directed cooperation between nucleoid-associated proteins

Nucleoid associated proteins (NAPs) are a class of highly abundant DNA binding proteins in bacteria and archaea. While the composition and relative abundance of the NAPs change during the bacterial growth cycle, surprisingly little is known about their crosstalk in mutually binding to the bacterial chromosome and stabilising higher-order nucleoprotein complexes. Here, we use atomic force microscopy and solid-state nanopores to investigate long-range nucleoprotein structures formed by the binding of two major NAPs, FIS and H-NS, to DNA molecules with distinct binding-site arrangements. We find that spatial organisation of the protein binding sites can govern the higher-order architecture of the nucleoprotein complexes. Based on sequence arrangement the complexes differed in their global shape and compaction, as well as the extent of FIS and H-NS binding. Our observations highlight the important role the DNA sequence plays in driving structural differentiations within the bacterial chromosome.

biophysics↗

bioRxiv2019

The phytopathogenic nature of Dickeya aquatica 174/2 and the dynamic early evolution of Dickeya pathogenicity

Originality-Significance statementAlthough the reach of large-scale comparative studies has spread exponentially over the years, the phytopathogenic Dickeya group remains overlooked. In this work, we sequence the complete genome of Dickeya aquatica type strain, a species isolated from water that was first assumed to be non-phytopathogenic. We show that the proteome of D. aquatica contains a wide number of proteins involved in Dickeya virulence, including plant cell wall degrading enzymes, suggesting that this species could be in fact pathogenic. Using experimental approaches, we confirm this prediction and uncover the particular affinity of D. aquatica for acidic fruits. In-depth phylogenomic analyses reveal that Dickeya species display a great degree of genetic plasticity in the pathogenicity determinants, explaining how this bacterial group was able to colonize a wide variety of plants growing in different climates. These observations greatly advance our understanding of how bacteria adapt to new ecological niches.\n\nSummaryDickeya is a genus of phytopathogenic enterobacterales causing soft rot in a variety of plants (e.g. potato, chicory, maize). Among the species affiliated to this genus, Dickeya aquatica, described in 2014, remained particularly mysterious because it had no known host. Furthermore, while D. aquatica was proposed to represent a deep-branching species among Dickeya genus, its precise phylogenetic position remained elusive.\n\nHere, we report the complete genome sequence of the D. aquatica type strain 174/2. We demonstrate the affinity of D. aquaticaT for acidic fruits such as tomato and cucumber, and show that exposure of this bacterium to acidic pH induces twitching motility. An in-depth phylogenomic analysis of all available Dickeya proteomes pinpoints D. aquatica as the second deepest branching lineage within this genus and reclassifies two lineages that likely correspond to new genomospecies (gs.): Dickeya gs. poaceaephila (Dickeya sp NCPPB 569) and Dickeya gs. undicola (Dickeya sp 2B12), together with a new putative genus, tentatively named Prodigiosinella. Finally, from comparative analyses of Dickeya proteomes we infer the complex evolutionary history of this genus, paving the way to study the adaptive patterns and processes of Dickeya to different environmental niches and hosts. In particular, we hypothetize that the lack of xylanases and xylose degradation pathways in D. aquatica could reflects adaptation to aquatic charophyte hosts which, in contrast to land plants, do not contain xyloglucans.

genomics↗

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↗

bioRxiv2018

xAtlas: Scalable small variant calling across heterogeneous next-generation sequencing experiments

MotivationThe rapid development of next-generation sequencing (NGS) technologies has lowered the barriers to genomic data generation, resulting in millions of samples sequenced across diverse experimental designs. The growing volume and heterogeneity of these sequencing data complicate the further optimization of methods for identifying DNA variation, especially considering that curated highconfidence variant call sets commonly used to evaluate these methods are generally developed by reference to results from the analysis of comparatively small and homogeneous sample sets.\n\nResultsWe have developed xAtlas, an application for the identification of single nucleotide variants (SNV) and small insertions and deletions (indels) in NGS data. xAtlas is easily scalable and enables execution and retraining with rapid development cycles. Generation of variant calls in VCF or gVCF format from BAM or CRAM alignments is accomplished in less than one CPU-hour per 30x short-read human whole-genome. The retraining capabilities of xAtlas allow its core variant evaluation models to be optimized on new sample data and user-defined truth sets. Obtaining SNV and indels calls from xAtlas can be achieved more than 40 times faster than established methods while retaining the same accuracy.\n\nAvailabilityFreely available under a BSD 3-clause license at https://github.com/jfarek/xatlas.\n\nContactfarek@bcm.edu\n\nSupplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗