Biology subjects

Nicolas, P.

Publications and source records attributed to Nicolas, P..

11 recordsLinked to original sources

bioRxiv2026

Inference on paths of yeast transcription pre-initiation complex assembly by model-based analysis of altered occupancy profiles

Dysfunctions in transcription cause severe human diseases. In eukaryotes, transcription initiation by RNA polymerase (Pol) II requires the formation of the pre-initiation complex (PIC) composed of general transcription factors (GTFs), Pol II, and an essential multiprotein coactivator, Mediator. In vitro experiments with GTFs and Pol II have depicted a linear sequence of PIC assembly. However, PIC assembly paths in vivo at the genomic scale and Mediator's role in these paths remain poorly understood. One strategy characterizes ChIP-seq occupancy alterations in Mediator mutants, but analysis of these data still lacks a formalized interpretation framework. In this study, we address this need by developing a quantitative framework that scores candidate assembly paths according to their ability to explain these alterations. This framework combines a model of PIC assembly based on a system of differential equations with parameter optimization based on a penalized criterion. We applied this new framework to datasets from two yeast Mediator mutants, in the Med17 and Med10 subunits. Our results show that the best-supported PIC assembly paths differ from those inferred in vitro. In particular, we show that it is important to distinguish the core TFIIH module from the TFIIK kinase module, and that similar paths starting with Mediator or TBP receive different levels of support. Furthermore, although multiple paths fit the data equally well, they differ markedly for specific gene groups. Taken together, the results show that the path can depend on the gene, and are also compatible with more than one path being operational in vivo for a single gene. This work improves our understanding of PIC assembly paths and transcription initiation, and opens perspectives for future modeling and experimental studies.

bioinformatics↗

bioRxiv2026

CRISPR interference functional genomics of coding and non-coding determinants of Bacillus subtilis biofilms

The architecture of Bacillus subtilis biofilms is influenced by the coordinated regulation of cellular specialization, matrix assembly, and metabolism. B. subtilis can form different types of biofilm in diverse physical and chemical environments. Understanding the molecular mechanisms that drive biofilm heterogeneity and adaptation to different environmental niches is crucial for developing more effective strategies to control their formation. In this study, we developed a tightly dual-regulated CRISPR interference (CRISPRi) system and employed multi-scale imaging to investigate the functions of individual genes in two distinct biofilm models: the floating pellicle and the intricate, three-dimensionally structured macrocolony, which develop at the liquid-air and solid-air interfaces, respectively. Our findings validated the CRISPRi approach as a powerful method for studying biofilm development over extended periods and revealed that numerous small non-coding RNAs are involved in regulating biofilm growth dynamics and architecture. The CRISPRi approach was also applied to a pool of 507 genes and transcription units, including protein-coding genes and non-coding RNAs, to screen for cell fitness in these two biofilm models. We discovered that, while both biofilm forms rely on fundamental processes such as cell wall synthesis and nucleotide metabolism, they exhibit different genetic dependencies with regard to matrix composition, motility, and signaling. Exopolysaccharide production, motility, and chemotaxis are crucial for pellicle formation. In contrast, macrocolony development is influenced by {gamma}-polyglutamate synthesis and nutrient acquisition functions. Genes of unknown function were also identified to play a differentially important role in the two biofilm forms. Additionally, the CRISPRi screens revealed further non-coding RNAs regulating biofilm architecture and growth dynamics, adding to the existing layers of post-transcriptional control. Collectively, these results demonstrate that biofilm formation at different physical interfaces is governed by a combination of shared and unique genetic pathways tailored to the specific biofilm environment, thereby opening research avenues into the molecular mechanisms specific to the solid-air and liquid-air interfaces.

microbiology↗

bioRxiv2026

Allele-specific expression reveals complex cis- and trans-regulatory divergence underlying fruit phenotypic differences between cultivated and wild tomato species

Tomato (Solanum lycopersicum) is one of the most important agricultural crops and serves as a model system for fleshy fruit biology. However, domestication bottlenecks have led to limited genetic diversity for further crop improvement. Wild relatives represent a rich reservoir of phenotypic diversity, yet the molecular basis underlying such phenotypic diversity remains largely unknown. As gene expression variation is a major driver of phenotypic diversity, we performed genome-wide allele-specific expression analyses in F1 hybrids between cultivated tomato and three wild relatives spanning a range of evolutionary distances, across three fruit tissues and multiple developmental stages. Our analysis generated a multi-species map of cis - and trans-regulatory variation, revealing a predominant role of cis-regulatory effects in expression divergence across species, tissues, and stages. The majority of cis effects were tissue- and/or stage-specific, underscoring the importance of tissue context in regulatory variation. Regulatory mechanisms and inheritance patterns shifted with evolutionary distance, with more distantly related species showing increased cis-regulatory contributions. Finally, we identified extensive regulatory divergence in biosynthetic pathways related to fruit nutritional value and flavor quality, including carotenoids, flavonoids, alkaloids, sugars, and volatiles. This study presents a high-resolution map of regulatory variation underlying tomato fruit development and provides evolutionary insights into the regulation of fruit nutrition and flavor traits.

genomics↗

bioRxiv2025

ModuloStat: An Internet of Things' Path to Continuous Cultures in Mini-Bioreactors

In continuous culture, a population of microorganisms is propagated in a stable environment over many generations. This is particularly relevant for experimental evolution and metabolic studies. However, continuous culture protocols are difficult to implement, so they are not commonly used in microbiology laboratories. Here, we present the ModuloStat, a modular, open-source framework that facilitates continuous culture in mini-bioreactors. The ModuloStat system is grounded on digital fabrication tools easily accessible in FabLabs and programmable electronics. Maintaining a culture is divided into tasks assigned to dedicated printed circuit boards with a microcontroller connected to a Wi-Fi network. According to Internet of Things principles, each board operates a set of sensors and actuators autonomously and can receive and send information. The boards are stacked to implement complex behaviors and can be modified to accommodate new features. A thermoregulated box holds the components and can be placed on a laboratory bench or transported under a sterile hood for inoculation. Sterility is ensured by autoclaving, after assembly, all components that will come into contact with the culture medium. In-situ optical density monitoring combined with modularity and computer control enables many cultivation modes. Additionally, we present the construction of the Bacillus subtilis strain ZB designed for bioreactor culture that exhibits a zero-biofilm phenotype. To demonstrate the systems versatility, we performed several experimental cultures with this model organism, including chemostat, turbidostat, medium swap, and a cascade of bioreactors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/669317v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@16b3bbborg.highwire.dtl.DTLVardef@11ea708org.highwire.dtl.DTLVardef@1681769org.highwire.dtl.DTLVardef@d91946_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

The mutational landscape of Bacillus subtilis conditional hypermutators suggests how proofreading inherently skews polymerase error rates

Polymerase errors during DNA replication are a major source of point mutations in genomes. The resulting rate of spontaneous mutation also depends on the counteracting activity of DNA repair mechanisms, with mutator phenotypes appearing constantly and allowing for periods of rapid evolution in nature and in the laboratory. Here, we use the Gram-positive model bacterium Bacillus subtilis to disentangle the contributions of DNA polymerase initial nucleotide selectivity, DNA polymerase proofreading, and mismatch repair (MMR) to the mutation rate. To achieve this, we constructed several conditional hypermutators with a proofreading-deficient allele of polC and/or a deficient allele of mutL and performed mutation accumulation experiments. With their wide range of mutation rates and contrasting mutation profiles, these conditional hypermutators enrich the B. subtilis synthetic biology toolbox for directed evolution. Using mathematical models, we investigated how to interpret the apparent probabilities with which errors escape MMR and proofreading, highlighting the difficulties of working with counts that aggregate potentially heterogeneous mutations and with unknowns about the pathways leading to mutations in the wild-type. Aware of these difficulties, the analysis shows that proofreading prevents partial saturation of the MMR in B. subtilis and that an inherent drawback of proofreading is to skew the net polymerase error rates by amplifying intrinsic biases in nucleotide selectivity.

genomics↗

bioRxiv2023

Diving into bacterial dormancy: emergence of osmotically stable wall-less forms in an aquatic environment

Bacteria can respond to environmental stresses by entering a dormant state, called viable but non-culturable (VBNC) state, in which they no longer grow in routine culture media. VBNC pathogens pose thus a significant risk for human and animal health as they are not detected by standard growth-based techniques and can "wake up" back into a vegetative and virulent state. Although hundreds of species were reported to become VBNC in response to different stresses, the molecular mechanisms governing this phenotypic switch remain largely elusive. Here, we characterized the VBNC state transition process in the Gram-positive pathogen Listeria monocytogenes in response to nutritional deprivation. By combining fluorescence microscopy, cryo-electron tomography and analytical biochemistry, we found that starvation in mineral water drives L. monocytogenes into a VBNC state via a mechanism of cell wall (CW) shedding that generates osmotically stable CW-deficient (CWD) coccoid forms. This phenomenon occurs in multiple L. monocytogenes strains and in other Listeria species, suggesting it may be a stress-adapting process transversal to the Listeria genus. Transcriptomic and gene-targeted approaches revealed the stress response regulator SigB and the autolysin NamA as major moderators of CW loss and VBNC state transition. Finally, we show that this CWD dormant state is transient as VBNC Listeria revert back to a walled, vegetative and virulent state after passage in embryonated eggs. Our findings provide unprecedented detail on the mechanisms governing the transition to a VBNC state, and reveal that dormant CWD bacterial forms can naturally arise in aquatic environments without osmotic stabilization. This may represent an alternative strategy for bacterial survival in oligotrophic conditions, which can potentially generate public health-threatening reservoirs of undetectable pathogens.

microbiology↗

bioRxiv2023

Multi-scale transcriptome unveils spatial organisation and temporal dynamics of Bacillus subtilis biofilms

Bacillus subtilis has been extensively used to study the molecular mechanisms behind the development and dispersal of surface bacterial multicellular communities. Well-structured spatially organised communities (colony, pellicle, and submerged biofilm) share some similarities, but also display considerable differences at the structural, chemical and biological levels. To unveil the spatial transcriptional heterogeneity between the different communities, we analysed by RNA-seq nine spatio-physiological populations selected from planktonic and spatially organised communities. This led to a global landscape characterisation of gene expression profiles uncovering genes specifically expressed in each compartmental population. From this mesoscale analysis and using fluorescent transcriptional reporter fusions, 17 genes were selected and their patterns of expression reported at single cell scale with time-lapse confocal laser scanning microscopy (CLSM). Derived kymographs allowed to emphasise spectacular mosaic gene expression patterns within a biofilm. A special emphasis on oppositely regulated carbon metabolism genes (gapA and gapB) permitted to pinpoint the coexistence of spatially segregated bacteria under either glycolytic or gluconeogenic regime in a same biofilm population. Altogether, this study gives novel insights on the development and dispersal of B. subtilis surface-associated communities.

microbiology↗

bioRxiv2022

Investigation of the genus Flavobacterium as a reservoir for fish-pathogenic bacterial species: the case of Flavobacterium collinsii

Bacteria of the genus Flavobacterium are recovered from a large variety of environments. Among the described species, Flavobacterium psychrophilum and Flavobacterium columnare are causing considerable losses in fish farms. Alongside these well-known fish-pathogenic species, isolates belonging to the same genus recovered from diseased or apparently healthy wild, feral, and farmed fish have been suspected to be pathogenic. Here, we report the identification and genomic characterization of a F. collinsii isolate (TRV642) retrieved from rainbow trout spleen. A phylogenetic tree of the genus built by aligning the core genome of 195 Flavobacterium species revealed that F. collinsii is standing within a cluster of species associated to diseased fish, the closest one being F. tructae which was recently confirmed as pathogenic. We evaluated the pathogenicity of F. collinsii TRV642 as well as of F. bernardetii F-372T, another recently described species reported as a possible emerging pathogen. Following intramuscular injection challenges in rainbow trout, no clinical signs nor mortalities were observed. However, F. collinsii was isolated from the internal organs of wounded fish, suggesting that the bacterium could invade fish under compromised conditions such as stress and/or wounds. Our results suggest that some fish-associated Flavobacterium species should be considered as opportunistic fish pathogens causing disease under specific circumstances. IMPORTANCEAquaculture has expanded significantly worldwide in the last decades and accounts for half of human fish consumption. However, infectious fish diseases are a major bottleneck for its sustainable development and an increasing number of bacterial species from diseased fish raise a great concern. The current study revealed phylogenetic associations with ecological niches among the Flavobacterium species. We also focused on Flavobacterium collinsii that belongs to a group of putative pathogenic species. The genome contents revealed a versatile metabolic repertoire suggesting the use of diverse nutrient sources, a characteristic of saprophytic or commensal bacteria. In a rainbow trout experimental challenge, the bacterium colonized only oppressed fish facing stressful conditions suggesting opportunistic pathogenic behavior. This study highlights the importance of experimentally evaluating the pathogenicity of the numerous bacterial species retrieved from diseased fish.

microbiology↗

bioRxiv2022

Identification and functional characterization of two heme uptake systems required for virulence of the fish pathogen Flavobacterium psychrophilum

Bacterial pathogens have a critical impact on aquaculture, a sector that accounts for half of the human fish consumption. Flavobacterium psychrophilum (phylum Bacteroidetes) is responsible for bacterial cold-water disease in salmonids worldwide. The molecular factors involved in host invasion, colonization and hemorrhagic septicaemia are mostly unknown. In this study, we identified two new TonB-dependent receptors, HfpR and BfpR, that are required for adaptation to iron conditions encountered during infection and for virulence in rainbow trout. Transcriptional analyses revealed that their expression is tightly controlled and upregulated under specific iron sources and concentrations. Characterization of deletion mutants showed that they act without redundancy: BfpR is required for optimal growth in the presence of high hemoglobin level, while HfpR confers the capacity to acquire nutrient iron from heme or hemoglobin under iron scarcity. The gene hfpY, co-transcribed with hfpR, encodes a protein related to the HmuY family. We demonstrated that HfpY binds heme and contributes significantly to host colonization and disease severity. Overall, these results are consistent with a model in which both BfpR and Hfp systems promote heme uptake and respond to distinct signals to adapt iron acquisition to the different stages of pathogenesis. Our findings give insight into the molecular basis of pathogenicity of a serious pathogen belonging to the understudied family Flavobacteriaceae and point to the newly identified heme receptors as promising targets for antibacterial development.

microbiology↗

bioRxiv2022

Termination factor Rho mediates transcriptional reprogramming of Bacillus subtilis stationary phase.

Reprogramming of gene expression during transition from exponential growth to stationary phase is crucial for bacterial survival. In the model Gram-positive bacterium Bacillus subtilis, this process is mainly governed by the activity of the global transcription regulators AbrB, CodY and Spo0A. We recently showed that the transcription termination factor Rho, known for its ubiquitous role in the inhibition of antisense transcription, is involved in Spo0A-mediated regulation of differentiation programs specific to the stationary phase in B. subtilis. To identify other aspects of the regulatory role of Rho during adaptation to starvation, we have constructed a B. subtilis strain that expresses rho at a relatively stable high level in order to circumvent its decrease occurring in the wild-type cells entering the stationary phase. We show that B. subtilis cells stably expressing Rho fail to sporulate and to develop genetic competence, which is largely, but not exclusively, due to abnormally low expression of the master regulator Spo0A. Moreover, in addition to a global decrease of antisense transcription, these cells exhibit genome-wide alterations of sense transcription. A significant part of these alterations affects genes from global regulatory networks of cellular adaptation to the stationary phase and reflects the attenuated de-repression of the AbrB and CodY regulons and the weakened stringent response. Accordingly, stabilization of Rho level reprograms stationary phase-specific physiology of B. subtilis cells, negatively affects cellular adaptation to nutrient limitations and alters cell-fate decision-making to such an extent that it blocks development of genetic competence and sporulation. Taken together, these results indicate that the activity of termination factor Rho constitutes a previously unknown layer of control over the stationary phase and post-exponential adaptive strategies in B. subtilis, from the adjustment of cellular metabolism to the activation of survival programs.

microbiology↗

bioRxiv2019

Statistical modeling of bacterial promoter sequences for regulatory motif discovery with the help of transcriptome data: application to Listeria monocytogenes

Automatic de novo identification of the main regulons of a bacterium from genome and transcriptome data remains a challenge. To address this task, we propose a statistical model of promoter DNA sequences that can use information on exact positions of the transcription start sites and condition-dependent expression profiles. Two main novelties are to allow overlaps between motif occurrences and to incorporate covariates summarising expression profiles (e.g. coordinates in projection spaces or hierarchical clustering trees). All parameters are estimated using a dedicated trans-dimensional Markov chain Monte Carlo algorithm that adjusts, simultaneously, for many motifs and many expression covariates: the width and palindromic properties of the corresponding position-weight matrices, the number of parameters to describe position with respect to the transcription start site, and the choice of relevant expression covariates. A data-set of transcription start sites and expression profiles available for the Listeria monocytogenes is analysed. The results validate the approach and provide a new global view of the transcription regulatory network of this important model food-borne pathogen. A previously unreported motif that may play an important role in the regulation of growth was found in promoter regions of ribosomal protein genes.

bioinformatics↗