Biology subjects

Val, M.-E.

Publications and source records attributed to Val, M.-E..

6 recordsLinked to original sources

bioRxiv2025

Evolutionary Constraints on RNA Polymerase Gene Positioning in the Genome of Fast-Growing Bacteria..

How gene order along chromosomes affects cellular homeostasis and genome evolution remains poorly understood. Bacterial chromosomes are organized along the replication origin (oriC)-terminus (ter) axis. The spatial arrengement of genes within this axis may influence cellular physiology, genome evolution, and transcriptional regulation. The catalytic core of the sole bacterial RNA polymerase (RNAP) is encoded by rpoB and rpoC within the universally conserved rplKAJL-rpoBC locus. In fast growing bacteria this locus is located near oriC potentially linking genome organization to cellular physiology. Here, we investigated the functional relevance of rplKAJL-rpoBC chromosomal positioning by relocating it within Vibrio cholerae genome. While viable, strains harboring rplKAJL-rpoBC far from oriC exhibited nutrient-dependent fitness defects. These phenotypes correlated with reduced locus copy number and RNAP abundance, without changes in its subcellular distribution. Introducing a second copy of rpIKAJL-rpoBC at a distal genomic location from oriC abolished the phenotypes, demonstrating that replication-associated gene dosage effects were the main mechanism behind the observed physiological changes. Further uncoupling rpoB and rpoC from the rplKAJL-rpoBC locus revealed that fitness costs were specifically linked to RNAP gene relocation. Our results reveal that selective pressures act to maintain RNAP-encoding genes near oriC to optimize replication-associated dosage effects, ensuring efficient RNAP production during exponential growth. We discuss the relationship between gene order and ecological strategies linked to bacterial growth. Our work underscores gene order as an underestimated but critical factor shaping cellular physiology and genome evolution. SignificanceBacterial chromosomes have a highly plastic gene content but the importance of their spatial organization and gene order is started to be appreciated as a driver of cellular physiology. Growing evidence suggests that the spatial organization of bacterial genomes plays a critical role in gene expression, replication dynamics, and fitness. In this study, we demonstrate that the conserved positioning of rpoB and rpoC, encoding the catalytic core of RNA polymerase (RNAP), near the replication origin (oriC) is essential for optimal bacterial growth. By relocating the rplKAJL-rpoBC locus in Vibrio cholerae, we show that its chromosomal position directly impacts nutrient-dependent fitness, RNAP abundance, and replication-associated gene dosage effects. These findings suggest that selective pressures maintain genes encoding RNAP near oriC to ensure efficient transcriptional output during exponential growth. More broadly, our study highlights gene order as an underappreciated factor in bacterial genome evolution, beyond its known roles in operon structure and transcriptional regulation. Given the fundamental role of RNAP, our results have implications for understanding bacterial ecological strategies, adaptation, and synthetic genome design. By integrating positional genomics with physiological studies, we provide a theoretical and experimental framework for investigating how genome architecture shapes cellular function.

microbiology↗

bioRxiv2025

Replication coordination marks the domestication of large extrachromosomal replicons in bacteria

Bacterial genomes often include extrachromosomal replicons (ERs), ranging from small plasmids to nearly chromosome-sized elements, that foster genome plasticity and adaptation. Despite their prevalence, the mechanisms underlying ER domestication and their long-term adaptation within bacterial hosts remain largely unexplored. By analyzing over 40,000 complete bacterial genomes, we identified two main ER categories: small ERs with diverse GC content and large ERs ([≥]10% the size of the main chromosome) that closely match the GC content of the chromosome. Across multiple phyla, marker frequency analyses showed that large ERs maintain a 1:1 copy number with the chromosome. Another key finding of this study is that large ERs terminate replication in synchrony with the chromosome. Hi-C contact maps revealed consistent ori-ori interactions between chromosomes and ERs. In large ERs, inter-replichore and ter-ter interactions, along with the recruitment of key chromosomal segregation motifs, suggest the co-option of chromosome-associated replication and segregation machineries. Together, our findings indicate that as ERs become larger, they become increasingly reliant on chromosome-driven processes for stable inheritance, potentially explaining why they do not exceed the size of the chromosome.

microbiology↗

bioRxiv2025

Genetic Determinants of pOXA-48 Plasmid Maintenance and Propagation in Escherichia coli

Conjugative plasmids are the main drivers of antibiotic resistance dissemination contributing to the emergence and extensive spread of multidrug resistance clinical bacterial pathogens. pOXA-48 plasmids, belonging to the IncL group, have emerged as the primary vehicle for carbapenem resistance in Enterobacteriaceae. Despite the problematic prevalence of pOXA-48, most research has focused on epidemiology and genomics, leaving gaps in our understanding of the mechanisms behind its propagation. In this study, we used a transposon sequencing approach to identify genetic elements critical for plasmid stability, replication, and conjugative transfer. Our results identified a novel type I toxin-antitoxin system, new essential maintenance factors, and components of the type IV secretion system and regulatory elements crucial for conjugation. This study advances our understanding of pOXA-48 biology, providing key insights into the genetic factors underlying its successful maintenance and spread in bacterial populations.

microbiology↗

bioRxiv2024

MatP delays the segregation of chromosome termini independently of cell division and tetramer formation in Vibrio cholerae

Vibrio cholerae harbours a primary chromosome derived from the monochromosomal ancestor of the Vibrionales (ChrI) and a secondary chromosome derived from a megaplasmid (ChrII). The coordinated segregation of the replication terminus of both chromosomes (TerI and TerII) determines when and where cell division occurs. ChrI encodes a homolog of Escherichia coli MatP, a protein that binds to a DNA motif (matS) that is overrepresented in replication termini. Here, we use a combination of deep sequencing and fluorescence microscopy techniques to show that V. cholerae MatP structures TerI and TerII into macrodomains, targets them to mid-cell during replication, and delays their segregation, thus supporting that ChrII behaves as a bona fide chromosome. We further show that the extent of the segregation delay mediated by MatP depends on the number and local density of matS sites, and is independent of its assembly into tetramers and any interaction with the divisome, in contrast to what has been previously observed in E. coli.

microbiology↗

bioRxiv2023

Identification of the active mechanism of aminoglycoside entry in V. cholerae through characterization of sRNA ctrR, regulating carbohydrate utilization and transport.

The possible active entry of aminoglycosides in bacterial cells has been debated since the development of this antibiotic family. Here we report the identification of their active transport mechanism in Vibrio species. We combined genome-wide transcriptional analysis and fitness screens to identify alterations driven by treatment of V. cholerae with sub-minimum inhibitory concentrations (sub-MIC) of the aminoglycoside tobramycin. RNA-seq data showed downregulation of the small non-coding RNA ncRNA586 during such treatment, while Tn-seq revealed that inactivation of this sRNA was associated with improved fitness in the presence of tobramycin. This sRNA is located near sugar transport genes and previous work on a homologous region in Vibrio tasmaniensis suggested that this sRNA stabilizes gene transcripts for carbohydrate transport and utilization, as well as phage receptors. The role for ncRNA586, hereafter named ctrR, in the transport of both carbohydrates and aminoglycosides, was further investigated. Flow cytometry on cells treated with a fluorescent aminoglycoside confirmed the role of ctrR and of carbohydrate transporters in differential aminoglycoside entry. Despite sequence diversity, ctrR showed functional conservation across the Vibrionales. This system in directly modulated by carbon sources, suggesting regulation by carbon catabolite repression, a widely conserved mechanism in Gram-negative bacteria, priming future research on aminoglycoside uptake by sugar transporters in other bacterial species.

microbiology↗

bioRxiv2019

Macromolecular crowding links ribosomal protein gene dosage to growth rate in Vibrio cholerae.

Ribosomal protein (RP) genes locate near the replication origin (oriC) in fast-growing bacteria, which is thought to have been selected as a translation optimization strategy. Relocation of S10-spc- locus (S10), which codes for most of the RP, to ectopic genomic positions shows that its relative distance to the oriC correlates to a reduction on its dosage, its expression, and bacterial growth rate. Deep-sequencing revealed that S10 relocation altered chromosomal replication dynamics and genome-wide transcription. Such changes increased as a function of oriC-S10 distance. Strikingly, in this work we observed that protein production capacity was independent of S10 position. Since RP constitute a large proportion of cell mass, lower S10 dosage could lead to changes in macromolecular crowding, impacting cell physiology. Accordingly, cytoplasm fluidity was higher in mutants where S10 is most distant from oriC. In hyperosmotic conditions, when crowding differences are minimized, the growth rate and replication dynamics were highly alleviated in these strains. Therefore, on top of its essential function in translation, RP genomic location contributes to sustain optimal macromolecular crowding. This is a novel mechanism coordinating DNA replication with bacterial growth.

microbiology↗