Biology subjects

Couturier, A.

Publications and source records attributed to Couturier, A..

5 recordsLinked to original sources

bioRxiv2026

RecA structure kinetics determine SOS dynamics and predict spontaneous cell death

Even under optimal growth conditions, bacterial cells experience endogenous stresses that threaten cellular function. Individual cells sporadically activate stress responses to restore homeostasis, but some cells ultimately die. The SOS response to DNA damage is particularly heterogeneous, but the origin of its spontaneous activation and its relationship to cell fate remain unclear. Here, we imaged fluorescently labelled RecA in single Escherichia coli cells under steady-state unperturbed growth using microfluidics. We find that RecA* structures assemble frequently near replication forks and precede SOS response pulses. RecA* structure lifetime quantitatively encodes the magnitude of SOS induction: longer-lived structures promote greater LexA degradation and larger SOS pulses. Thus, cell-to-cell variability in spontaneous SOS activation reflects differences in the repair kinetics of endogenous DNA damage events. By tracking cell lineages over time, we further show that RecA* structures are strong and early predictors of cell fate. Elevated RecA* structure presence emerges up to six generations before spontaneous cell death in over 75% of cases, before other physiological disturbances become detectable. Together, our results establish spontaneous SOS activation as a quantitative readout of endogenous DNA damage processing and identify failure to resolve such damage as a major route to spontaneous bacterial cell death.

biophysics↗

bioRxiv2025

Exclusion Systems Preserve Host Cell Homeostasis and fitness, Ensuring Successful Dissemination of Conjugative Plasmids and Associated Resistance Genes

Plasmid conjugation is a major driver of antibiotic resistance dissemination in bacteria. In addition to genes required for transfer and maintenance, conjugative plasmids encode exclusion systems that prevent host cells from acquiring identical or redundant plasmids. Despite their ubiquity, the biological impact of these systems remains poorly understood. Here, we investigate the importance of the exclusion mechanism for plasmid dynamics and bacterial physiology at the single-cell level. Using real-time microscopy, we directly visualize how the absence of exclusion results in plasmid unregulated self-transfer, causing continuous and repeated plasmid exchange among host cells. This runaway conjugation severely compromises cell integrity, viability, and fitness, a largely undescribed phenomenon termed lethal zygosis. We demonstrate that lethal zygosis is associated with membrane stress, activation of the SOS response and potential reactivation of SOS-inducible prophages, as well as chromosome replication and segregation defects. This study highlights how exclusion systems maintain host cell homeostasis by limiting plasmid transfer. Paradoxically, this restriction is critical to the successful dissemination of conjugative plasmids by conferring a selective advantage, which explains their evolutionary conservation and underscores their role in the spread of antibiotic resistance among pathogenic bacteria.

microbiology↗

bioRxiv2024

A palette of bright and photostable monomeric fluorescent proteins for bacterial time-lapse imaging

Fluorescent proteins (FPs) are pivotal for examining protein production, localization, and dynamics in live bacterial cells. However, the use of FPs in time-lapse imaging is frequently constrained by issues such as oligomerization or limited photostability. Here, we report the engineering of four new fluorescent proteins: mChartreuse (green), mJuniper (cyan), mLemon (yellow), and mLychee (red), using site-directed mutagenesis. These fluorophores outperform current standards for photostability and aggregation properties while retaining high levels of brightness in living E. coli bacteria. Starting with superfolder GFP (sfGFP), we developed mChartreuse, which shows significantly enhanced brightness, stability, and monomericity compared to other GFP variants. From mChartreuse, we derived mJuniper, a cyan fluorescent protein with rapid maturation and high photostability, and mLemon, a yellow fluorescent protein with improved photostability and brightness. We also identified a mutation that eliminates residual oligomerization in red fluorescent proteins derived from Discosoma species, such as mCherry and mApple. Incorporating this mutation into mApple, along with other substitutions, resulted in mLychee, a bright and photostable monomeric red fluorescent protein. These novel fluorescent proteins advance fluorescence time-lapse analysis in bacteria and their spectral properties match current imaging standards, ensuring seamless integration into existing research workflows.

molecular biology↗

bioRxiv2022

Protective role of podocytic IL-15/STAT5 pathway in experimental focal and segmental glomerulosclerosis

During glomerular diseases, podocyte-specific pathways can modulate the intensity of the lesions and prognosis. The therapeutic targeting of these pathways could thus improve the management and prognosis of chronic kidney diseases. The Janus Kinase/ Signal Transducer and Activator of Transcription (JAK/STAT) pathway, classically described in immune cells, has been recently described in intrinsic kidney cells. Here, we show, for the first time, that STAT5 is activated in human podocytes in focal segmental glomerulosclerosis (FSGS). Additionally, Stat5 podocyte-specific inactivation aggravates the functional and structural alterations in a mouse model of FSGS. This could be due, at least in part, to an inhibition of the autophagic flux. Finally, Interleukin 15 (IL-15), a classical activator of STAT5 in immune cells, increases STAT5 phosphorylation in human podocytes and its administration alleviates glomerular injury in vivo by maintaining the autophagy flux in podocytes. In conclusion, activating podocytic STAT5 with commercially available IL-15 represents a new therapeutic avenue with the potential for FSGS.

pathology↗

bioRxiv2022

Real-time visualisation of conjugation reveals the molecular strategy evolved by the conjugative F plasmid to ensure the sequential production of plasmid factors during establishment in the new host cell

DNA conjugation is a contact-dependent horizontal gene transfer mechanism responsible for disseminating drug resistance among bacterial species. Conjugation remains poorly characterised at the cellular scale, particularly regarding the reactions occurring after the plasmid enters the new host cell. Here, we use live-cell microscopy to visualise the intracellular dynamics of conjugation in real time. We reveal that the transfer of the plasmid in single-stranded DNA (ssDNA) form followed by its conversion into double-stranded DNA (dsDNA) are fast and efficient processes that occur with specific timing and subcellular localisation. Notably, the ss-to-dsDNA conversion is the critical step that governs the timing of plasmid-encoded protein production. The leading region that first enters the recipient cell carries single-stranded promoters that allow the early and transient synthesis of leading proteins immediately upon entry of the ssDNA plasmid. The subsequent ss-to-dsDNA conversion turns off leading gene expression and licences the expression of the other plasmid genes under the control of conventional double-stranded promoters. This elegant molecular strategy evolved by the conjugative plasmid allows for the timely production of factors sequentially involved in establishing, maintaining and disseminating the plasmid.

microbiology↗