Biology subjects

Lesterlin, C.

Publications and source records attributed to Lesterlin, C..

14 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↗

bioRxiv2026

Antibiotic inhibition uncouples growth from horizontal gene transfer

Conjugative plasmids often encode resistance to multiple antibiotics, allowing a single mobile genetic element to transfer across diverse selective environments. This co-selection is often invoked to explain the persistence and spread of multidrug resistance, with antibiotic resistance genes encoded on the same plasmid frequently treated as equivalent routes of plasmid maintenance. However, accumulating evidence suggests that different antibiotics can produce distinct co-selection outcomes. These differences have been interpreted through the lens of fitness and population-level selection, while the potential effects on plasmid transfer itself are largely unknown. Here, we show that different antibiotics generate significantly different transfer dynamics for the exact same multidrug-resistant plasmid. Surprisingly, much of this variation arises from continued conjugation after antibiotic exposure begins, whereby a transient residual conjugation window persists despite strong growth inhibition, allowing new transconjugants to continue forming for hours after treatment in an antibiotic dependent manner. A simple model for describing antibiotic effect on transfer competence of both donors and recipients quantitatively explains these antibiotic-specific dissemination dynamics. Moreover, different recipient genetic backgrounds exhibit distinct residual conjugation windows, demonstrating that the capacity for continued plasmid transfer during antibiotic exposure is both drug- and host-dependent. Together, our results show that antibiotics suppress plasmid dissemination on different timescales, creating drug-specific residual conjugation windows that are largely decoupled from growth inhibition. These findings identify conjugation dynamics as a previously overlooked contributor to co-selection outcomes and suggest that antibiotics that select for the same multidrug-resistant plasmid are not equivalent in their ability to promote plasmid persistence.

microbiology↗

bioRxiv2026

Model-based evaluation of Targeted-Antibacterial-Plasmids (TAPs) transfer kinetics and resensitization of pOXA-48 carbapenem-resistant Escherichia coli

Background Targeted-Antibacterial Plasmids (TAPs) are engineered mobile genetic elements that use bacterial conjugation to deliver selective CRISPR/Cas9 antibacterial activity against a specific target strain. Yet, the efficiency of TAPs is typically evaluated at a single time point, whereas the success of TAP-mediated resensitization critically depends on the dynamics of plasmid transfer and the complex interactions between bacterial subpopulations. This is the first study to evaluate the efficiency of a conjugation-based antibacterial approach at the subpopulation level, using an analytical framework analogous to that used for conventional antibiotics. Here, we investigate which process limits resensitization by TAPF-dCas9-OXA48: plasmid delivery, dCas9 activity, or the emergence of refractory and escape populations. Methods We fitted a mechanistic model of five interacting subpopulations (donors, recipients, transconjugants, escapers, and recusants) to 44 longitudinal conjugation experiments and used the fitted model to explore a range of biologically relevant scenarios. Results Using longitudinal conjugation data spanning 24 h, we show that up to 24% of recipients become recusants within 24h, refractory to further conjugation via entry exclusion, while secondary transconjugant emergence stays below 0.01%. Overall resensitization efficiency reaches up to 80%. Conclusion Plasmid transfer, rather than dCas9 repression, therefore appears to be the main bottleneck limiting the efficiency of TAPF-dCas9-OXA48 efficiency. These results identify plasmid delivery as a key engineering target for improving the performance of future TAPs.

bioinformatics↗

bioRxiv2026

Plasmid-encoded host reprogramming promotes plasmid dissemination

Conjugative plasmids are major drivers of antibiotic resistance dissemination, yet how newly transferred plasmids establish in recipient cells remains poorly understood. Here we investigate YfjB, a previously uncharacterized conserved leading-region protein, which is zygotically induced immediately after plasmid entry and acts specifically during the earliest post-transfer stages. Multi-omics analyses reveal that YfjB reprograms host transcription, triggering extensive metabolic rewiring that compensates the transient fitness cost of plasmid acquisition. Structural analyses show that YfjB is a ParB-like protein containing a CTP-binding domain and a helix-turn-helix DNA-binding motif, linked to a previously uncharacterized dimerization module that forms a V-shaped clamp-like architecture compatible with DNA loading. Consistently, live-cell imaging reveals nucleoid-associated foci in transconjugants, and ChIP-seq identifies multiple chromosomal binding sites. We therefore rename the protein HerB (Host Expression Reprogrammer, ParB-like). More broadly, our findings reveal how mobile genetic elements facilitate their dissemination by transiently subverting host physiology.

microbiology↗

bioRxiv2026

Widespread immune systems protect bacteria against conjugative plasmids

Conjugative plasmids are a class of mobile genetic elements capable of efficient transfer between bacterial cells. Although they can introduce beneficial traits such as antibiotic resistance to recipients, they may also behave as genetic parasites. Bacteria would thus be expected to have evolved barriers to plasmid conjugation. However, the distribution of these barriers and their underlying mechanisms remain poorly understood. Here, we performed a large-scale analysis of 364 diverse strains of the opportunistic pathogen Acinetobacter baumannii as recipients of the broad-host-range conjugative plasmids R388 and RP4. Major variations in host susceptibilities to conjugation, with limited phylogenetic association, suggested multiple and fast-evolving plasmid-specific barriers. Functional genetic analyses revealed a role for core genes, pointing to epistasis or genetic background effects. This is illustrated by the previously unrecognized role of H-NS expression in alleviating conjugation barriers in a strain-dependent manner. Most importantly, we identified three novel immune systems protecting bacteria against conjugation by R388 and RP4. Their patchy distribution within the species, and that of their homologs across bacteria, indicate that they are part of a dynamic repertoire of immune systems against conjugation. While the Ishtar system promotes plasmid loss through putative HEPN nuclease domains, Namtar and Attar sense distinct components of the R388 type IV secretion system (T4SS) to trigger a non-proliferative, energetically depleted state, analogously to the abortive infection response of anti-phage defenses. Live imaging of conjugation showed Namtar halting cell division in Escherichia coli recipients, conferring population-level immunity against plasmid spread via horizontal and vertical transmission. The existence of immune systems specifically targeting T4SS components suggests that conjugative plasmids impose a selective disadvantage greater than previously thought. This work reveals an additional layer of bacterial immunity directed at a class of genetic elements driving dissemination of antibiotic resistance.

microbiology↗

bioRxiv2026

Coupling DNA processing to early gene expression drives antibiotic resistance plasmid dissemination

The rapid global spread of antibiotic resistance is mediated by conjugative plasmids, yet how these elements establish immediately after entering new bacterial hosts remains poorly understood. Here we show that plasmids actively coordinate DNA processing with early gene expression to promote their own establishment. Using single-cell measurements of plasmid conversion dynamics, we demonstrate that the F plasmid selectively delays complementary strand synthesis within its leading region, creating a transient single-stranded DNA window that prolongs zygotic induction. This delay is imposed by a single-stranded DNA promoter that forms a structural roadblock to complementary strand DNA synthesis and is resolved by the host helicase UvrD. By extending the single-stranded state of the leading region, plasmids enhance early expression of protective genes that counter host defence responses and promote survival during entry, particularly in non-isogenic hosts. Together, these findings identify early post-entry regulation as a critical determinant of plasmid establishment and reveal how coupling DNA conversion to transient protective gene expression enables conjugative plasmids to overcome host barriers and disseminate antibiotic resistance.

microbiology↗

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↗

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

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↗

bioRxiv2023

The F pilus serves as a conduit for the DNA during conjugation between physically distant bacteria

Horizontal transfer of F-like plasmids by bacterial conjugation is responsible for disseminating antibiotic resistance and virulence determinants among pathogenic Enterobacteriaceae species, a growing health concern worldwide. Central to this process is the conjugative F pilus, a long extracellular filamentous polymer that extends from the surface of plasmid donor cells, allowing it to probe the environment and make contact with the recipient cell. It is well established the F pilus can retract to bring mating pair cells in tight contact before DNA transfer. However, whether DNA transfer can occur through the extended pilus has been a subject of active debate. In this study, we use live-cell microscopy to show that the F pilus can indeed serve as a conduit for the DNA during transfer between physically distant cells. Our findings enable us to propose a new model for conjugation that revises our understanding of the DNA transport mechanism and the dissemination of drug resistance in complex bacterial communities. One-Sentence SummaryPlasmid DNA passes through the F pilus during conjugational transfer between physically distant bacteria.

microbiology↗

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↗

bioRxiv2020

Targeted-Antibacterial-Plasmids (TAPs) combining conjugation and CRISPR /Cas systems achieve strain-specific antibacterial activity

The global emergence of drug-resistant bacteria leads to the loss of efficacy of our antibiotics arsenal and severely limits the success of currently available treatments. Here, we developed an innovative strategy based on Targeted-Antibacterial-Plasmids (TAPs) that use bacterial conjugation to deliver CRISPR/Cas systems exerting a strain-specific antibacterial activity. TAPs are highly versatile as they can be directed against any specific genomic or plasmid DNA using the custom algorithm (CSTB) that identifies appropriate targeting spacer sequences. We demonstrate the ability of TAPs to induce strain-selective killing by introducing lethal double strand breaks (DSBs) into the targeted genomes. TAPs directed against a plasmid-born carbapenem resistance gene efficiently resensitise the strain to the drug. This work represents an essential step towards the development of an alternative to antibiotic treatments, which could be used for in situ microbiota modification to eradicate targeted resistant and/or pathogenic bacteria without affecting other non-targeted bacterial species.

microbiology↗

bioRxiv2020

Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Towards identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli. The mobility of these proteins during the target search was dictated by DNA interactions, rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid does not pose a physical barrier for protein diffusion, but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58-99%) of their search time bound to DNA and occupy as much as [~]30% of the chromosomal DNA at any time. Chromosome-crowding likely has important implications for the function of all DNA-binding proteins.

cell biology↗

bioRxiv2020

Bacterial filament division dynamics allows rapid post-stress cell proliferation

Many bacterial species grow into filaments under stress conditions. Initially regarded as an indicator of cell death, filamentation is now proposed to be a transient morphological change that improves bacterial survival in hostile environments. However, the mechanism of filament recovery remains poorly characterized. Using real-time microscopy in live-cells, we analysed the fate of filamentous Escherichia coli induced by antibiotic-mediated specific inhibition of cell division, or by UV-induced DNA-damage that additionally perturbs chromosome segregation. Both filament types recover by successive and accelerated rounds of divisions, which are preferentially positioned asymmetrically at the tip of the cell by the Min system. Such division dynamics allows the rapid production of daughter cells with normal size, which DNA content depends on the progression of chromosome segregation prior to division. In most filaments, nucleoid segregation precedes tip-division, which produces nucleated daughter cells that resume normal growth. However, when segregation is deficient, tip-division occurs in the absence of DNA and produces anucleated cells. These findings uncover the mechanism by which bacterial filamentation allows efficient post-stress cell proliferation. One Sentence SummaryBacterial filaments recover by successive, frequent and Min-dependent asymmetric tip-divisions that rapidly produce multiple daughter cells with normal size and viability

microbiology↗