Biology subjects

Uphoff, S.

Publications and source records attributed to Uphoff, S..

15 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

Single-molecule imaging of DNA repair and cytoplasmic rigidification in intracellular bacteria

DNA damage is an important component of the antibacterial response of phagocytes, but which DNA repair mechanisms are active in intracellular bacteria remains unclear. We developed a live-cell single-molecule tracking approach to directly measure DNA repair activity in Escherichia coli within macrophages. Phagocytosis activates bacterial base excision and nucleotide excision repair pathways and increases DNA mismatch repair foci indicative of DNA replication errors. Phagocyte-generated stresses also cause a general slowdown in protein diffusion within bacteria, consistent with a transition of the cytoplasm towards a glass-like state, which is associated with reduced metabolic activity. At the single-cell level, DNA repair activity is highly heterogeneous, with metabolically inactive bacteria showing the greatest engagement of repair proteins. Together, these findings reveal how distinct DNA repair pathways are deployed during macrophage infection and link repair activity to the metabolic and biophysical states of individual intracellular bacteria.

microbiology↗

bioRxiv2026

Replication-coupled search positions MutH for strand incision in DNA mismatch repair

DNA mismatch repair (MMR) safeguards genome stability by correcting replication errors, yet how its components coordinate as an efficient pathway in living cells remains unclear. In Escherichia coli, MutS and MutL detect mismatches and must rapidly activate MutH to incise the nascent DNA strand at distantly located GATC sites. Here, we used live-cell single-molecule tracking to resolve the target-search dynamics of MMR proteins in vivo. We find that MutH binds DNA at hemimethylated GATC sites independently of mismatch recognition and without MutS or MutL. The autonomous recruitment of MutH is facilitated by a switch from fast diffusion to a slow-search mode near DNA replication forks. This guidance allows MutH to transiently occupy potential incision sites in the wake of replication forks, awaiting activation by MutS-MutL. The parallelised search mechanism of MMR proteins facilitates rapid and targeted repair of replication errors in cells.

molecular biology↗

bioRxiv2025

Selective interaction of the protein SMCHD1 with specific chromatin regions is governed by the loading factor LRIF1 and SMCHD1 ATPase activity

The chromosomal protein SMCHD1 is a GHKL ATPase that plays important roles in epigenetic silencing, including on the inactive X chromosome (Xi) and at the D4Z4 macrosatellite linked to regulation of DUX4 expression in the disorders facioscapulohumeral muscular dystrophy (FSHD2) and Bosma arrhinia micropthalmia syndrome (BAMS). In this study we use live-cell and single-molecule imaging approaches to investigate SMCHD1 interactions with chromatin and its function in epigenetic silencing. We show that chromatin binding of SMCHD1 genome-wide, including on the Xi, is critically dependent on the protein LRIF1 that mediates interaction with H3K9me2/3 modified nucleosomes. Using engineered mutations in the GHKL ATPase domain we show that ATP hydrolysis is required for selective enrichment of SMCHD1 at specific chromatin regions, which is critical for gene silencing on the Xi. A gain-of-function mutation, G137E, that occurs in BAMS patients, results in accelerated Xi recruitment and greater Xi chromosome compaction. Together, our findings advance mechanistic understanding of SMCHD1 function on the Xi and at other target sites in the genome.

molecular biology↗

bioRxiv2024

A simple regulatory network coordinates a bacterial stress response in space and time

Bacteria employ diverse gene regulatory networks to protect themselves from stressful environments. While transcriptomics and proteomics show that the expression of different genes can shift strongly in response to stress, the underlying logic of large regulatory networks is difficult to understand from bulk measurements performed at discrete time points. As a result, it remains challenging to predict how these regulatory networks function at a system level. Here we use time-resolved single-cell imaging to explore the functioning of a key bacterial stress response: The Escherichia coli response to oxidative stress. Our work reveals a striking diversity in the expression dynamics of genes in the regulatory network, with differences in the timing, magnitude, and direction of expression changes. Nevertheless, we find that these patterns have a simple underlying logic. Firstly, all genes exhibit a transient increase in their protein levels simply due to the slowing down of cell growth under stress. Controlling for this effect reveals three classes of gene regulation driven by the transcription factor OxyR. Downregulated genes drop in expression level, while upregulated genes either show pulsatile expression that decays rapidly or gradual induction, dependent upon transcription factor binding dynamics. These classes appear to serve distinct functional roles in cell populations. Pulsatile genes are stress-sensitive and activate rapidly and transiently in a few cells, which provides an initial protection for cell groups. Gradually upregulated genes are less sensitive and induce more evenly generating a lasting protection that involves a larger number of cells. Our study shows how bacterial populations use simple regulatory principles to coordinate a stress response in space and time.

systems biology↗

bioRxiv2022

Regulatory fine-tuning and horizontal gene transfer stabilize mobile colistin resistance

Antibiotic resistance tends to carry fitness costs, making it difficult to understand how resistance can be stably maintained in pathogen populations over the long-term. Here, we investigate this problem in the context of mcr-1, a fitness-costly gene that confers resistance to the last-resort antibiotic, colistin. Here we show that regulatory evolution has fine-tuned the expression of mcr-1, allowing E. coli to reduce the cost of mcr-1 while simultaneously increasing colistin resistance. Conjugative plasmids have transferred low cost/high resistance mcr-1 alleles across an incredible diversity of E. coli strains, further stabilizing mcr-1 at the species level. Crucially, regulatory mutations were associated with increased mcr-1 stability in pig farms following a ban on the use of colistin as a growth promoter that decreased colistin consumption by 90%. Our study shows how the rapid evolution and horizontal transmission of resistance genes can combine to stabilize resistance and reduce the efficiency of interventions aimed at reducing AMR by limiting antibiotic consumption.

evolutionary biology↗

bioRxiv2022

Environmental feedback drives oxidative stress response heterogeneity in bacterial populations

Induction of phenotypic heterogeneity is a ubiquitous consequence of bacterial stress responses. It is commonly postulated that isogenic cells exploit stochastic molecular fluctuations to generate phenotypic heterogeneity as a population survival strategy (termed bet-hedging). However, it is also possible that each cell attempts to maximise its own chances of survival. In that case, the apparent heterogeneity could either be caused by inevitable molecular noise or by underlying deterministic mechanisms which have escaped observation. Here, we investigated the sources and consequences of gene expression heterogeneity in the response of Escherichia coli cells to hydrogen peroxide (H2O2) stress. A machine-learning model accurately predicted the variable responses of individual cells growing in structured populations, indicating that the phenotypic heterogeneity has a deterministic origin. The model further showed that spatio-temporal dynamics in H2O2 concentration were dictated by the H2O2 scavenging capacity and morphology of cells in the local environment. Hence, oxidative stress response fluctuations were in fact the result of a precise and rapid feedback between each cell and its immediate environment. Although single cells optimise their individual responses, the formation of short-range H2O2 gradients by their scavenging activities increases stress tolerance at the population level and leads to spatial variation in mutation rates.

microbiology↗

bioRxiv2022

Adaptation delay causes a burst of mutations in bacteria responding to oxidative stress

Understanding the interplay between phenotypic plasticity and genetic adaptation is a long-standing focus of evolutionary biology. In bacteria, the oxidative stress response limits the formation of mutagenic reactive oxygen species (ROS) under diverse stress conditions. This suggests that the dynamics of the oxidative stress response are closely tied to the timing of the mutation supply that fuels genetic adaptation to stress. Here, we explored how mutation rates change in real-time in Escherichia coli cells during continuous hydrogen peroxide treatment in microfluidic channels. By visualising nascent DNA replication errors, we uncovered that sudden oxidative stress causes a burst of mutations. We developed a range of single-molecule and single-cell microscopy assays to determine how these mutation dynamics arise from phenotypic adaptation mechanisms. Signalling of peroxide stress by the transcription factor OxyR rapidly induces ROS scavenging enzymes. However, an adaptation delay leaves cells vulnerable to the mutagenic and toxic effects of hydroxyl radicals generated by the Fenton reaction. Resulting DNA damage is counteracted by a spike in DNA repair activities during the adaptation delay. Prior stress exposure or constitutive OxyR induction allowed cells to avoid the burst of mutations. Similar observations for alkylation stress show that mutation bursts are a general phenomenon associated with adaptation delays.

microbiology↗

bioRxiv2021

Cellular heterogeneity in DNA alkylation repair as a trade-off between cell survival and genetic plasticity

DNA repair mechanisms fulfil a dual role, as they are essential for cell survival and genome maintenance. Here, we studied how cells regulate the interplay between DNA repair and mutation. We focused on the Escherichia coli adaptive response that increases resistance to DNA alkylation damage. Combination of single-molecule imaging and microfluidic-based single-cell microscopy showed that noise in the gene activation timing of the master regulator Ada is accurately propagated to generate a distinct subpopulation of cells in which all proteins of the adaptive response are absent. Although lack of these proteins causes extreme sensitivity to alkylation stress, cellular heterogeneity in DNA alkylation repair provides a functional benefit by increasing the evolvability of the whole population. We demonstrated this by monitoring the dynamics of nascent mutations during alkylation stress as well as the frequency of fixed mutations that are generated by the distinct subpopulations of the adaptive response. This highlighted that evolvability is a trade-off between mutability and cell survival. Stochastic modulation of DNA repair capacity by the adaptive response solves this trade-off through the generation of a viable hypermutable subpopulation of cells that acts as a source of genetic diversity in a clonal population.

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

Non-random segregation of sister chromosomes by Escherichia coli MukBEF axial cores

The Escherichia coli structural maintenance of chromosomes complex, MukBEF, forms axial cores to chromosomes that determine their spatio-temporal organization. Here, we show that axial cores direct chromosome arms to opposite poles and generate the translational symmetry between newly replicated sister chromosomes. MatP, a replication terminus (ter) binding protein prevents chromosome rotation around the longitudinal cell axis by displacing MukBEF from ter, thereby maintaining the linear shape of axial cores. During DNA replication, MukBEF action directs lagging strands towards the cell center, marked by accumulation of DNA-bound {beta}2-clamps in the wake of replisomes, in a process necessary for the translational symmetry of sister chromosomes. Finally, the ancestral ( immortal) template DNA strand, propagated from previous generations, is preferentially inherited by the cell forming at the old pole, dependent on MukBEF-MatP. The work demonstrates how chromosome organization-segregation can foster non-random inheritance of genetic material and provides a framework for understanding how chromosome conformation and dynamics shape subcellular organization.

microbiology↗

bioRxiv2020

Imaging LexA degradation in cells explains regulatory mechanisms and heterogeneity of the SOS response

The SOS response functions as the central regulator of DNA repair and mutagenesis in most bacteria and stands as a paradigm of gene networks controlled by a master transcriptional regulator, LexA. We developed a single-molecule imaging approach to directly monitor the LexA repressor inside live Escherichia coli cells, demonstrating key mechanisms by which DNA-binding and degradation of LexA regulates the SOS response in vivo. Our approach revealed that self-cleavage of LexA occurs frequently during unperturbed growth and causes substantial heterogeneity in LexA abundances across cells. LexA variability underlies SOS gene expression heterogeneity and triggers spontaneous SOS pulses, which enhance bacterial survival in anticipation of stress.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Gene expression noise randomizes the adaptive response to DNA alkylation damage in E. coli

DNA damage caused by alkylating chemicals induces an adaptive response in Escherichia coli cells that increases their tolerance to further damage. Signalling of the response occurs through methylation of the Ada protein which acts as a damage sensor and induces its own gene expression through a positive feedback loop. However, random fluctuations in the abundance of Ada jeopardize the reliability of the induction signal. I developed a quantitative model to test how gene expression noise and feedback amplification affect the fidelity of the adaptive response. A remarkably simple model accurately reproduced experimental observations from single-cell measurements of gene expression dynamics in a microfluidic device. Stochastic simulations showed that delays in the adaptive response are a direct consequence of the very low number of Ada molecules present to signal DNA damage. For cells that have zero copies of Ada, response activation becomes a memoryless process that is dictated by an exponential waiting time distribution between basal Ada expression events. Experiments also confirmed the model prediction that the strength of the adaptive response drops with increasing growth rate of cells.

biophysics↗

bioRxiv2018

Choosing the right label for single-molecule tracking in live bacteria: Side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes

Visualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins. An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and single-molecule localization and tracking results. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.

biophysics↗

bioRxiv2017

Scc2/Nipbl Hops Between Chromosomal Cohesin Rings After Loading

The cohesin complex mediates DNA-DNA interactions both between (sister chromatid cohesion) and within chromosomes (DNA looping) via a process thought to involve entrapment of DNAs within its tripartite ring. It has been suggested that intra- chromosome loops are generated through processive extrusion of DNAs through the lumen of cohesins ring. Scc2 (Nipbl) is essential for loading cohesin onto chromosomes but not for maintaining sister chromatid cohesion following DNA replication. It has therefore been assumed that Scc2 is involved exclusively in the cohesin loading process. However, it is possible that the stimulation of cohesins ABC-like ATPase by Scc2 also has a post-loading function, for example driving loop extrusion. Using fluorescence recovery after photobleaching (FRAP) and single-molecule tracking, we show that Scc2 binds dynamically to chromatin, principally through an association with cohesin. Scc2s movement within chromatin is consistent with a \"stop-and-go\" or \"hopping\" motion. We suggest that a low diffusion coefficient, a low stoichiometry relative to cohesin, and a high affinity for chromosomal cohesin enables Scc2 to move rapidly from one chromosomal cohesin complex to another, performing a function distinct from loading.

cell biology↗