Biology subjects

Fange, D.

Publications and source records attributed to Fange, D..

8 recordsLinked to original sources

bioRxiv2025

The lac repressor strikes a balance between inducibility and strength of repression

Transcription factors (TFs) efficiently locate their target DNA sequences by combining three-dimensional diffusion and one-dimensional sliding on nonspecific DNA. To balance rapid sliding with strong specific binding, TFs were proposed to switch between search and recognition conformations. For E. coli lac repressor (LacI), the folding of the hinge helices has been implicated in the conformational switch. Here, we tested how mutations in the hinge region impact the search speed and binding stability. Based on molecular dynamics simulations, we selected two LacI mutants favoring either search or recognition conformation. We measured the binding kinetics of the mutants both in vitro on DNA microarrays with 2,479 different Lac operators and in vivo via single-molecule experiments. We conclude that a hinge region mutation causing less helix propensity enhances the specificity but reduces binding strength globally, while a hinge region mutation causing higher helix propensity has opposite effects. However, altered specificity impacts the search time less than expected. Instead, the major effect was impaired dissociation in response to IPTG induction for the strongly binding mutant. Together with earlier reports of affinity-inducibility trade-offs in LacI, our data support the model in which the hinge region governs a trade-off between binding stability and inducibility rather than between speed and binding stability.

molecular biology↗

bioRxiv2025

Phenotypic Antibiotic Susceptibility Testing at the limit of one bacterial cell

The blood of a patient with bacteremia contains only a few bacteria per milliliter. Recently, techniques have been developed for extracting these few bacteria from whole blood. Here, we evaluate if a phenotypic Antibiotic Susceptibility Test (AST) can be performed on a single bacterial cell by monitoring changes in its phenotypic distributions sampled over time rather than across populations, while also accounting for experimental noise and cell-to-cell variability. We use the method to estimate the time required to correctly classify single susceptible or resistant bacteria when treated with antibiotics that affect growth, cell division, or cause lysis. We find that due to heterogeneous single cell responses, it is necessary to consider all bacteria close to the susceptibility breakpoint as resistant. However, it is possible to follow up resistance calls by sequentially exposing the cell or its lineage to new antibiotics until a susceptible phenotype is detected.

microbiology↗

bioRxiv2024

A dynamic 3D polymer model of the Escherichia coli chromosome driven by data from optical pooled screening

The DNA of bacterial cells is organized in a highly dynamic chromosome structure. To guarantee its propagation, the chromosome must replicate, segregate, and accommodate other biological processes like gene expression. Therefore, to understand the causal relationship between chromosome organization and biological function, it is essential to follow chromosome dynamics. Live-cell imaging of fluorescent labels allows the tracking of specific genomic locations, but the current imaging-based approaches to study bacterial chromosome organization over the cell cycle are limited in their throughput. Even with multicolor fluorescent locus labeling, the genomic resolution is insufficient to gain insights about the whole chromosome structure from a single experiment. Although sequencing-based methods like chromosome conformation capture provide high genomic coverage, they can only be done in bulk, providing an averaged static view of the chromosome structure. In this study, we address these limitations and investigate the bacterial chromosome organization by imaging fluorescently labeled loci in a library of Escherichia coli strains, following the 3D locations of 68 different loci in live cells in a single experiment. The resulting location distributions along the cells longitudinal and radial axes are used to inform a dynamic polymer model of the chromosome over the cell cycle. We show a global reorganization of the E. coli chromosome, at both the longitudinal and radial axes. Our model reproduces the known chromosomal architecture of four macrodomains and demonstrates how these domains form dynamically over the cell cycle.

systems biology↗

bioRxiv2023

Pooled optical screening in bacteria using chromosomally expressed barcodes

Optical pooled screening is an important tool to study dynamic phenotypes for libraries of genetically engineered cells. However, the desired engineering often requires that the barcodes used for in situ genotyping are expressed from the chromosome. This has not been possible in bacteria. Here we describe a method for in situ genotyping of libraries with genomic barcodes in Escherichia. coli. The method is applied to measure the intracellular maturation time of 81 red fluorescent proteins.

biophysics↗

bioRxiv2023

The Escherichia coli chromosome moves to the replisome

The replisome, a large multi-subunit assembly, carries out the process of chromosome replication, connecting the unwrapping of the parental DNA with the creation of new daughter strands. In Escherichia coli, it is debated whether the two replisomes move independently along the two chromosome arms during replication or if they remain in close proximity, with the DNA being pulled toward the replisomes as replication progresses. Here, we use fluorescence microscopy to determine the location and diffusivity of the replisome and various chromosomal loci throughout the cell cycle of the model organism E. coli. We find that (i) the two replisomes are confined to a region of 250 nm and 120 nm along the cell long and short axis respectively, and the chromosomal loci move through this region sequentially based on distance from the origin of replication. (ii) When a locus is being replicated, its diffusivity slows down. (iii) There is no indication that replication initiation occurs close to the cell membrane as has been proposed in a few previous studies. In conclusion, our data supports a model with DNA moving towards stationary replisomes at replication.

microbiology↗

bioRxiv2021

The spatial organization of replication is determined by cell size independently of chromosome copy number

Escherichia coli coordinates replication and division cycles by initiating replication at approximately the same size per chromosome at all growth rates. By tracking replisomes in individual cells through thousands of division cycles, we have dissected the mechanism behind this precise process. We have characterized wild-type cells grown under different conditions and also many mutants related to the expression and binding states of the initiator protein DnaA. This rich data set allowed us to compare the relative importance of all previously described control systems. We found that the replication initiation size regulation is not strongly dependent on the absolute concentration of DnaA, nor does it depend on active dnaA expression. Replication initiation is also not consistently triggered by cell division or replication termination. In contrast, some of the factors that convert DnaA between its ATP- and ADP-bound states have a strong effect on initiation size. We suggest a plausible model for DnaA-ATP mediated triggering of initiation at fast growth, where regulatory inactivation of DnaA (RIDA) is the main system for monitoring the number of chromosomes during active replication.

microbiology↗

bioRxiv2019

A time-resolved imaging-based CRISPRi screening method

Our ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live cell microscopy and library-scale genomic engineering. Specifically, this has restricted studies of intracellular dynamics to one strain at a time and thus, generally, to the impact of genes with known function. Here we show how in situ genotyping of a library of E. coli strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference (CRISPRi) knockdowns impact the coordination of the replication and division cycles of E. coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes, and replication initiation volumes. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation in order to determine which genes are important for cell cycle control. The technology presented in this study enables genome-scale screens of virtually all live-cell microscopy assays and, therefore, constitutes a qualitatively new approach to cellular biophysics.

biophysics↗

bioRxiv2017

In Situ Genotyping Of A Pooled Strain Library After Characterizing Complex Phenotypes

So far, it has not been possible to perform advanced microscopy on pool generated strain libraries and at the same time know each strains genotype. We have overcome this barrier by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single molecule fluorescence imaging of E. coli strains harboring barcoded plasmids that express a sgRNA which suppress different genes through dCas9.

systems biology↗