Transcription factors regulate gene expression through their binding to DNA. In a living Escherichia coli cell, we directly observed specific of lac repressor, labeled with fluorescent protein, chromosomal operator. Using single-molecule detection techniques, measured the kinetics and dissociation repressor in response metabolic signals. Furthermore, characterized nonspecific DNA, one-dimensional (1D) diffusion along DNA segments, 3D translocation among segments cytoplasm at level. searching...

Superresolution imaging in sharper focus An optical microscope cannot distinguish objects separated by less than half the wavelength of light. techniques have broken this “diffraction limit” and provided exciting new insights into cell biology. Still, such hit a limit at resolution about 10 nm. Balzarotti et al. describe another way localizing single molecules called MINFLUX (see Perspective Xiao Ha). As photoactivated localization microscopy stochastic reconstruction microscopy,...

Biochemical networks in single cells can display large fluctuations molecule numbers, making mesoscopic approaches necessary for correct system descriptions. We present a general method that allows rapid characterization of the stochastic properties intracellular networks. The starting point is macroscopic description identifies system's elementary reactions terms rate laws and stoichiometries. From this formulation follows directly stationary solution linear noise approximation (LNA) Master...

Summary With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function behavior, microscopy has re‐emerged as a primary tool in bacterial research during last decade. However, bottleneck today's single‐cell studies is quantitative image analysis of fluorescent signals. Here, we address current limitations through development Oufti, stand‐alone, open‐source software package automated measurements...

Transcription factors (TFs) are proteins that regulate the expression of genes by binding sequence-specific sites on chromosome. It has been proposed to find these fast and accurately, TFs combine one-dimensional (1D) sliding DNA with 3D diffusion in cytoplasm. This facilitated mechanism demonstrated vitro, but it not shown experimentally be exploited living cells. We have developed a single-molecule assay allows us investigate process bacteria. Here we show lac repressor slides 45 ± 10 base...

Bi-stable chemical systems are the basic building blocks for intracellular memory and cell fate decision circuits. These circuits built from molecules, which present at low copy numbers slowly diffusing in complex geometries. The stochastic reaction-diffusion kinetics of a double-negative feedback system MAPK phosphorylation-dephosphorylation is analysed with Monte-Carlo simulations master equation. results show geometry reaction compartments to be important both duration locality...

Significance Antibiotic resistance is a global threat to human health. The problem aggravated by unnecessary and incorrect use of broad spectrum antibiotics. One way provide correct treatment slow down the development antibiotic assay susceptibility profile infecting bacteria before initiated let this information guide choice antibiotic. Here, we present an test that sufficiently fast be used at point care. We show it possible determine if urinary tract infection caused resistant within 30...

The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in cell cycle virulence. RelA integrates several environmental cues synthesizes alarmone ppGpp, which globally reprograms transcription, translation, replication. We have developed implemented novel single-molecule tracking methodology characterize intracellular catalytic RelA. Our experiments show that on ribosome under nonstarved conditions individual enzyme molecule...

Biochemical and genetic data show that ribosomes closely follow RNA polymerases are transcribing protein-coding genes in bacteria. At the same time, electron fluorescence microscopy have revealed excluded from Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can reconciled if of mRNAs start throughout nucleoid before they relocate periphery. However, this mechanism requires free ribosomal subunits not...

How fast can a cell locate specific chromosomal DNA sequence specified by single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of Cas9 protein, which be programmed guide RNA to bind essentially any sequence. This targeting flexibility requires unwind double helix test for correct base pairing RNA. Here study mechanisms catalytically inactive (dCas9) in living

Summary: MesoRD is a tool for stochastic simulation of chemical reactions and diffusion. In particular, it an implementation the next subvolume method, which exact method to simulate Markov process corresponding reaction-diffusion master equation.

We modeled how the charged levels of different transfer RNAs (tRNAs) that carry same amino acid (isoacceptors) respond when this becomes growth-limiting. The will approach zero for some isoacceptors (such as \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{2}^{\mathrm{Leu}}\) \end{document} ) and remain high others \(\mathrm{tRNA}_{4}^{\mathrm{Leu}}\) ), determined by...

The spatiotemporal oscillations of the Escherichia coli proteins MinD and MinE direct cell division to region between chromosomes. Several quantitative models Min system have been suggested before, but no one them accounts for behavior all documented mutant phenotypes. We analyzed stochastic reaction-diffusion kinetics several E. mutants compared results corresponding deterministic mean-field description. found that wild-type (wt) filamentous (ftsZ-) cells are well characterized by model, a...

Hfq, a protein required for small RNA (sRNA)-mediated regulation in bacteria, binds with low-nanomolar K d values and long half-lives of complexes (>100 min). This cannot be reconciled the 1- 2-min response time vivo. We show that RNAs displace each other on Hfq short scale by concentration-driven (active) cycling. Already at submicromolar concentrations competitor RNA, RNA–Hfq are ≈1 min. propose associates transiently complexes, exchange binding sites, one eventually dissociates. solves...

Quantitative analysis of biochemical networks often requires consideration both spatial and stochastic aspects chemical processes. Despite significant progress in the field, it is still computationally prohibitive to simulate systems involving many reactants or complex geometries using a microscopic framework that includes finest length time scales diffusion-limited molecular interactions. For this reason, spatially temporally discretized simulations schemes are commonly used when modeling...

Abstract Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs) 1 . Initially, RecBCD complex 2 resects ends DSB into 3′ single-stranded on which a RecA filament assembles 3 Next, locates homologous template sister chromosome 4 Here we directly visualize DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, Escherichia coli , between segregated loci completed 15 ± 5 min (mean s.d.) with minimal fitness...

Abstract Antimicrobial resistance is an increasing problem on a global scale. Rapid antibiotic susceptibility testing (AST) urgently needed in the clinic to enable personalized prescriptions high-resistance environments and limit use of broad-spectrum drugs. Current rapid phenotypic AST methods do not include species identification (ID), leaving time-consuming plating or culturing as only available option when ID make sensitivity call. Here we describe method perform at single-cell level...