Biology subjects

El Sayyed, H.

Publications and source records attributed to El Sayyed, H..

5 recordsLinked to original sources

bioRxiv2025

Unraveling single-molecule reactions via multiplexed in-situ DNA sequencing

DNA sequence regulates complex reactions and protein-DNA interactions, yet sequence effects remain poorly understood due to the lack of direct, high-throughput approaches to study sequence-dependence at the single-molecule level. Here, we introduce Single-molecule Phenotyping and In-Situ Sequencing (SPIN-Seq), a DNA-based, protein-free method that links functional and structural properties of a single DNA molecule with the sequence of that same molecule. After performing functional assays on immobilized DNA molecules, SPIN-Seq uses sequencing-by-transient-hybridization on the same surface and instrument to read sequences in each immobilized DNA molecule, directly linking phenotype and genotype. Applying SPIN-Seq, we dissected the interaction of a transcription factor with its target sequence, and revealed the sequence-dependence of RNA polymerase pausing and reaction-path branching during initial transcription. Our method provides powerful, systematic ways to understand complex molecular mechanisms and their sequence dependence.

biophysics↗

bioRxiv2025

Structure of the conjugation surface exclusion protein TraT

Conjugal transfer of plasmids between bacteria is a major route for the spread of antimicrobial resistance. Many conjugative plasmids encode exclusion systems that inhibit redundant conjugation. In incompatibility group F (IncF) plasmids surface exclusion is mediated by the outer membrane protein TraT. Here we report the cryoEM structure of the TraT exclusion protein complex from the canonical F plasmid of Escherichia coli. TraT is a hollow homodecamer shaped like a chefs hat. In contrast to most outer membrane proteins, TraT spans the outer membrane using transmembrane helices. We use a novel microscopy-based conjugation assay to probe the effects of directed mutagenesis on TraT. Our analysis provides no support for the idea that TraT has specific interactions with partner proteins. Instead, we infer that TraT is most likely to function by physical interference with conjugation.

microbiology↗

bioRxiv2025

Single-molecule observation of multi-scale dynamic heterogeneity in the molecular bearing of the bacterial flagellum

The bacterial flagellar motor (BFM) is a protein-based rotary machine that drives bacterial motility. It comprises a stator anchored to the cell wall and a rotor in the cytoplasmic membrane, linked via the flagellar rod to the extracellular hook and filament. We describe high-speed polarization microscopy of gold nanorods attached to the hook, allowing unprecedented continuous recording of single BFMs with [~]0.5{degrees} resolution at 250 kHz for tens of minutes. The angular distribution of actively rotating motors exhibited multiple periodicities that are consistent with structure of the bearing between the rod and LP-ring. We observed passive diffusional rotation of this bearing, revealing complex dynamics at odds with the expected classical Ornstein-Uhlenbeck process in a smooth potential. Resolved transitions between the main 26-fold bearing states exhibited highly non-Poissonian kinetics spanning four orders of magnitude in timescale. Using the average transition times, we estimated the interaction barrier in the bearing to be close to 13 kBT, suggesting that the previously estimated torque generated by a single stator unit is just sufficient to overcome this barrier. At sub-millisecond timescales we observed anomalous ultra-slow diffusion typically associated with multi-scale disordered systems, despite the ordered crystalline atomic structure of the bearing revealed by cryo-Electron Microscopy. Over longer periods, we observed dynamic shifts in the preferred angular positions, indicating that the motors potential energy landscape evolves over time. We speculate that these newly discovered rich dynamics may be a universal feature of protein machines.

biophysics↗

bioRxiv2022

RNA polymerase redistribution and increased gene dosage support growth in E. coli strains with a minimal number of ribosomal RNA operons

Bacterial transcription by RNA polymerase (RNAP) is spatially organised. RNAPs transcribing highly expressed genes locate in the nucleoid periphery, and form clusters in rich media, with several studies linking RNAP clustering and transcription of ribosomal RNA (rrn). However, the nature of RNAP clusters and their association with rrn transcription remains unclear. Here we address these questions by using single-molecule tracking to monitor the subcellular distribution of mobile and immobile RNAP in strains with a heavily reduced number of chromosomal rrn operons ({Delta}rrn strains). Strikingly, we find that the fraction of chromosome-associated RNAP (which is mainly engaged in transcription) is robust to deleting 5 or 6 of the 7 chromosomal rrn operons. Spatial analysis in{Delta} rrn strains showed substantial RNAP redistribution during moderate growth, with clustering increasing at the cell end-caps, where the remaining rrn operons reside. These results support a model where RNAPs in{Delta} rrn strains relocate to copies of the remaining rrn operons. We also show that{Delta} rrn strains experience increased rrn gene dosage in rich media, minimising growth defects due to rrn deletions. Our study further links RNAP clusters and rrn transcription, and offers insight on how bacteria maintain growth in the presence of only 1-2 rrn operons.

microbiology↗

bioRxiv2021

Genomic contacts reveal the control of sister chromosome decatenation in E. coli

In bacteria, chromosome segregation occurs progressively, from the origin to the terminus, a few minutes after the replication of each locus. In-between replication and segregation, sister loci are maintained in an apparent cohesive state by topological links. Whereas topoisomerase IV (Topo IV), the main bacteria decatenase, controls segregation, little is known regarding the influence of the cohesion step on chromosome folding. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, a massive chromosome reorganization takes place, associated with increases in trans-contacts between catenated sister chromatids and in long-range cis-contacts between the terminus and distant loci on the genome. A genetic analysis of these signals allowed us to decipher specific roles for Topo IV and Topo III, an accessory decatenase. Moreover we revealed the role of MatP, the terminus macrodomain organizing system and MukB, the E. coli SMC in organizing sister chromatids tied by persistent catenation links. We propose that large-scale conformation changes observed in these conditions reveal a defective decatenation hub located in the terminus area. Altogether, our findings support a model of spatial and temporal partition of the tasks required for sister chromosome segregation.

molecular biology↗