Biology subjects

Ghilarov, D.

Publications and source records attributed to Ghilarov, D..

4 recordsLinked to original sources

bioRxiv2025

Structural analysis of the NifL-NifA complex reveals the molecular basis of anti-activation of nitrogen fixation gene expression in Azotobacter vinelandii

Understanding the molecular basis of regulated nitrogen (N2) fixation is essential for engineering N2-fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system NifL-NifA controls the expression of nif genes that encode the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability. The nitrogen fixation machinery is thereby only activated under strictly favorable conditions, enabling diazotrophs to thrive in competitive environments. Whilst genetic and biochemical studies have enlightened our understanding of how NifL represses NifA, the molecular basis of NifA sequestration by NifL depends on structural information on their interaction. Here, we present mechanistic insights into how nitrogen fixation is regulated by combining biochemical and genetic approaches with a low-resolution cryo-EM map of the oxidized NifL-NifA complex. Our findings define the interaction surface between NifL and NifA and reveal how this interaction can be manipulated to generate bacterial strains with increased nitrogen fixation rates able to secrete surplus nitrogen outside the cell, a crucial step in engineering improved nitrogen delivery to crop plants.

microbiology↗

bioRxiv2024

Structure of Escherichia coli DNA gyrase with chirally wrapped DNA supports ratchet-and-pawl mechanism for an ATP-powered supercoiling motor

Gyrase is essential for replication and transcription in bacteria, and as such is an important target for antibiotics including fluoroquinolones. Gyrase is a molecular machine that channels the energy of ATP hydrolysis into negative supercoiling of DNA. The mechanism proposed more than 40 years ago involves stabilising and inverting a chiral DNA loop; however, the molecular basis for this is poorly understood, as the loop was never directly observed. We present high-resolution cryoEM structures of the Escherichia coli gyrase - 217 bp DNA holocomplex, and of the moxifloxacin-bound gyrase complex with cleaved 217 bp DNA. Each structure constrains an intact figure-of-eight positively supercoiled DNA loop, poised for strand passage. The loop is stabilised by a GyrA {beta}-pinwheel domain which how we here show has a structure of a flat disc, and functions akin to a mini-nucleosome. Our data implies that during catalysis the ATPase domains of the enzyme undergo a large movement to push the transported DNA segment through the break in DNA. By comparing the catalytic site between native drug-free and moxifloxacin-bound gyrase structures that both contain a single metal ion we demonstrate that the enzyme is observed in a native pre-catalytic state. Finally, we propose a ratchet and pawl mechanism for energy coupling in gyrase. These unexpected findings call for re-evaluation of existing data and offer a framework for further experiments designed to dissect the details of how gyrase molecular motor converts chemical energy into mechanical tension.

molecular biology↗

bioRxiv2024

Molecular basis of foreign DNA recognition by BREX anti-phage immunity system

Anti-phage systems of the BREX (BacteRiophage EXclusion) superfamily rely on epigenetic DNA methylation to discriminate between the host and invading DNA, but their mechanism of protection remains enigmatic. We demonstrate that in Type I BREX systems, both defense and methylation are based on site-specific DNA recognition by the BrxX (PglX) methyltransferase and require the S-adenosyl methionine cofactor. We present a 2.2-[A] cryoEM structure of Escherichia coli BrxX bound to target dsDNA, which reveals the molecular details of DNA recognition by BREX and paves the way for rational engineering of BREX specificity. We show that BrxX alone does not support methylation, and BREX activity requires an assembly of a supramolecular BrxBCXZ immune complex. Finally, we present a cryoEM structure of BrxX bound to a phage-encoded inhibitor Ocr that sequesters an inactive dimeric form of BrxX. Together, these results allow us to propose a model of BREX-mediated DNA sensing and anti-phage defense.

molecular biology↗

bioRxiv2022

Gene amplifications cause high-level resistance against albicidin in Gram-negative bacteria

Antibiotic resistance is a continuously increasing concern for public health care. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against the antibiotic albicidin. We show that this mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of the GyrI-like transcription regulator STM3175 (YgiV) which binds albicidin. X-ray crystallography and molecular docking studies reveal a conserved binding motif that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in Gram-negative bacteria and our experiments confirm that STM3175 homologs can convey resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

microbiology↗