Biology subjects

Japaridze, A.

Publications and source records attributed to Japaridze, A..

9 recordsLinked to original sources

bioRxiv2023

Prospects and Challenges for Graphene Drums As Sensors Of Individual Bacteria

Graphene-drum-enabled nanomotion detection can play an important role in probing life at the nanoscale. By combining micro- and nanomechanical systems with optics, nanomotion sensors bridge the gap between mechanics and cellular biophysics. They have allowed investigation of processes involved in metabolism, growth, and structural organisation of a large variety of microorganisms, ranging from yeasts to bacterial cells. Using graphene drums, these processes can now be resolved at the single-cell level. In this perspective, we discuss the key achievements of nanomotion spectroscopy, and peek forward into the prospects for application of this single-cell technology in clinical settings. Furthermore, we discuss the steps required for implementation and look into applications beyond microbial sensing.

microbiology↗

bioRxiv2023

Microwell-enhanced optical detection of single bacteria

Bacteria that are resistant to antibiotics present an increasing burden on healthcare. To address this emerging crisis, novel rapid Antibiotic Susceptibility Testing (AST) methods are eagerly needed. Here, we present an optical AST technique that can determine the bacterial viability within one hour down to a resolution of single bacteria. The method is based on measuring intensity fluctuations of a reflected laser focused on a bacterium in reflective microwells. Using numerical simulations, we show that both refraction and absorption of light by the bacterium contribute to the observed signal. By administering antibiotics that kill the bacteria, we show that the variance of the detected fluctuations vanishes within one hour, indicating the potential of this technique for rapid sensing of bacterial antibiotic susceptibility. We envisage the use of this method for massively parallelizable AST tests and fast detection of drug resistant pathogens.

bioengineering↗

bioRxiv2023

Direct observation of a crescent-shape chromosome in Bacillus subtilis

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genomic information. Direct visualization of the chromosomal shape within bacterial cells is hampered by cell-wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high-resolution fluorescence microscopy techniques, and genetic engineering to visualize the intrinsic shape of the bacterial chromosome in real-time in live Bacillus subtilis cells. We show that the chromosome exhibits a crescent shape with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins - the key drivers of chromosomal organization - along the contour of the crescent chromosome, showing the highest density near oriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

microbiology↗

bioRxiv2022

MukBEF-dependent chromosomal organization in widened Escherichia coli

The bacterial chromosome is spatially organized through protein-mediated compaction, supercoiling, and cell-boundary confinement. Structural Maintenance of Chromosomes (SMC) complexes are a major class of chromosome-organizing proteins present throughout all domains of life. Here, we study the role of the Escherichia coli SMC complex MukBEF in chromosome architecture and segregation. Using quantitative live-cell imaging of shape-manipulated cells, we show that MukBEF is crucial to preserve the toroidal topology of the E. coli chromosome and that it is non-uniformly distributed along the chromosome: it prefers locations towards the origin and away from the terminus of replication, and it is unevenly distributed over the origin of replication along the two chromosome arms. Using an ATP hydrolysis-deficient MukB mutant, we find that MukBEF translocation along the chromosome is ATP-dependent, in contrast to its loading onto DNA. MukBEF and MatP are furthermore found to be essential for sister chromosome decatenation. We propose a model that explains how MukBEF, MatP, and their interacting partners organize the chromosome and contribute to sister segregation and recombination. The combination of bacterial cell-shape modification and quantitative fluorescence microscopy paves way to investigating chromosome-organization factors in vivo.

microbiology↗

bioRxiv2022

Extracting and characterizing protein-free megabasepair DNA for in vitro experiments

Chromosome structure and function is studied in cells using imaging and chromosome-conformation-based methods as well as in vitro with a range of single-molecule techniques. Here we present a method to obtain genome-size (megabasepair length) deproteinated DNA for in vitro studies, which provides DNA substrates that are two orders of magnitude longer than typically studied in single-molecule experiments. We isolated chromosomes from bacterial cells and enzymatically digested the native proteins. Mass spectrometry indicated that 97-100% of DNA-binding proteins are removed from the sample. Upon protein removal, we observed an increase in the radius of gyration of the DNA polymers, while quantification of the fluorescence intensities showed that the length of the DNA objects remained megabasepair sized. In first proof-of-concept experiments using these deproteinated long DNA molecules, we observed DNA compaction upon adding the DNA-binding protein Fis or PEG crowding agents and showed that it is possible to track the motion of a fluorescently labelled DNA locus. These results indicate the practical feasibility of a genome-in-a-box approach to study chromosome organization from the bottom up.

biophysics↗

bioRxiv2021

Probing nanomotion of single bacteria with graphene drums

Motion is a key characteristic of every form of life1. Even at the microscale, it has been reported that colonies of bacteria can generate nanomotion on mechanical cantilevers2, but the origin of these nanoscale vibrations has remained unresolved3,4. Here, we present a novel technique using drums made of ultrathin bilayer graphene, where the nanomotion of single bacteria can be measured in its aqueous growth environment. A single E. coli cell is found to generate random oscillations with amplitudes of up to 60 nm, exerting forces of up to 6 nN to its environment. Using mutant strains, we are able to pinpoint the bacterial flagella as the main source of nanomotion. By real-time tracing of changes in nanomotion upon administering antibiotics, we demonstrate that graphene drums can perform antibiotic susceptibility testing with single-cell sensitivity. These findings deepen our understanding of processes underlying cellular dynamics, and pave the way towards high throughput and parallelized rapid screening of the effectiveness of antibiotics in bacterial infections with graphene devices.

biophysics↗

bioRxiv2020

DNA sequence-directed cooperation between nucleoid-associated proteins

Nucleoid associated proteins (NAPs) are a class of highly abundant DNA binding proteins in bacteria and archaea. While the composition and relative abundance of the NAPs change during the bacterial growth cycle, surprisingly little is known about their crosstalk in mutually binding to the bacterial chromosome and stabilising higher-order nucleoprotein complexes. Here, we use atomic force microscopy and solid-state nanopores to investigate long-range nucleoprotein structures formed by the binding of two major NAPs, FIS and H-NS, to DNA molecules with distinct binding-site arrangements. We find that spatial organisation of the protein binding sites can govern the higher-order architecture of the nucleoprotein complexes. Based on sequence arrangement the complexes differed in their global shape and compaction, as well as the extent of FIS and H-NS binding. Our observations highlight the important role the DNA sequence plays in driving structural differentiations within the bacterial chromosome.

biophysics↗

bioRxiv2019

Direct observation of independently moving replisomes in Escherichia coli

The replication and transfer of genomic material from a cell to its progeny are vital processes in all living systems. Here we visualize the process of chromosome replication in E. coli cells with an increased width. Monitoring the replication of single chromosomes yields clear examples of replication bubbles that reveal that the two replisomes move independently from the origin to the terminus of replication along each of the two arms of the circular chromosome, providing direct support for the so-called train-track model, and against a factory model for replisomes. The origin of replication duplicates near midcell, initially splitting to random directions and subsequently towards the poles. The probability of successful segregation of chromosomes significantly decreases with increasing cell width, indicating that chromosome confinement by the cell boundary is an important driver of DNA segregation. Our findings resolve long standing questions in bacterial chromosome organization.

cell biology↗

bioRxiv2018

Direct Imaging of the circular chromosome of a live bacterium

New assays for quantitative imaging1-6 and sequencing7-11 have yielded great progress towards understanding the organizational principles of chromosomes. Yet, even for the well-studied model bacterium Escherichia coli, many basic questions remain unresolved regarding chromosomal (sub-)structure2,11, its mechanics1,2,12 and dynamics13,14, and the link between structure and function1,15,16. Here we resolve the spatial organization of the circular chromosome of bacteria by directly imaging the chromosome in live E. coli cells with a broadened cell shape. The chromosome was observed to exhibit a torus topology with a 4.2 m toroidal length and 0.4 m bundle thickness. On average, the DNA density along the chromosome shows dense right and left arms that branch from a lower-density origin of replication, and are connected at the terminus of replication by an ultrathin flexible string of DNA. At the single-cell level, the DNA density along the torus is found to be strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. We show that prominent domain boundaries at the terminus and origin of replication are induced by MatP proteins, while weaker transient domain boundaries are facilitated by the global transcription regulators HU and Fis. These findings provide an architectural basis for the understanding of the spatial organization of bacterial genomes.

microbiology↗