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 organization 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.

Correction to: Nature Communicationhttps://doi.org/10.1038/s41467-024-47094-x, published online 28 March 2024 The original version of this article contained an error in the “Acknowledgement “section. The original version read “We also acknowledge funding for the work in S.G. lab by the Swiss National Science Foundation (grant number: 310030L_170242).” This has been amended to “We also acknowledge funding for the work in S.G. lab by the Swiss National Science Foundation (grant number: 310030_197770).” This has now been corrected in both the PDF and HTML versions of the Article.

Bacterial chromosomes are folded into tightly regulated three-dimensional structures to ensure proper transcription, replication, and segregation of the genetic information. Direct visualization of 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 shape of unconfined bacterial chromosome in real-time in live Bacillus subtilis cells that are expanded in volume. We show that the chromosomes predominantly exhibit crescent shapes 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.

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 Escherichia coli chromosome and that it is non-uniformly distributed along the chromosome: it prefers locations toward 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 confirm 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. The combination of bacterial cell-shape modification and quantitative fluorescence microscopy paves way to investigating chromosome-organization factors in vivo.

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 1 h 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 1 h, 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.

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.

Chromosome structure and function is studied using various cell-based methods as well as with a range of in vitro single-molecule techniques on short DNA substrates. Here, we present a method to obtain megabase-pair-length deproteinated DNA for in vitro studies. 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. Fluorescence microscopy analysis showed an increase in the radius of gyration of the DNA polymers, while the DNA length remained megabase-pair sized. In 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 labeled DNA locus. These results indicate the practical feasibility of a “genome-in-a-box” approach to study chromosome organization from the bottom up.

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 unresolved^3,4. Here, we present a new technique using drums made of ultrathin bilayer graphene, where the nanomotion of single bacteria can be measured in its aqueous growth environment. A single Escherichia 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 that differ by single gene deletions that affect motility, we are able to pinpoint the bacterial flagella as the main source of nanomotion. By real-time tracing of changes in nanomotion on 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.

The process of DNA segregation, the redistribution of newly replicated genomic material to daughter cells, is a crucial step in the life cycle of all living systems. Here, we review DNA segregation in bacteria which evolved a variety of mechanisms for partitioning newly replicated DNA. Bacterial species such as Caulobacter crescentus and Bacillus subtilis contain pushing and pulling mechanisms that exert forces and directionality to mediate the moving of newly synthesized chromosomes to the bacterial poles. Other bacteria such as Escherichia coli lack such active segregation systems, yet exhibit a spontaneous de-mixing of chromosomes due to entropic forces as DNA is being replicated under the confinement of the cell wall. Furthermore, we present a synopsis of the main players that contribute to prokaryotic genome segregation. We finish with emphasizing the importance of bottom-up approaches for the investigation of the various factors that contribute to genome segregation.

Nucleoid-associated proteins (NAPs) are a class of highly abundant DNA-binding proteins in bacteria and archaea. While both the composition and relative abundance of the NAPs change during the bacterial growth cycle, surprisingly little is known about their crosstalk in mutually binding and stabilizing higher-order nucleoprotein complexes in the bacterial chromosome. 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 organization 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 differentiation within the bacterial chromosome.

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 widened E. coli cells. 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.

The fine interplay between the simultaneous stretching and confinement of amyloid fibrils is probed by combining a microcapillary setup with atomic force microscopy. Single-molecule statistics reveal how the stretching of fibrils changed from force to confinement dominated at different length scales. System order, however, is solely ruled by confinement. Coarse-grained simulations support the results and display the potential to tailor system properties by tuning the two effects. These findings may further help shed light on in vivo amyloid fibril growth and transport in highly confined environments such as blood vessels.

Although the physical properties of chromosomes, including their morphology, mechanics, and dynamics are crucial for their biological function, many basic questions remain unresolved. Here we directly image the circular chromosome in live E. coli with a broadened cell shape. We find that it exhibits a torus topology with, on average, a lower-density origin of replication and an ultrathin flexible string of DNA at the terminus of replication. At the single-cell level, the torus is strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. Our data show a domain organization underlying the chromosome structure of E. coli, where MatP proteins induce site-specific persistent domain boundaries at Ori/Ter, while transcription regulators HU and Fis induce weaker transient domain boundaries throughout the genome. These findings provide an architectural basis for the understanding of the dynamic spatial organization of bacterial genomes in live cells.