DNA adopts different conformations based on its environment. We reveal conditions that either preserve the DNA’s physiological B-conformation, even upon its placement in UHV, or lead to a partial B-form to A-form reorganization upon DNA’s deposition on a surface. We use high-resolution AFM to image DNA with a well-defined number of base pairs deposited on mica. To enable the DNA’s adhesion, we either add divalent cations to the DNA solution or functionalize the surface with a silane layer. The contour length of DNA on the silane is always in perfect agreement with the B-form conformation, whereas cation-deposited DNA is always, in some cases up to 20% shorter. We varied the equilibration time, the DNA length, and sequence and compared nicked to non-nicked molecules, thus identifying several factors controlling the DNA’s length. We performed TERS measurements confirming spectroscopically that cation-deposited DNA undergoes a partial B-form to A-form conformational transition upon drying and pinpointed positions along the DNA where this transition was more probable, namely the ends of the molecules. Controlling the conformation of DNA is essential for its nanotechnology applications such as nanotemplating. Our findings could also shed a whole new light on DNA polymer physics, the mechanisms of DNA binding to surfaces, or the abundant contradictory data on DNA’s electrical behavior.

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.

Mycobacterium tuberculosis secretes multiple virulence factors during infection via the general Sec and Tat pathways, and via specialized ESX secretion systems, also referred to as type VII secretion systems. The ESX-1 secretion system is an important virulence determinant because deletion of ESX-1 leads to attenuation of M. tuberculosis. ESX-1 secreted protein B (EspB) contains putative PE (Pro-Glu) and PPE (Pro-Pro-Glu) domains, and a C-terminal domain, which is processed by MycP1 protease during secretion. We determined the crystal structure of PE–PPE domains of EspB, which represents an all-helical, elongated molecule closely resembling the structure of the PE25–PPE41 heterodimer despite limited sequence similarity. Also, we determined the structure of full-length EspB, which does not have interpretable electron density for the C-terminal domain confirming that it is largely disordered. Comparative analysis of EspB in cell lysate and culture filtrates of M. tuberculosis revealed that mature secreted EspB forms oligomers. Electron microscopy analysis showed that the N-terminal fragment of EspB forms donut-shaped particles. These data provide a rationale for the future investigation of EspB's role in M. tuberculosis pathogenesis.

New assays for quantitative imaging [1][2][3][4][5][6] and sequencing [7][8][9][10][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-)structure 2,11 , its mechanics 1,2,12 and dynamics 13,14 , and the link between

DNA in bacterial chromosomes and bacterial plasmids is supercoiled. DNA supercoiling is essential for DNA replication and gene regulation. However, the density of supercoiling in vivo is circa twice smaller than in deproteinized DNA molecules isolated from bacteria. What are then the specific advantages of reduced supercoiling density that is maintained in vivo? Using Brownian dynamics simulations and atomic force microscopy we show here that thanks to physiological DNA–DNA crowding DNA molecules with reduced supercoiling density are still sufficiently supercoiled to stimulate interaction between cis-regulatory elements. On the other hand, weak supercoiling permits DNA molecules to modulate their overall shape in response to physiological changes in DNA crowding. This plasticity of DNA shapes may have regulatory role and be important for the postreplicative spontaneous segregation of bacterial chromosomes.

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.

Bacterial chromosome has a compact structure that dynamically changes its shape in response to bacterial growth rate and growth phase. Determining how chromatin remains accessible to DNA binding proteins, and transcription machinery is crucial to understand the link between genetic regulation, DNA structure, and topology. Here, we study very large supercoiled dsDNA using high-resolution characterization, theoretical modeling, and molecular dynamics calculations. We unveil a new type of highly ordered DNA organization forming in the presence of attractive DNA-DNA interactions, which we call hyperplectonemes. We demonstrate that their formation depends on DNA size, supercoiling, and bacterial physiology. We compare structural, nanomechanic, and dynamic properties of hyperplectonemes bound by three highly abundant nucleoid-associated proteins (FIS, H-NS, and HU). In all these cases, the negative supercoiling of DNA determines molecular dynamics, modulating their 3D shape. Overall, our findings provide a mechanistic insight into the critical role of DNA topology in genetic regulation.

Fluorescent dyes are widely used for staining and visualization of DNA in optical microscopy based methods. Even though for some dyes the mechanism of binding is known, how this binding affects DNA remains poorly understood. Here we present a novel experimental study of the influence of staining dyes on DNA properties. We use atomic force microscopy which allows quantification and measurement of structural properties of stained DNA with nanometer resolution. We studied the influence of dyes on the persistence length, the total contour length, and the morphology of individual DNA molecules. We tested three widely used dyes known to differently bind DNA molecules, namely PicoGreen, Dapi, and DRAQ5. Based on our measurements, when imaged at typical concentrations (manufacturer suggested concentrations used for cell imaging), PicoGreen dye showed little effect, Dapi dye decreased the DNA persistence length, and DRAQ5 decreased the persistence length and elongated the DNA. When used at high concentrations, all of the dyes induced drastic changes in the DNA morphology. Our study clearly shows that DNA-binding dyes, irrespective of their DNA binding mechanisms, strongly influence the physical properties of DNA. These changes are strongly dose and dye type dependent and therefore should be taken into consideration when conducting experiments with DNA. ■ INTRODUCTIONFluorescence microscopy is a widely used technique in cell biology, biochemistry, and medicine among other fields of scientific research. The need to label and visualize DNA and chromatin is gaining importance in different fields of biological research, with experiments ranging from measurements on isolated DNA 1−6,11−13 to experiments in fixed cells and live cells. [7][8][9][10]14 There is a large variety of DNA labeling dyes: some binding to the minor/major grooves of double-stranded DNA, others intercalating into DNA, and some doing both. 1,8−20 The influence of DNA labeling dyes on the DNA structure and its properties is still a topic of debate, since no clear data exist on how strongly they influence physical properties. 11−19 Nevertheless, several works revealed that parameters such as the contour length and the persistence length of bare DNA can be significantly changed by fluorescent dyes upon binding. 11−20On the example of YOYO-1 dyes, Gunther et al. showed that the intercalation of the dye in double-stranded DNA modified its mechanical and structural properties. 11 Different studies showed that upon binding of YOYO dye the contour length of the DNA increased roughly linearly, reaching 30−40% elongation compared to bare DNA. In experiments performed with optical tweezers Sischka et al. compared different DNA binding agents with different binding modes (such as ethidium bromide, YO-1, distamycin-A, and YOYO), revealing changes in the mechanical response of DNA pulling as the function of the corresponding dye binding mechanisms. 16 Wojcik et al. showed that DRAQ5 dye detached H1 and H2B histones in a concentration dependent manner in live cells. 14 Hig...