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

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.