Genome architecture studied by nanoscale imaging: analyses among bacterial phyla and their implication to eukaryotic genome folding

Abstract

The proper function of the genome largely depends on the higher order architecture of the chromosome. Our previous application of nanotechnology to the questions regarding the structural basis for such macromolecular dynamics has shown that the higher order architecture of the Escherichia coli genome (nucleoid) is achieved via several steps of DNA folding (Kim et al., 2004). In this study, the hierarchy of genome organization was compared among E. coli, Staphylococcus aureus and Clostridium perfringens. A one-molecule-imaging technique, atomic force microscopy (AFM), was applied to the E. coli cells on a cover glass that were successively treated with a detergent, and demonstrated that the nucleoids consist of a fundamental fibrous structure with a diameter of 80 nm that was further dissected into a 40-nm fiber. An application of this on-substrate procedure to the S. aureus and the C. perfringens nucleoids revealed that they also possessed the 40- and 80-nm fibers that were sustainable in the mild detergent solution. The E. coli nucleoid dynamically changed its structure during cell growth; the 80-nm fibers releasable from the cell could be transformed into a tightly packed state depending upon the expression of Dps. However, the S. aureus and the C. perfringens nucleoids never underwent such tight compaction when they reached stationary phase. Bioinformatic analysis suggested that this was possibly due to the lack of a nucleoid protein, Dps, in both species. AFM analysis revealed that both the mitotic chromosome and the interphase chromatin of human cells were also composed of 80-nm fibers. Taking all together, we propose a structural model of the bacterial nucleoid in which a fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.

Nuclear architecture and chromatin dynamics revealed by atomic force microscopy in combination with biochemistry and cell biology

“…The recent development of atomic force microscopy (AFM) [33] has been a breakthrough in the study of nuclear architecture [16,34,35]. The instrumentation and application of AFM have been enormously fruitful due to the development of sharp cantilevers for molecular imaging [36][37][38], cantilever modification techniques for single-molecule force measurement [39][40][41][42][43], and fast-scanning devices for realtime imaging [44][45][46][47][48].…”

“…These lines of evidence suggest that the 30-to 40-nm thin fiber and the 70-to 80-nm granular fiber are the fundamental structural units of eukaryotic chromosomes; these fibers are relatively stable in a wide range of experimental conditions. Recent reports have demonstrated that these hierarchical structures exist in both eukaryotes and prokaryotes [35] and that nascent single-stranded RNAs are part of the 30-80 nm fiber structures [62,63].…”

Development of Nano-Biology with Atomic Force Microscopy

“…However, in terms of "structure", biochemical analyses are somewhat limited due to their reliance on inherent indirect detection systems. By contrast, AFM has the potential to visualize such structures directly [12,13]. Genomic DNA interacts with an assortment of proteins and forms a hierarchical higher-order structure called "chromatin".…”

“…The current model of the genomic DNA hierarchy includes the step-wise formation of higher-order folding of the genome with different nuclear proteins [12]. However, the folding mechanisms beyond 30 nm fibers are still completely open questions.…”

Adaptation beyond the Stress Response: Cell Structure Dynamics and Population Heterogeneity in Staphylococcus aureus

“…Furthermore, they are highly variable depending on growth phase: roughly 70% are replaced from the log to stationary phase in S. aureus. The nucleoid from lysed cells is usually a fibrous structure varying in thickness when observed by the 'on-substrate lysis' method with atomic force microscopy regardless of the growth phase (84), but the physical characteristics of the nucleoid change in response to oxidative stress, where the MrgA protein plays a key role (57,58).…”