A small container of several to a few hundred microm3 (i.e. bacterial cells and eukaryotic nuclei) contains extremely long genomic DNA (i.e. mm and m long, respectively) in a highly organized fashion. To understand how such genomic architecture could be achieved, Escherichia coli nucleoids were subjected to structural analyses under atomic force microscopy, and found to change their structure dynamically during cell growth, i.e. the nucleoid structure in the stationary phase was more tightly compacted than in the log phase. However, in both log and stationary phases, a fundamental fibrous structure with a diameter of approximately 80 nm was found. In addition to this '80 nm fiber', a thinner '40 nm fiber' and a higher order 'loop' structure were identified in the log phase nucleoid. In the later growth phases, the nucleoid turned into a 'coral reef structure' that also possessed the 80 nm fiber units, and, finally, into a 'tightly compacted nucleoid' that was stable in a mild lysis buffer. Mutant analysis demonstrated that these tight compactions of the nucleoid required a protein, Dps. From these results and previously available information, we propose a structural model of the E.coli nucleoid.

A single-molecule-imaging technique, atomic force microscopy (AFM) was applied to the analyses of the genome architecture of Staphylococcus aureus . The staphylococcal cells on a cover glass were subjected to a mild lysis procedure that had maintained the fundamental structural units in Escherichia coli . The nucleoids were found to consist of fibrous structures with diameters of 80 and 40 nm. This feature was shared with the E. coli nucleoid. However, whereas the E. coli nucleoid dynamically changed its structure to a highly compacted one towards the stationary phase, the S. aureus nucleoid never underwent such a tight compaction under a normal growth condition. Bioinformatic analysis suggested that this was attributable to the lack of IHF that regulate the expression of a nucleoid protein, Dps, required for nucleoid compaction in E. coli . On the other hand, under oxidative conditions, MrgA (a staphylococcal Dps homolog) was over-expressed and a drastic compaction of the nucleoid was detected. A knock-out mutant of the gene encoding the transcription factor ( perR ) constitutively expressed mrgA , and its nucleoid was compacted without the oxidative stresses. The regulatory mechanisms of Dps/MrgA expression and their biological significance were postulated in relation to the nucleoid compaction.

In bacteria, Dps is one of the critical proteins to build up a condensed nucleoid in response to the environmental stresses. In this study, we found that the expression of Dps and the nucleoid condensation was not simply correlated in Escherichia coli, and that Fis, which is an E. coli (gamma-Proteobacteria)-specific nucleoid protein, interfered with the Dps-dependent nucleoid condensation. Atomic force microscopy and Northern blot analyses indicated that the inhibitory effect of Fis was due to the repression of the expression of Topoismerase I (Topo I) and DNA gyrase. In the Dfis strain, both topA and gyrA/B genes were found to be upregulated. Overexpression of Topo I and DNA gyrase enhanced the nulceoid condensation in the presence of Dps. DNA-topology assays using the cell extract showed that the extracts from the Dfis and Topo I-/DNA gyrase-overexpressing strains, but not the wildtype extract, shifted the population toward relaxed forms. These results indicate that the topology of DNA is dynamically transmutable and that the topology control is important for Dps-induced nucleoid condensation.

The circular bacterial genome DNA exists in cells in the form of nucleoids. In the present study, using genetic, molecular and structural biology techniques, we show that nascent single-stranded RNAs are involved in the step-wise folding of nucleoid fibers. In Escherichia coli , RNase A degraded thicker fibers (30 and 80 nm wide) into thinner fibers (10 nm wide), while RNase III and RNase H degraded 80-nm fibers into 30-nm (but not 10-nm) fibers. Similarly in Staphylococcus aureus , RNase A treatment resulted in 10-nm fibers. Treatment with the transcription inhibitor, rifampicin, in the absence of RNase A changed most nucleoid fibers to 10-nm fibers. Proteinase-K treatment of nucleoids exposed DNA. Thus, the smallest structural unit is an RNase A-resistant 10-nm fiber composed of DNA and proteins, and the hierarchical structure of the bacterial chromosome is controlled by transcription itself. In addition, the formation of 80-nm fibers from 30-nm fibers requires double-stranded RNA and RNA-DNA hetero duplex. RNA is evident in the architecture of log-phase uncondensed and stationary-phase condensed nucleoids.

The proper function of the genome largely depends on the higher-order architecture of the chromosome. To understand the detailed chromosome structure in a native state, we developed an on-substrate procedure of subcellular fractionation suitable for the observation by atomic force microscopy (AFM). HeLa cells on a coverslip were successively treated with a detergent and a high-salt solution to remove the cytoplasmic and nucleoplasmic materials. A closer observation of the nucleus by AFM revealed that the interphase chromosome is composed of a granular unit of approximately 80 nm in diameter. Subsequent mild treatment with deoxyribonuclease I (10 U ml(-1)) exposed these units more clearly, which enabled us to uncover the 80-nm granules forming a fibre of approximately 80 nm width. In the cytoplasmic regions, cytoskeletal fibres with varying widths (10-70 nm) were observed. These observations suggest that the 80 nm granular fibre is a fundamental structural unit of the interphase chromosome. This on-substrate procedure was also applied to Escherichia coli. Cells attached on a coverslip were successively treated with lysozyme and detergent to partially release the nucleoid onto the substrate. The AFM observation revealed that the approximately 80 nm fundamental structural unit forms a granular fibre similar to that of HeLa cells. These results suggest that the fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.