CEMOVIS revealed aspects of D. radiodurans
structure which are not resolved by conventional electron microscopy embedding and sectioning techniques. For example, the periodic structure and hexagonal order of the S-layer of the cell envelope are directly seen in vitreous sections, whereas they were previously observed only on biochemically isolated S-layers or on freeze-etched cells (5
Another characteristic aspect of native D. radiodurans
structure is the dense spherical granules (electron-dense granules). Recent structural studies did not report electron-dense granules (9
), although early freeze-etching studies described spherical structures reminiscent of electron-dense granules in their size and location (37
). Thornley et al. also observed dense granules in resin sections on a few occasions, but their content was often lost during cutting (39
). It seems, therefore, that electron-dense granules do exist in D. radiodurans
but they are optimally preserved only in frozen-hydrated material.
Electron-dense granules are located in the central part of exponentially growing bacteria in the nucleoid region. This addresses a question about the possible chromatin nature of electron-dense granule. However, their high sensitivity to beam-induced bubbling suggests that they do not have a high nucleic acid concentration, since aromatic-rich materials are radiation resistant (11
). Due to structural similarity, Thornley et al. (39
) associated electron-dense granules with polyphosphate granules observed in other species of bacteria. Phosphate, in combination with proteins, would account for the high density and radiation sensitivity of the granules. The precise composition of electron-dense granules, however, remains to be identified.
CEMOVIS successfully revealed aspects of the molecular arrangement of DNA within D. radiodurans nucleoids. The diffuse coralline nucleoids of exponentially growing cells do not show any particular order. We attribute this to the high transcriptional and replication activity required for active growth. A local order first appears in stationary-phase cells in the form of bundles of locally parallel DNA filaments with an average interfilament distance of 4.8 nm. Upon prolonged starvation the aspect of cholesteric liquid crystalline order is observed and the average interfilament distance shortens to 4.0 nm. The gradual increase in the local order together with reduction of the interfilament distance suggests a liquid crystalline organization of the D. radiodurans nucleoid.
It is known that liquid crystalline phases of DNA spontaneously assemble in vitro with increasing DNA concentration (26
). In bacteria, the increased DNA concentration leading to liquid crystallization may result from accumulation of high-copy plasmids (34
), but this is not the case for stationary-phase cells of D. radiodurans
, in which DNA content per cell is lower than in exponentially growing cells (18
). Since at stationary phase the nucleoid segregates to a compact round domain excluding ribosomes, the crowding of DNA can be originated by its redistribution into the confined part of the cell volume. This effect can be related to the fact that cholesteric liquid crystalline nucleoids were found in starving Escherichia coli
lacking Dps, an abundant starvation-induced unspecific DNA binding protein (16
). In contrast, wild-type E. coli
and strains overexpressing Dps show nucleoid compaction by formation of DNA-Dps cocrystals with a specific structural appearance different from that observed in D. radiodurans
). It can therefore be concluded that the segregation and compaction of the D. radiodurans
nucleoid can be driven by a decrease of protein-DNA binding in the stationary phase. In addition, an unusually high concentration of Mn2+
ions found in D. radiodurans
) can facilitate liquid crystalline compaction by compensating for repulsive forces between DNA molecules.
A minority of cells in stationary-phase cultures of D. radiodurans
have the typical morphology of exponentially growing cells. We attribute this morphological polymorphism to the presence of mutants gaining a growth advantage during the extended stationary phase (14
Nucleoid shapes that can be interpreted as rings have been found in only a few cases. This observation argues against the results of other researchers showing an abundance of ring-like nucleoids, which were considered DNA toroids (29
). We suspect that conventional studies have misinterpreted the dispersed nucleoids as DNA toroids when the central electron-dense granule is lost during sample preparation and the resolution is insufficient to define the molecular arrangement. Our high-resolution analysis reveals that the DNA arrangement in nucleoids of D. radiodurans
differs from toroidal DNA spooling both in actively growing and in stationary-phase cultures.
Diffuse coralline nucleoids without a specific molecular order observed in exponentially growing D. radiodurans
are similar to those of nonradioresistant bacteria (7
). Nevertheless, exponentially growing cells of D. radiodurans
are known to tolerate 5,000 Gy (33
). Stationary-phase cultures have a radioresistance approximately three times higher, but this increase already occurs at the beginning of the stationary-phase (24
) and is therefore independent of the ordered genome compaction which appears with the aging of the culture. This suggests that the arrangement of the nucleoid does not play a key role in the radioresistance of D. radiodurans
. Even if it does, the mechanism must differ from the one proposed by Levin-Zaidman et al. (29
), in which the dense toroidal package prevents separation of double-stranded DNA break ends, thus favoring efficient repair.
We have found that the dense toroidal package probably does not exist in D. radiodurans
. Furthermore, we observed that the ordered condensation of DNA, leading to cholesteric organization, always remains changing and dynamic. It might well be that the diffusibility of DNA fragments is reduced in liquid crystals but that is the nature of this type of order; they still remain mobile. This is confirmed by experiments in λ phage DNA cyclization in the presence of polyamines, which show that DNA ends remains mobile in most condensed liquid crystalline phases (22
). The hypothesis that the liquid crystalline order of DNA is not directly related to radioresistance is also supported by the fact that dinoflagellata, whose genome is normally in the form of cholesteric liquid crystal (35
), are not unusually radioresistant.
The fact that the segregation of the nucleoid from the ribosome-rich cytoplasm is already complete at the early phase of DNA ordering suggests that nucleoid separation is the basic structural change accompanying the transition to the stationary phase. Therefore it could be relevant to the increase in radioresistance at the beginning of stationary phase. This idea is supported by fact that the nucleoids of radiation-sensitive bacteria remain coralline and dispersed at stationary phase (17
), whereas the nucleoids of radioresistant bacterial species are more localized (41
). We speculate that the segregation of nucleoids reduces the damage caused by free radicals generated in the cytoplasm by radiation (17
The molecular arrangement of DNA revealed in the nucleoid of D. radiodurans cannot directly serve as a structural support for DNA repair. We believe that the unusual efficiency of the latter in D. radiodurans is more likely to have a physiological than a structural basis. Nevertheless, nucleoid segregation at the stationary phase can be protective and requires further study.