Study of the Deinococcus radiodurans Nucleoid by Cryoelectron Microscopy of Vitreous Sections: Supplementary Comments

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Abstract
Recently, we reported results of the structural analysis of Deinococcus radiodurans nucleoid observed by cryoelectron microscopy of vitreous sections (CEMOVIS) (15). We were able to visualize the arrangement of DNA molecules in the nucleoid of this extraordinary radioresistant bacterium. In pre- vious work, Minsky and colleagues proposed that the nucleoid of D. radiodurans is organized as a densely packed DNA toroid. According to them, this structure could play a crucial role in the radiation resistance of D. radiodurans and, poten- tially, other species of Deinococcaceae (16, 24). According to our observations, the DNA arrangement in the nucleoid of D. radiodurans Sark differed from a dense toroidal spooling, sug- gesting that the model of Minsky and colleagues is not gener- ally correct for this bacterium. Soon after publication, our article faced criticism from Dr. Minsky. His major points, as we understand them, were the following: (i) the quality of the images obtained by our method is insufficient to draw any serious conclusions and (ii) there was inadequate discussion of some of our results. We attribute these criticisms to the newness of our technique and to the attractiveness of his toroidal model, which our results do not support. We see that supplementary explanation and discus- sion are necessary. Below we explain why the micrographs of vitreous sections we have presented are faithful and detailed representations of native biological material but must be interpreted in a manner different than that for conventionally fixed, stained, and em- bedded sections. Furthermore, we discuss several aspects of our results in relation to what has previously been published by other researchers. CEMOVIS Micrographs obtained by CEMOVIS must be viewed using criteria different from those we have been accustomed to using during 50 years of electron microscopy of stained and dry specimens. We will try to convince the reader that the micro- graphs we present contain more, and more reliable, informa- tion than that obtained from resin-embedded sections. We will also explain why the cutting artifacts inherent to CEMOVIS are well controlled in good sections; they do not impair image interpretation.