The DNA of Escherichia coli is largely confined to one or a few compact bodies known as nucleoids. The nucleoids occupy only a small fraction of the nominal cellular volume. The factors causing their compaction have been difficult to characterize, in part because of the lack of suitable experimental systems. We here use lysed cell preparations to examine the effects of release of cytoplasm on nucleoid compaction. Lysis was accomplished with retention of sufficient residual cell wall material to form a rod-shaped boundary that continued to enclose the DNA. DNA fluorescence and phase contrast microscopy were compared in order to localize the DNA inside this boundary. Lysis of exponential phase cells of E. coli allowed expansion of the DNA to fill the volume within the boundary. This expansion supports our earlier suggestion of the powerful DNA compaction associated with macromolecular crowding from the bacterial cytoplasm. In contrast, lysis of cells that had been preexposed to chloramphenicol or nalidixic acid to generate the characteristic DNA structures associated with those drugs was not accompanied by an expansion of the DNA to fill the boundaries of those cells. The restricted expansion of the DNA upon lysis of chloramphenicol- or nalidixic acid-treated cells indicated the presence of alternative, more stable DNA structures; in accord with this interpretation, dilatancy assays of spermidine nucleoids from nalidixic acid-treated cells showed increased resistance to unfolding in urea solutions, as had been found earlier for nucleoids from chloramphenicol-treated cells.
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