Release of Compact Nucleoids with Characteristic Shapes from Escherichia coli

Abstract

The genomic DNA of bacteria is contained in one or a few compact bodies known as nucleoids. We describe a simple procedure that retains the general shape and compaction of nucleoids from Escherichia coli upon cell lysis and nucleoid release from the cell envelope. The procedure is a modification of that used for the preparation of spermidine nucleoids (nucleoids released in the presence of spermidine) (T. Kornberg, A. Lockwood, and A. Worcel, Proc. Natl. Acad. Sci. USA 71:3189-3193, 1974). Polylysine is added to prevent the normal decompaction of nucleoids which occurs upon cell lysis. Nucleoids retained their characteristic shapes in lysates of exponential-phase cells or in lysates of cells treated with chloramphenicol or nalidixate to alter nucleoid morphology. The notably unstable nucleoids of rifampin-treated cells were obtained in compact, stable form in such lysates. Nucleoids released in the presence of polylysine were easily processed and provided well-defined DNA fluorescence and phase-contrast images. Uniform populations of nucleoids retaining characteristic shapes could be isolated after formaldehyde fixation and heating with sodium dodecyl sulfate.The genomic DNA of Escherichia coli is localized in one or two compact bodies, known as nucleoids, per cell (3,15,31,36,38,40). There have been many attempts to isolate representative nucleoids outside of cells (24,25). However, the various DNA-containing structures that have been isolated generally have failed to meet this goal in two important ways. First, the nucleoid DNA underwent the partial decompaction that is associated with cell lysis (21). Second, large amounts of residual envelope remained with the nucleoid DNA, so that many such preparations are more accurately described as lysed or broken cell preparations than as isolated nucleoids (see footnote 6 of reference 20).Evaluation of the released nucleoids is made difficult by a lack of information on the structure of the original nucleoids within cells. Here we use retention of the general shape of nucleoids in initial cells as a criterion for evaluation of a new procedure for nucleoid isolation. There have been few reports on the shapes of isolated nucleoids; the doublet-shaped highsalt nucleoids (nucleoids released in the presence of high salt concentrations) described by Hecht et al. (10) and Van Ness and Pettijohn (33) and the large DNA double structure found in high-salt nucleoids of a DNA gyrase mutant by Steck and Drlica (28) are the only instances of which we are aware.We have used the distinctively shaped nucleoids formed in cells that have been exposed to antibiotics (4, 5, 11, 12) as test objects. The three antibiotics used here, chloramphenicol, nalidixate, and rifampin, all promote the coalescence of nucleoids in cells and can cause changes in nucleoid morphology and stability:The nucleoid coalescence caused by chloramphenicol (17, 32) has been attributed to a loss of cotranslational insertion linkages between the DNA and the cell envelope when protein synthesis is inhibited; the los...

Active Transcription of rRNA Operons Condenses the Nucleoid inEscherichia coli: Examining the Effect of Transcription on Nucleoid Structure in the Absence of Transertion

“…Transcription and/or transcription-induced supercoiling could also interfere with the binding of nucleoid-associated proteins to DNA (1,17), which in turn could affect DNA supercoiling. The supercoiling introduced by transcription, however, is unlikely to account for the effect of transcription on the structure of the nucleoid described in this study, because the nucleoid of the cell remains compact when treated with either coumermycin or nalidixate, an inhibitor of gyrase (35,36,52), in contrast to fully expanded nucleoids induced by rifampin or the stringent response. The E. coli chromosome consists of ϳ50 large distinctive domains of supercoiling, each of which is topologically independent of the others (31,45).…”

“…In support of this concept, it is consistently reported that chloramphenicol, a translation inhibitor, induces nucleoid compaction in the cell (41,50). However, there are conflicting results regarding the effect of rifampin on nucleoid structure: both rifampin-induced nucleoid expansion (6,13,26,36) and nucleoid compaction (3,40,52,53) have been reported. Thus, the exact role of transcription in the structure of the nucleoid remains poorly understood and understudied (28).…”

Nucleoid remodeling by an altered HU protein: Reorganization of the transcription program

“…Intact nucleoids were extracted from strain DM0100 (⌬hupAhupB), following the procedure of Zimmerman and Murphy (10). Isolated nucleoids were incubated with wild-type HU␣ and HU␣ mutant at a (HU␣) 2 :nucleoid DNA molar ratio of 4 nM:0.3 fM in a buffer containing 10 mM Tris (pH 8.0) and 50 mM KCl for 10 min on ice.…”

From Water and Ions to Crowded Biomacromolecules:In VivoStructuring of a Prokaryotic Cell

“…on April 30, 2019 by guest http://mmbr.asm.org/ one could attempt to isolate multiplexes and nucleoids through a controlled break-up of bacterial L forms in a properly formulated medium, analogous to the isolation of nucleoids (266).…”