Studies on the compaction of isolated nucleoids from Escherichia coli

Cooperative transitions of isolated Escherichia coli nucleoids: Implications for the nucleoid as a cellular phase

“…Alternatively, to avoid protein rebinding during the removal of urea, samples of control nucleoids were washed with 3.5 M urea, a Transitions of isolated nucleoids. The two transitions of nucleoids in lysates marked with asterisks were described in Zimmerman (2004); the compaction caused by incubation at 37°C is further analyzed in the present study and appears to be a result of protein rearrangements. Circles represent average thresholded nucleoid areas of the indicated nucleoid species drawn to scale; dark gray and light gray circles are the compact and open conformations, respectively, described in the text.…”

“…The changes applied here are an increase in the lysozyme exposure to reduce the amounts of envelope-associated materials, the use of a lower molecular weight preparation of polylysine to decrease aggregation of nucleoids and aggregation between nucleoids and envelope material (Zimmerman, 2004), dilution of the cell lysates before centrifugation to decrease nucleoid aggregation, and use of a glycerol step gradient to obtain a partial puriWcation and removal of components added to cause lysis. Other gradient media (metrizamide, iodixanol, and sucrose) were less eVective in removing unlysed cells and free ribosomes.…”

“…3A, triangles vs circles, inverted triangles, and squares, respectively), undoubtedly because most nucleoids mounted in either manner were rapidly adsorbed onto the surfaces of either the slide or coverslip. Automatic threephase thresholding was used to determine apparent areas (Zimmerman, 2004); such areas are systematically »50% smaller than those determined with a two-phase thresholding procedure. The rounded nucleoids from chloramphenicol-treated cells were invariably treated by the thresholding routine as discs of the same diameter, i.e., the hollow center of the chloramphenicol nucleoids was ignored.…”

Growth phase dependent changes in the structure and protein composition of nucleoid in Escherichia coli

“…The nucleoids were stained with 5 μL 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (5 μg mL 1 ) which bind specifically to DNA, using the method developed by Hiraga et al [33]. Average nucleoids area was estimated as described previously [34].…”

Replication Demands an Amendment of the Double Helix

“…In a rapidly growing E.coli, there are more than 6 replication forks (Skarstad et al 1986). The positive supercoiling generated during the synthesis of new DNA is very difficult to transfer to the terminal along the highly twisted chromosomal DNA confined in the nucleoid (Zimmerman, 2004). b) At the same time, many tRNAs, ribosomal RNAs and mRNAs were being actively transcribed from the same chromosomal DNA.…”