Cooperative transitions of isolated Escherichia coli nucleoids: implications for the nucleoid as a cellular phase.
The genomic DNA of Escherichia coli occurs in compact bodies known as nucleoids. Organization and structure of nucleoids are poorly understood. Compact, characteristically shaped, nucleoids isolated by the polylysine-spermidine procedure were visualized by DNA fluorescence microscopy. Treatment with urea or trypsin converted compact nucleoids to partially expanded forms. The transition in urea solutions was accompanied by release of most DNA-associated proteins; the transition point between compact and partially expanded forms was not changed by the loss of the proteins nor was it changed in nucleoids isolated from cells after exposure to chloramphenicol or from cells in which Dps, Fis, or H-NS and StpA had been deleted. Partially expanded forms became dispersed upon RNase exposure, indicating a role of RNA in maintaining the partial expansion. Partially expanded forms that had been stripped of most DNA-associated proteins were recompacted by polyethylene glycol 8,000, a macromolecular crowding agent, in a cooperative transition. DNA-associated proteins are suggested to have relatively little effect on the phase-like behavior of the cellular nucleoid. Changes in the urea transition indicate that a previously described procedure for compaction of polylysine-spermidine nucleoids may have an artifactual basis, and raise questions about reports of repetitive local structures involving the DNA of lysed cells.
Zimmerman
Steven B
SB
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0560, USA.
D002701:Chloramphenicol / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D005456:Fluorescent Dyes; D007211:Indoles; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D009154:Mutation; D011073:Polyamines / Q000737:chemistry; D011092:Polyethylene Glycols / Q000494:pharmacology; D011107:Polylysine / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D012996:Solutions / Q000737:chemistry; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D007211:Indoles; D011073:Polyamines; D012996:Solutions; D011107:Polylysine; D011092:Polyethylene Glycols; D012965:Sodium Chloride; C007293:DAPI; D002701:Chloramphenicol; D014508:Urea; D012259:Ribonuclease, Pancreatic; D014357:Trypsin