Nucleoid partitioning and the division plane in Escherichia coli

Abstract

Escherichia coli nucleoids were visualized after the DNA of OsO4-fixed but hydrated cells was stained with the fluorochrome DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate). In slowly growing cells, the nucleoids are rod shaped and seem to move along the major cell axis, whereas in rapidly growing, wider cells they consist of two-to four-lobed structures that often appear to advance along axes lying perpendicular or oblique to the major axis of the cell. To test the idea that the increase in cell diameter following nutritional shift-up is caused by the increased amount of DNA in the nucleoid, the cells were subjected to DNA synthesis inhibition. In the absence of DNA replication, the nucleoids continued to move in the growing filaments and were pulled apart into small domains along the length of the cell. When these cells were then transferred to a richer medium, their diameters increased, especially in the region enclosing the nucleoid. It thus appears that the nucleoid motive force does not depend on DNA synthesis and that cell diameter is determined not by the amount of DNA per chromosome but rather by the synthetic activity surrounding the nucleoid. Under the non-steady-state but balanced growth conditions induced by thymine limitation, nucleoids become separated into small lobules, often lying in asymmetric configurations along the cell periphery, and oblique and asymmetric division planes occur in more than half of the constricting cells. We suggest that such irregular DNA movement affects both the angle of the division plane and its position.A bacterial cell maintains its DNA in a central space called the nucleoid. In living cells, this domain can be visualized either by phase contrast in the presence of gelatin (25) or by fluorescence microscopy with a fluorochrome (13). Fixing the cells with osmium tetroxide results in nucleoids that are more compact (5, 39) but that still retain characteristic shapes which seem to reflect the replication and segregation states of the chromosome (see also references 7, 16, and 32). These distinct nucleoid shapes indicate that the DNA is not dispersed randomly throughout the cytoplasm but that most of it is confined to a region separated from the cytoplasmic phase by some unknown chemical property (38). This phenomenon does not preclude the presence throughout the cytoplasm of short loops of DNA that extend towards the plasma membrane and are actively involved in transcription and translation (14,33). That such connections are indeed effective in shaping the nucleoid is suggested by the old observation that treatment of cells with chloramphenicol causes the nucleoid to round off (21).Several models for the various mechanisms and forces involved in the movement and positioning of the chromosomes within the cell have been advanced. The classical replicon hypothesis (20) considered envelope growth between postulated sites of chromosomal attachment to the plasma membrane. However, the rigid peptidoglycan layer has been shown to be synthesized in a dispersed mode ...

Macromolecular crowding and the mandatory condensation of DNA in bacteria

“…The term 'condensation' is used to indicate adoption of a relatively concentrated, compact state occupying a fraction of the volume available. The DNA of the bacterial nucleoid is estimated to have a local concentration of ,'-50-100 mg DNA/ml (see footnote 6 of [3]) and to occupy 1/8 to 1/5 of the volume within the cell envelope [1].Condensation of DNA within bacterial ceils has been observed by light microscopy of both living and fixed cells, as well as by electron microscopy [4][5][6][7][8] (reviewed in [9,10]). Condensation of DNA in isolated nucleoids is indicated by microscopy as well as by hydrodynamic properties [11][12][13].…”

Perpendicular planes of FtsZ arcs in spheroidal Escherichia coli cells

“…Cooper [1] presumes that the peptidoglycan 'constrained-hoop' structure of rod-shaped bacteria such as Escherichia coli forces them to divide in one dimension, while Begg and Donachie [2] demonstrated that they could do so in two. We have argued [3] that the rigid peptidoglycan forces the two replicating nucleoids to segregate along cell length and that division ring is placed at the envelope adjacent to and between them, irrespective of the previous location upon termination of a chromosome replication cycle [4]. Our hypothesis implies three-dimensional randomization of division planes in spheroidal cells.…”

“…It was inserted in pBAD (from Invitrogene) and introduced into the thyA strain CR34 of E. coli [3] by the λInCh system [11]. One of the clones (LMC1492) was found most suitable and displays FtsZ-Gfp fluorescent rings following addition of L-arabinose.…”

Visualizing multiple constrictions in spheroidal Escherichia coli cells

“…coli K12 (CR34; thr-1 leuB6 thyA [26,32]) was the major strain used in this study. One series of experiments was performed with E. coli 15T -(555-7; trp met arg thy drm [20,33]).…”

“…One series of experiments was performed with E. coli 15T -(555-7; trp met arg thy drm [20,33]). Cultures were grown at 37°C in glucose minimal medium [32], with or without casamino acids (1% w/v; Difco), and thymine as indicated. For fast replication, 2'-deoxyguanosine (dG) (100 µg mL -1 ; Sigma) was added [34].…”