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Chapter 10 : Structure of DNA within the Bacterial Cell: Physics and Physiology

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Abstract:

This chapter is an interdisciplinary attempt to bring together physical and biological perspectives on the organization of DNA within the bacterial cell. In living bacteria the nucleoid can be observed by phase-contrast microscopy when the cells are immersed in a medium with a high refractive index, such as aqueous gelatin or polyvinylpyrrolidone, adjusted to give isoosmotic conditions. The negative superhelical tension in the chromosome is maintained through the combined action of topoisomerases I and IV and DNA gyrase. The latter enzyme actively supercoils the DNA at the expense of free energy of ATP hydrolysis. Anchoring of DNA to a fixed structure like the plasma membrane could occur if envelope proteins are being transcriptionally and cotranslationally inserted into the membrane. A DNA model that has been of considerable use in polymer theory is that of the wormlike chain. Chemical details, including the base pair sequence, are smoothed out completely. Linear, double-stranded DNA in solution is viewed as a homogeneous elastic rod undulating in a heat bath at temperature T. Solvent molecules are continually buffeting the rod, which adopts wormlike configurations. The majority of cytoplasmic particles consist of small globular proteins, and it is these that we need to account for within an approximate picture based on statistical physics.

Citation: Woldringh C, Odijk T. 1999. Structure of DNA within the Bacterial Cell: Physics and Physiology, p 171-187. In Charlebois R (ed), Organization of the Prokaryotic Genome. ASM Press, Washington, DC. doi: 10.1128/9781555818180.ch10

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Plasma Membrane
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Chromosomal DNA
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Transmission Electron Microscope
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Figures

Image of FIGURE 1
FIGURE 1

Light microscope images of the nucleoid in E. coli. (A) CSLM image (absorption contrast; resolution, 130 nm [ ]) of E. coli B/r (H266) cells grown in alanine minimal medium with a doubling time of 150 min. The smaller cell can be assumed to represent a newborn cell, containing an unreplicated chromosome. (B) Fluorescence microscopy of an OsO,-fixed and DAPI-stained E. coli MC4100 rf/mX(Ts) cell, grown in glucose minimal medium at permissive temperature (30C). (C) Cells from the same culture as in panel ? but grown at restrictive temperature (42°C) for two mass doublings. The nucleoid(s) can be seen to be pulled out into small lobules ( ). (D) Cell from the same population as in panel С but treated with 300 μg of chloramphenicol/ml to inhibit protein synthesis. The DNA has retracted into confined regions ( ). It should be emphasized that there is no indication of an increased concentration (packing density) or a different organizational state of this DNA, as previously noted by Kellenberger ( ). Bars, 1 μm (A) and 5 /Am (B through D).

Citation: Woldringh C, Odijk T. 1999. Structure of DNA within the Bacterial Cell: Physics and Physiology, p 171-187. In Charlebois R (ed), Organization of the Prokaryotic Genome. ASM Press, Washington, DC. doi: 10.1128/9781555818180.ch10
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Image of FIGURE 2
FIGURE 2

Electron microscope images of the nucleoid in E. . (A) Whole-mount transmission electron micrograph of an B/r A cell, prefixed only in growth medium (minimal alanine medium; doubling time, 126 min) with 0.1% Os0 and directly dehydrated with alcohol for critical-point drying. Note that this strain differs in size and shape from the B/r (H266) cell in Fig. 1A . Within the region of the nucleoplasm of the transparent cell, the DNA, containing few bound proteins and not properly fixed, can be seen to have precipitated into an undulating structure. Note also that the diameter of the cell (350 nm) has shrunk considerably compared to that of nondehydrated cells (640 nm [ ]). (B) Thin section of an B/r A cell, grown in minimal alanine medium (doubling time, 90 min), and fixed with Os0 under Ryter-Kellenberger conditions (see the text). Within the region of the nucleoplasm, the DNA is now visible as a fibrillar network, which has resisted the collapse by dehydration visible in panel A. (C) Thin section of an K-12 cell, grown in broth (doubling time, 22 min) and prefixed with 2.5% glutaraldehyde. The nucleoid contains sparse fibrils and is dispersed throughout the cytoplasm. (D) Thin section of an B cell prepared by CFS. The dispersed nucleoid contains only a fine-grained plasm (from reference 16 with permission). All magnification bars represent 0.5 /Am.

Citation: Woldringh C, Odijk T. 1999. Structure of DNA within the Bacterial Cell: Physics and Physiology, p 171-187. In Charlebois R (ed), Organization of the Prokaryotic Genome. ASM Press, Washington, DC. doi: 10.1128/9781555818180.ch10
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Image of FIGURE 3
FIGURE 3

(A) Typical configuration of linear wormlike DNA. The two unit vectors u(t1,) and u(t2) are tangential to the chain at respective positions tx and t2. The persistence length, P, is indicated as being 40 nm. (B) Typical configuration of a plectonemic DNA supercoil showing one branch. The persistence length, Ps, is unknown (see the text).

Citation: Woldringh C, Odijk T. 1999. Structure of DNA within the Bacterial Cell: Physics and Physiology, p 171-187. In Charlebois R (ed), Organization of the Prokaryotic Genome. ASM Press, Washington, DC. doi: 10.1128/9781555818180.ch10
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Image of FIGURE 4
FIGURE 4

(A) Cross section of perpendicular to the long axis of the cell. If the superhelical DNA (here visible in the form of several sections within the left circle) were dispersed throughout the cell, the free energy would be (see equation 5 ), but now pertaining to the total volume, . According to the phase separation theory ( ), the dispersed state is unstable because the free energy is higher than that when the DNA is confined within the nucleoid, as shown in the right circle (i.e., when the total free energy is + F, as discussed in the text). (B) The nucleoid is stabilized by the osmotic pressure arising from the excess concentration of proteins in the cytoplasm. The small circles within the two cubes depict the small proteins discussed in the text. Conforming to the concentrations indicated in see Table 1 , the upper cube (50 nm) contains 392 proteins and 2.2 ribosomes, reflecting the cytoplasm; the cube reflecting the nucleoid contains 152 proteins and 1 μηι of supercoiled DNA. Each configuration is actually a snapshot of spheres distributed randomly via a computer program. The cross section area reflects the numbers of proteins and ribosomes of a slice of arbitrary depth, indicating the phase separation between cytoplasm and nucleoid.

Citation: Woldringh C, Odijk T. 1999. Structure of DNA within the Bacterial Cell: Physics and Physiology, p 171-187. In Charlebois R (ed), Organization of the Prokaryotic Genome. ASM Press, Washington, DC. doi: 10.1128/9781555818180.ch10
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Tables

Generic image for table
TABLE 1

Macromolecular compositions of cell, cytoplasm, and nucleoid in B/r (H266) cells grown in alanine medium = 150 min)

Citation: Woldringh C, Odijk T. 1999. Structure of DNA within the Bacterial Cell: Physics and Physiology, p 171-187. In Charlebois R (ed), Organization of the Prokaryotic Genome. ASM Press, Washington, DC. doi: 10.1128/9781555818180.ch10
Generic image for table
TАBLE 2

Input variables used in the theoretical analysis ( )

Citation: Woldringh C, Odijk T. 1999. Structure of DNA within the Bacterial Cell: Physics and Physiology, p 171-187. In Charlebois R (ed), Organization of the Prokaryotic Genome. ASM Press, Washington, DC. doi: 10.1128/9781555818180.ch10
Generic image for table
TABLE 3

Variables computed from theory and derived from the coexistence equations ( )

Citation: Woldringh C, Odijk T. 1999. Structure of DNA within the Bacterial Cell: Physics and Physiology, p 171-187. In Charlebois R (ed), Organization of the Prokaryotic Genome. ASM Press, Washington, DC. doi: 10.1128/9781555818180.ch10

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