Internal structure and dynamics of isolated Escherichia coli nucleoids assessed by fluorescence correlation spectroscopy

Abstract

The morphology and dynamics of DNA in a bacterial nucleoid affects the kinetics of such major processes as DNA replication, gene expression. and chromosome segregation. In this work, we have applied fluorescence correlation spectroscopy to assess the structure and internal dynamics of isolated Escherichia coli nucleoids. We show that structural information can be extracted from the amplitude of fluorescence correlation spectroscopy correlation functions of randomly labeled nucleoids. Based on the developed formalism we estimate the characteristic size of nucleoid structural units for native, relaxed, and positively supercoiled nucleoids. The degree of supercoiling was varied using the intercalating agent chloroquine and evaluated from fluorescence microscopy images. The relaxation of superhelicity was accompanied by 15-fold decrease in the length of nucleoid units (from approximately 50 kbp to approximately 3 kbp).

FIGURE 1

Typical images of isolated nucleoids as seen in fluorescent microscope: (A) natively supercoiled, volume 18 ± 4 μm³ (mean ± SD over 74 nucleoids), (B) relaxed, volume 52 ± 18 μm³ (85 nucleoids), and (C) positively supercoiled, volume 19 ± 4 μm³ (67 nucleoids). Bar = 1 μm.

FIGURE 2

Dependence of nucleoid volume on chloroquine concentration: as negative supercoiling relaxes, the nucleoids expand, reaching maximum volume at full relaxation. Further increase in chloroquine concentration leads to the accumulation of positive supercoiling and associated contraction of nucleoids.

FIGURE 3

Examples of correlation functions G₂ measured on the same isolated nucleoid at different label densities. Nucleoid is in its native state. G₂ has units of photon count rate and is given in the units of kilocounts per second. Bulk concentrations of TOTO-1: (bottom to top) 0.1 μM, 0.25 μM, and 0.75 μM, respectively.

FIGURE 4

Dependence of the amplitude of FCS correlation function G₂(t → 0) on the average emission rate 〈I_em〉 for native (solid circles), relaxed (open circles), and positively supercoiled (triangles) nucleoids. Lines are linear fits to the data.