Abstract
Bacterial chromosomal DNA is a highly compact nucleoid. The organization of this nucleoid is poorly understood due to limitations in the methods used to monitor the complexities of DNA organization in live bacteria. Here, we report that circular plasmid DNA is auto-packaged into a uniform dual-toroidal-spool conformation in response to mechanical stress stemming from sharp bending and un-winding by atomic force microscopic analysis. The mechanism underlying this phenomenon was deduced with basic physical principles to explain the auto-packaging behaviour of circular DNA. Based on our observations and previous studies, we propose a dynamic model of how chromosomal DNA in E. coli may be organized during a cell division cycle. Next, we test the model by monitoring the development of HNS clusters in live E. coli during a cell cycle. The results were in close agreement with the model. Furthermore, the model accommodates a majority of the thus-far-discovered remarkable features of nucleoids in vivo.
Highlights
Given the comparable amount of divalent Pt atoms detected in C and C+T samples by inductively coupled plasma mass spectrometry (ICP-MS), insufficient mechanical stress injected by C alone compared with C+T might underlie the marked difference in DNA conformation between the two groups[20]
Based on our observations and other related work, such as the findings that the replication factory is located at the centre of the E. coli cell, that the DNA strands were pulled through the replication machinery during DNA replication[27,28] and that the oriC and dif loci have distinct cytoplasmic locations[16,29,30], we further propose a dynamic model for chromosome organization during a cell division cycle in live E. coli (Fig. 2e)
Extensive theoretical and experimental studies have reported that NAPs in bacteria, or histones in mammal cells, play an important role in initiating the wrapping of DNA through electrostatic interactions with its phosphate backbone[23,37,38,39], and further result in DNA compaction via protein–protein interactions[9,14,40,41]
Summary
The nucleoid had an eight-shaped scaffold with DNA coiled onto itself in each spool to form a buckled balanced dual-toroidal-spool conformation (Fig. 2b) In this model, the centre core of each spool corresponds to the HNS cluster observed by photoactivated localization microscopy (PALM) in vivo[7]. The HNS cluster distance in middle aged and old cells averaged ~0.89 ± 0.30 μ m (mean ± SD, N = 2 39), with one peak centred at approximately 0.7 μ m for adjacent clusters and another peak centred at approximately 1.37 μ m for distal clusters (Fig. 4e) These observations agree with our model that chromosomal DNA is organized into a single-spool docking on replication forks prior to the completion of DNA replication
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