Dynamic Escherichia coli SeqA complexes organize the newly replicated DNA at a considerable distance from the replisome

Helgesen, Emily; Fossum-Raunehaug, Solveig; Sætre, Frank; Schink, Kay Oliver; Skarstad, Kirsten

doi:10.1093/nar/gkv146

Cited by 36 publications

(46 citation statements)

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“…The script is run under Fiji and uses Find Maxima as a tool for detecting the center of mass of foci within regions of interest, i.e., within cells. It is possible to distinguish foci that are closer than the resolution limit of fluorescence microscopes when measuring the distance between spots in two different channels (53,77,78). Thus, in this study, we were able to present measurements of focus distances below the resolution limit of our microscopy system.…”

Section: Methodsmentioning

confidence: 87%

“…We have previously shown that there is an average distance of 200 to 300 nm between the replisome and the large SeqA structure trailing it, which may correspond to several thousands of base pairs of DNA (53). Since H-NS is able to form hairpin loops on DNA in vitro, we wondered whether an increased distance between SeqA and the replisome could be observed in H-NS mutant cells in vivo as a result of decondensed DNA between the two structures.…”

Section: Resultsmentioning

confidence: 99%

“…Since H-NS is able to form hairpin loops on DNA in vitro, we wondered whether an increased distance between SeqA and the replisome could be observed in H-NS mutant cells in vivo as a result of decondensed DNA between the two structures. In order to investigate this, we used a Python-based script that automatically measures distances between the nearest neighboring foci in two separate channels (53), in our case between foci of SeqA and the replisome. Images of wild-type cells (EH02 in glycerol medium) and H-NS mutant cells (SF154 in glucose-CAA medium) were subjected to extensive analysis with this script.…”

Section: Resultsmentioning

confidence: 99%

“…We have developed a Python-based script for automatic measurements of the distance between neighboring fluorescent spots/foci that are detected in two different fluorescence channels, as described previously (53). The script is run under Fiji and uses Find Maxima as a tool for detecting the center of mass of foci within regions of interest, i.e., within cells.…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, SeqA sequesters newly formed origins for one-third of the cell cycle (49) and contributes to ensure that no more than one initiation occurs per origin per cell cycle (50)(51)(52). Fluorescence imaging indicates that SeqA structures trail the replication forks at a considerable distance of ϳ200 to 300 nm, whereas the two sister SeqA structures behind the same fork are situated closer than 30 nm together (53).…”

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confidence: 99%

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Lack of the H-NS Protein Results in Extended and Aberrantly Positioned DNA during Chromosome Replication and Segregation in Escherichia coli

2016

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The architectural protein H-NS binds nonspecifically to hundreds of sites throughout the chromosome and can multimerize to stiffen segments of DNA as well as to form DNA-protein-DNA bridges. H-NS has been suggested to contribute to the orderly folding of the Escherichia coli chromosome in the highly compacted nucleoid. In this study, we investigated the positioning and dynamics of the origins, the replisomes, and the SeqA structures trailing the replication forks in cells lacking the H-NS protein.In H-NS mutant cells, foci of SeqA, replisomes, and origins were irregularly positioned in the cell. Further analysis showed that the average distance between the SeqA structures and the replisome was increased by ϳ100 nm compared to that in wild-type cells, whereas the colocalization of SeqA-bound sister DNA behind replication forks was not affected. This result may suggest that H-NS contributes to the folding of DNA along adjacent segments. H-NS mutant cells were found to be incapable of adopting the distinct and condensed nucleoid structures characteristic of E. coli cells growing rapidly in rich medium. It appears as if H-NS mutant cells adopt a "slow-growth" type of chromosome organization under nutrient-rich conditions, which leads to a decreased cellular DNA content. IMPORTANCE It is not fully understood how and to what extent nucleoid-associated proteins contribute to chromosome folding and organization during replication and segregation in Escherichia coli. In this work, we find in vivo indications that cells lacking the nucleoid-associated protein H-NS have a lower degree of DNA condensation than wild-type cells. Our work suggests that H-NS is in-volved in condensing the DNA along adjacent segments on the chromosome and is not likely to tether newly replicated strands of sister DNA. We also find indications that H-NS is required for rapid growth with high DNA content and for the formation of a highly condensed nucleoid structure under such conditions. A cross all domains of life, it is crucial that genomes are structurally organized in a way that compacts DNA to fit inside the confined space of a cell and at the same time allows for interaction with key proteins performing replication, transcription, recombination, and repair (1-7). Unlike eukaryotic cells, bacterial cells do not possess an envelope-enclosed organelle for storage and handling of genomic DNA. The DNA is instead organized into compact bodies called nucleoids (3)(4)(5)8). These nucleoids are highly complex, and the underlying organizational mechanisms appear to be remarkably similar to that of eukaryotic cells (3, 9). The nucleoid occupies the central part of the bacterial cell (8), and its shape is dependent on a variety of factors, such as environmental conditions or genetic mutations (7, 10-13). For example, significant nucleoid compaction occurs after exposure of Escherichia coli to UV light, due to a global reorganization in response to DNA damage and the activation of the SOS response (12, 13).Certain types of proteins, called nucle...

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Section: Methodsmentioning

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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