Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.

The nucleoid of Escherichia coli comprises DNA, nucleoid associated proteins (NAPs) and RNA, whose role is unclear. We found that lysing bacterial cells embedded in agarose plugs in the presence of RNases caused massive fragmentation of the chromosomal DNA. This RNase-induced chromosomal fragmentati...

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Main Authors: Sharik R Khan, Andrei Kuzminov
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2017-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC5739488?pdf=render
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author Sharik R Khan
Andrei Kuzminov
author_facet Sharik R Khan
Andrei Kuzminov
author_sort Sharik R Khan
collection DOAJ
description The nucleoid of Escherichia coli comprises DNA, nucleoid associated proteins (NAPs) and RNA, whose role is unclear. We found that lysing bacterial cells embedded in agarose plugs in the presence of RNases caused massive fragmentation of the chromosomal DNA. This RNase-induced chromosomal fragmentation (RiCF) was completely dependent on the presence of RNase around lysing cells, while the maximal chromosomal breakage required fast cell lysis. Cell lysis in plugs without RNAse made the chromosomal DNA resistant to subsequent RNAse treatment. RiCF was not influenced by changes in the DNA supercoiling, but was influenced by growth temperature or age of the culture. RiCF was partially dependent on H-NS, histone-like nucleoid structuring- and global transcription regulator protein. The hupAB deletion of heat-unstable nucleoid protein (HU) caused increase in spontaneous fragmentation that was further increased when combined with deletions in two non-coding RNAs, nc1 and nc5. RiCF was completely dependent upon endonuclease I, a periplasmic deoxyribonuclease that is normally found inhibited by cellular RNA. Unlike RiCF, the spontaneous fragmentation in hupAB nc1 nc5 quadruple mutant was resistant to deletion of endonuclease I. RiCF-like phenomenon was observed without addition of RNase to agarose plugs if EDTA was significantly reduced during cell lysis. Addition of RNase under this condition was synergistic, breaking chromosomes into pieces too small to be retained by the pulsed field gels. RNase-independent fragmentation was qualitatively and quantitatively comparable to RiCF and was partially mediated by endonuclease I.
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spelling doaj.art-d5bda4c60f714deda4513bb73d0f2fec2022-12-21T23:23:40ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-011212e019017710.1371/journal.pone.0190177Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.Sharik R KhanAndrei KuzminovThe nucleoid of Escherichia coli comprises DNA, nucleoid associated proteins (NAPs) and RNA, whose role is unclear. We found that lysing bacterial cells embedded in agarose plugs in the presence of RNases caused massive fragmentation of the chromosomal DNA. This RNase-induced chromosomal fragmentation (RiCF) was completely dependent on the presence of RNase around lysing cells, while the maximal chromosomal breakage required fast cell lysis. Cell lysis in plugs without RNAse made the chromosomal DNA resistant to subsequent RNAse treatment. RiCF was not influenced by changes in the DNA supercoiling, but was influenced by growth temperature or age of the culture. RiCF was partially dependent on H-NS, histone-like nucleoid structuring- and global transcription regulator protein. The hupAB deletion of heat-unstable nucleoid protein (HU) caused increase in spontaneous fragmentation that was further increased when combined with deletions in two non-coding RNAs, nc1 and nc5. RiCF was completely dependent upon endonuclease I, a periplasmic deoxyribonuclease that is normally found inhibited by cellular RNA. Unlike RiCF, the spontaneous fragmentation in hupAB nc1 nc5 quadruple mutant was resistant to deletion of endonuclease I. RiCF-like phenomenon was observed without addition of RNase to agarose plugs if EDTA was significantly reduced during cell lysis. Addition of RNase under this condition was synergistic, breaking chromosomes into pieces too small to be retained by the pulsed field gels. RNase-independent fragmentation was qualitatively and quantitatively comparable to RiCF and was partially mediated by endonuclease I.http://europepmc.org/articles/PMC5739488?pdf=render
spellingShingle Sharik R Khan
Andrei Kuzminov
Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.
PLoS ONE
title Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.
title_full Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.
title_fullStr Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.
title_full_unstemmed Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.
title_short Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.
title_sort degradation of rna during lysis of escherichia coli cells in agarose plugs breaks the chromosome
url http://europepmc.org/articles/PMC5739488?pdf=render
work_keys_str_mv AT sharikrkhan degradationofrnaduringlysisofescherichiacolicellsinagaroseplugsbreaksthechromosome
AT andreikuzminov degradationofrnaduringlysisofescherichiacolicellsinagaroseplugsbreaksthechromosome