Association of the chromosome replication initiator DnaA with the Escherichia coli inner membrane in vivo: quantity and mode of binding.

PloS One
Tomer RegevItzhak Fishov

Abstract

DnaA initiates chromosome replication in most known bacteria and its activity is controlled so that this event occurs only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and the resulting ADP is replaced with ATP on the verge of the next initiation. Two putative recycling mechanisms depend on the binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation. While there is no doubt that DnaA interacts with artificial membranes in vitro, it is still controversial as to whether it binds the cytoplasmic membrane in vivo. In this work we looked for DnaA-membrane interaction in E. coli cells by employing cell fractionation with both native and fluorescent DnaA hybrids. We show that about 10% of cellular DnaA is reproducibly membrane-associated. This small fraction might be physiologically significant and represent the free DnaA available for initiation, rather than the vast majority bound to the datA reservoir. Using the combination of mCherry with a variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the surface of the protein's domain III, rather than confined to a particular sequence. We propose a new...Continue Reading

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Citations

Jun 18, 2015·Frontiers in Microbiology·Alan C Leonard, Julia E Grimwade
Jul 1, 2015·Frontiers in Microbiology·Kouji MatsumotoVic Norris
Jan 1, 2012·Life·Vic Norris, Patrick Amar
Jul 17, 2012·Journal of Bacteriology·Geoffrey S BriggsPanos Soultanas
Nov 28, 2020·Research in Microbiology·Masamichi Kohiyama

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Methods Mentioned

BETA
optical microscopy
dissection
nucleotide exchange
PCR
flow cytometry
Fluorescence
electrophoresis

Software Mentioned

ImageJ
TASSER
Elements Basic
NIS
PyMol
PeakFit

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