Association of the Chromosome Replication Initiator DnaA with the Escherichia coli Inner Membrane In Vivo: Quantity and Mode of Binding

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 binding-bending mechanism to explain the membrane-induced nucleotide release from DnaA. This mechanism would be fundamental to the initiation of replication.

Crosstalk between DnaA Protein, the Initiator of Escherichia coli Chromosomal Replication, and Acidic Phospholipids Present in Bacterial Membranes

“…Moreover, problems with photobleaching prevented visualization of the DnaA-EYFP protein by confocal microscopy [124]. A plasmid-born mcherry-tagged DnaA protein did not reveal helical structures, but that is not surprising given the high level of overexpression of mcherry-DnaA [125]. Interestingly, the overall global helical pattern formed by internally-tagged GFP-DnaA protein remains unaltered in the bacteria containing significantly reduced levels of acidic phospholipids (unpublished results), suggesting that localization of DnaA protein is not influenced by CL domains within the cell.…”

Chromosomal Replication Initiation Machinery of Low-G+C-Content Firmicutes

“…In E. coli, DnaA is thought to bind directly to membranes (18,54), although membrane association of oriC is mediated primarily via SeqA and SeqB (60). In contrast, membrane association in B. subtilis is dependent on DnaB (23,66,74), and there is no evidence for a direct association between DnaA and the membrane.…”

Centrifugal sedimentation immunoassays for multiplexed detection of enteric bacteria in ground water

“…No significant photobleaching was observed throughout our experiments due to relatively photostable probes, large working distance (7.5 mm), and illumination of samples through 10Â (low-power) objective. [25][26][27][28] The exposure of sample to the light was minimized to 1-2 s during focusing. An area of 400 000 pixels (0.33 mm 2 , approximately 1/3 of the total bead area) was designated as "reading area" to record the mean intensity of each individual sample.…”