Invisible Binding: DnaA, the Initiator of Chromosome Replication in Bacteria, Associates with the Escherichia Coli Inner Membrane In Vivo Academic Article uri icon

abstract

  • DnaA initiates chromosome replication in most known bacteria and its activity is controlled to execute only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and ADP is replaced back to ATP on the verge of next initiation. Thus DnaA acts as a molecular switch, in which the nucleotide recycling couples key processes in the cell. Two putative recycling mechanisms presume 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. We sought after DnaA-membrane interaction in E. coli cells employing fluorescent microscopy and cell fractionation with both native and fluorescent DnaA hybrids. A small (5-10%) but reliable portion of DnaA is indeed membrane associated, though invisible in fluorescent cell images. This small fraction is physiologically significant as representing the free DnaA available for initiation. Using combination of mCherry with variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the protein structure. Analysis of E. coli DnaA structure model reveals a hydrophobic continuity on the protein surface, supporting a concerted interaction of distant residues with the membrane, rather than by an individual amphiphilic helix. A binding-bending mechanism is suggested, explaining the membrane-induced nucleotide release from DnaA. We have suggested previously that the enigmatic ‘initiation mass’ phenomenon may result from a highly cooperative inter-conversion between two functional states of DnaA driven by its membrane surface occupancy (Aranovich et al., 2006, Aranovich et al., 2007). Our present results provide a strong basis for extrapolation of this phenomenon to in vivo situation.

publication date

  • January 1, 2012