Restoration of Growth to Acidic Phospholipid-deficient Cells by DnaA(L366K) Is Independent of Its Capacity for Nucleotide Binding and Exchange and Requires DnaA

Abstract

In the absence of adequate levels of cellular acidic phospholipids, Escherichia coli remain viable but are arrested for growth. Expression of a DnaA protein that contains a single amino acid substitution in the membrane-binding domain, DnaA(L366K), in concert with expression of wild-type DnaA protein, restores growth. DnaA protein has high affinity for ATP and ADP, and in vitro lipid bilayers that are fluid and contain acidic phospholipids reactivate inert ADP-DnaA by promoting an exchange of ATP for ADP. Here, nucleotide and lipid interactions and replication activity of purified DnaA(L366K) were examined to gain insight into the mechanism of how it restores growth to cells lacking acidic phospholipids. DnaA(L366K) behaved like wildtype DnaA with respect to nucleotide binding affinities and hydrolysis properties, specificity of acidic phospholipids for nucleotide release, and origin binding. Yet, DnaA(L366K) was feeble at initiating replication from oriC unless augmented with a limiting quantity of wildtype DnaA, reflecting the in vivo requirement that both wild-type and a mutant form of DnaA must be expressed and act together to restore growth to acidic phospholipid deficient cells.

Remodeling of Nucleoprotein Complexes Is Independent of the Nucleotide State of a Mutant AAA+ Protein

“…Expression and Purification of Recombinant Proteins-Recombinant wild-type DnaA and DnaA(L366K) proteins were expressed and purified as described previously (30,37). Protein concentrations were determined according to Bradford (38).…”

“…The stoichiometry of DnaA bound to oriC is still debatable, varying from as low as five DnaA molecules per origin as detected by electro-phoretic mobility shift assays (31) and gel filtration (32), to 15 when measured by filter retention (10), to even 20 -30 as suggested by electron microscopy (8,34). Filter retention data (30) suggest that DnaA and DnaA(L366K) bind oriC comparably.…”

“…However, growth can be restored by overexpressing DnaA proteins that contain certain mutations within the DNA-binding or membrane-binding domains, including DnaA(L366K) (29). Although DnaA(L366K) as the sole form of DnaA is unable to initiate DNA synthesis in vivo (29) or in vitro (30), purified DnaA(L366K) is similar to wild-type DnaA for nucleotide binding affinities, ATP hydrolysis, and specificity for acidic phospholipids promoting nucleotide exchange (30). The stoichiometry of DnaA bound to oriC is still debatable, varying from as low as five DnaA molecules per origin as detected by electro-phoretic mobility shift assays (31) and gel filtration (32), to 15 when measured by filter retention (10), to even 20 -30 as suggested by electron microscopy (8,34).…”

Functional Taxonomy of Bacterial Hyperstructures

“…The result is an initiation hyperstructure that contains several different sorts of protein, RNA, and oriC. The involvement of the membrane in the initiation of replication has a long history (77), and it is tempting to speculate that the initiation hyperstructure may also contain cardiolipin, given that the conversion of DnaA-ADP into DnaA-ATP in vitro requires acidic phospholipids in the bilayer in a fluid state (28,42,144). (It is conceivable that more than one initiation hyperstructure may exist when minichromosomes are present [192].)…”

The initiator protein DnaA, chromosomal origin oriC and their interaction to generate origin recognition complex and pre-replication complex like nucleoprotein structures in Escherichia coli

“…Beside the specific arrangement of DnaA binding elements present within oriC, a mutation, DnaA(L366K) 46 , present in the membrane binding domain of DnaA protein (Figure 1) also prevents ORC to pre-RC conversion 32 . In-fact in vitro, an ADP-DnaA ORC can not support the loading of ATP-DnaA(L366K) to low affinity sites 32 .…”