The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP‐DnaA protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino‐termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.

The toxicity of a peptide derived from the amino-terminal portion of 33-kDa TrfA, one of the initiation proteins encoded by the broad-host-range plasmid RK2, was suppressed by a host protein related to DnaA, the initiation protein of Escherichia coli. The newly identified 28.4-kDa protein, termed a DnaA paralog (Dp) because it is similar to a region of DnaA but likely has a different function in initiation of plasmid RK2 replication, interacts physically with the 33-kDa TrfA initiation protein, including the initiation-active monomeric form. The Dp has a cellular distribution similar to that of the 33-kDa TrfA initiation protein, being found primarily in the inner membrane fraction, with lesser amounts detected in the outer membrane fraction and almost none in the soluble fraction of E. coli. Maintenance and inner membrane-associated replication of plasmid RK2 were enhanced in a Dp knockout strain and inhibited in strains containing extra copies of the Dp gene or in membrane extracts to which a tagged form of Dp was added. Recently, the Dp was independently shown to help prevent overinitiation in E. coli and was termed Hda (S. Kato and T. Katayama, EMBO J. 20:4253-4262, 2001).The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. Carrying resistance genes to three antibiotics (ampicillin, kanamycin, and tetracycline), it has been isolated from a number of different medical environments (4). Despite the diversity of host cells, only two plasmid-carried loci are necessary to initiate unidirectional replication, the cis-acting origin of replication (oriV) and the trans-acting initiator gene trfA, which codes for two proteins of 44 and 33 kDa, the latter of which is expressed from an internal translational start within trfA (35). Recently, specific binding of TrfA and host initiation protein DnaA of Escherichia coli to oriV was found to be required for initiation of plasmid replication, consistent with the presence of four DnaA boxes within the oriV sequence (15). It appears that the DnaA protein cannot by itself form an open complex in oriV but rather enhances formation of the complex by TrfA. Nevertheless, numerous experiments have failed to demonstrate a direct physical interaction between TrfA and the DnaA protein although a physical association between TrfA and the ClpX chaperone protein has been observed (17). The latter interaction is important in converting the dimer form of TrfA, which is inactive in initiating DNA replication, to the active monomer form (16,38).RK2 replication is associated with the inner membranes of a number of gram-negative hosts (1). The TrfA initiation proteins were detected in both inner and outer membrane fractions of the hosts (1, 25), but further studies of E. coli revealed that oriV, the TrfA initiator proteins, and replication inhibited by specific anti-TrfA antibody were enriched in a specific subdomain found in the inner membrane fraction, representing less than 10% of the total membrane (13, 23)....