Identification of a Novel Membrane-Associated Gene Product That Suppresses Toxicity of a TrfA Peptide from Plasmid RK2 and Its Relationship to the DnaA Host Initiation Protein

Abstract

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)....

Interaction of the Sliding Clamp β-Subunit and Hda, a DnaA-Related Protein

“…Hda protein is one-half the size of DnaA and has high-level sequence homology to the domain III region of DnaA, which contains the ATP binding site (22). Hda also has a role in plasmid initiation, as it is found to interact with initiation protein TrfA of the broad-host-range plasmid RK2 (23).…”

Overexpression of the Hda DnaA-Related Protein in Escherichia coli Inhibits Multiplication, Affects Membrane Permeability, and Induces the SOS Response

“…By interacting physically with the plasmid-encoded initiation protein (TrfA), it acted as a steric inhibitor of either or both of TrfA's two functions: cooperating with the DnaA protein (which is also required by RK2) to open the replication bubble and guiding the DnaB-DnaC complex into the open site (8,9,10). The protein is identical to the Hda protein ("homologous to DnaA" protein) that is responsible for controlling overinitiation in E. coli by accelerating the ability of the ␤-clamp subunit of DNA polymerase III to convert the active form of DnaA (ATP-bound DnaA) to its inactive form (ADP-bound DnaA) (1,7,11).…”

“…We recently identified a small membrane-associated protein (28.4 kDa) in Escherichia coli that is related to the DnaA host initiation protein and that affected the initiation of the broadhost-range plasmid RK2 (8). By interacting physically with the plasmid-encoded initiation protein (TrfA), it acted as a steric inhibitor of either or both of TrfA's two functions: cooperating with the DnaA protein (which is also required by RK2) to open the replication bubble and guiding the DnaB-DnaC complex into the open site (8,9,10).…”

“…The protein is identical to the Hda protein ("homologous to DnaA" protein) that is responsible for controlling overinitiation in E. coli by accelerating the ability of the ␤-clamp subunit of DNA polymerase III to convert the active form of DnaA (ATP-bound DnaA) to its inactive form (ADP-bound DnaA) (1, 7, 11). Hda has a high sequence homology to the domain III ATPase region of DnaA (1,7,8,13) and is important as an accessory component for initiation and, subsequently, replication in prokaryotes.In further assessing the role of Hda protein in RK2 metabolism, we previously constructed a compatible plasmid that placed hda under the control of an inducible promoter and monitored the effects of increasing levels of Hda induction in vivo. Profound inhibitory effects on both maintenance and replication of RK2 were observed (8).…”

“…The protein is identical to the Hda protein ("homologous to DnaA" protein) that is responsible for controlling overinitiation in E. coli by accelerating the ability of the ␤-clamp subunit of DNA polymerase III to convert the active form of DnaA (ATP-bound DnaA) to its inactive form (ADP-bound DnaA) (1, 7, 11). Hda has a high sequence homology to the domain III ATPase region of DnaA (1,7,8,13) and is important as an accessory component for initiation and, subsequently, replication in prokaryotes.…”