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

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Abstract
The Hda protein, a recently identified DnaA-related protein from Escherichia coli, is part of the AAA ATPase family known to be involved with various aspects of initiation of DNA replication in prokaryotes. We report here that overexpression of this membrane-associated protein inhibits multiplication, affects membrane permeability, and is also an unexpected ini- tiator of the bacterial SOS response, which may represent a major new pathway for inducing DNA damage repair mecha- nisms 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 broad- host-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 abil- ity 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 metab- olism, 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). Of additional interest, Hda overexpression also inhibited cell multiplication, with only a limited effect on optical density profiles. In this study, we investigated the basis for these inhibitory effects and deter- mined that they involve induction of the SOS response system that may be actuated by perturbation of membrane integrity and/or permeability. Effects of Hda on growth and viability of E. coli. BL21(DE3)/ pLysS, which contains an IPTG (isopropyl-(3-D-thiogalacto-