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

Abstract

The Hda protein, a recently identified DnaA-related protein from Escherichia coli, is part of the AAA ϩ 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 initiator of the bacterial SOS response, which may represent a major new pathway for inducing DNA damage repair mechanismsWe 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). 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 determined 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-thiogalactoside)]-inducible T7 RNA polymerase gene (21), was transformed with plasmid pPK101 or the pET17B vector, as described by Hanahan (6), resulting in strain 1921 or 1110, respectively. pPK101 is a pET17B derivative (Novogen) that expresses a functional N-terminal T7 epitope-tagged Hda protein that was constructed previously (8). These two strains were grown to mid-logarithmic phase, induced with different IPTG concentrations, and viable cells were quantified. Whereas IPTG induction had a significant inhibitory effect on the viability of strain 1921 at all concentrations utilized (Fig. 1a), there was only a modest effect on the viability of the control 1110 strain (Fig. 1b). Of note, there was a threshold level of inhibition of cell viability ...

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