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J. Biol. Chem., Vol. 280, Issue 11, 9796-9801, March 18, 2005

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

Zhenya Li, Jennifer L. Kitchen, Kelly Boeneman¶, Priyanka Anand, and Elliott Crooke||

From the Department of Biochemistry and Molecular Biology and Program in Tumor Biology, Georgetown University Medical Center, Washington, D. C. 20007

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 wild-type 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 wild-type 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.

Received for publication, December 10, 2004

^* This work was supported in part by National Institutes of Health Grant R01GM49700 (to E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported in part by National Institutes of Health Predoctoral Training Grant T32CA009686 in tumor biology.

^|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd. NW, Washington, D. C. 20007. Tel.: 202-687-1644; Fax: 202-687-7186; E-mail: crooke{at}georgetown.edu.

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