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Originally published In Press as doi:10.1074/jbc.M204525200 on May 17, 2002

J. Biol. Chem., Vol. 277, Issue 32, 28959-28971, August 9, 2002
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Expression Cloning and Characterization of the C28 Acyltransferase of Lipid A Biosynthesis in Rhizobium leguminosarum*

Shib Sankar Basu, Mark J. KarbarzDagger , and Christian R. H. Raetz§

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

An unusual feature of lipid A from plant endosymbionts of the Rhizobiaceae family is the presence of a 27-hydroxyoctacosanoic acid (C28) moiety. An enzyme that incorporates this acyl chain is present in extracts of Rhizobium leguminosarum, Rhizobium etli, and Sinorhizobium meliloti but not Escherichia coli. The enzyme transfers 27-hydroxyoctacosanate from a specialized acyl carrier protein (AcpXL) to the precursor Kdo2 ((3-deoxy-D-manno-octulosonic acid)2)-lipid IVA. We now report the identification of five hybrid cosmids that direct the overexpression of this activity by screening ~4000 lysates of individual colonies of an R. leguminosarum 3841 genomic DNA library in the host strain S. meliloti 1021. In these heterologous constructs, both the C28 acyltransferase and C28-AcpXL are overproduced. Sequencing of a 9-kb insert from cosmid pSSB-1, which is also present in the other cosmids, shows that acpXL and the lipid A acyltransferase gene (lpxXL) are close to each other but not contiguous. Nine other open reading frames around lpxXL were also sequenced. Four of them encode orthologues of fatty acid and/or polyketide biosynthetic enzymes. AcpXL purified from S. meliloti expressing pSSB-1 is fully acylated, mainly with 27-hydroxyoctacosanoate. Expression of lpxXL in E. coli behind a T7 promoter results in overproduction in vitro of the expected R. leguminosarum acyltransferase, which is C28-AcpXL-dependent and utilizes (3-deoxy-D-manno-octulosonic acid)2-lipid IVA as the acceptor. These findings confirm that lpxXL is the structural gene for the C28 acyltransferase. LpxXL is distantly related to the lauroyltransferase (LpxL) of E. coli lipid A biosynthesis, but highly significant LpxXL orthologues are present in Agrobacterium tumefaciens, Brucella melitensis, and all sequenced strains of Rhizobium, consistent with the occurrence of long secondary acyl chains in the lipid A molecules of these organisms.

* This work was supported in part by National Institutes of Health Grant R37-GM-51796 (to C. R. H. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF510733.

Dagger Supported by National Institutes of Health Training Grant GM-08558.

§ To whom correspondence should be addressed: Dept. of Biochemistry, Duke University Medical Center, P. O. Box 3711, Durham, NC 27710. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz@biochem.duke.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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