HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 18, 12865-12878, May 5, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/18/12865    most recent M513864200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kanjilal-Kolar, S. Articles by Raetz, C. R. H. Search for Related Content PubMed PubMed Citation Articles by Kanjilal-Kolar, S. Articles by Raetz, C. R. H. Social Bookmarking                      What's this?

Expression Cloning of Three Rhizobium leguminosarum Lipopolysaccharide Core Galacturonosyltransferases^*

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

The lipid A and core regions of the lipopolysaccharide in Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, are strikingly different from those of Escherichia coli. In R. leguminosarum lipopolysaccharide, the inner core is modified with three galacturonic acid (GalA) moieties, two on the distal 3-deoxy-D-manno-octulosonic acid (Kdo) unit and one on the mannose residue. Here we describe the expression cloning of three novel GalA transferases from a 22-kb R. leguminosarum genomic DNA insert-containing cosmid (pSGAT). Two of these enzymes modify the substrate, Kdo₂-[4'-³²P]lipid IV₂ and its 1-dephosphorylated derivative on the distal Kdo residue, as indicated by mild acid hydrolysis. The third enzyme modifies the mannose unit of the substrate mannosyl-Kdo₂-1-dephospho-[4'-³²P]lipid IV₂. Sequencing of a 7-kb subclone derived from pSGAT revealed three putative membrane-bound glycosyltransferases, now designated RgtA, RgtB, and RgtC. Transfer by tri-parental mating of these genes into Sinorhizobium meliloti 1021, a strain that lacks these particular GalA residues, results in the heterologous expression of the GalA transferase activities seen in membranes of cells expressing pSGAT. Reconstitution experiments with the individual genes demonstrated that the activity of RgtA precedes and is necessary for the subsequent activity of RgtB, which is followed by the activity of RgtC. Electrospray ionization-tandem mass spectrometry and gas-liquid chromatography of the product generated in vitro by RgtA confirmed the presence of a GalA moiety. No in vitro activity was detected when RgtA was expressed in Escherichia coli unless Rhizobiaceae membranes were also included.

Received for publication, December 29, 2005 , and in revised form, February 22, 2006.

^* 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Supported by the LIPID MAPS Large Scale Collaborative Grant GM-069338 from the National Institutes of Health.

² To whom correspondence should be addressed: Duke University Medical Center, Durham, NC, 27710. Tel.: 919-684-5326; Fax: 919-684-8885; E-mail: raetz{at}biochem.duke.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				E. Mileykovskaya, A. C. Ryan, X. Mo, C.-C. Lin, K. I. Khalaf, W. Dowhan, and T. A. Garrett Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol J. Biol. Chem., January 30, 2009; 284(5): 2990 - 3000. [Abstract] [Full Text] [PDF]

				M. J. Karbarz, D. A. Six, and C. R. H. Raetz Purification and Characterization of the Lipid A 1-Phosphatase LpxE of Rhizobium leguminosarum J. Biol. Chem., January 2, 2009; 284(1): 414 - 425. [Abstract] [Full Text] [PDF]

				S. Kanjilal-Kolar and C. R. H. Raetz Dodecaprenyl Phosphate-Galacturonic Acid as a Donor Substrate for Lipopolysaccharide Core Glycosylation in Rhizobium leguminosarum J. Biol. Chem., May 5, 2006; 281(18): 12879 - 12887. [Abstract] [Full Text] [PDF]