Cloning and Overexpression of Glycosyltransferases That Generate the Lipopolysaccharide Core of Rhizobium leguminosarum

Kadrmas, Julie L.; Allaway, David; Studholme, Rachael E.; Sullivan, John T.; Ronson, Clive W.; Poole, Philip S.; Raetz, Christian R. H.

doi:10.1074/jbc.273.41.26432

Cited by 40 publications

(51 citation statements)

References 52 publications

(50 reference statements)

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“…Isolation and Preparation of Lipid Substrates-Lipid IV A and (Kdo) 2 -lipid IV A were isolated and purified as described (14,24,25 (25)(26)(27), except that membranes of the 4Ј-kinase overproducing strain BLR(DE3)pLysS/pJK2 (28) were used to increase the yield of [4Ј-32 P]lipid IV A . Lauroyl, myristoyl, palmitoyl, R-3-hydroxymyristoyl, and palmitoleoyl-ACP were synthesized from the corresponding fatty acids and commercial acyl carrier protein, using solubilized membranes from the acyl-ACP synthase overproducing strain E. coli LCH109/ pLCH5/pGP1-2, as described previously (10,29).…”

Section: Materials-[␥-mentioning

confidence: 99%

Effect of Cold Shock on Lipid A Biosynthesis inEscherichia coli

Carty¹,

Sreekumar²,

Raetz³

1999

Journal of Biological Chemistry

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153

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Palmitoleate is not present in lipid A isolated fromEscherichia coli grown at 30°C or higher, but it comprises ϳ11% of the fatty acyl chains of lipid A in cells grown at 12°C. The appearance of palmitoleate at 12°C is accompanied by a decline in laurate from ϳ18% to ϳ5.5%. We now report that wild-type E. coli shifted from 30°C to 12°C acquire a novel palmitoleoyl-acyl carrier protein (ACP)-dependent acyltransferase that acts on the key lipid A precursor Kdo 2 -lipid IV A . The palmitoleoyl transferase is induced more than 30-fold upon cold shock, as judged by assaying extracts of cells shifted to 12°C. The induced activity is maximal after 2 h of cold shock, and then gradually declines but does not disappear. Strains harboring an insertion mutation in the lpxL(htrB) gene, which encodes the enzyme that normally transfers laurate from lauroyl-ACP to Kdo 2 -lipid IV A (Clementz, T., Bednarski, J. J., and Raetz, C. R. H. (1996) J. Biol. Chem. 271, 12095-12102) are not defective in the cold-induced palmitoleoyl transferase. Recently, a gene displaying 54% identity and 73% similarity at the protein level to lpxL was found in the genome of E. coli. This lpxL homologue, designated lpxP, encodes the cold shock-induced palmitoleoyl transferase. Extracts of cells containing lpxP on the multicopy plasmid pSK57 exhibit a 10-fold increase in the specific activity of the cold-induced palmitoleoyl transferase compared with cells lacking the plasmid. The elevated specific activity of the palmitoleoyl transferase under conditions of cold shock is attributed to greatly increased levels of lpxP mRNA. The replacement of laurate with palmitoleate in lipid A may reflect the desirability of maintaining the optimal outer membrane fluidity at 12°C.

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Section: Materials-[␥-mentioning

confidence: 99%

Effect of Cold Shock on Lipid A Biosynthesis inEscherichia coli

Carty¹,

Sreekumar²,

Raetz³

1999

Journal of Biological Chemistry

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153

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“…3). There was no dependence of activity on the addition of cytosolic components (data not shown), in contrast to the behavior of the R. leguminosarum core glycosyltransferases LpcA, LpcB, and LpcC, which are membranebound enzymes requiring cytosolic sugar nucleotide donors (15).…”

Section: Subcellular Localization and Substrate Preference Of The Putmentioning

confidence: 55%

“…5). Unlike the membrane-bound R. leguminosarum core glycosyltransferases LpcA, LpcB, and LpcC, which require a cytosolic sugar nucleotide donor (15), the proposed GalA transferases require only membrane components (Fig. 3).…”

Section: Discussionmentioning

confidence: 99%

“…Order of possible enzymatic reactions for the attachment of GalA moieties to the R. leguminosarum LPS core. The enzymes responsible for the 1-dephosphorylation of lipid A and for the incorporation of the mannose have been reported elsewhere (15,22). RgtA and RgtB are proposed to add the two GalA units to the outer Kdo.…”

Section: Discussionmentioning

confidence: 99%

“…The supplementary components added for mannosylation were 50 mM HEPES, pH 7.5, 0.1% Triton X-100, 1 mM GDP-mannose, and 0.3-0.5 mg/ml pIJ1848/S. meliloti 1021 membranes (15,40) in a total volume of 20 l, making the final reaction volume 120 l. The reaction was allowed to proceed at 30°C for 30 min. This was followed by the addition of another 0.3-0.5 mg/ml membranes with 30 min of incubation at 30°C.…”

Section: Chemicals and Materials-[␥-mentioning

confidence: 99%

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Expression Cloning of Three Rhizobium leguminosarum Lipopolysaccharide Core Galacturonosyltransferases

Kanjilal-Kolar¹,

Basu²,

Kanipes³

et al. 2006

Journal of Biological Chemistry

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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, Kdo 2 -[4- 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.

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