Lipopolysaccharide Biosynthesis in Rhizobium leguminosarum

Self Cite

The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizobium leguminosarum is more amenable to enzymatic study than that of Escherichia coli because much of it is synthesized from readily available sugar nucleotides. The inner portion of the R. leguminosarum core contains mannose, galactose, and three We have also discovered the new gene (lpcC) that encodes the mannosyltransferase. The gene is separated by several kilobase pairs from the lpcAB cluster. All three glycosyltransferases are carried on cosmid pIJ1848, which contains at least 20 kilobase pairs of R. leguminosarum DNA. Transfer of pIJ1848 into R. meliloti 1021 results in heterologous expression of all three enzymes, which are not normally present in strain 1021. Expression of the lpc genes individually behind the T7 promoter results in the production of each R. leguminosarum glycosyltransferase in E. coli membranes in a catalytically active form, demonstrating that lpcA, lpcB, and lpcC are structural genes.

supporting

confidence: 86%

Section: Sequential Addition Of Mannose Galactose and Kdo To The Acmentioning

confidence: 71%

Section: Sequential Addition Of Mannose Galactose and Kdo To The Acmentioning

confidence: 99%

See 1 more Smart Citation

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

Kadrmas¹,

Allaway²,

Studholme³

et al. 1998

Self Cite

“…The R. leguminosarum lpxE structural gene encoding the 1-phosphatase has recently 2 M. Karbarz and C. R. H. Raetz, manuscript in preparation. (43,44,(55)(56)(57). The only exceptions are the reactions that incorporate the 4Ј-galacturonic acid and the ␤-hydroxybutyrate residues.…”

Section: Discussionmentioning

confidence: 99%

Origin of the 2-Amino-2-deoxy-gluconate Unit inRhizobium leguminosarum Lipid A

Que-Gewirth

Karbarz

Kalb

et al. 2003

An unusual feature of the lipid A from the plant endosymbionts Rhizobium etli and Rhizobium leguminosarum is the presence of a proximal sugar unit consisting of a 2-amino-2-deoxy-gluconate moiety in place of glucosamine. An outer membrane oxidase that generates the 2-amino-2-deoxy-gluconate unit from a glucosaminecontaining precursor is present in membranes of R. leguminosarum and R. etli but not in S. meliloti or Escherichia coli. We now report the identification of a hybrid cosmid that directs the overexpression of this activity by screening 1800 lysates of individual colonies of a R. leguminosarum 3841 genomic DNA library in the host strain R. etli CE3. Two cosmids (p1S11D and p1U12G) were identified in this manner and transferred into S. meliloti, in which they also directed the expression of oxidase activity in the absence of any chromosomal background. Subcloning and sequencing of the oxidase gene on a 6.5-kb fragment derived from the ϳ20-kb insert in p1S11D revealed that the enzyme is encoded by a gene (lpxQ) that specifies a protein of 224 amino acid residues with a putative signal sequence cleavage site at position 28. Heterologous expression of lpxQ using the T7lac promoter system in E. coli resulted in the production of catalytically active oxidase that was localized in the outer membrane. A new outer membrane protein of the size expected for LpxQ was present in this construct and was subjected to microsequencing to confirm its identity and the site of signal peptide cleavage. LpxQ expressed in E. coli generates the same products as seen in R. leguminosarum membranes. LpxQ is dependent on O 2 for activity, as demonstrated by inhibition of the reaction under strictly anaerobic conditions. An ortholog of LpxQ is present in the genome of Agrobacterium tumefaciens, as shown by heterologous expression of oxidase activity in E. coli.As demonstrated in the accompanying article (1), the outer membranes of Rhizobium leguminosarum and Rhizobium etli contain an unusual oxidase that converts the proximal glucosamine unit of 1-dephosphorylated lipid A (or related molecules) to a novel 2-aminogluconate moiety. The membranes of Sinorhizobium meliloti and Escherichia coli do not normally contain such an oxidase activity. Although the function of this unusual covalent modification of lipid A is unknown (2-4), the existence of an oxidative enzyme in the outer membrane of a Gram-negative bacterium is without precedent. The few outer membrane enzymes described to date are all phospholipases (5, 6), acyltransferases (7, 8), or proteases (9). Other characterized outer membrane proteins function either as porins or specialized transporters (10 -12). The presence of the oxidase in outer membranes of certain strains of Rhizobium suggests that lipid A oxidation (1), when it occurs, is a late event in lipopolysaccharide assembly.Given the considerable progress that has recently been made with the structural biology of outer membrane proteins by x-ray crystallography (12) and NMR spectroscopy (8, 13) and the great interest in li...

“…The presence of this common lipid A precursor also implied that these rhizobial cells possess unique enzyme activities, which process their Kdo 2 lipid-IVa analog into the mature rhizobial lipid A structure. Several of these enzyme activities have been reported, including a membranebound phosphatase that removes the 4Ј-phosphate from Kdo 2 lipid-IVa and requires the presence of the Kdo residues for maximum activity (21), a second phosphatase that removes the 1-phosphate (22), and a unique acyl carrier protein required for the transfer of the 27-hydroxyoctacosanoyl residue to the lipid A (23). In addition, a rhizobial-specific mannosyl transferase activity has been identified that transfers mannose from GDPMan to Kdo 2 lipid-IVa (24).…”

mentioning

confidence: 99%

The Structures of the Lipopolysaccharides from Rhizobium etli Strains CE358 and CE359

Forsberg

Carlson

1998

The structural arrangement of oligosaccharides comprising the core region of Rhizobium etli CE3 lipopolysaccharide (LPS) has been elucidated through the characterization of the LPSs from two R. etli mutants. One mutant, CE358, completely lacks the O-chain polysaccharide, while the second mutant, CE359, contains a truncated portion of this polysaccharide. This structural arrangement of the core oligosaccharides in these LPSs was determined using electrospray ionization mass spectrometry, tandem mass spectrometry, and methylation analysis. Mild acid hydrolysis of the CE359 LPS produces two major core oligosaccharides: a tetrasaccharide (1) with the structure ␣-D-Galp- (136) Bacteria belonging to the family Rhizobiaceae are Gram-negative and are able to form nitrogen-fixing symbiotic relationships with legume plants. The surface polysaccharides, including the lipopolysaccharides (LPSs), 1 are involved in the normal infection process. Mutants that lack the O-chain polysaccharide portion of their LPSs are symbiotically defective in that they are unable to form normal infection threads (1, 2), and/or they cannot invade the root nodule cells (3-5). In addition, it has been shown that structural changes in the LPS occur during symbiotic infection and that most of these changes appear to take place in the O-chain polysaccharide portion of the LPS (6 -14). These structural adaptations in response to the environment of the host plant are likely to be important in order for the symbiont bacterium to induce a nitrogen-fixing nodule.In addition to the importance of determining the symbiotic "virulence" of these bacteria, rhizobial LPSs can have unique structural features compared with the LPSs from enteric bacteria. The most studied LPSs, the topic of this report, are those from Rhizobium etli and Rhizobium leguminosarum. All of the LPSs examined from wild-type or parent strains of these species are devoid of phosphate. Their lipid A region has a trisaccharide backbone consisting of one each of galacturonosyl (GalA), GlcN, and 2-aminogluconosyl (GlcN-onate) residues, the latter two residues being O-and N-acylated with ␤-hydroxy fatty acids and one very long chain fatty acid, 27-hydroxyoctacosanoic acid (15). This lipid A also appears not to carry any acyloxylacyl substituents. The core region has been found to consist of two oligosaccharides (16, 17) as shown below.