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

Abstract

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...

Lipid A Modification Systems in Gram-Negative Bacteria

“…The involvement of a dodecaprenyl phosphate-linked galacturonic acid donor, instead of a sugar nucleotide, is likewise consistent with the addition of galacturonate residues to core-lipid A occurring on the outer surface of the inner membrane (Figure 15) (232,233), analogous to L-Ara4N in polymyxin-resistant E. coli (Figures 8 and 9) (48). PagL and LpxQ are recovered in the outer membrane when expressed in E. coli (49,230,231 The biological significance of the lipid A modification systems of Rhizobium has been evaluated by genetics. In each instance studied to date, the relevant enzymes were first identified by the development of in vitro biochemical assays, followed by the expression cloning of the corresponding structural genes (59, 225, 227-229, 231, 232).…”

“…The Rgt proteins require a polyisoprene donor as their cosubstrate, consistent with a periplasmic localization (232,233). PagL and LpxQ are known to be outer membrane proteins (47,231). The X-ray structure of PagL shows that its active site is oriented toward the outside (209).…”

“…The X-ray structure of PagL shows that its active site is oriented toward the outside (209). The orientation of LpxQ is unknown (231). PagP is not present in Rhizobium.…”

“…R. leguminosarum and R. etli also contain the phosphatases LpxE (58,229) and LpxF (59), which catalyze the removal of the 1-and 4 -phosphate groups respectively (Figure 14), as in Francisella. Following removal of the 1-phosphate moiety, LpxQ (230,231) can oxidize the proximal glucosamine of Rhizobium lipid A in the presence of O 2 to form an aminogluconate unit. Removal of the 4 -phosphate group by LpxF is necessary for the incorporation of the 4 -galacturonic acid moiety (Figure 14), which appears to involve a polyisoprene phosphate sugar donor (rather than a sugar nucleotide) (B.O.…”

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…14D) (58). (2-Amino-2-deoxygluconic acid is generated by an oxidase located in the outer membrane [521,522].) In the closely related R. etli, even the core region is devoid of phosphoryl substituents and the negative charges are provided by uronic acids and KDO (207).…”