Structures of the lipopolysaccharides from Rhizobium leguminosarum RBL5523 and its UDP-glucose dehydrogenase mutant (exo5)

Abstract

Rhizobial lipopolysaccharide (LPS) is required to establish an effective symbiosis with its host plant. An exo5 mutant of Rhizobium leguminosarum RBL5523, strain RBL5808, is defective in UDP-glucose (Glc) dehydrogenase that converts UDP-Glc to UDP-glucuronic acid (GlcA). This mutant is unable to synthesize either UDP-GlcA or UDP-galacturonic acid (GalA) and is unable to synthesize extracellular and capsular polysaccharides, lacks GalA in its LPS and is defective in symbiosis (Laus MC, Logman TJ, van Brussel AAN, Carlson RW, Azadi P, Gao MY, Kijne JW. 2004. Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp. nigra. J Bacteriol. 186:6617-6625). Here, we determined and compared the structures of the RBL5523 parent and RBL5808 mutant LPSs. The parent LPS core oligosaccharide (OS), as with other R. leguminosarum and Rhizobium etli strains, is a Gal(1)Man(1)GalA(3)Kdo(3) octasaccharide in, which each of the GalA residues is terminally linked. The core OS from the mutant lacks all three GalA residues. Also, the parent lipid A consists of a fatty acylated GlcNGlcNonate or GlcNGlcN disaccharide that has a GalA residue at the 4'-position, typical of other R. leguminosarum and R. etli lipids A. The mutant lipid A lacks the 4'-GalA residue, and the proximal glycosyl residue was only present as GlcNonate. In spite of these alterations to the lipid A and core OSs, the mutant was still able to synthesize an LPS containing a normal O-chain polysaccharide (OPS), but at reduced levels. The structure of the OPS of the mutant LPS was identical to that of the parent and consists of an O-acetylated →4)-α-d-Glcp-(1→3)-α-d-QuipNAc-(1→ repeating unit.

Fig. 1.

Structures of the LPSs reported for R. etli CE3 (Forsberg et al. 2000) and R. leguminosarum biovar viciae 3841 (Forsberg and Carlson 2008). The core–lipid A portion of the LPS is identical in structure in these strains and the structure of each OPS is as shown.

Fig. 2.

DOC-PAGE analysis of R. leguminosarum LPSs extracted with hot phenol/water and purified by ultracentrifugation. Samples (2 µg each) were loaded onto gel and stained as follows. The gel was fixed in the absence (A) or presence (B) of Alcian blue, followed by silver staining. Lanes: R. leguminosarum biovar viciae 5523 LPS obtained from (1) the water layer and (2) the phenol layer; mutant 5808 LPS obtained from (3) the water layer and (4) the phenol layer.

Fig. 3.

DOC-PAGE analysis of the R. leguminosarum biovar viciae 5523 (top) and the mutant 5808 LPS (bottom) fractions eluting during S100 gel filtration in the presence of DOC buffer. HMW LPS, high-molecular weight LPS that contains the OPS; LMW LPS, low-molecular weight LPS that lacks or may contain truncated OPS.

Fig. 4.

Comparative DIONEX HPAEC analysis of the OSs released by mild acid hydrolysis of the LMW LPS from R. etli CE3 (A), R. leguminosarum biovar viciae 5523 (B, solid line) and the mutant 5808 (B, dashed line). The peaks are: 1, Kdo; 2, GalA; 3, tetrasaccharide; 4 and 5, anhydroKdo versions of the Gal(GalA)ManKdo tetrasaccharide; 6, GalA(GalA)Kdo trisaccharide; 7, GalManKdo trisaccharide as reported by Carlson et al. (1989); 8, possibly an anhydroKdo version of 7.

Fig. 5.

MALDI-TOF-MS analysis of lipid A isolated from the R. leguminosarum biovar viciae 5523 LPS (A) and the 5808 mutant (B). Each lipid A shows two clusters of ions. The smaller molecular weight cluster differs from the larger one due to the lack of a β-ΟHC14:0 fatty acyl residue. The ion masses for the mutant 5808 lipid A are less than those of the parent 5523 lipid A due to the lack of a GalA residue. Proposed compositions for the ions observed are given in Table II.

Fig. 6.

The proposed structures of the major ions observed in MALDI-TOF-MS analysis of the lipid A from the R. leguminosarum biovar viciae 5523 (structures A–D) and its exo5 mutant, strain 5808 (structures E and F). Proposed compositions of all the ionic species are given in Table II.

Fig. 7.

MALDI-TOF-MS analysis of de-O-acetylated OPSs isolated from the R. leguminosarum biovar viciae 5523 (A) and the 5808 mutant (B). Spectra were acquired in a negative mode.

Fig. 8.

The proton spectra for the OPS from (A) the R. leguminosarum biovar viciae RBL5523 and (B) its RBL5808 mutant.

Fig. 9.

The TOCSY (A) and NOESY (B) spectra for the OPS from the R. leguminosarum biovar viciae 5523. The assignments are as indicated. The OPS from the 5808 mutant gave an identical spectrum. The proton chemical shift assignments are shown in Table III.

Fig. 10.

The HSQC spectrum for the OPS from the R. leguminosarum biovar viciae 5523. The OPS from the 5808 mutant gave an identical spectrum. The carbon chemical shift assignments are as indicated and are listed in Table III.

Fig. 11.

The structures of the LPS from the R. leguminosarum bv. viciae RBL5523 and its mutant 5808. The OPS for both RBL5523 and 5808 have the same structure as shown. The glycosyl residue that attached the OPS to Kdo III of the core region has not been determined but, based on the R. etli CE3 structure shown in Figure 1, is hypothesized to be the QuiNAc residue. The OPS is also highly O-acetylated (the location of the O-acetyl groups has not been determined). The LPS from the mutant 5808 differs from that of 5523 in that it is devoid of all GalA residues, that is, the circled residues in the structures shown.