Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC. Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants

Abstract

The lpcC gene of Rhizobium leguminosarum and the lpsB gene of Sinorhizobium meliloti encode protein orthologs that are 58% identical over their entire lengths of about 350 amino acid residues. LpcC and LpsB are required for symbiosis with pea and Medicago plants, respectively. S. meliloti lpsB complements a mutant of R. leguminosarum defective in lpcC, but the converse does not occur. LpcC encodes a highly selective mannosyl transferase that utilizes GDP-mannose to glycosylate the inner 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the lipopolysaccharide precursor Kdo(2)-lipid IV(A). We now demonstrate that LpsB can also efficiently mannosylate the same acceptor substrate as does LpcC. Unexpectedly, however, the sugar nucleotide selectivity of LpsB is greatly relaxed compared with that of LpcC. Membranes of the wild-type S. meliloti strain 2011 catalyze the glycosylation of Kdo(2)-[4'-(32)P]lipid IV(A) at comparable rates using a diverse set of sugar nucleotides, including GDP-mannose, ADP-mannose, UDP-glucose, and ADP-glucose. This complex pattern of glycosylation is due entirely to LpsB, since membranes of the S. meliloti lpsB mutant 6963 do not glycosylate Kdo(2)-[4'-(32)P]lipid IV(A) in the presence of any of these sugar nucleotides. Expression of lpsB in E. coli using a T7lac promoter-driven construct results in the appearance of similar multiple glycosyl transferase activities seen in S. meliloti 2011 membranes. Constructs expressing lpcC display only mannosyl transferase activity. We conclude that LpsB, despite its high degree of similarity to LpcC, is a much more versatile glycosyltransferase, probably accounting for the inability of lpcC to complement S. meliloti lpsB mutants. Our findings have important implications for the regulation of core glycosylation in S. meliloti and other bacteria containing LpcC orthologs.

Fig. 1. Sequence similarity comparison of R. leguminosarum LpcC and S. meliloti LpsB

The middle line indicates identities and similarities. The proteins show 58% identity and 72% similarity over 350 of the 352 amino acid residues in LpcC. The E value (28) determined against the nonredundant data base is ~10⁻¹⁰³.

Fig. 2. Distinct patterns of Kdo₂-lipid IV_A glycosylation with membranes of R. leguminosarum 3841 versus S. meliloti 1021

Reactions were carried out under standard conditions at pH 7.5 using a 1 mM concentration of each of the sugar nucleotides, as indicated. Membranes were used at 1 mg/ml. The incubation was carried out at 30 °C for 60 min.

Fig. 3. Glycosylation of Kdo₂-lipid IV_A catalyzed by membranes of S. meliloti 2011

Reactions were carried out under standard conditions at pH 7.5 using a 1 mM concentration of each of the sugar nucleotides, as indicated. Membranes were used at 1 mg/ml. The incubation was carried out at 30 °C for 60 min.

Fig. 4. Restoration of Kdo₂-lipid IV_A mannosylation in membranes of lpsB mutant 6963 expressing either R. leguminosarum lpcC or S. meliloti lpsB.

Reactions were carried out under standard conditions at pH 7.5 using 1 mM GDP-mannose as the sugar donor. Membranes were used at 1 mg/ml. The incubation was carried out at 30 °C for 60 min. Lane 1, no enzyme; lane 2, S. meliloti 2011; lane 3, S. meliloti 6963; lane 4, S. meliloti 6963/pJBlpsSme (lpsB); lane 5, S. meliloti 6963/pPN120 (lpcC).

Fig. 5. Restoration of other Kdo₂-lipid IV_A glycosylation reactions in membranes of lpsB mutant 6963 expressing S. meliloti lpsB.

Reactions were carried out under standard conditions at pH 7.5 using a 1 mM concentration of each of the sugar nucleotides, as indicated. Membranes were used at 1 mg/ml. The incubation was carried out at 30 °C for 60 min.

Fig. 6. No Kdo₂-lipid IV_A glycosylation with sugars other than mannose in membranes of lpsB mutant 6963 expressing R. leguminosarum lpcC

Reactions were carried out under standard conditions at pH 7.5 using a 1 mM concentration of each of the sugar nucleotides, as indicated. Membranes were used as the enzyme source at 1 mg/ml. The incubation was carried out at 30 °C for 60 min.

Fig. 7. Glycosylations of Kdo₂-lipid IV_A catalyzed by membranes of E. coli expressing lpsB behind the T7lac promoter

Reactions were carried out under standard conditions at pH 7.5 using a 1 mM concentration of each of the sugar nucleotides, as indicated. Enzyme was used at 0.02 mg/ml. The incubation was carried out at 30 °C for 60 min. A, membranes of BLR(DE3)/pLysS/pET28b; B, membranes of BLR(DE3)/pLysS/pMKRM.

Fig. 8. Negative mode MALDI-TOF mass spectrum of LPS purified from mutant 6963 by chloroform/methanol extraction

The proposed LPS molecular species that could account for the major peaks observed in A is shown in B. This structure is consistent with the overall fatty acid composition recently reported for lipid A of Sinorhizobium sp. NGR234 (22).