Activity of Sinorhizobium meliloti NodAB and NodH enzymes on thiochitooligosaccharides

Abstract

Rhizobium bacteria synthesize signal molecules called Nod factors that elicit responses in the legume root during nodulation. Nod factors, modified N-acylated beta-(1,4)-N-acetylglucosamine, are synthesized by the nodulation (nod) gene products. We tested the ability of three Sinorhizobium meliloti nod gene products to modify Nod factor analogs with thio linkages instead of O-glycosidic bonds in the oligosaccharide backbone.

Figures

FIG. 1.

(A) Structure of the major S. meliloti Nod factor and the biosynthetic role of specific nod genes. The Nod factor β-(1,4)-N-acetylglucosamine oligosaccharide backbone is synthesized by NodC. NodB deacetylates the nonreducing-end glucosamine to accept the fatty acyl group from the acyltransferase NodA. NodH is a sulfotransferase which catalyzes the sulfation of the reducing-end C-6 position. (B to D) Structures of N-acetyl chitotetraose (B); N-acetyl thiochitobiose (n = 0), thiochitotriose (n = 1), and thiochitotetraose (n = 2) (C); and the α- and β-methyl glycosides of N-acetyl thiochitooligosaccharides (D).

FIG. 2.

NodH sulfation activity on reducing thiooligosaccharides. Chitooligosaccharides and thiochitooligosaccharides were incubated with NodH and [35S]PAPS. The reaction products were analyzed by TLC on PEI-cellulose and by autoradiography and were identified as previously described (8). Lanes: 1, no substrate; 2, chitotetraose; 3 to 5, α-methyl glycosides (3, thiochitobiose; 4, thiochitotriose; 5, thiochitotetraose); 6 to 8, reducing thio-oligosaccharides (6, thiochitobiose; 7, thiochitotriose; 8, thiochitotetraose).

FIG. 3.

The α- and β-methyl glycosides of thiochitotriose are not substrates for NodH. The oligosaccharides were incubated with NodH and [35S]PAPS. The reaction products were analyzed by TLC on PEI-cellulose and by autoradiography. Lanes: 1, no substrate; 2, thiochitotriose α-methyl glycoside; 3, thiochitotriose β-methyl glycoside; 4, thiochitotetraose; 5, chitotetraose.

FIG. 4.

NodH sulfation activity on chitotriose is not inhibited by thiochitotriose methyl glycosides. Chitotriose and thiochitotriose methyl glycosides were incubated with NodH and [35S]PAPS. The reaction products were analyzed by TLC on PEI-cellulose. Lanes: 1, thiochitotriose β-methyl glycoside (β-MTG3); 2, thiochitotriose α-methyl glycoside (α-MTG3); 3, chitotriose (C3); 4, β-MTG3:C3 (1:1); 5, α-MTG3:C3 (1:1); 6, β-MTG3:C3 (10:1); 7, α-MTG3:C3 (10:1); 8, no substrate.

FIG. 5.

Acylation of thiochitotetraose. Acylation of 35S-labeled chitotetraose or thiochitooligosaccharides was assayed using permeabilized S. meliloti cells. The S. meliloti cells used were from a nodC::Tn5 mutant (TJ170/pE65) and the common nod gene deletion strain SL44. The reaction products were extracted and analyzed on Silica-Gel 60 HPTLC plates. Multiple bands near the origin may represent variable anomeric and/or acetylated substrate forms (see the text). The TJ170 extracts assayed included thiochitobiose (lane 1), thiochitotriose (lane 2), thiochitotetraose (lane 3), and chitotetraose (lane 4). The SL44 extracts assayed included thiochitobiose (lane 5), thiochitotriose (lane 6), thiochitotetraose (lane 7), and chitotetraose (lane 8). The 35S-labeled Nod factor (indicated by asterisk) is also shown (lane 9).