Abstract
In the symbiosis formed between Mesorhizobium loti strain R7A and Lotus japonicus Gifu, rhizobial exopolysaccharide (EPS) plays an important role in infection thread formation. Mutants of strain R7A affected in early exopolysaccharide biosynthetic steps form nitrogen-fixing nodules on L. japonicus Gifu after a delay, whereas mutants affected in mid or late biosynthetic steps induce uninfected nodule primordia. Recently, it was shown that a plant receptor-like kinase, EPR3, binds low molecular mass exopolysaccharide from strain R7A to regulate bacterial passage through the plant's epidermal cell layer (Kawaharada, Y., Kelly, S., Nielsen, M. W., Hjuler, C. T., Gysel, K., Muszyński, A., Carlson, R. W., Thygesen, M. B., Sandal, N., Asmussen, M. H., Vinther, M., Andersen, S. U., Krusell, L., Thirup, S., Jensen, K. J., et al. (2015) Nature 523, 308–312). In this work, we define the structure of both high and low molecular mass exopolysaccharide from R7A. The low molecular mass exopolysaccharide produced by R7A is a monomer unit of the acetylated octasaccharide with the structure (2,3/3-OAc)β-d-RibfA-(1→4)-α-d-GlcpA-(1→4)-β-d-Glcp-(1→6)-(3OAc)β-d-Glcp-(1→6)-*[(2OAc)β-d-Glcp-(1→4)-(2/3OAc)β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-β-d-Galp]. We propose it is a biosynthetic constituent of high molecular mass EPS polymer. Every new repeating unit is attached via its reducing-end β-d-Galp to C-4 of the fourth glucose (asterisked above) of the octasaccharide, forming a branch. The O-acetylation occurs on the four glycosyl residues in a non-stoichiometric ratio, and each octasaccharide subunit is on average substituted with three O-acetyl groups. The availability of these structures will facilitate studies of EPR3 receptor binding of symbiotically compatible and incompatible EPS and the positive or negative consequences on infection by the M. loti exo mutants synthesizing such EPS variants.
Highlights
20946 JOURNAL OF BIOLOGICAL CHEMISTRY octasaccharide subunit is on average substituted with three Oacetyl groups
The bacteria entrapped in the curled root hairs form microcolonies and enter the host through infection threads that are formed by localized digestion of the plant cell wall within the curl, followed by inverted growth of the cell wall/plasma membrane down the length of the root hair (7, 8)
Exopolysaccharides (EPS)[4] in particular are required for the formation and development of infection threads in indeterminate symbioses, of which the best characterized are those that occur between Sinorhizobium meliloti and Medicago species and between Rhizobium leguminosarum and Vicia species
Summary
We have previously reported that GC/MS composition analysis of R7A EPS showed the presence of Glc, Gal, GlcpA, and RibfA (38). The PMAA linkage analysis with methylation done after carboxyl reduction indicated the presence of 6-linked Glcp, 4-linked Glcp, terminal RibfA, and 4-linked GlcpA in an approximate ratio of 2:3:1:1, with some terminal GlcpA and traces of a 3-linked Galp residue. The latter was detected in a smaller quantity and underwent partial degradation and modification in that it was converted to its furanose form, and in addition, the C1 of Gal was partially converted to a methyl glycoside during carboxyl esterification by methanolysis. The inter-residue connections between O-1 of RibfA and O-4 of GlcpA, as well as that from O-1 of GlcpA to O-4 of a -Glc residue
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