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Exo-Oligosaccharides of Rhizobium sp. Strain NGR234 Are Required for Symbiosis with Various Legumes

Christian Staehelin,^1,2* Lennart S. Forsberg,³ Wim D'Haeze,³ Mu-Yun Gao,³ Russell W. Carlson,³ Zhi-Ping Xie,¹ Brett J. Pellock,⁴ Kathryn M. Jones,⁴ Graham C. Walker,⁴ Wolfgang R. Streit,^5, and William J. Broughton²

State Key Laboratory of Biocontrol, School of Life Sciences, SunYat-Sen (Zhongshan) University, Guangzhou 510275, China,¹ LBMPS, Université de Genève, Sciences III, 1211 Genève 4, Switzerland,² Complex Carbohydrate Research Center, The University of Georgia, Athens, Georgia 30602-4712,³ Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,⁴ Molekulare Enzymtechnologie, Universität Duisburg-Essen, D-47057 Duisburg, Germany⁵

Received 14 March 2006/ Accepted 9 June 2006

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with ß-1,3, ß-1,4, ß-1,6, -1,3, and -1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGRexoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGRexoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)₅(GlcA)₂PvGal. When used as inoculants, both the exo-deficient mutants and NGRexoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, 50 µg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-ß-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

Present address: Biozentrum Klein Flottbek, Universität Hamburg, D-22609 Hamburg, Germany.

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