Sinorhizobium fredii and Sinorhizobium meliloti produce structurally conserved lipopolysaccharides and strain-specific K antigens

Abstract

Lipopolysaccharides (LPS) and capsular polysaccharides (K antigens) may influence the interaction of rhizobia with their specific hosts; therefore, we conducted a comparative analysis of Sinorhizobium fredii and Sinorhizobium meliloti, which are genetically related, yet symbiotically distinct, nitrogen-fixing microsymbionts of legumes. We found that both species typically produce strain-specific K antigens that consist of 3-deoxy-D-manno-2-octulosonic acid (Kdo), or other 1-carboxy-2-keto-3-deoxy sugars (such as sialic acid), and hexoses. The K antigens of each strain are distinguished by glycosyl composition, anomeric configuration, acetylation, and molecular weight distribution. One consistent difference between the K antigens of S. fredii and those of S. meliloti is the presence of N-acetyl groups in the polysaccharides of the latter. In contrast to the K antigens, the LPS of Sinorhizobium spp. are major common antigens. Rough (R) LPS is the predominant form of LPS produced by cultured cells, and some strains release almost no detectable smooth (S) LPS upon extraction. Sinorhizobium spp. are delineated into two major RLPS core serogroups, which do not correspond to species (i.e., host range). The O antigens of the SLPS, when present, have similar degrees of polymerization and appear to be structurally conserved throughout the genus. Interestingly, one strain was found to be distinct from all others: S. fredii HH303 produces a unique K antigen, which contains galacturonic acid and rhamnose, and the RLPS did not fall into either of the RLPS core serogroups. The results of this study indicate that the conserved S- and RLPS of Sinorhizobium spp. lack the structural information necessary to influence host specificity, whereas the variable K antigens may affect strain-cultivar interactions.

FIG. 1

PAGE analysis of the K antigens. The gels were prestained with alcian blue, a cationic dye that binds the acidic polysaccharides, prior to silver staining. (A) Lanes: 1, S. fredii USDA205; 2, S. fredii USDA257; 3, S. fredii USDA201; 4, S. fredii USDA208; 5, Sinorhizobium sp. strain NGR234; 6, S. fredii HH103; 7, S. fredii HH303; 8, S. fredii USDA192; 9, S. fredii USDA191; 10, S. meliloti AK631. (B) Lanes: 1, S. meliloti NRG133; 2, S. meliloti NRG23; 3, S. meliloti NRG185; 4, S. meliloti NRG247; 5, S. meliloti NRG286; 6, S. meliloti NRG53; 7, S. meliloti AK631.

FIG. 2

¹H NMR spectra of the K antigens from S. meliloti NRG247 (A), S. meliloti NRG185 (B), S. fredii USDA208 (C), S. fredii USDA201 (D), Sinorhizobium sp. strain NGR234 (E), and S. fredii HH303 (F). The chemical shift of the ²HOH resonance (4.55 or 4.75 ppm) is temperature dependent. See the text for detailed descriptions.

FIG. 3

PAGE analysis of the LPS. The gels were silver stained; the omission of the alcian blue prestain precludes the staining of the K antigens (compare Fig. 1) and allows the LPS to be clearly visualized. (A) Lanes: 1, S. fredii USDA205; 2, S. fredii USDA257; 3, S. fredii USDA201; 4, S. fredii USDA208; 5, Sinorhizobium sp. strain NGR234; 6, S. fredii HH103; 7, S. fredii HH303; 8, S. fredii USDA192; 9, S. fredii USDA191; 10, S. meliloti AK631. (B) Lanes: 1, S. meliloti NRG133; 2, S. meliloti NRG23; 3, S. meliloti NRG185; 4, S. meliloti NRG247; 5, S. meliloti NRG286; 6, S. meliloti NRG53; 7, S. meliloti AK631 (included on both gels for comparison).

FIG. 4

Serotype analysis of the RLPS. The immunoblots were probed with polyclonal antisera raised against whole cells of S. meliloti Rm41 (anti-Rm41) (A) and S. fredii USDA205 (anti-Rf205) (B). Lanes: 1, S. fredii USDA205; 2, S. fredii USDA257; 3, S. fredii USDA201; 4, S. fredii USDA208; 5, Sinorhizobium sp. strain NGR234; 6, S. fredii HH103; 7, S. fredii HH303; 8, S. fredii USDA192; 9, S. fredii USDA191; 10, S. meliloti AK631. The dark areas in panel A, lanes 5 and 10, show cross-reaction of the anti-Rm41 with the Sinorhizobium sp. strain NGR234 and S. meliloti AK631 K antigens, both of which contain Pse (Table 4).

FIG. 5

HPAEC-PAD analysis of the LPS core oligosaccharides released from RLPS of S. meliloti Rm1021 (anti-Rm41 serogroup) (A) and S. fredii USDA205 (anti-Rf205 serogroup) (B) after mild acid hydrolysis. The core oligosaccharides elute between 24 and 45 min. Each strain produces serogroup-specific and common oligosaccharides. See the text for details.