Note Structure of the O-polysaccharide of Azorhizobium caulinodans HAMBI 216; identification of 3-C-methyl-D-rhamnose as a component of bacterial polysaccharides Evelina L. Zdorovenko a,⇑ , Olga A. Valueva a , Vadim V. Kachala a , Alexander S. Shashkov a , Yuriy A. Knirel a , Iwona Komaniecka b , Adam Choma b a N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 119991 Moscow, Russia b Maria Curie-Sklodowska University, Department of Genetics and Microbiology, 20-033 Lublin, Poland article info Article history: Received 29 May 2012 Accepted 19 June 2012 Available online 29 June 2012 Keywords: Azorhizobium caulinodans Bacterial polysaccharide structure Lipopolysaccharide 3-C-Methyl-D-rhamnose 2-O-Methyl-D-rhamnose abstract The O-polysaccharide was obtained from the lipopolysaccharide of the stem-nodulating nitrogen-fixing bacterium Azorhizobium caulinodans HAMBI 216 and studied by sugar and methylation analyses along with 1 H and 13 C NMR spectroscopy. The polysaccharide was found to have a linear pentasaccharide repeating unit containing D-rhamnose and its rarely occurring 2-O-methyl (Rha2OMe) and 3-C-methyl (Rha3CMe) derivatives and having the following structure: ?3)-a-D-Rhap2OMe-(1?2)-b-D-Rhap3CMe-(1?3)-a-D-Rhap-(1?2)-b-D-Rhap3CMe-(1?3)-a-D-Rhap-(1? Ó 2012 Elsevier Ltd. All rights reserved. As a result of the symbiotic interaction, (azo)rhizobia provide an easily available nitrogen source to the plant in exchange for access to the plants photosynthesis products. Two species, Azorhizobium caulinodans and Azorhizobium doebereinerae, have been described in the Azorhizobium genus. 1,2 A. caulinodans is a microsymbiont of the tropical legume Sesbania rostrata—a water-tolerant plant. 1 The bacteria can induce nitrogen-fixing nodules both on stems and roots of the host plant. 3 Carbohydrate polymers, such as lipo- polysaccharides (LPS), capsular polysaccharides (CPS), and exo- polysaccharides (EPS), play an important role during the infection process. 4 LPS is an integral component of Gram-negative bacterial cell walls. It consists of three domains: lipid A, a glycolipid that anchors the LPS molecule in bacterial outer membrane, a core oli- gosaccharide, and an O-specific polysaccharide (O-polysaccharide, O-chain, O-antigen). Recently, the structure of lipid A of A. caulinodans HAMBI 216 has been elucidated in detail. 5 In this work, we report on the structure of the O-polysaccharide of this strain consisting of D-rhamnose and its O- and C-methylated derivatives. Mild acid degradation of the LPS of A. caulinodans HAMBI 216 resulted in the O-polysaccharide, which was isolated by GPC on Sephadex G-50. Sugar analysis by GLC–MS of the alditol acetates derived after full acid hydrolysis of the polysaccharide revealed rhamnose, a 6-deoxy-2-O-methylhexose and a 6-deoxy-3-C-meth- ylhexose. The electron-impact mass spectrum of the alditol acetate from 6-deoxy-3-C-methylhexose showed ion peaks at m/z 331, 203, 143, 129, and 101, which were assigned to secondary fragments derived from primary fragment ion peaks at m/z 391 ([M+H] + ), 245 (C-3  C-6 fragment) and 231 (C-1  C-3 fragment) (Fig. 1). The D configuration of rhamnose was determined by GLC of the acetylated (S)-2-octyl glycosides. The O- and C-methylated sugars were identified as the D-rhamnose derivatives D-Rha2OMe and D-Rha3CMe, respectively (see below). The 13 C NMR spectrum of the polysaccharide (Table 1) contained signals for five anomeric carbons at d 97.3–103.6, seven CH 3 -C groups (C-6 of Rha and derivatives and Me of Rha3CMe) at d 18.1–19.3, one CH 3 -O group of Rha2OMe at d 59.3 and other carbons (C-2  C-5 of Rha and derivatives) at d 68.7–84.0. Accord- ingly, the 1 H NMR spectrum (Table 1) showed signals for five anomeric protons at d 4.97–5.25, seven CH 3 -C groups at d 1.29– 1.37, one CH 3 -O group at d 3.47 and other protons at d 3.47–4.34. Three typical sugar spin systems were revealed by 2D 1 H, 1 H COSY, TOCSY, ROESY, 1 H, 13 C HSQC, and HMBC experiments and assigned to residues of Rha and Rha2OMe based on characteristic 3 J H,H coupling constants (Table 1). All sugars showed correlations within each pair of neighboring protons in the COSY spectrum as well as H-1/H-2,3 and H-6/H-5,4,3,2 correlations in the TOCSY spectrum. Rha2OMe (unit A) was distinguished from the Rha residues (units C and E) by correlations of the CH 3 -O group with 0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carres.2012.06.012 ⇑ Corresponding author. Tel.: +7 499 1376148; fax: +7 495 1355328. E-mail address: zdorovenkoe@mail.ru (E.L. Zdorovenko). Carbohydrate Research 358 (2012) 106–109 Contents lists available at SciVerse ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres