Carbohydrate Research 404 (2015) 93–97
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
Structure of the O-specific polysaccharides from planktonic and biofilm cultures of Pseudomonas chlororaphis 449 Evelina L. Zdorovenko a,⇑, Alexander S. Shashkov a, Marina V. Zhurina b, Vladimir K. Plakunov b, Yuriy A. Knirel a a b
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow 119991, Russia S.N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow 117312, Russia
a r t i c l e
i n f o
Article history: Received 23 September 2014 Received in revised form 20 October 2014 Accepted 21 October 2014 Available online 31 October 2014 Keywords: Pseudomonas chlororaphis Lipopolysaccharide O-Polysaccharide structure Biofilm Planktonic form
a b s t r a c t O-Specific polysaccharides were obtained from the lipopolysaccharides isolated from the planktonic and biofilm cultures of Pseudomonas chlororaphis 449 and studied by composition analysis and 1D and 2D 1H and 13C NMR spectroscopy. The following structure was established:
where the degree of non-stoichiometric 6-O-acetylation of GalNAc is 60% in the planktonic form or 10% in biofilm. Ó 2014 Elsevier Ltd. All rights reserved.
Lipopolysaccharide (LPS) is built up of three structural domains, including the most structurally conserved part called lipid A, a core oligosaccharide, and an O-polysaccharide (O-antigen) composed of oligosaccharide repeats (O-units). The O-antigen contacts directly with the environment and varies in composition and structure depending on the microorganism type and environmental conditions.1 Therefore, it can be expected that the planktonic microbial cells and those attached to the surface (biofilm) may express different O-antigen structures. Indeed, most strains of Pseudomonas aeruginosa studied produce two types of polysaccharides, one being a D-rhamnose homopolymer (common polysaccharide antigen, CPA) and the other heteropolysaccharide O-antigen.2 The latter predominates in planktonic cells of P. aeruginosa PAO1, whereas the shorter and less immunogenic CPA is preferably present in the biofilm.3 This shift to CPA may facilitate protection against the host immune system and improve the ability of the microbe to attach to epithelial cells of the respiratory tract.4 The O-polysaccharide localized on the cell surface of Pseudomonas affects the size and composition of outer membrane vesicles, which are important components of biofilms and play a significant role in their formation and maturation.5 Therefore, comparative studies of O-polysaccharide structures of plankton and biofilm microorganisms may help understanding the role and mechanisms ⇑ Corresponding author. Tel.: +7 (499) 1376148; fax: +7 (499) 1355328. E-mail address: [email protected] (E.L. Zdorovenko). http://dx.doi.org/10.1016/j.carres.2014.10.020 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
of bacterial adaptation to different environmental conditions. In this work, we compared the O-polysaccharide structures of Pseudomonas chlororaphis 449 grown as planktonic cells and biofilm. Mild acid degradation of the LPS from both bacterial forms afforded O-polysaccharides, which were isolated by GPC on Sephadex G-50 and studied by sugar analysis using GLC of the alditol acetates derived after full acid hydrolysis. As a result, Glc, 2-acetamido-2,6-dideoxyglucose (N-acetylquinovosamine, QuiNAc), GlcNAc, and GalNAc in the ratio 1:1:2:1, respectively, were identified in both polysaccharides. Later studies showed that Glc was not a component of the O-unit and, most likely, derived from the LPS core that was linked to the O-polysaccharide.6,7 Determination of the absolute configurations by GLC of the acetylated glycosides of (S)-2-octanol showed that GlcNAc is D. The D configuration of GalNAc and QuiNAc was determined on the basis of known effects of glycosylation on 13C NMR chemical shifts.8,9 The 1H NMR spectrum of the O-polysaccharide from planktonic cells indicated a structural irregularity, most likely, owing to nonstoichiometric O-acetylation (there was a signal for an O-acetyl group at d 2.15). Indeed, O-deacylation resulted in a structurally homogeneous polysaccharide. Its 1H NMR spectrum showed the presence of signals for four anomeric protons at d 4.48–5.49, one CH3–C group (H-6 of QuiNAc) at d 1.33, and four N-acetyl groups at d 2.00, 2.08, and 2.10 (double intensity). The 13C NMR spectrum contained signals for four anomeric carbons at d 98.1–104.6, one CH3–C group (C-6 of QuiNAc) at d 17.9, three HOCH2–C groups
94
E. L. Zdorovenko et al. / Carbohydrate Research 404 (2015) 93–97
(C-6 of GlcNAc and GalNAc) at d 60.7–69.1, four nitrogen-bearing carbons (C-2 of the amino sugars) at d 49.9–56.5, other sugar ring carbons in the region d 71.0–78.5, and four N-acetyl groups at d 23.1–23.6 (Me) and d 174.9–175.6 (CO). Four sugar spin systems were identified by 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC (Fig. 1), and HMBC experiments and assigned by tracing connectivities to residues of GalNAc (A), QuiNAc (B), and GlcNAc (C and D) (Tables 1 and 2). Particularly, there were correlations of H-1 with H-2-H-4 for GalNAc, H-1 with H-2-H-6 and H-6 with H-5-H-2 for QuiNAc and GlcNAc in the TOCSY spectrum, and H-5 with H-4 and H-3 for GalNAc in the ROESY spectrum. The assignment within each spin system was performed using COSY. The absence of signals from the region of d 83–88 characteristic of furanosides10 showed that all monosaccharide residues are in the pyranose form. The b configuration of QuiNAc B and GlcNAc D residues was inferred by relatively low-field positions of the C-1 signals at d 102.5 and 104.6 as well as the C-5 signals at d 73.0 and 77.1, respectively, as compared with published data for the corresponding a- and b -pyranosides.11,12 The a configuration of GalNAc A and GlcNAc C followed from relatively high-field positions of the C-5 signals at d 72.9 and 71.3, respectively.
Significant downfield displacements of the signals for C-3 and C-4 of GalNAc A, C-3 of QuiNAc B, and C-6 of GlcNAc C at d 77.1, 75.1, 78.5, and 69.1, respectively, from their positions in the spectra of the corresponding non-substituted monosaccharides,11,12 defined the positions of substitution of the monosaccharides. Therefore, the polysaccharide is branched with GalNAc A at the branching point and GlcNAc D in the side chain. Sequence analysis was performed by 1H,13C HMBC, and ROESY experiments, which showed interresidue correlations of the anomeric atoms with the linkage carbons or protons at the linkage carbons of the neighboring sugar residues (Table 2). The data obtained showed that the O-deacetylated polysaccharide has the tetrasaccharide O-unit with the following structure:
In addition to the major signals tabulated in Table 1, the NMR spectra showed a number of minor signals, e.g. a minor H-1 signal at d 5.00 for a-GlcpNAc, which may belong to monosaccharides from the terminal O-units. The 1H and 13C NMR spectra of the O-polysaccharide were assigned as described above for the O-deacetylated polysaccharide
Figure 1. Part of a 1H, 13C HSQC spectrum of the O-deacetylated O-polysaccharide of P. chlororaphis 449 (planktonic form). The corresponding parts of the 1H and spectra are shown along the horizontal and vertical axis, respectively.
13
C NMR
Table 1 H and 13C NMR chemical shifts (d, ppm) of the O-deacetylated O-polysaccharide of P. chlororaphis 449
1
Sugar residue ?3,4)-a-D-GalpNAc-(1?
A
?3)-b-D-QuipNAc-(1?
B
?6)-a-D-GlcpNAc-(1?
C
b-D-GlcpNAc-(1?
D
H-1 C-1
H-2 C-2
H-3 C-3
H-4 C-4
H-5 C-5
H-6 (6a, 6b) C-6
5.49 98.2 4.59 102.5 4.93 98.1 4.48 104.6
4.37 49.9 3.86 55.7 3.88 55.3 3.56 56.5
3.83 77.1 3.69 78.5 3.88 71.7 3.55 75.0
4.33 75.1 3.46 77.9 3.50 71.0 3.39 71.8
3.93 72.9 3.51 73.0 4.43 71.3 3.42 77.1
3.66 60.7 1.33 17.9 4.00, 4.16 69.1 3.75, 3.95 62.4
13 C NMR chemical shifts are shown in italics. Signals for the N-acetyl groups are at dH 2.00, 2.08, and 2.10 (double intensity); dC 23.1, 23.4, 23.5, and 23.6 (all Me), d 174.9, 175.2, 175.5, and 175.6 (all CO).
95
E. L. Zdorovenko et al. / Carbohydrate Research 404 (2015) 93–97 Table 2 Correlations for H-1 and C-1 in the 2D ROESY and 1H,
13
C HMBC spectra of the O-deacetylated O-polysaccharide of P. chlororaphis 449
Anomeric atom in sugar residue (d)
A H-1 (5.49) A C-1 (98.2) B H-1 (4.59) B C-1 (102.5) C H-1 (4.93) C C-1 (98.1) D H-1 (4.48) D C-1 (104.6)
Correlation(s) to atoms in sugar residue(s) (d) ROESY
HMBC
B H-3 (3.69), B H-4 (3.46), A H-2 (4.37)
B C-3 (78.5), A C-3 (77.1), A C-5 (72.9) B H-3 (3.69), A H-2 (4.37) C C-6 (69.1) B H-2 (3.86), B H-5 (3.51) A C-4 (75.1), C C-5 (71.3) A H-4 (4.33) A C-3 (77.1), D C-5 (77.1) A H-3 (3.83), D H-3 (3.55)
C H-6a (4.00), B H-3 (3.69), B H-5 (3.51) A H-4 (4.33), C H-2 (3.88), C H-3 (3.88) A H-3 (3.83), D H-3 (3.55), D H-5 (3.42)
Figure 2. Part of a 1H, 13C HSQC spectrum of the O-polysaccharide of P. chlororaphi 449 (planktonic form). The corresponding parts of the 1H and 13C NMR spectra are shown along the horizontal and vertical axis, respectively. A0 indicates 6-O-acetylated unit A.
(Supplementary Tables 1S and 2S). A comparison of the 1H,13C HSQC spectra of the initial and O-deacetylated polysaccharides (Figs. 1 and 2) showed a downfield displacement of a part of the GalNAc H-6/C-6 cross-peak from d 3.66/60.7 in the latter spectrum to d 4.02/62.9 and 4.27/62.9 in the former spectrum, thus indicating O-acetylation of GalNAc at position 6. This conclusion was confirmed by an upfield displacement from d 72.9–70.1 of a part of the C-5 signal of this residue (b-effect of O-acetylation13). As judged by relative intensities of the NMR signals for GalNAc6Ac and GalNAc, the degree of O-acetylation in the plankton O-polysaccharide was 60%. Similar studies by NMR spectroscopy of the O-polysaccharide from biofilm (for the 1H,13C HSQC spectrum see Figure 3) showed that it had the same structure but the degree of 6-O-acetylation of GalNAc was significantly lower (10%). O-Acetylation impacts the rheological properties of polysaccharides and protects them from enzymatic breakdown presumably through steric hindrance and/or conformational changes of the polymer.14 It is possible that fine-tuning the acetylation level of polysaccharides impacts non-covalent interaction of polymers and hinders their degradation.15 The established structure of the O-polysaccharide of P. chlororaphis 449 is unique among bacterial polysaccharides. Particularly, it
differs from the O-polysaccharide structure of another strain of this bacterium, P. chlororaphis subsp. aereofaciens M71 (planktonic form), which has a trisaccharide O-unit consisting of one residue each of L-Rha, L-QuiNAc, and L-FucNAc.16 1. Experimental 1.1. Bacterial strain, cultivation, and isolation of the LPS P. chlororaphis 449 isolated from maize rhizosphere was obtained from the microbial-culture collection held at the Institute of Molecular Genetics, Russian Academy of Sciences (Moscow). The culture was grown on Luria-Bertani (LB) Lennox medium at 29 °C. Planktonic culture was grown in liquid LB medium under stirring (150 rpm) for 2 days. Biofilms were grown on LB-agar on plates. Surface of the medium was inoculated with 2 mL of overnight culture, distributed on the surface, and incubated during 5 days. Cells (planktonic form) were separated by centrifugation and dried with acetone. Biofilm was ultrasonicated with glass beads, after ultracentrifugation (45,000g, 1,5 h) cells were dried with acetone. The dried cells (4 g planktonic form or 2 g biofilm) were extracted using the phenol–water method,17 the aqueous layer was dialyzed, and the crude extract was freed from nucleic acids
96
E. L. Zdorovenko et al. / Carbohydrate Research 404 (2015) 93–97
Figure 3. Part of a 1H, 13C HSQC spectrum of the O-polysaccharide of P. chlororaphis 449 (biofilm form). The corresponding parts of the 1H and along the horizontal and vertical axis, respectively. A0 indicates 6-O-acetylated unit A.
and proteins by treatment with 40% aq CCl3CO2H to pH 2.5 at 4 °C. The precipitate was removed by centrifugation, the supernatant was dialyzed and lyophilized to give LPS. 1.2. Isolation and O-deacetylation of the O-polysaccharide A LPS sample from planktonic cells or biofilm (100 and 60 mg, respectively) was hydrolyzed with aq 2% HOAc at 100 °C for 1.5 h, the lipid precipitate was removed by centrifugation (13,000g, 20 min), and the carbohydrate portion was fractionated by GPC on a column (56 2.6 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer, pH 4.5, monitored with a differential refractometer (Knauer, Germany) to give O-polysaccharide preparations (14 and 8 mg, respectively). An O-polysaccharide sample from planktonic cells was O-deacetylated with aq 12% ammonia at 37 °C for 16 h. 1.3. Sugar analyses An O-polysaccharide sample (0.5 mg) was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were analyzed as the alditol acetates18 by GLC on a HP-5 capillary column using a Maestro (Agilent 7820) chromatograph (Interlab, Russia) and a temperature gradient of 160 °C (1 min) to 290 °C at 7 °C min1. The absolute configuration of GlcNAc was determined by GLC of the acetylated (S)-2-octyl glycosides as described.19 1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying twice from D2O and then examined as solution in 99.95% D2O. 1H and 13 C NMR spectra were recorded using a Bruker Avance II 600 MHz spectrometer (Germany) at 30 °C using internal sodium
13
C NMR spectra are shown
3-trimethylsilylpropanoate-2,2,3,3-d4 (dH 0.0 ppm) and external acetone (dC 31.45 ppm) as references. 2D experiments were performed using standard Bruker software, and Bruker TopSpin 2.1 program was used to acquire and process the NMR data. A mixing time of 100 ms was used in TOCSY and ROESY experiments. The HMBC spectrum was recorded with a 60-ms delay for evolution of long-range couplings.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carres.2014.10. 020.
References 1. Lerouge, I.; Vanderleyden, J. FEMS Microbiol. Rev. 2002, 26, 17–47. 2. Lam, J. S.; Taylor, V. L.; Islam, S. T.; Hao, Y.; Kocíncová, D. Front. Microbiol. 2011, 2, 118. 3. Beveridge, T. J.; Makin, S. A.; Kadurugamuwa, J. L.; Li, Z. FEMS Microbiol. Rev. 1997, 20, 291–303. 4. Matewish, J. M. The functional role of lipopolysaccharide in the cell envelope and surface proteins of Pseudomonas aeruginosa. 2004. PhD thesis. University of Guelph, Guelph, Canada. 5. Murphy, K.; Park, A. J.; Hao, Y.; Brever, D.; Lam, J. S.; Khursigara, C. M. J. Bacteriol. 2014, 196, 1306–1317. 6. Bystrova, O. V.; Knirel, Y. A.; Lindner, B.; Kocharova, N. A.; Kondakova, A. N.; Zähringer, U.; Pier, G. B. FEMS Immunol. Med. Microbiol. 2006, 46, 85–99. 7. Silipo, A.; Leone, S.; Molinaro, A.; Lanzetta, R.; Parrilli, M. Carbohydr. Res. 2004, 339, 2241–2248. 8. Shashkov, A. S.; Lipkind, G. M.; Knirel, Y. A.; Kochetkov, N. K. Magn. Reson. Chem. 1988, 26, 735–747. 9. Knirel, Y. A.; Kocharova, N. A.; Shashkov, A. S.; Kochetkov, N. K.; Kholodkova, E. V.; Stanislavsky, E. S. Eur. J. Biochem. 1987, 166, 189–197. 10. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983, 41, 27–66. 11. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59–75.
E. L. Zdorovenko et al. / Carbohydrate Research 404 (2015) 93–97 12. Knirel, Y. A.; Vinogradov, E. V.; Shashkov, A. S.; Dmitriev, B. A.; Kochetkov, N. K.; Stanislavsky, E. S.; Mashilova, G. M. Eur. J. Biochem. 1987, 163, 627–637. 13. Jansson, P.-E.; Kenne, L.; Schweda, E. J. Chem. Soc., Perkin Trans. 1 1987, 377– 383. 14. Biely, P.; Mackenzie, C.; Puls, J.; Schneider, H. Biotechnology 1986, 4, 731–733. 15. Gille, S.; Pauly, M. Front. Plant Sci. 2012, 31, 3–12.
97
16. Pieretti, G.; Puopolo, G.; Carillo, S.; Zoina, A.; Lanzetta, R.; Parrilli, M.; Evidente, A.; Corsaro, M. M. Carbohydr. Res. 2011, 346, 2705–2709. 17. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91. 18. Sawardeker, J. S.; Sloneker, J. H.; Jeanes, A. Anal. Chem. 1965, 37, 1602–1603. 19. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87–89.
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Note
Structure of the O-specific polysaccharides from planktonic and biofilm cultures of Pseudomonas chlororaphis 449 Evelina L. Zdorovenko a,⇑, Alexander S. Shashkov a, Marina V. Zhurina b, Vladimir K. Plakunov b, Yuriy A. Knirel a a b
N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow 119991, Russia S.N. Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow 117312, Russia
a r t i c l e
i n f o
Article history: Received 23 September 2014 Received in revised form 20 October 2014 Accepted 21 October 2014 Available online 31 October 2014 Keywords: Pseudomonas chlororaphis Lipopolysaccharide O-Polysaccharide structure Biofilm Planktonic form
a b s t r a c t O-Specific polysaccharides were obtained from the lipopolysaccharides isolated from the planktonic and biofilm cultures of Pseudomonas chlororaphis 449 and studied by composition analysis and 1D and 2D 1H and 13C NMR spectroscopy. The following structure was established:
where the degree of non-stoichiometric 6-O-acetylation of GalNAc is 60% in the planktonic form or 10% in biofilm. Ó 2014 Elsevier Ltd. All rights reserved.
Lipopolysaccharide (LPS) is built up of three structural domains, including the most structurally conserved part called lipid A, a core oligosaccharide, and an O-polysaccharide (O-antigen) composed of oligosaccharide repeats (O-units). The O-antigen contacts directly with the environment and varies in composition and structure depending on the microorganism type and environmental conditions.1 Therefore, it can be expected that the planktonic microbial cells and those attached to the surface (biofilm) may express different O-antigen structures. Indeed, most strains of Pseudomonas aeruginosa studied produce two types of polysaccharides, one being a D-rhamnose homopolymer (common polysaccharide antigen, CPA) and the other heteropolysaccharide O-antigen.2 The latter predominates in planktonic cells of P. aeruginosa PAO1, whereas the shorter and less immunogenic CPA is preferably present in the biofilm.3 This shift to CPA may facilitate protection against the host immune system and improve the ability of the microbe to attach to epithelial cells of the respiratory tract.4 The O-polysaccharide localized on the cell surface of Pseudomonas affects the size and composition of outer membrane vesicles, which are important components of biofilms and play a significant role in their formation and maturation.5 Therefore, comparative studies of O-polysaccharide structures of plankton and biofilm microorganisms may help understanding the role and mechanisms ⇑ Corresponding author. Tel.: +7 (499) 1376148; fax: +7 (499) 1355328. E-mail address: [email protected] (E.L. Zdorovenko). http://dx.doi.org/10.1016/j.carres.2014.10.020 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
of bacterial adaptation to different environmental conditions. In this work, we compared the O-polysaccharide structures of Pseudomonas chlororaphis 449 grown as planktonic cells and biofilm. Mild acid degradation of the LPS from both bacterial forms afforded O-polysaccharides, which were isolated by GPC on Sephadex G-50 and studied by sugar analysis using GLC of the alditol acetates derived after full acid hydrolysis. As a result, Glc, 2-acetamido-2,6-dideoxyglucose (N-acetylquinovosamine, QuiNAc), GlcNAc, and GalNAc in the ratio 1:1:2:1, respectively, were identified in both polysaccharides. Later studies showed that Glc was not a component of the O-unit and, most likely, derived from the LPS core that was linked to the O-polysaccharide.6,7 Determination of the absolute configurations by GLC of the acetylated glycosides of (S)-2-octanol showed that GlcNAc is D. The D configuration of GalNAc and QuiNAc was determined on the basis of known effects of glycosylation on 13C NMR chemical shifts.8,9 The 1H NMR spectrum of the O-polysaccharide from planktonic cells indicated a structural irregularity, most likely, owing to nonstoichiometric O-acetylation (there was a signal for an O-acetyl group at d 2.15). Indeed, O-deacylation resulted in a structurally homogeneous polysaccharide. Its 1H NMR spectrum showed the presence of signals for four anomeric protons at d 4.48–5.49, one CH3–C group (H-6 of QuiNAc) at d 1.33, and four N-acetyl groups at d 2.00, 2.08, and 2.10 (double intensity). The 13C NMR spectrum contained signals for four anomeric carbons at d 98.1–104.6, one CH3–C group (C-6 of QuiNAc) at d 17.9, three HOCH2–C groups
94
E. L. Zdorovenko et al. / Carbohydrate Research 404 (2015) 93–97
(C-6 of GlcNAc and GalNAc) at d 60.7–69.1, four nitrogen-bearing carbons (C-2 of the amino sugars) at d 49.9–56.5, other sugar ring carbons in the region d 71.0–78.5, and four N-acetyl groups at d 23.1–23.6 (Me) and d 174.9–175.6 (CO). Four sugar spin systems were identified by 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC (Fig. 1), and HMBC experiments and assigned by tracing connectivities to residues of GalNAc (A), QuiNAc (B), and GlcNAc (C and D) (Tables 1 and 2). Particularly, there were correlations of H-1 with H-2-H-4 for GalNAc, H-1 with H-2-H-6 and H-6 with H-5-H-2 for QuiNAc and GlcNAc in the TOCSY spectrum, and H-5 with H-4 and H-3 for GalNAc in the ROESY spectrum. The assignment within each spin system was performed using COSY. The absence of signals from the region of d 83–88 characteristic of furanosides10 showed that all monosaccharide residues are in the pyranose form. The b configuration of QuiNAc B and GlcNAc D residues was inferred by relatively low-field positions of the C-1 signals at d 102.5 and 104.6 as well as the C-5 signals at d 73.0 and 77.1, respectively, as compared with published data for the corresponding a- and b -pyranosides.11,12 The a configuration of GalNAc A and GlcNAc C followed from relatively high-field positions of the C-5 signals at d 72.9 and 71.3, respectively.
Significant downfield displacements of the signals for C-3 and C-4 of GalNAc A, C-3 of QuiNAc B, and C-6 of GlcNAc C at d 77.1, 75.1, 78.5, and 69.1, respectively, from their positions in the spectra of the corresponding non-substituted monosaccharides,11,12 defined the positions of substitution of the monosaccharides. Therefore, the polysaccharide is branched with GalNAc A at the branching point and GlcNAc D in the side chain. Sequence analysis was performed by 1H,13C HMBC, and ROESY experiments, which showed interresidue correlations of the anomeric atoms with the linkage carbons or protons at the linkage carbons of the neighboring sugar residues (Table 2). The data obtained showed that the O-deacetylated polysaccharide has the tetrasaccharide O-unit with the following structure:
In addition to the major signals tabulated in Table 1, the NMR spectra showed a number of minor signals, e.g. a minor H-1 signal at d 5.00 for a-GlcpNAc, which may belong to monosaccharides from the terminal O-units. The 1H and 13C NMR spectra of the O-polysaccharide were assigned as described above for the O-deacetylated polysaccharide
Figure 1. Part of a 1H, 13C HSQC spectrum of the O-deacetylated O-polysaccharide of P. chlororaphis 449 (planktonic form). The corresponding parts of the 1H and spectra are shown along the horizontal and vertical axis, respectively.
13
C NMR
Table 1 H and 13C NMR chemical shifts (d, ppm) of the O-deacetylated O-polysaccharide of P. chlororaphis 449
1
Sugar residue ?3,4)-a-D-GalpNAc-(1?
A
?3)-b-D-QuipNAc-(1?
B
?6)-a-D-GlcpNAc-(1?
C
b-D-GlcpNAc-(1?
D
H-1 C-1
H-2 C-2
H-3 C-3
H-4 C-4
H-5 C-5
H-6 (6a, 6b) C-6
5.49 98.2 4.59 102.5 4.93 98.1 4.48 104.6
4.37 49.9 3.86 55.7 3.88 55.3 3.56 56.5
3.83 77.1 3.69 78.5 3.88 71.7 3.55 75.0
4.33 75.1 3.46 77.9 3.50 71.0 3.39 71.8
3.93 72.9 3.51 73.0 4.43 71.3 3.42 77.1
3.66 60.7 1.33 17.9 4.00, 4.16 69.1 3.75, 3.95 62.4
13 C NMR chemical shifts are shown in italics. Signals for the N-acetyl groups are at dH 2.00, 2.08, and 2.10 (double intensity); dC 23.1, 23.4, 23.5, and 23.6 (all Me), d 174.9, 175.2, 175.5, and 175.6 (all CO).
95
E. L. Zdorovenko et al. / Carbohydrate Research 404 (2015) 93–97 Table 2 Correlations for H-1 and C-1 in the 2D ROESY and 1H,
13
C HMBC spectra of the O-deacetylated O-polysaccharide of P. chlororaphis 449
Anomeric atom in sugar residue (d)
A H-1 (5.49) A C-1 (98.2) B H-1 (4.59) B C-1 (102.5) C H-1 (4.93) C C-1 (98.1) D H-1 (4.48) D C-1 (104.6)
Correlation(s) to atoms in sugar residue(s) (d) ROESY
HMBC
B H-3 (3.69), B H-4 (3.46), A H-2 (4.37)
B C-3 (78.5), A C-3 (77.1), A C-5 (72.9) B H-3 (3.69), A H-2 (4.37) C C-6 (69.1) B H-2 (3.86), B H-5 (3.51) A C-4 (75.1), C C-5 (71.3) A H-4 (4.33) A C-3 (77.1), D C-5 (77.1) A H-3 (3.83), D H-3 (3.55)
C H-6a (4.00), B H-3 (3.69), B H-5 (3.51) A H-4 (4.33), C H-2 (3.88), C H-3 (3.88) A H-3 (3.83), D H-3 (3.55), D H-5 (3.42)
Figure 2. Part of a 1H, 13C HSQC spectrum of the O-polysaccharide of P. chlororaphi 449 (planktonic form). The corresponding parts of the 1H and 13C NMR spectra are shown along the horizontal and vertical axis, respectively. A0 indicates 6-O-acetylated unit A.
(Supplementary Tables 1S and 2S). A comparison of the 1H,13C HSQC spectra of the initial and O-deacetylated polysaccharides (Figs. 1 and 2) showed a downfield displacement of a part of the GalNAc H-6/C-6 cross-peak from d 3.66/60.7 in the latter spectrum to d 4.02/62.9 and 4.27/62.9 in the former spectrum, thus indicating O-acetylation of GalNAc at position 6. This conclusion was confirmed by an upfield displacement from d 72.9–70.1 of a part of the C-5 signal of this residue (b-effect of O-acetylation13). As judged by relative intensities of the NMR signals for GalNAc6Ac and GalNAc, the degree of O-acetylation in the plankton O-polysaccharide was 60%. Similar studies by NMR spectroscopy of the O-polysaccharide from biofilm (for the 1H,13C HSQC spectrum see Figure 3) showed that it had the same structure but the degree of 6-O-acetylation of GalNAc was significantly lower (10%). O-Acetylation impacts the rheological properties of polysaccharides and protects them from enzymatic breakdown presumably through steric hindrance and/or conformational changes of the polymer.14 It is possible that fine-tuning the acetylation level of polysaccharides impacts non-covalent interaction of polymers and hinders their degradation.15 The established structure of the O-polysaccharide of P. chlororaphis 449 is unique among bacterial polysaccharides. Particularly, it
differs from the O-polysaccharide structure of another strain of this bacterium, P. chlororaphis subsp. aereofaciens M71 (planktonic form), which has a trisaccharide O-unit consisting of one residue each of L-Rha, L-QuiNAc, and L-FucNAc.16 1. Experimental 1.1. Bacterial strain, cultivation, and isolation of the LPS P. chlororaphis 449 isolated from maize rhizosphere was obtained from the microbial-culture collection held at the Institute of Molecular Genetics, Russian Academy of Sciences (Moscow). The culture was grown on Luria-Bertani (LB) Lennox medium at 29 °C. Planktonic culture was grown in liquid LB medium under stirring (150 rpm) for 2 days. Biofilms were grown on LB-agar on plates. Surface of the medium was inoculated with 2 mL of overnight culture, distributed on the surface, and incubated during 5 days. Cells (planktonic form) were separated by centrifugation and dried with acetone. Biofilm was ultrasonicated with glass beads, after ultracentrifugation (45,000g, 1,5 h) cells were dried with acetone. The dried cells (4 g planktonic form or 2 g biofilm) were extracted using the phenol–water method,17 the aqueous layer was dialyzed, and the crude extract was freed from nucleic acids
96
E. L. Zdorovenko et al. / Carbohydrate Research 404 (2015) 93–97
Figure 3. Part of a 1H, 13C HSQC spectrum of the O-polysaccharide of P. chlororaphis 449 (biofilm form). The corresponding parts of the 1H and along the horizontal and vertical axis, respectively. A0 indicates 6-O-acetylated unit A.
and proteins by treatment with 40% aq CCl3CO2H to pH 2.5 at 4 °C. The precipitate was removed by centrifugation, the supernatant was dialyzed and lyophilized to give LPS. 1.2. Isolation and O-deacetylation of the O-polysaccharide A LPS sample from planktonic cells or biofilm (100 and 60 mg, respectively) was hydrolyzed with aq 2% HOAc at 100 °C for 1.5 h, the lipid precipitate was removed by centrifugation (13,000g, 20 min), and the carbohydrate portion was fractionated by GPC on a column (56 2.6 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer, pH 4.5, monitored with a differential refractometer (Knauer, Germany) to give O-polysaccharide preparations (14 and 8 mg, respectively). An O-polysaccharide sample from planktonic cells was O-deacetylated with aq 12% ammonia at 37 °C for 16 h. 1.3. Sugar analyses An O-polysaccharide sample (0.5 mg) was hydrolyzed with 2 M CF3CO2H (120 °C, 2 h). Monosaccharides were analyzed as the alditol acetates18 by GLC on a HP-5 capillary column using a Maestro (Agilent 7820) chromatograph (Interlab, Russia) and a temperature gradient of 160 °C (1 min) to 290 °C at 7 °C min1. The absolute configuration of GlcNAc was determined by GLC of the acetylated (S)-2-octyl glycosides as described.19 1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying twice from D2O and then examined as solution in 99.95% D2O. 1H and 13 C NMR spectra were recorded using a Bruker Avance II 600 MHz spectrometer (Germany) at 30 °C using internal sodium
13
C NMR spectra are shown
3-trimethylsilylpropanoate-2,2,3,3-d4 (dH 0.0 ppm) and external acetone (dC 31.45 ppm) as references. 2D experiments were performed using standard Bruker software, and Bruker TopSpin 2.1 program was used to acquire and process the NMR data. A mixing time of 100 ms was used in TOCSY and ROESY experiments. The HMBC spectrum was recorded with a 60-ms delay for evolution of long-range couplings.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carres.2014.10. 020.
References 1. Lerouge, I.; Vanderleyden, J. FEMS Microbiol. Rev. 2002, 26, 17–47. 2. Lam, J. S.; Taylor, V. L.; Islam, S. T.; Hao, Y.; Kocíncová, D. Front. Microbiol. 2011, 2, 118. 3. Beveridge, T. J.; Makin, S. A.; Kadurugamuwa, J. L.; Li, Z. FEMS Microbiol. Rev. 1997, 20, 291–303. 4. Matewish, J. M. The functional role of lipopolysaccharide in the cell envelope and surface proteins of Pseudomonas aeruginosa. 2004. PhD thesis. University of Guelph, Guelph, Canada. 5. Murphy, K.; Park, A. J.; Hao, Y.; Brever, D.; Lam, J. S.; Khursigara, C. M. J. Bacteriol. 2014, 196, 1306–1317. 6. Bystrova, O. V.; Knirel, Y. A.; Lindner, B.; Kocharova, N. A.; Kondakova, A. N.; Zähringer, U.; Pier, G. B. FEMS Immunol. Med. Microbiol. 2006, 46, 85–99. 7. Silipo, A.; Leone, S.; Molinaro, A.; Lanzetta, R.; Parrilli, M. Carbohydr. Res. 2004, 339, 2241–2248. 8. Shashkov, A. S.; Lipkind, G. M.; Knirel, Y. A.; Kochetkov, N. K. Magn. Reson. Chem. 1988, 26, 735–747. 9. Knirel, Y. A.; Kocharova, N. A.; Shashkov, A. S.; Kochetkov, N. K.; Kholodkova, E. V.; Stanislavsky, E. S. Eur. J. Biochem. 1987, 166, 189–197. 10. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983, 41, 27–66. 11. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59–75.
E. L. Zdorovenko et al. / Carbohydrate Research 404 (2015) 93–97 12. Knirel, Y. A.; Vinogradov, E. V.; Shashkov, A. S.; Dmitriev, B. A.; Kochetkov, N. K.; Stanislavsky, E. S.; Mashilova, G. M. Eur. J. Biochem. 1987, 163, 627–637. 13. Jansson, P.-E.; Kenne, L.; Schweda, E. J. Chem. Soc., Perkin Trans. 1 1987, 377– 383. 14. Biely, P.; Mackenzie, C.; Puls, J.; Schneider, H. Biotechnology 1986, 4, 731–733. 15. Gille, S.; Pauly, M. Front. Plant Sci. 2012, 31, 3–12.
97
16. Pieretti, G.; Puopolo, G.; Carillo, S.; Zoina, A.; Lanzetta, R.; Parrilli, M.; Evidente, A.; Corsaro, M. M. Carbohydr. Res. 2011, 346, 2705–2709. 17. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91. 18. Sawardeker, J. S.; Sloneker, J. H.; Jeanes, A. Anal. Chem. 1965, 37, 1602–1603. 19. Leontein, K.; Lönngren, J. Methods Carbohydr. Chem. 1993, 9, 87–89.
