Accepted Manuscript Note Structural studies of the polysaccharides from the lipopolysaccharides of Azospirillum brasilense Sp246 and SpBr14 Elena N. Sigida, Yuliya P. Fedonenko, Alexander S. Shashkov, Vyacheslav S. Grinev, Evelina L. Zdorovenko, Svetlana A. Konnova, Vladimir V. Ignatov, Yuriy A. Knirel PII: DOI: Reference:
S0008-6215(14)00200-6 http://dx.doi.org/10.1016/j.carres.2014.05.008 CAR 6747
To appear in:
Carbohydrate Research
Received Date: Revised Date: Accepted Date:
3 April 2014 2 May 2014 8 May 2014
Please cite this article as: Sigida, E.N., Fedonenko, Y.P., Shashkov, A.S., Grinev, V.S., Zdorovenko, E.L., Konnova, S.A., Ignatov, V.V., Knirel, Y.A., Structural studies of the polysaccharides from the lipopolysaccharides of Azospirillum brasilense Sp246 and SpBr14, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres. 2014.05.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Note
Structural studies of the polysaccharides from the lipopolysaccharides of Azospirillum brasilense Sp246 and SpBr14 Elena N. Sigida,a,* Yuliya P. Fedonenko,a Alexander S. Shashkov,b Vyacheslav S. Grinev,a Evelina L. Zdorovenko,b Svetlana A. Konnova,a,c Vladimir V. Ignatov,a Yuriy A. Knirelb
a
Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of
Sciences, Prospekt Entuziastov 13, 410049 Saratov, Russia b
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Leninsky Prospekt 47, 119991 Moscow, Russia c
Chernyshevsky Saratov State University, 83 Ulitsa Astrakhanskaya, Saratov 410012, Russia
*
Corresponding author. Tel.: +7 8452 970444; Fax: +7 8452 970383.
E-mail address: [email protected] (E.N. Sigida)
Abstract Lipopolysaccharides from closely related Azospirillum brasilense strains, Sp246 and SpBr14, were obtained by phenol-water extraction. Mild acid hydrolysis of the lipopolysaccharides followed by GPC on Sephadex G-50 resulted in polysaccharide mixtures. On the basis of sugar and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy data, it was concluded that both bacteria possess the same two distinct polysaccharides having structures 1 and 2:
Structure 1 has been reported earlier for a polysaccharide of A. brasilense 54 [Fedonenko, Y. P.; Boiko, A. S.; Zdorovenko, E. L.; Konnova, S. A.; Shashkov, A. S.; Ignatov, V. V.; Knirel, Y. A. Biochemistry (Moscow) 2011, 76, 797–802] whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides.
Key words: Lipopolysaccharide; Bacterial polysaccharide structure; Azospirillum brasilense.
Surface polysaccharides of soil bacteria are an important communication factor allowing microorganisms to build various forms of interactions in biocenosis. Mutualistic plant-bacterial associations increase the survival chances of both partners, and the success of their formation depends on the “molecular dialogue” at the initial stages of the interaction. The Gram-negative N2fixing bacteria Azospirillum are well-known growth-promoting microorganisms that interact with a broad host range.1, 2 Lipopolysaccharides (LPSs) are amphiphilic macromolecules that form the outer layer of the outer membrane of Gram-negative bacteria. In addition to structural and barrier functions, Azospirillum LPSs are involved in mechanisms of host recognition and adsorption and in induction of host responses.3, 4 The LPS molecule is composed of three moieties: lipid A, a hydrophobic domain that anchors the LPS molecule into the membrane and is responsible for biological activities of LPS; a core oligosaccharide; and an O-specific polysaccharide (OPS), which protrudes into the environment and carries antigenic determinants. S-form LPS has all three moieties, whereas in Rform LPS, the carbohydrate portion is limited to the core oligosaccharide. Recently, structures of the OPSs of Azospirillum have been studied intensively. Several groups of the bacteria have been identified whose OPSs either have identical repeating units or share a common backbone structure.5-7 Although azospirilla are not strictly specific toward their plant hosts, a selectivity in identifying a partner has been demonstrated for several strains,8,9 which may be due to a convergent development of the bacteria residing in similar ecological niches. In this paper, we report structures of the OPSs isolated from the LPSs of two closely related A. brasilense strains, Sp246 and SpBr14.10 The LPSs were extracted from bacterial biomass by aqueous phenol and degraded under mild acidic conditions. Lipid A sediments were removed by centrifugation, the OPS-containing supernatants were separated from low-molecular fractions by GPC on Sephadex G-50. Sugar analysis by GLC of the alditol acetates derived after full acid hydrolysis of the OPSs from both strains revealed rhamnose, glucose, 2-acetamido-2-deoxymannose (ManNAc), and 3-acetamido-3deoxyfucose (Fuc3NAc) in the ratio ~3.7 : 0.7 : 1 : 0.6. GLC of the acetylated (S)-2-octyl glycosides revealed the D configuration of Glc and the L configuration of Rha. The absolute configurations of ManN and Fuc3N were determined by 13C NMR spectroscopy (see below). GLC-MS analysis of the partially methylated alditol acetates derived from the methylated OPSs indicated the presence of 2,3,4,6-tetra-O-methyl-glucose, 2-O-methylrhamnose, 3-Omethylrhamnose, 3,4-di-O-methylrhamnose, 2,4-di-O-methylrhamnose, 2-deoxy-4,6-di-O-methyl-2-(Nmethyl)acetamidomannose (from ManNAc), and 3-deoxy-2,4-di-O-methyl-3-(Nmethyl)acetamidofucose (from Fuc3NAc). Therefore, each OPS contains 2-substituted, 3-substituted,
2,4-disubstituted, and 3,4-disubstituted Rha, 3-substituted ManNAc, terminal Glc, and terminal Fuc3NAc. The 1H NMR spectra of both OPSs were essentially identical (Fig 1). This finding and the results of the chemical analyses suggested the presence of repeating units of the same structure. Further studies were performed with the OPS from A. brasilense Sp246. The 13C NMR spectrum of the OPS (Fig. 2) showed signals of seven anomeric carbons. Analysis using 2D NMR spectroscopy, including ROESY and 1H,13C HSQC experiments, revealed the presence of two distinct repeating units. One of them was tetrasaccharide 1 (Chart 1), whose structure had been established earlier in the OPS of A. brasilense 54.6 In order to determine the structure of the other repeating unit, Smith degradation of the OPS was performed. As a result, repeating units 1 were destroyed and a polysaccharide (PS) was obtained. A comparison of 1D and 2D NMR spectra of the PS and OPS showed that the remaining repeating unit 2 was not affected by Smith degradation. The 13C NMR spectrum of the PS (Fig. 3) contained signals for three anomeric carbons at δ 95.1-100.8, two CH3-C groups (C-6 of Rha and Fuc3NAc) at δ 16.5 and 18.0, one HOCH2-C group (C-6 of ManNAc) at δ 61.4, two nitrogen-bearing carbons (C-2 of ManNAc and C-3 of Fuc3NAc) at δ 50.7 and 52.4, other sugar ring carbons in the region of δ 66.1-80.8, and two N-acetyl groups (CH3 at δ 23.0 and 23.3, CO at δ 175.7 and 176.2). The absence of signals in the region of δ 83-88 that are characteristic of furanosides11 confirmed the pyranosidic form of all monosaccharide residues. Accordingly, the 1H NMR spectrum of the PS showed signals for three anomeric protons at δ 4.99-5.08, two CH3-C groups (H-6 of Rha and Fuc3NAc) at δ 1.19 and 1.33, other sugar protons at 3.41-4.71, and two N-acetyl groups at δ 2.06 and 2.07. The 1H and 13C NMR signals for three monosaccharide residues were assigned using 2D 1
H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments (Tables 1 and 2). The
TOCSY spectrum showed H-1/H-2, H-2/H-3,4,5,6, and H-6/H-5,4,3,2 cross-peaks for Rha and ManNAc residues, as well as H-1/H-2,3,4, H-4/H-5, and H-6/H-5 cross-peaks for Fuc3NAc. The assignment of signals within each spin system was performed using the COSY spectrum. The α configuration of Rha and Fuc3NAc was inferred from relatively high-field positions of the C-1 signals at δ 95.1-98.6 and C-5 signals at δ 68.2-68.8 in the 13C-NMR spectrum as compared with published data of the corresponding α- and β-isomers.11 The β configuration of ManNAc was established by a relatively low-field position of the C-5 signal at δ 77.3. Strong H-1/H-3 and H-1/H-5 correlations for ManNAc and H-1/H-2 correlations for Rha and Fuc3NAc in the ROESY spectrum confirmed that ManNAc is β-linked, whereas Rha and Fuc3NAc are α-linked. The ROESY spectrum also showed interresidue cross-peaks between the following anomeric protons and protons at the linkage carbons: Rha H-1/ManNAc H-3 at δ 5.08/3.86; ManNAc H-1/Rha H-4 at δ 5.03/3.81, and Fuc3NAc H-1/Rha H-2 at δ 4.99/3.81. The 1H,13C HMBC spectrum showed
the respective correlations between the following anomeric protons and transglycosidic carbons: Rha H-1/ManNAc C-3 at δ 5.08/77.0; ManNAc H-1/Rha C-4 at δ 5.03/80.8, and Fuc3NAc H-1/Rha C-2 at δ 4.99/77.7, and vice versa. These data defined the glycosylation pattern and the monosaccharide sequence in the repeating unit. The positions of substitution of the monosaccharides were confirmed by downfield displacements of the signals for C-3 of ManNAc, C-2 and C-4 of Rha to δ 77.0, 77.7 and 80.8, respectively, as compared with their positions in corresponding unsubstituted monosaccharides.12 Correlations of CO groups of NAc with ManNAc H-2 at δ 176.2/4.71 and Fuc3NAc H-3 at δ 175.7/4.23 in the 1H,13C HMBC spectrum enabled assignment of the signals for each NAc group. Taking into account the L configuration of Rha established by GLC analysis, a relatively high negative β-glycosylation effect on C-2 of β-ManNAc (-4.6 ppm) and a relatively low α-glycosylation effect on C-1 of α-Fuc3NAc (~5 ppm), as compared with published data of free monosaccharides,12 indicated the D configuration of both amino sugars13 (in case of the same configuration of Rha and the amino sugars, the glycosylation effects would be ~0 ppm and ~8.5 ppm, respectively). Therefore, the PS is built up of branched trisaccharide repeating units 2, and the OPSs from A. brasilense Sp246 and SpBr14 consist of two types of repeating units having structures 1 and 2 (Chart 1). As judged by relative integral intensities of the 1H NMR signals, in strains Sp246 and SpBr14 these structures were present in the ratio ~1:1.2 or ~1:1.3, respectively. In both cases it remains unclear if LPS contains structurally different independent chains or repetitive blocks within the single chain. Earlier, the occurrence of repeating units of different types has been reported for A. brasilense S17, SR80, Sp7, and 54.6,14-16 The latter strain also produces the OPS having the repeating unit 16 but the structure of the other polysaccharide(s) in A. brasilense 54 differs from that established in this work and remains obscure. Polysaccharides composed of L-rhamnose in the main chain and Dglucose in the side chain are widespread in Azospirillum sp. and provide the chemical basis for serological cross-reactivity of strains classified into serogroup III.6 To our knowledge, the repeating unit 2 has not been found in bacterial polysaccharides earlier. It is distinguished by the presence of terminal Fuc3NAc, which is common for OPSs of plantassociated bacteria Pseudomonas syringae17 and has been found in an OPS of Xanthomonas campestris.18 A disaccharide repeating unit composed of 4-substituted Rha and 3-substituted ManNAc is characteristic for OPS of some plant pathogens, including Burkholderia (Pseudomonas) cepacia 19 and Burkholderia plantarii,20.as well as a human pathogen Vibrio fluvialis21 and a fish pathogen Aeromonas salmonicida.22
1. Experimental 1.1. Bacterial strain, growth, isolation and degradation of the lipopolysaccharides A. brasilense Sp24623 and SpBr1424 isolated from roots of Triticum sp. in Brazil were obtained from the microbial culture collection of the Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences (Saratov). The bacteria were cultivated at 30 °C in a liquid malate medium25 to late exponential phase, and cells were separated by centrifugation. Capsular polysaccharides were removed by repeated washing bacterial biomass with 0.15 M NaCl. Finally, cells were washed with acetone and dried on air. The biomass (10 g) was extracted by the Westphal procedure,26 proteins and nucleic acids were precipitated by CCl3CO2 H as described27 and removed by centrifugation. After dialysis of the supernatant, LPS preparations were obtained in yields 8.3 % and 4.2 % of the dry mass of strains Sp246 and SpBr14, respectively. LPS samples (~100 mg each) were hydrolysed with aq 2 % AcOH at 100 °C for 4 h, the lipid precipitates were removed by centrifugation (13,000×g, 20 min), and the carbohydrate portions were fractionated by GPC on a column (56 × 2.6 cm) of Sephadex G-50 Superfine in 0.05 M pyridinium acetate buffer, pH 4.5. The elution was monitored by the phenol-sulfuric acid assay of fractions. High-molecular-mass OPS preparations were obtained in yields 23.3 % and 31.0 % of the LPS mass, respectively. 1.2. Chemical analyses and methylation Hydrolysis of the OPS was conducted with 2 M CF3CO2H (120 °C, 2 h). The monosaccharides were analysed by GLC as the alditol acetates28 on an HP-5ms capillary column using an Agilent 7820A GC system and a temperature gradient of 160 °C (1 min) to 290 °C at 7 °C min-1. The absolute configurations of the monosaccharides were determined by GLC of the acetylated glycosides with (S)-2-octanol as described.29 An OPS sample was methylated with CH3I in dimethyl sulfoxide in the presence of sodium methylsulphinylmethanide.30 The methylated polysaccharide was hydrolysed with 2 M CF3CO2H (100 °C, 2 h), and the partially methylated monosaccharides were conventionally reduced with NaBH4, acetylated and analysed by GLC−MS on an Agilent MSD 5975C instrument equipped with an HP-5ms column using a temperature gradient of 150 °C (3 min) to 320 °C at 5 °C min-1. 1.3. Smith degradation An OPS sample (24 mg) was oxidised with 0.1 M NaIO4 (1.5 mL) in the dark at 20 °C for 48 h. After addition of ethylene glycol (0.1 mL), reduction with NaBH4 and desalting on a column (80 × 1.6 cm) of TSK HW-40 (S) in aq 1 % AcOH, the products were hydrolysed with aq 2 % AcOH at
100 °C for 2 h and fractionated by GPC on TSK HW-40 (S) in aq 1 % AcOH to yield a PS preparation (9 mg). 1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying from 99.9% D2O. NMR spectra were obtained using a Bruker Avance II 600 MHz instrument (Germany) at 30 °C in 99.95 % D2O using 3-trimethylsilylpropanoate-d4 (δH 0.0) and acetone (δC 31.45) as internal calibration standards. 2D NMR experiments were performed using standard Bruker software. A mixing time in the TOCSY and ROESY spectra was set to 200 ms. The HMBC spectrum was recorded with a 60-ms delay for evolution of long-range couplings.
Acknowledgements This work was funded in part by the Russian Foundation for Basic Research (project 14-0401658).
References 1. Steenhoudt, O.; Vanderleyden, J. FEMS Microbiol Rev. 2000, 24, 487–506. 2. Fibach-Paldi, S.; Burdman, S.; Okon, Y. FEMS Microbiol. Lett. 2012, 326, 99–108. 3. Fedonenko, Iu. P.; Egorenkova, I. V.; Konnova, S. A.; Ignatov, V. V. Microbiology 2001, 70, 329–334. 4. Evseeva, N. V.; Matora, L. Yu.; Burygin, G. L.; Dmitrienko, V. V.; Shchyogolev, S. Yu. Plant Soil. 2011, 346, 181–188. 5. Boiko, A. S.; Smol'kina, O. N.; Fedonenko, Y. P.; Zdorovenko, E. L.; Kachala, V. V.; Konnova, S.A.; Ignatov, V. V. Microbiology 2010, 79, 197–205. 6. Fedonenko, Y. P.; Boiko, A. S.; Zdorovenko, E. L.; Konnova, S. A.; Shashkov, A. S.; Ignatov, V. V.; Knirel, Y. A. Biochemistry (Moscow) 2011, 76, 797–802. 7. Sigida, E. N.; Fedonenko, Y. P.; Zdorovenko, E. L.; Burygin, G. L.; Konnova, S. A.; Ignatov, V. V. Microbiology 2014, 83, in press. 8. Baldani, V .L. D.; Dobereiner, J. Soil Biol. Biochem. 1980, 12, 433–439. 9. Egorenkova, I. V.; Konnova, S. A.; Skvortsov, I. M.; Ignatov, V. V. Microbiology 2000, 69, 103– 108. 10. De Mot, R.; Vanderleyden, J. Can. J. Microbiol. 1989, 35, 960–967. 11. Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983, 41, 27–66. 12. Lipkind, G. M.; Shashkov, A. S.; Knirel, Y. A.; Vinogradov, E. V.; Kochetkov, N. K. Carbohydr. Res. 1988, 175, 59-75. 13. Shashkov, A. S.; Lipkind, G. M.; Knirel, Y. A.; Kochetkov, N. K. Magn. Reson. Chem. 1988, 26, 735–747. 14. Fedonenko, Yu. P.; Konnova, O. N.; Zdorovenko, E. L.; Konnova, S. A.; Zatonsky, G. V.; Shashkov, A. S.; Ignatov, V. V.; Knirel, Y. A. Carbohydr. Res. 2008, 343, 810−816. 15. Sigida, E. N.; Fedonenko, Y. P.; Zdorovenko, E. L.; Konnova, S. A.; Shashkov, A. S.; Ignatov, V. V.; Knirel, Y. A. Carbohydr. Res. 2013, 371, 40−44. 16. Sigida, E. N.; Fedonenko, Yu. P.; Shashkov, A. S.; Zdorovenko, E. L.; Konnova, S. A.; Ignatov, V. V.; Knirel, Y. A. Carbohydr. Res. 2013, 380, 76−80. 17. Zdorovenko, G. M.; Zdorovenko, E. L. Microbiology 2010, 79, 47–57. 18. Molinaro, A.; Silipo, A.; Lanzetta, R.; Newman, M. A.; Dow, J. M.; Parrilli, M. Carbohydr. Res. 2003, 338, 277−281. 19. Cox, A. D.; Wilkinson, S. G. Carbohydr. Res. 1989, 195, 123–129. 20. Zahringer, U.; Rettenmaier, H.; Moll, H.; Senchenkova, S. N.; Knirel, Y. A. Carbohydr. Res. 1997, 300, 143–151.
21. Nazarenko, E. L.; Zubkov, V. A.; Ivanova, E. P.; Gorshkova, R. P. Bioorg. Khim. 1992, 18, 418– 421. 22. Wang, Z.; Liu, X.; Dacanay, A.; Harrison, B. A.; Fast, M.; Colquhoun, D. J.; Lund, V.; Brown, L. L.; Li, J.; Altman, E. Fish Shellfish Immunol. 2007, 23, 1095–1106 23. Baldani, V. L. D.; Alvarez, M. A. de B.; Baldani, J. I.; Dobereiner, J. Plant Soil. 1986, 90, 35–46. 24. Tarrand, J. J.; Krieg, N. R.; Dobereiner J. Can. J. Microbiol. 1978, 24, 967–980. 25. Konnova, S.A.; Makarov, O.E.; Skvortsov, I.M.; Ignatov, V.V. FEMS Microbiol. Lett. 1994, 118, 93–94. 26. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91. 27. Arbatsky, N. P.; Wang, M.; Shashkov, A. S.; Feng, L.; Knirel, Y. A.; Wang, L. Carbohydr. Res. 2010, 345, 2095-2098. 28. Sawardeker, J. S.; Sloneker, J. H.; Jeanes, A. Anal. Chem. 1965, 37, 1602–1603. 29. Leontein, K.; Lindberg, B.; Lönngren, J. Carbohydr. Res. 1978, 62, 359–362. 30. Conrad, H. E. Methods Carbohydr. Chem. 1972, 6, 361–364.
Legends to Figures
Chart 1. Structures of the repeating units 1 and 2 of the OPSs from A. brasilense Sp246 and SpBr14 and repeating unit 2 of the PS obtained after Smith degradation of the OPS. Fig. 1. 1H NMR spectra of the OPSs from A. brasilense Sp246 (A) and SpBr14 (B). Arabic numerals refer to protons in sugar residues denoted as follows: G, Glc; RI, RhaI; RII, RhaII; RIII, RhaIII in the repeating unit 1; R, Rha; M, ManNAc; F, FucNAc in the repeating unit 2. Fig. 2. 13C NMR spectrum of the OPS from A. brasilense Sp246. Arabic numerals refer to carbons in sugar residues denoted as follows: G, Glc; RI, RhaI; RII, RhaII; RIII, RhaIII in the repeating unit 1; R, Rha; M, ManNAc; F, FucNAc in the repeating unit 2. Fig. 3. 13C NMR spectrum of the PS obtained after Smith degradation of the OPS from A. brasilense Sp246. Arabic numerals refer to carbons in sugar residues denoted as follows: R, Rha; M, ManNAc; F, FucNAc.
Table 1. 1H NMR chemical shifts of the PS (δ, ppm)
Monosaccharide residue
Sugar H-1
H-2
H-3
2,4)--L-Rhap-(1
5.08
3.81
3.98
3)--D-ManpNAc -(1
5.03
4.71
-D-Fucp3NAc-(1
4.98
3.85
H-4
NAc H-5
H-6 (a; b)
H-2
3.81
3.99
1.33
3.86
3.60
3.41
3.87; 3.93
2.06
4.23
3.77
4.46
1.19
2.07
Table 2. 13C NMR chemical shifts of the PS (δ, ppm)
Monosaccharide residue
Sugar
NAc
С-1
С-2
С-3
С-4
С-5
С-6
С-1
C-2
2,4)--L-Rhap-(1
95.1
77.7
70.8
80.8
68.8
18.0
3)--D-ManpNAc -(1
100.8
50.7
77.0
66.1
77.3
61.4
176.2
23.0
-D-Fucp3NAc-(1
98.6
67.2
52.4
71.7
68.2
16.5
175.7
23.3
•
Lipopolysaccharides of A. brasilense Sp246 and SpBr14 were degraded with mild acid.
•
The O-specific polysaccharides (OPS) were obtained.
•
Structure of the repeating units of the OPS was established by 2D NMR spectroscopy.
•
The OPS of both strains contained the same two distinct repeating units.
Graphical abstract
S0008-6215(14)00200-6 http://dx.doi.org/10.1016/j.carres.2014.05.008 CAR 6747
To appear in:
Carbohydrate Research
Received Date: Revised Date: Accepted Date:
3 April 2014 2 May 2014 8 May 2014
Please cite this article as: Sigida, E.N., Fedonenko, Y.P., Shashkov, A.S., Grinev, V.S., Zdorovenko, E.L., Konnova, S.A., Ignatov, V.V., Knirel, Y.A., Structural studies of the polysaccharides from the lipopolysaccharides of Azospirillum brasilense Sp246 and SpBr14, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres. 2014.05.008
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Note
Structural studies of the polysaccharides from the lipopolysaccharides of Azospirillum brasilense Sp246 and SpBr14 Elena N. Sigida,a,* Yuliya P. Fedonenko,a Alexander S. Shashkov,b Vyacheslav S. Grinev,a Evelina L. Zdorovenko,b Svetlana A. Konnova,a,c Vladimir V. Ignatov,a Yuriy A. Knirelb
a
Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of
Sciences, Prospekt Entuziastov 13, 410049 Saratov, Russia b
N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
Leninsky Prospekt 47, 119991 Moscow, Russia c
Chernyshevsky Saratov State University, 83 Ulitsa Astrakhanskaya, Saratov 410012, Russia
*
Corresponding author. Tel.: +7 8452 970444; Fax: +7 8452 970383.
E-mail address: [email protected] (E.N. Sigida)
Abstract Lipopolysaccharides from closely related Azospirillum brasilense strains, Sp246 and SpBr14, were obtained by phenol-water extraction. Mild acid hydrolysis of the lipopolysaccharides followed by GPC on Sephadex G-50 resulted in polysaccharide mixtures. On the basis of sugar and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy data, it was concluded that both bacteria possess the same two distinct polysaccharides having structures 1 and 2:
Structure 1 has been reported earlier for a polysaccharide of A. brasilense 54 [Fedonenko, Y. P.; Boiko, A. S.; Zdorovenko, E. L.; Konnova, S. A.; Shashkov, A. S.; Ignatov, V. V.; Knirel, Y. A. Biochemistry (Moscow) 2011, 76, 797–802] whereas to our knowledge structure 2 has not been hitherto found in bacterial polysaccharides.
Key words: Lipopolysaccharide; Bacterial polysaccharide structure; Azospirillum brasilense.
Surface polysaccharides of soil bacteria are an important communication factor allowing microorganisms to build various forms of interactions in biocenosis. Mutualistic plant-bacterial associations increase the survival chances of both partners, and the success of their formation depends on the “molecular dialogue” at the initial stages of the interaction. The Gram-negative N2fixing bacteria Azospirillum are well-known growth-promoting microorganisms that interact with a broad host range.1, 2 Lipopolysaccharides (LPSs) are amphiphilic macromolecules that form the outer layer of the outer membrane of Gram-negative bacteria. In addition to structural and barrier functions, Azospirillum LPSs are involved in mechanisms of host recognition and adsorption and in induction of host responses.3, 4 The LPS molecule is composed of three moieties: lipid A, a hydrophobic domain that anchors the LPS molecule into the membrane and is responsible for biological activities of LPS; a core oligosaccharide; and an O-specific polysaccharide (OPS), which protrudes into the environment and carries antigenic determinants. S-form LPS has all three moieties, whereas in Rform LPS, the carbohydrate portion is limited to the core oligosaccharide. Recently, structures of the OPSs of Azospirillum have been studied intensively. Several groups of the bacteria have been identified whose OPSs either have identical repeating units or share a common backbone structure.5-7 Although azospirilla are not strictly specific toward their plant hosts, a selectivity in identifying a partner has been demonstrated for several strains,8,9 which may be due to a convergent development of the bacteria residing in similar ecological niches. In this paper, we report structures of the OPSs isolated from the LPSs of two closely related A. brasilense strains, Sp246 and SpBr14.10 The LPSs were extracted from bacterial biomass by aqueous phenol and degraded under mild acidic conditions. Lipid A sediments were removed by centrifugation, the OPS-containing supernatants were separated from low-molecular fractions by GPC on Sephadex G-50. Sugar analysis by GLC of the alditol acetates derived after full acid hydrolysis of the OPSs from both strains revealed rhamnose, glucose, 2-acetamido-2-deoxymannose (ManNAc), and 3-acetamido-3deoxyfucose (Fuc3NAc) in the ratio ~3.7 : 0.7 : 1 : 0.6. GLC of the acetylated (S)-2-octyl glycosides revealed the D configuration of Glc and the L configuration of Rha. The absolute configurations of ManN and Fuc3N were determined by 13C NMR spectroscopy (see below). GLC-MS analysis of the partially methylated alditol acetates derived from the methylated OPSs indicated the presence of 2,3,4,6-tetra-O-methyl-glucose, 2-O-methylrhamnose, 3-Omethylrhamnose, 3,4-di-O-methylrhamnose, 2,4-di-O-methylrhamnose, 2-deoxy-4,6-di-O-methyl-2-(Nmethyl)acetamidomannose (from ManNAc), and 3-deoxy-2,4-di-O-methyl-3-(Nmethyl)acetamidofucose (from Fuc3NAc). Therefore, each OPS contains 2-substituted, 3-substituted,
2,4-disubstituted, and 3,4-disubstituted Rha, 3-substituted ManNAc, terminal Glc, and terminal Fuc3NAc. The 1H NMR spectra of both OPSs were essentially identical (Fig 1). This finding and the results of the chemical analyses suggested the presence of repeating units of the same structure. Further studies were performed with the OPS from A. brasilense Sp246. The 13C NMR spectrum of the OPS (Fig. 2) showed signals of seven anomeric carbons. Analysis using 2D NMR spectroscopy, including ROESY and 1H,13C HSQC experiments, revealed the presence of two distinct repeating units. One of them was tetrasaccharide 1 (Chart 1), whose structure had been established earlier in the OPS of A. brasilense 54.6 In order to determine the structure of the other repeating unit, Smith degradation of the OPS was performed. As a result, repeating units 1 were destroyed and a polysaccharide (PS) was obtained. A comparison of 1D and 2D NMR spectra of the PS and OPS showed that the remaining repeating unit 2 was not affected by Smith degradation. The 13C NMR spectrum of the PS (Fig. 3) contained signals for three anomeric carbons at δ 95.1-100.8, two CH3-C groups (C-6 of Rha and Fuc3NAc) at δ 16.5 and 18.0, one HOCH2-C group (C-6 of ManNAc) at δ 61.4, two nitrogen-bearing carbons (C-2 of ManNAc and C-3 of Fuc3NAc) at δ 50.7 and 52.4, other sugar ring carbons in the region of δ 66.1-80.8, and two N-acetyl groups (CH3 at δ 23.0 and 23.3, CO at δ 175.7 and 176.2). The absence of signals in the region of δ 83-88 that are characteristic of furanosides11 confirmed the pyranosidic form of all monosaccharide residues. Accordingly, the 1H NMR spectrum of the PS showed signals for three anomeric protons at δ 4.99-5.08, two CH3-C groups (H-6 of Rha and Fuc3NAc) at δ 1.19 and 1.33, other sugar protons at 3.41-4.71, and two N-acetyl groups at δ 2.06 and 2.07. The 1H and 13C NMR signals for three monosaccharide residues were assigned using 2D 1
H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments (Tables 1 and 2). The
TOCSY spectrum showed H-1/H-2, H-2/H-3,4,5,6, and H-6/H-5,4,3,2 cross-peaks for Rha and ManNAc residues, as well as H-1/H-2,3,4, H-4/H-5, and H-6/H-5 cross-peaks for Fuc3NAc. The assignment of signals within each spin system was performed using the COSY spectrum. The α configuration of Rha and Fuc3NAc was inferred from relatively high-field positions of the C-1 signals at δ 95.1-98.6 and C-5 signals at δ 68.2-68.8 in the 13C-NMR spectrum as compared with published data of the corresponding α- and β-isomers.11 The β configuration of ManNAc was established by a relatively low-field position of the C-5 signal at δ 77.3. Strong H-1/H-3 and H-1/H-5 correlations for ManNAc and H-1/H-2 correlations for Rha and Fuc3NAc in the ROESY spectrum confirmed that ManNAc is β-linked, whereas Rha and Fuc3NAc are α-linked. The ROESY spectrum also showed interresidue cross-peaks between the following anomeric protons and protons at the linkage carbons: Rha H-1/ManNAc H-3 at δ 5.08/3.86; ManNAc H-1/Rha H-4 at δ 5.03/3.81, and Fuc3NAc H-1/Rha H-2 at δ 4.99/3.81. The 1H,13C HMBC spectrum showed
the respective correlations between the following anomeric protons and transglycosidic carbons: Rha H-1/ManNAc C-3 at δ 5.08/77.0; ManNAc H-1/Rha C-4 at δ 5.03/80.8, and Fuc3NAc H-1/Rha C-2 at δ 4.99/77.7, and vice versa. These data defined the glycosylation pattern and the monosaccharide sequence in the repeating unit. The positions of substitution of the monosaccharides were confirmed by downfield displacements of the signals for C-3 of ManNAc, C-2 and C-4 of Rha to δ 77.0, 77.7 and 80.8, respectively, as compared with their positions in corresponding unsubstituted monosaccharides.12 Correlations of CO groups of NAc with ManNAc H-2 at δ 176.2/4.71 and Fuc3NAc H-3 at δ 175.7/4.23 in the 1H,13C HMBC spectrum enabled assignment of the signals for each NAc group. Taking into account the L configuration of Rha established by GLC analysis, a relatively high negative β-glycosylation effect on C-2 of β-ManNAc (-4.6 ppm) and a relatively low α-glycosylation effect on C-1 of α-Fuc3NAc (~5 ppm), as compared with published data of free monosaccharides,12 indicated the D configuration of both amino sugars13 (in case of the same configuration of Rha and the amino sugars, the glycosylation effects would be ~0 ppm and ~8.5 ppm, respectively). Therefore, the PS is built up of branched trisaccharide repeating units 2, and the OPSs from A. brasilense Sp246 and SpBr14 consist of two types of repeating units having structures 1 and 2 (Chart 1). As judged by relative integral intensities of the 1H NMR signals, in strains Sp246 and SpBr14 these structures were present in the ratio ~1:1.2 or ~1:1.3, respectively. In both cases it remains unclear if LPS contains structurally different independent chains or repetitive blocks within the single chain. Earlier, the occurrence of repeating units of different types has been reported for A. brasilense S17, SR80, Sp7, and 54.6,14-16 The latter strain also produces the OPS having the repeating unit 16 but the structure of the other polysaccharide(s) in A. brasilense 54 differs from that established in this work and remains obscure. Polysaccharides composed of L-rhamnose in the main chain and Dglucose in the side chain are widespread in Azospirillum sp. and provide the chemical basis for serological cross-reactivity of strains classified into serogroup III.6 To our knowledge, the repeating unit 2 has not been found in bacterial polysaccharides earlier. It is distinguished by the presence of terminal Fuc3NAc, which is common for OPSs of plantassociated bacteria Pseudomonas syringae17 and has been found in an OPS of Xanthomonas campestris.18 A disaccharide repeating unit composed of 4-substituted Rha and 3-substituted ManNAc is characteristic for OPS of some plant pathogens, including Burkholderia (Pseudomonas) cepacia 19 and Burkholderia plantarii,20.as well as a human pathogen Vibrio fluvialis21 and a fish pathogen Aeromonas salmonicida.22
1. Experimental 1.1. Bacterial strain, growth, isolation and degradation of the lipopolysaccharides A. brasilense Sp24623 and SpBr1424 isolated from roots of Triticum sp. in Brazil were obtained from the microbial culture collection of the Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences (Saratov). The bacteria were cultivated at 30 °C in a liquid malate medium25 to late exponential phase, and cells were separated by centrifugation. Capsular polysaccharides were removed by repeated washing bacterial biomass with 0.15 M NaCl. Finally, cells were washed with acetone and dried on air. The biomass (10 g) was extracted by the Westphal procedure,26 proteins and nucleic acids were precipitated by CCl3CO2 H as described27 and removed by centrifugation. After dialysis of the supernatant, LPS preparations were obtained in yields 8.3 % and 4.2 % of the dry mass of strains Sp246 and SpBr14, respectively. LPS samples (~100 mg each) were hydrolysed with aq 2 % AcOH at 100 °C for 4 h, the lipid precipitates were removed by centrifugation (13,000×g, 20 min), and the carbohydrate portions were fractionated by GPC on a column (56 × 2.6 cm) of Sephadex G-50 Superfine in 0.05 M pyridinium acetate buffer, pH 4.5. The elution was monitored by the phenol-sulfuric acid assay of fractions. High-molecular-mass OPS preparations were obtained in yields 23.3 % and 31.0 % of the LPS mass, respectively. 1.2. Chemical analyses and methylation Hydrolysis of the OPS was conducted with 2 M CF3CO2H (120 °C, 2 h). The monosaccharides were analysed by GLC as the alditol acetates28 on an HP-5ms capillary column using an Agilent 7820A GC system and a temperature gradient of 160 °C (1 min) to 290 °C at 7 °C min-1. The absolute configurations of the monosaccharides were determined by GLC of the acetylated glycosides with (S)-2-octanol as described.29 An OPS sample was methylated with CH3I in dimethyl sulfoxide in the presence of sodium methylsulphinylmethanide.30 The methylated polysaccharide was hydrolysed with 2 M CF3CO2H (100 °C, 2 h), and the partially methylated monosaccharides were conventionally reduced with NaBH4, acetylated and analysed by GLC−MS on an Agilent MSD 5975C instrument equipped with an HP-5ms column using a temperature gradient of 150 °C (3 min) to 320 °C at 5 °C min-1. 1.3. Smith degradation An OPS sample (24 mg) was oxidised with 0.1 M NaIO4 (1.5 mL) in the dark at 20 °C for 48 h. After addition of ethylene glycol (0.1 mL), reduction with NaBH4 and desalting on a column (80 × 1.6 cm) of TSK HW-40 (S) in aq 1 % AcOH, the products were hydrolysed with aq 2 % AcOH at
100 °C for 2 h and fractionated by GPC on TSK HW-40 (S) in aq 1 % AcOH to yield a PS preparation (9 mg). 1.4. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying from 99.9% D2O. NMR spectra were obtained using a Bruker Avance II 600 MHz instrument (Germany) at 30 °C in 99.95 % D2O using 3-trimethylsilylpropanoate-d4 (δH 0.0) and acetone (δC 31.45) as internal calibration standards. 2D NMR experiments were performed using standard Bruker software. A mixing time in the TOCSY and ROESY spectra was set to 200 ms. The HMBC spectrum was recorded with a 60-ms delay for evolution of long-range couplings.
Acknowledgements This work was funded in part by the Russian Foundation for Basic Research (project 14-0401658).
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21. Nazarenko, E. L.; Zubkov, V. A.; Ivanova, E. P.; Gorshkova, R. P. Bioorg. Khim. 1992, 18, 418– 421. 22. Wang, Z.; Liu, X.; Dacanay, A.; Harrison, B. A.; Fast, M.; Colquhoun, D. J.; Lund, V.; Brown, L. L.; Li, J.; Altman, E. Fish Shellfish Immunol. 2007, 23, 1095–1106 23. Baldani, V. L. D.; Alvarez, M. A. de B.; Baldani, J. I.; Dobereiner, J. Plant Soil. 1986, 90, 35–46. 24. Tarrand, J. J.; Krieg, N. R.; Dobereiner J. Can. J. Microbiol. 1978, 24, 967–980. 25. Konnova, S.A.; Makarov, O.E.; Skvortsov, I.M.; Ignatov, V.V. FEMS Microbiol. Lett. 1994, 118, 93–94. 26. Westphal, O.; Jann, K. Methods Carbohydr. Chem. 1965, 5, 83–91. 27. Arbatsky, N. P.; Wang, M.; Shashkov, A. S.; Feng, L.; Knirel, Y. A.; Wang, L. Carbohydr. Res. 2010, 345, 2095-2098. 28. Sawardeker, J. S.; Sloneker, J. H.; Jeanes, A. Anal. Chem. 1965, 37, 1602–1603. 29. Leontein, K.; Lindberg, B.; Lönngren, J. Carbohydr. Res. 1978, 62, 359–362. 30. Conrad, H. E. Methods Carbohydr. Chem. 1972, 6, 361–364.
Legends to Figures
Chart 1. Structures of the repeating units 1 and 2 of the OPSs from A. brasilense Sp246 and SpBr14 and repeating unit 2 of the PS obtained after Smith degradation of the OPS. Fig. 1. 1H NMR spectra of the OPSs from A. brasilense Sp246 (A) and SpBr14 (B). Arabic numerals refer to protons in sugar residues denoted as follows: G, Glc; RI, RhaI; RII, RhaII; RIII, RhaIII in the repeating unit 1; R, Rha; M, ManNAc; F, FucNAc in the repeating unit 2. Fig. 2. 13C NMR spectrum of the OPS from A. brasilense Sp246. Arabic numerals refer to carbons in sugar residues denoted as follows: G, Glc; RI, RhaI; RII, RhaII; RIII, RhaIII in the repeating unit 1; R, Rha; M, ManNAc; F, FucNAc in the repeating unit 2. Fig. 3. 13C NMR spectrum of the PS obtained after Smith degradation of the OPS from A. brasilense Sp246. Arabic numerals refer to carbons in sugar residues denoted as follows: R, Rha; M, ManNAc; F, FucNAc.
Table 1. 1H NMR chemical shifts of the PS (δ, ppm)
Monosaccharide residue
Sugar H-1
H-2
H-3
2,4)--L-Rhap-(1
5.08
3.81
3.98
3)--D-ManpNAc -(1
5.03
4.71
-D-Fucp3NAc-(1
4.98
3.85
H-4
NAc H-5
H-6 (a; b)
H-2
3.81
3.99
1.33
3.86
3.60
3.41
3.87; 3.93
2.06
4.23
3.77
4.46
1.19
2.07
Table 2. 13C NMR chemical shifts of the PS (δ, ppm)
Monosaccharide residue
Sugar
NAc
С-1
С-2
С-3
С-4
С-5
С-6
С-1
C-2
2,4)--L-Rhap-(1
95.1
77.7
70.8
80.8
68.8
18.0
3)--D-ManpNAc -(1
100.8
50.7
77.0
66.1
77.3
61.4
176.2
23.0
-D-Fucp3NAc-(1
98.6
67.2
52.4
71.7
68.2
16.5
175.7
23.3
•
Lipopolysaccharides of A. brasilense Sp246 and SpBr14 were degraded with mild acid.
•
The O-specific polysaccharides (OPS) were obtained.
•
Structure of the repeating units of the OPS was established by 2D NMR spectroscopy.
•
The OPS of both strains contained the same two distinct repeating units.
Graphical abstract
