2 Department of Genetics, Faculty of Biology, University of Bielefeld, D-33615 Bielefeld, Germany; 3 Division of Biophysics, Research Center Borstel, Leibniz Center for Medicine and Biosciences, D-23845 Borstel, Germany; 4 Division of Structural Biochemistry, Research Center Borstel, Leibniz Center for Medicine and Biosciences, D-23845 Borstel, Germany; and 5 Laboratoire des IMRCP, Universite Paul Sabatier, F-31062 Toulouse, France
Received on July 20, 2004; revised on August 19, 2004; accepted on August 23, 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: capsular polysaccharides / Fourier transform ion cyclotron resonance / phospholipic anchor / QTOF MS / Sinorhizobium meliloti
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This work focuses on the CPS (or K-antigen polysaccharides, or KPS) of S. meliloti strain 1021. They have initially been structurally characterized in S. meliloti and S. fredii (Reuhs et al., 1993) and are analogous to group II KPSs found in Escherichia coli, so that the K-antigen appellation, or KPS, is mostly used for this class of polysaccharides. To date, all K-antigens found in Rhizobia exhibit a dimeric repeating unit of one hexose (that can be diversely substituted) and a 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) or a related sugar (Fraysse et al., 2003
). The biological importance of rhizobial CPS (or KPS) has been investigated in detail in the symbiotic couple S. meliloti/alfalfa (Pellock et al., 2000
).
The nature and the size of KPS are variable within the S. meliloti genus from strain to strain (Rheus et al., 1998). Rm 41 exhibits two types of KPS, a low- (LMM) and a high-molecular-mass (HMM) form. In the corresponding exoB mutant, AK631, the HMM KPS can replace the deficiency of EPS and lead to a time-delayed normal nitrogen-fixing phenotype. A mutation of the rkpZ gene involved in the biosynthesis of KPS (more precisely on its molecular size) results in a non-nitrogen fixating (Fix) phenotype (Putnoky et al., 1990
; Williams et al., 1990
). This gene enables the K-antigen to increase to an average molecular mass of 2026 kDa, which is necessary to render it symbiotically efficient. S. meliloti strain 1021 does not possess such a gene, and an exoB mutation, resulting in a lack of EPS, leads to a Fix phenotype. Indeed, when only a LMM KPS is synthesized, the EPS deficiency is not overcome. The nitrogen-fixation phenotype is restored by a complementation with rkpZ (Reuhs et al., 1995
). Nevertheless, this restoration is not complete and only the introduction of the complete pSymB megaplasmid results in a total restoration of a nitrogen-fixing phenotype. This means that the molecular size of KPS is a determinant of biological activity, but not the only one. Some other structural features are still probably harbored or lacking in the KPS of the RkpZ-complemented S. meliloti 1021 but also in the 1021 wild type, and this is responsible for a noncompletely restored nitrogen-fixing phenotype.
The goal of this investigation was to characterize the structure of the KPS produced by S. meliloti 1021, which, unlike to those of S. meliloti strain 41 and several related strains (Reuhs et al., 1998), has not been determined. For the first time, it is reported that S. meliloti strain 1021 is able to synthesize a KPS that is unique with regard to its structure (purely constituted of Kdo) and to the presence of a phospholipid anchor. The presence in any Rhizobium strain of this phospholipid anchor, even if it was already demonstrated for other Gram-negative bacteria, such as E. coli (Fischer et al., 1982
; Gotschlich et al., 1981
; Schmidt and Jann, 1984
), has been discussed based on genetic approaches but without being structurally demonstrated.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
MS analyses of the crude polysaccharide extract
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of the crude polysaccharide extract revealed a complex mixture of a series of poorly resolved ion peaks with mass differences of 220 amu, indicating oligosaccharides carrying different numbers of Kdo (data not shown). Furthermore, the spectrum is made up of ion peaks with slightly higher abundance around m/z 2030, 1463, and 3979. The first two ions are consistent with the lipid A structures described by Kanipes et al. (2003), and the third may be a complete rLPS II carrying the complete core oligosaccharide. However, due to presence of numerous salt adducts, which could not be reduced significantly by cation exchanger, the mass spectra did not allow a detailed interpretation.
High-resolution Fourier transform ion cyclotron resonance (FT-ICR) MS using electrospray ionization (ESI) was applied. The negative-ion mass spectrum of the crude extract revealed a complex mixture of ions, each one present in multiple charge states. To facilitate the interpretation, the mass spectrum was charge-deconvoluted, and mass numbers given refer to the monoisotopic peaks of the neutral molecule. Consistent with molecular species characterized by MALDI-TOF MS, a series of compounds in the range of 2800 to 5100 amu with mass differences of 220.06 amu (one Kdo residue) were observed. However, no ions corresponding to rLPS II and the lipid A moiety could be identified. This might be due to the lower solubility of the highly hydrophobic LPS in the electrospray solution, leading to a reduced ionization efficiency. As is demonstrated by the enlargement of the most abundant group of ions around 3926 amu (see Figure 2), each group of ions consists of two molecular species, differing by 28 amu. The other ions within in each group originate from multiple cationization with a combination of sodium and potassium adducts. The mass difference of 28 amu might point to heterogeneity with respect to the chain length of a fatty acid residue and thus may indicate that the Kdo polysaccharide was covalently linked to a lipid anchor.
|
|
|
|
|
ESI-MS analysis of the crude polysaccharides extract from S. meliloti strain 1021 cultivated in Vincent minimal liquid medium
ESI-QTOF MS analysis was also performed on the crude polysaccharides extract obtained from S. meliloti strain 1021 cultivated in Vincent minimal liquid medium. The same Kdo homopolymer was identified (see Figure 6), possessing similar average molecular mass and polydispersity. The exact masses corresponded to the same phospholipid anchor (ion at 622.480 amu) substituted with a certain number of Kdo.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The average molecular mass of this Kdo polysaccharide was assumed to be very close to that of the rough LPS, the presence of which made clear observation of the polysaccharide in DOC PAGE more complicated. ESI-MS experiments confirmed the average molecular mass of 44.5 kDa.
The homopolymeric nature of this Kdo polysaccharide was characterized on the basis of the exact masses determined by ESI FT-ICR MS and by CSD. NMR analyses showed that the Kdo was in ß-pyranosyl configuration as it was demonstrated for S. meliloti strain AK631 (Reuhs et al., 1993). For the first time, a lipid anchor was identified in a K-antigen polysaccharide of S. meliloti based on the identification of two ions that corresponded to phospholipid moieties. These two compounds possessed a mass difference of 28.032 amu that corresponded to a CH2-CH2 moiety. For these compounds, we propose the formulas [C33O7NH69P] for the one with 622.480 amu and [C31O7NH65P] for that with 694.474 amu. The heterogeneity at the level of the phospholipid anchors gave rise to the two different series of polysaccharides. Thus the K-antigen polysaccharides should be anchored in the outer membrane by a phospholipid moiety. The detailed structures of the phospholipid anchor species have not been unequivocally identified, but the MS/MS experiments suggested the presence of a phosphoglycerol moiety and a C28:OH fatty acid that is typical for rhizobia species.
The presence of a lipid anchor has been discussed for some time in investigations dealing with rhizobial KPS structures. The rhizobial K-antigen polysaccharides were first described to be analogous to group II KPSs found in E. coli (Reuhs et al., 1993). This similarity is due not only to high contents of Kdo but also to the presence of a phospholipid anchor. The phospholipid anchors of rhizobial KPS have not been fully characterized. Their purification and characterization is difficult for many reasons, for example, fragility of the structure in acid hydrolysis or the strong physicochemical affinity with amphiphilic LPS that hampers the isolation. Also, direct MS analysis is difficult to pursue. However, cyclotron resonance or high-resolution QTOF mass spectrometers allowed the accurate determination of the complex polysaccharide structures in the mixture. Another difficulty in the structural characterization was the presence of a phospholipid anchor that renders the KPS amphiphilic. Reuhs et al. (1998)
reported the purification and the structural characterization of rhizobial KPS. They all consisted of simple dimeric repeating units, Hex-Kdx sugars, without a lipid anchor. It is possible that the observed nonpolymyxin bound KPS resulted from hydrolyzed saccharidic products whose anchor has been lost. This highly water-soluble hydrolysate by-product from KPS yielded much better immediate mass spectra than the entire KPS, which required a complex solvent system to obtain good MS data. The difficulties to detect and structurally characterize the lipid anchor might have also been due to noncovalent interactions with LPS.
The amphiphilic properties and the noncovalent interactions of KPS with the LPS suggest that this polysaccharide is bound to the outer membrane and does not represent an intracellular compound or a loosely attached CPS.
It has been previously shown that the absence of the rkpZ gene in S. meliloti strain AK631 led to a change in the molecular mass of the KPS, that is, to a decrease from 22 kDa to
6 kDa. The high size range mass was also expected for KPS of S. meliloti strain 1021 but was not identified, even if the presence of three other RkpZ homologs in the S. meliloti 1021 genome has been recently demonstrated (Sharypova et al., unpublished data). However, whether these were expressed remains to be determined. The KPS contained only Kdo, which could explain the fact that even when a RkpZ complementation was carried out, the usual phenotype made up of a hexose-Kdo disaccharide repeating unit was not completely restored, leading to a reduced nitrogen-fixation activity.
On the other hand, genetic studies on S. meliloti suggested that certain genes involved in KPS biosynthesis correspond to genes involved in the constitution of a KPS lipid anchor (Kiss et al., 1997, 2001
; Petrovics et al., 1993
). RkpG (sharing similarity with acyltransferases), RkpH (homologous to short-chain alcohol deydrogenase) proteins, and more generally fatty acid synthase genes from the fix23 region, are required for KPS biosynthesis and transport. Reuhs et al. (1995)
proposed a model for KPS expression in rhizobia: A short oligosaccharide (815 units) is synthesized, and such subunits are polymerized to HMM KPS, which is finally exported. The membrane-attached LMM oligosaccharides that were observed in our work may represent such KPS subunits. In the present case of S. meliloti 1021, some genes are missing (like RkpZ) and thus the complete HMM KPS could no longer be polymerized and transported through the membrane.
The fact that this LMM Kdo-rich polysaccharide was as well produced under conditions that favor the expression of nod genes suggests that expression of the genes involved in its biosynthesis are probably not dependent on environmental conditions. The structural characterization of LMM Kdo-rich polysaccharides (that can be assumed as LMM CPSs) from S.meliloti 1021 shows that the presence of a KPS phospholipid anchor in other Rhizobium species is possible. Consequently, using similar techniques to those described in this article, the characterized CPSs from other species should be reexamined.
![]() |
Material and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
S. meliloti strain 1021 was obtained from the collection of INRA, Auzeville, France, and cultured in liquid Vincent minimal medium complemented with the nod gene inducer luteoline (Fraysse et al., 2003). The cultures were grown at 36°C. Growth was stopped by the addition of sodium azide when the optical density observed at 600 nm reached 1.5.
Extraction and purification of KPS
Bacteria were harvested by washing each plate with 0.9% NaCl (2 ml) and centrifugation at 10,000 x g, 4°C, 20 min. The bacteria were recovered from liquid medium using the same centrifugation protocol. Both bacterial masses were extracted as described. Pellets were resuspended twice in 0.9% NaCl (500 ml) and centrifuged as described. The pellets were extracted following the hot-phenol water extraction protocol (Westphal and Jann, 1965
). The water phase (
700 ml) was reduced by evaporation to 200 ml, dialyzed three times against water (12,000 kDa molecular weight cutoff) to eliminate traces of phenol, and then digested by RNase, DNase, and Proteinase K (Carlson et al., 1978
). Samples were dialyzed and then freeze-dried. This material was dissolved in 20 ml water and ultracentrifuged at 100,000 x g for 18 h. Pellets were recuperated and freeze-dried.
PAGE of polysaccharides
DOC PAGE was performed as previously described (Reuhs et al., 1998) and stained with either alcian blue-silver (Corzo et al., 1991
) or silver nitrate (Tsai and Frasch, 1982
).
Semi-preparative TLC analysis and microextraction
TLC analysis was performed on 0.5 mm silica gel 60 plates with CHCl3:MeOH (2:1, v/v) as eluent. For staining, 15% sulfuric acid in ethanol was used. For the identification of sugars, 15% sulfuric acid in ethanol containing 0.5% anthrone was applied. Bands of interest were scrapped off and the silica (about 15 mg) was eluted with 10 ml CHCl3:MeOH (2:1, v/v). The solution was evaporated to dryness and the residue dissolved in 500 µl CHCl3:MeOH (2:1, v/v) for direct ESI-MS analysis.
ESI FT-ICR MS
ESI FT-ICR MS was performed in the negative ion mode using an APEX II Instrument (Bruker Daltonics, Billerica, MA) equipped with a 7 Tesla actively shielded superconducting magnet and an Apollo ion source. Mass spectra were acquired using standard experimental sequences as provided by the manufacturer. Samples were dissolved at a concentration of 10 ng/µl in a 50:50:0.001 (v/v/v) mixture of 2-propanol, water, and triethylamine and sprayed at a flow rate of 2 µl/min. Capillary entrance voltage was set to 3.8 kV and dry gas temperature to 150°C.
The spectra, which showed several charge states for each component, were charge-deconvoluted. The mass numbers given refer to the monoisotopic molecular masses.
CSD was induced by increasing the capillary exit voltage from 100 V to 350 V.
ESI QTOF MS
ESI QTOF MS was performed in the negative and positive ion mode using a QTOF Ultima Instrument (Waters, Milford, CT). Samples were dissolved at a concentration of about 10 ng/µl in a 50:50:0.001 (v/v/v) mixture of 2-propanol, water, and triethylamine and sprayed at a flow rate of 10 µl/min. Capillary entrance voltage was set to 3.0 kV, and dry gas temperature to 120°C, Cone: 100 V, Rf lens: 80 V, MS profile [150(10%), 900(80%), ramp 10%]. The collision-induced dissociation MS/MS experiment was performed selecting the precursor ion with the first quadrupole. Argon was used as the collision gas at a pressure of 3.5 x 105 mbar.
MALDI-TOF MS
Crude extracts were analyzed on a Bruker Reflex II (Bruker-Daltonics) in the negative-ion mode in the linear and in the reflector TOF. Configuration using gentisic acid matrix. For sample preparation, 1 µl saturated gentisic acid solution was mixed with 1 µl of 50 mg · ml1 crude extract lyophilisate. One microliter of this mixture was dropped onto the MALDI target.
NMR spectroscopy
NMR spectra were obtained with a Bruker DRX Avance 600 MHz spectrometer using standard Bruker software. All recordings were made at 305 K on crude polysaccharide extracts in D2O (8 mg · ml1) after three exchanges with D2O. Chemical shifts were reported relative to internal acetone (H 2.225;
C 31.45). Two-dimensional 1H-1H COSY was recorded with data sets (t1 x t2) of 512 x 2048 points. Two-dimensional total COSY was performed in a phase-sensitive manner using a data set of 512 x 2048 points and a mixing time of 100 ms. Heteronuclear 2D 1H-13C correlation was recorded in the 1H detection mode via multiple-quantum coherence by use of data sets of 2048 x 256. For homo- and heterocorrelation experiments, 64 and 128 scans were acquired for each t1 value, respectively.
Methylation analysis
The methylation analysis was carried out using method of Ciucanu and Kerek (1984). The chloroform extract was hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates were analyzed by gas chromatographyMS.
![]() |
Acknowledgements |
---|
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Carlson, R.W., Sanders, R.E., Napoli, C., and Albersheim, P. (1978) Host symbiont interaction III. Purification and partial characterisation of Rhizobium lipopolysaccharides. Plant Physiol., 62, 912917.[ISI]
Carlson, R.W., Reuhs, B.L., Forsberg, L.S., and Kannenberg E.L. (1999) Rhizobial cell surface carbohydrates: their structure, biosynthesis and functions. In Goldberg, J.B. (Ed.), Genetics of bacterial polysaccharides. CRC Press, Boca Raton, Fl.
Ciucanu, I. and Kerek, F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209217.[CrossRef][ISI]
Corzo, J., Perez-Galdona, R., Leon-Barrios, M., and Gutierrez-Navarro, A.M. (1991) Alcian blue fixation allows silver staining of the isolated polysaccharide component of bacterial lipopolysaccharides in polyacrylamide gels. Electrophoresis, 12(6), 439441.[ISI][Medline]
Fischer, W., Schmidt, M.A., Jan, B., and Jan, K. (1982) Structure of the Escherichia coli K2 capsular antigen. Stereochemical configuration of the glycerophosphate and distribution of the galactopyranosyl and galactofuranosyl residues. Biochemistry, 21(6), 12791281.[ISI][Medline]
Fraysse, N., Couderc, F., and Poinsot, V. (2003). Surface polysaccharide involvement in establishing the rhizobium-legume symbiosis. Eur. J. Biochem., 270, 13651380.
Gotschlich, E.C., Fraser, B.A., Nishimura, O., Robbins, J.B., and Liu, T.Y. (1981) Lipid on capsular polysaccharides of gram negative bacteria. J. Biol. Chem., 256, 89158921.
Kanipes, M.I., Kalb, S.R., Cotter, R.J., Hozbor, D.F., Lagares, A., and Raetz, C.R. (2003). Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC. Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants. J. Biol. Chem., 278, 1636516371.
Kiss, E., Reuhs, B.L., Kim, J.S., Kereszt, A., Petrovics, G., Putnoky, P., Dusha, I., Carlson, R.W., and Kondorosi, A. (1997) The rkpGHI and -J genes are involved in capsular polysaccharide production by Rhizobium meliloti. J. Bacteriol., 179(7), 21322140.
Kiss, E., Kereszt, A., Barta, F., Stephens, S., Reuhs, B.L., Kondorosi, A., and Putnoky, P. (2001) The rkp-3 gene region of Sinorhizobium meliloti Rm41 contains strain-specific genes that determine K antigen structure. Mol. Plant. Microbe Interact., 14(12), 13951403.[ISI][Medline]
Noel, K.D. and Duelli, D.M. (2000) Rhizobium polysaccharide and its role in symbiosis. In Triplett, E.W. (Ed.), Prokariotic nitrogen fixation: a model system for analyse of biological process. Horizon Scientific Press, Wymondham, UK, pp 415431.
Pellock, B.J., Cheng, H.P., and Walker, G.C. (2000) Alfalfa root nodule invasion efficiency is dependent on Sinorhizobium meliloti polysaccharides. J. Bacteriol., 182, 43104318.
Petrovics, G., Putnoky, P., Reuhs, B.L., Kim, J., Thorp, T.A., Noel, K.D., Carlson, R.W., and Kondorosi, A. (1993) The presence of a novel type of surface polysaccharide in Rhizobium meliloti requires a new fatty acid synthase like gene cluster involved in symbiotic nodule development. Mol. Microbiol., 8(6), 10831094.[ISI][Medline]
Pulfer, M. and Murphy, R.C. (2003) Electrospray mass spectrometry of phospholipids. Mass Spectrom. Rev., 22, 332364.[CrossRef][ISI][Medline]
Putnoky, P., Petrovics, G., Kereszt, A., Grosskopf, E., Ha, D.T.C., Banfalvi, Z., and Kondorosi, A. (1990) Rhizobium meliloti lipopolysaccharide and exopolysaccharide can have the same function in the plant-bacterium interaction. J. Bacteriol., 172, 54505458.[ISI][Medline]
Reuhs, B.L., Carlson, R.W., and Kim, J.S. (1993) Rhizobium fredii and Rhizobium meliloti produce 3-deoxy-D-manno-2-octulosonic acid-containing polysaccharides that are structurally analoguous to group II K antigens (capsular polysaccharides) found in Escherichia coli. J. Bact., 175(11), 35703580.[Abstract]
Reuhs, B.L., Kim, J.S., Badgett, A., and Carlson R.W. (1994) Production of cell-associated polysaccharides of Rhizobium fredii USDA205 is modulated by apigenin and host root extract. Mol. Plant. Microbe Interact., 7, 240247.[ISI][Medline]
Reuhs, B.L., Williams, M.N.V., Kim, J.S., Carlson, R.W., and Cote, F. (1995) Suppression of the Fix 2 phenotype of Rhizobium meliloti exoB mutants by lpsZ is correlated to a modified expression of the K-polysaccharide. J. Bacteriol., 177, 42894296.
Reuhs, B.L., Geller, D.P., Kim J.S., Fox J.E., Kolli V.S., and Pueppke S.G. (1998) Sinorhizobium fredii and Sinorhizobium meliloti produce structurally conserved lipopolysaccharides and strain-specific K antigens. Appl. Environ. Microbiol., 64, 49304938.
Reuhs, B.L., Stephens, S.B., Geller, D.P., Kim, J.S., Glenn, J., Przytycki, J., and Ojanen-Reuhs, T. (1999) Epitope identification for a panel of anti-Sinorizobium meliloti monoclonal antibodies and application to the analysis of K-antigen and LPS from bacteroids. Appl. Env. Microbiol., 65, 51865191.
Schmidt, M.A. and Jann, K. (1984) Phospholipid substitution of capsular (K) antigens from Escherichia coli causing extra-intestinal infections FEMS Microbiol. Lett., 14, 6974.
Schwudke, D., Linscheid, M., Strauch, E., Appel, B., Zähringer, U., Moll, H., Müller, M., Brecker, L., Gronow, S., and Lindner, B. (2003) The obligate predatory Bdellovibrio bacteriovorus possesses a neutral lipid A containing alpha-D-mannoses that replace phosphate residues: similarities and differences between the lipid As and the lipopolysaccharides of the wild type strain B. bacteriovorus HD100 and its host-independent derivative HI100. J. Biol. Chem., 278, 2750227512.
Spaink, H.P. (2000) Root nodulation and infection factors produced by rhizobial bacteria. Annu. Rev. Microbiol., 54, 257288.[CrossRef][ISI][Medline]
Tsai, C.M. and Frasch, C.E. (1982) A sensitive silver stain for detecting LPS in polyacrylamide gels. Anal. Biochem., 119, 115119.[CrossRef][ISI][Medline]
Unger, F.M. (1981) The chemistry and biological significance of 3-deoxy-D-manno-2-octulosonic acid (KDO). Adv. Carbohydr. Chem. Biochem., 38, 323388.
Westphal, O. and Jann, K. (1965) Bacterial lipopolysaccharides. Methods Carbohydr. Chem., 5, 8391.
Williams, M.N.V., Hollingsworth, R.I., Klein, S., and Signer, E.R. (1990) The symbiotic defect of Rhizobium meliloti exopolysaccharide mutants is suppressed by lpsZ+, a gene involved in lipopolysaccharide synthesis. J. Bacteriol., 172, 26222632.[ISI][Medline]
|