QUARTERLY Characterisation of Mesorhizobium huakuii cyclic �-glucan

Periplasmic and extracellular glucans of Mesorhizobium huakuii were isolated and characterized by compositional and MALDI-TOF analyses, as well as 1H and 13C NMR spectroscopy. It was shown that M. huakuii produces a cyclic beta-glucan composed entirely of nonbranched glucose chains and unmodified by nonsugar substituents. The degree of polymerisation of the cyclic oligosaccharides was estimated to be in the range from 17 to 28. The most abundant glucan molecules contained 22 glucose residues. Glucose residues within the glucan were connected by beta-(1,2) glycosidic linkages. The cyclic glucan produced by M. huakuii is quite similar to the periplasmic beta-(1,2) glucans synthesized by Agrobacterium and Sinorhizobium genera. The synthesis of beta-glucan in M. huakuii is osmoregulated and this glucan could function as an osmoprotectant in free living cells.

A specific compartment between the cytosol and the outer membrane, named periplasmic space, exists in Gram-negative bacteria.This compartment tends to remain isoosmotic with the cytosol over a range of extracellular conditions (Stock et al., 1977), a function that is accomplished by specific oligosaccharides.In E. coli, those oligosaccharides were named MDO (membrane-derived oligosaccharides) (Schulman & Kennedy, 1979).MDO are linear molecules composed of 6 to 8 glucose residues linked by b-(1,2) and branched by b-(1,6)-glycosidic bonds and substituted with phosphoglycerol, phosphoethanolamine and succinyl residues (Schneider et al., 1979).
The best known OPGs from Sinorhizobium meliloti and Agrobacterium tumefaciens are composed of 17 to 25 b-(1,2)-linked D-glucose residues that may be substituted with phosphoglycerol, succinate or methylmalonate groups.In S. meliloti cells sn-1-phosphoglycerol, linked to position 6 of glucose, is the predominant substituent of cyclic glucans.The sn-1-phosphoglycerol is derived from the head group of cytoplasmic membrane phosphatidylglycerol.A specific transferase acts during the transport of neutral glucans across the outer membrane.
The knowledge about mesorhizobial glucan structure and its biosynthesis is limited to a few publications.Slow growing strain of Rhizobium loti synthesises an oligosaccharide resembling the glucan from B. japonicum (Estrella et al., 2000).A fast growing strain of R. loti isolated from Lotus tenuis was found to synthesise b-(1,2) glucan typical for Rhizobium genera (Lepek et al., 1990).Rhizobium huakuii My6 produces and excretes oligosaccharides that are structurally identical to those of the fast growing R. loti (Hisamatsu et al., 1997).However, the results described in that paper, were based only on a comparison of the chromatographic profile of isolated My6 glucan to the chromatographic profiles of glucan preparations of known structure.
In this report we present the results of structural studies on the periplasmic and extracellular oligosaccharides from Mesorhizobium huakuii.The effect of osmotic strength of the growth medium on glucan biosynthesis is described.Our investigation of the b-glucan synthesised by M. huakuii IFO 12543 T completes the previous work of Hisamatsu et al. (1997) on mesorhizobial secreted polysaccharides.We show that M. huakuii accumulates b-glucan within the periplasm and exports some of it to the environment without biochemical modification.

MATERIALS AND METHODS
Mesorhizobium huakuii IFO 15243 T is the type strain from the Culture Collection of the Institute for Fermentation (Osaka, Japan).This strain was isolated from Astragalus sinicus in China and has the original denotation CCBAU 2603 (Nuswantara et al., 1997).
Bacteria were grown in liquid mannitol-yeast extract medium 79CA (Vincent, 1970) and were aerated by vigorous shaking.Cells and culture supernatant were separated by centrifugation.The cell pellet was washed twice with saline and once with distilled water.Cell-associated glucans were liberated from the periplasmic space by a modified Bligh and Dyer extraction procedure (Miller et al., 1988).Aqueous methanol fractions were concentrated by rotary evaporation and chromatographed on a Sephadex G50 column (1.6 ´80 cm).The column was washed with 0.15 M ammonium acetate (pH 7.0) containing 7% (v/v) propanol.The flow rate was 15 ml h -1 (Miller et al., 1990).Fractions (1 ml) were collected and assayed for total carbohydrate by the phenol-sulfuric acid method (Dubois et al., 1956).The fractions containing oligosaccharides were pooled, concentrated and desalted on a Bio-Gel P2 column (1.6 60 cm) (Bio-Rad) with 7% propanol in water as the eluent.The desalted samples were then separated on a DEAE-cellulose column (1 27 cm) (DE 23; Whatman).After application of the sample, the column was washed with 40 ml of 10 mM Tris/HCl (pH 8.4).Next, 100 ml gradient was applied, starting with 0 mM KCl and ending with 200 mM KCl in the same Tris/HCl buffer (Miller et al, 1988;1990).The flow rate was 24 ml h -1 .Fractions (2 ml) were assayed for total carbohydrate by the phenol-sulfuric acid method.Carbohydrate containing fractions were collected and dialysed against distilled water.
Culture supernatant was condensed fourfold by rotary evaporation.The high-molecular-mass polysaccharides (EPS) were precipitated by adding 3 vol. of ethanol, as described previously (Bhagwat et al., 1999;Lee et al., 2001), whereas low-molecular-mass oligosaccharides remained in the supernatant (75% alcohol).The material from the supernatant was concentrated and separated using Bio-Gel P2 column chromatography (1.6 ´57 cm) with 7% propanol in water (v/v) as the eluent, to remove medium salts.The flow rate was 15 ml h -1 .Fractions (3 ml) were collected and sugar compounds were determined.The glucan containing fraction was further analysed in the same way as cell-associated glucan.
In order to examine the effects of growth in a high-osmotic-strength medium on glucan synthesis, 0.25 M NaCl, 0.5 M mannitol or 0.5 M glucose was introduced into the 79CA medium.Bacterial cells from 6-day-old cultures were centrifuged, washed twice with saline and once with distilled water.The amount of total cellular protein was determined according to Lowry (Lowry et al., 1951) using bovine serum albumin as a standard.To determine the amount of periplasmic glucan cell pellets were subjected to the procedure described by Breedveld and co-workers (Breedveld et al., 1993).Finally, all 50% alcohol extracts that contained periplasmic glucans were quantified for carbohydrates by the phenol-sulfuric acid procedure.
For compositional analysis, glucan samples were hydrolysed with 2 M TFA (100°C, 4 h) and the liberated sugars were converted into alditol acetates.
Methylation analysis was done according to a modified Hakomori procedure, as described by York and co-workers (York et al., 1986).Part of the methylated sample was hydrolysed with 2 M TFA, reduced with NaBD 4 , peracetylated and then partially methylated alditol acetates were analysed by GC-MS.The remaining part of the material was analysed by MALDI-TOF (matrix-assisted laser desorption ionisation -time of flight mass spectrometry).
Alditol acetates and partially methylated alditol acetates were analysed using a Hewlett-Packard gas chromatograph (model HP 5890A) coupled to a mass selective detector (MSD HP 5971).The gas chromatograph was equipped with a capillary column (HP-5MS, 30 m ´0.25 mm) and helium was the carrier gas (flow rate was 0.7 ml min -1 ).For the alditol acetate analysis the temperature program was as follows: 150°C for 5 min, then raised to 310°C (5 o C min -1 ) and the final temp.was 310°C for 20 min.For analysis of partially methylated alditol acetates the temperature program was: 70°C for 2 min, then raised to 150°C (50°C min -1 ) followed by 3°C min -1 to the final temperature of 310°C.MALDI-TOF analysis was performed on a Voyager -Elite (PE Biosystems) instrument in positive-ion mode.The methylated samples of glucan were dissolved in chloroform/methanol (2/1: v/v), then mixed with a matrix of gentisic acid solution in acetonitrile (50%, w/v).The solvents were allowed to dry.Samples were desorbed with a nitrogen laser, and a 20 kV extraction voltage was used.The spectra obtained are the average of 256 (periplasmic glucan sample) or 122 (extracellular glucan preparation) scans.
1-D and 2-D 1 H NMR, as well as 13 C NMR experiments were carried out on a Bruker 300 instrument at 313°K using standard software.Samples were dissolved in D 2 O. Acetone was the internal standard (d H = 2.225 p.p.m., d C = 31.45p.p.m.).

RESULTS AND DISCUSSION
The purified oligosaccharides from the periplasm of M. huakuii cells and from culture supernatant were separated by ion-exchange chromatography using a DEAE-cellulose column.Fractions rich in glucose were found to elute prior to the applied KCl gradient.This is indicative of a neutral polymer (Fig. 1).In contrast to mesorhizobial periplasmic oligosaccharides, glucan molecules isolated from Agrobacterium or Sinorhizobium were found to be partly substituted by phosphoglycerol, phosphoethanolamine or succinate residues and, having a highly anionic character, were retarded by anionic chromatographic columns (Breedweld & Miller, 1994;Miller et al., 1988).In the extracellular glucan fraction, small quantities of galactose and ribose accompanying the main component, glucose, were identified.These contaminants could derive from LMW-EPS, as was described in R. huakuii My6 (Hisamatsu et al., 1997).
The range of molecular mass of the cell-associated glucan preparations was determined by positive-ion MALDI-TOF spectrum of the methylated sample (Fig. 2).This analysis re-vealed the presence of several sodium-cationized molecular ions [M+Na] + .The pseudomolecular ion species had masses identical to those expected for a cyclic form of glucan composed of 17 to 28 glucose residues.Among them, the ion at m/z 4517 was the most intense.This signal represented the main glucan species containing 22 glucose residues within the ring.The theoretically calculated mass for native glucan is 3567.102Da and for its methylated form it is 4492.884Da.The pattern of molecular masses obtained for the extracellular glucan fraction was identical to that obtained for the periplasmic glucan sample.Similar ranges of the ring size were observed in the case of Rhizobium, Sinorhizobium and Agrobacterium cyclic glucans.It is worth noticing that the major glucan species, containing 22 glucose residues, is close to the range typical for glucans dominating in Rhizobium and Agrobacterium (19-20 glucose residues per ring) (York et al., 1980;Batley et al., 1987;Hisamatsu et al., 1987).GC-MS analysis of permethylated alditol acetates derived from both periplasmic and extracellular glucan preparations indicated the presence of only 1,2-linked glucose residues (not shown).In GC analysis neither peaks from fully methylated nor from branched glucose residues were detected.Those results indicate that the periplasmic and extracellular glucans are not branched and that those molecules are cyclic.
In the anomeric region of the 1 H NMR spectrum, overlaping dublets at 4.9 p.p.m. were found.The shift values and their coupling constants (J 1,2 about 8 Hz) are characteristic for b-configuration of all glucose residues.Signals between 3.55 p.p.m. and 3.60 p.p.m. correspond to the H2 protons involved in b-(1,2) linkages.Chemical shifts attributed to other protons in the glucan molecule are listed in Table 1.All cross-peaks are indicated on the COSY spectrum (Fig. 3A).On the TOCSY spectrum (Fig. 3B), the glucose spin system starting from the anomeric proton is traced using a solid line. 13C NMR analysis of the periplasmic glucan sample revealed a peak cluster of C1 resonance at 102.7 p.p.m. (Fig. 4).The presence of signals with a chemical shift above 102 p.p.m. indicates b configuration of glucose (Usui et al., 1973).There are no signals at 92 to 96 p.p.m. characteristic for the C1 resonances of reducing glucose residues.These results confirm the previous observation that the glucan molecules are cyclic and all glucose residues are b-linked.Resonances below 60 p.p.m. were not observed, indicating the absence of nonsugar substituents, e.g.phosphoglycerol or acetyl residues.The resonances at 82-83 p.p.m. were assigned to C2 implicated in b-(1,2) glycosidic linkages.The absence of a signal at 73 to 74 p.p.m. indicates that all C2 carbons are involved in glycosidic bonds (Dell, 1983).The multiple resonances of C1 and C2 signals derive from cyclic glucan molecules of a wide size range (17 to 28 residues) and are due to the slightly different angles between the adjacent glucosyl residues.All assignments in 13 C NMR (Table 1, Fig. 4) were confirmed by a 13 C-decoupled, 1 H-detected HSQC analysis (spectrum not shown).
The above conclusion was confirmed by NOESY experiment.The NOESY spectrum revealed a strong transglycosidic correlation between H1 (4.89 p.p.m.) and H2 (3.57p.p.m.) (Fig. 3C).This correlation signal was accompanied by two cross-peaks, which represented intramolecular interaction between H1 and H3, as well as between H1 and H5 protons.
The results of 1 H and 13 C NMR experiments showed that mesorhizobial periplasmic glucan and the glucan exported to the environment represent the same type of oligosaccharides as the glucans from Rhizobium, Sinorhizobium and Agrobacterium species.However, in contrast to most rhizobial glucans, the glucans from M. huakuii were found to be unsubstituted.
It is well known that B. japonicum, A. tumefaciens, as well as most of S. meliloti and Rhizobium strains, when grow in a high-osmotic pressure medium, are strongly inhibited in the synthesis and accumulation of periplasmic cyclic glucans (Miller et al., 1986;Dylan et al., 1990).M. huakuii glucan synthesis in the presence of high concentration of different osmolites was examined.The results are listed in Table 2.The quantity of glucans determined in bacteria grown in a medium supplemented with 0.5 M glucose, 0.5 M mannitol or 0.25 M NaCl was considerably reduced.In the presence of glucose and NaCl even a two-fold reduction of the quantity of glucans was observed.It is known that the biosynthesis of periplasmic b-glucans in bacterial cells may increase when the osmolarity of the medium is low, and decrease, even in the presence of osmoprotectants, when the medium osmolarity is raised.This type of osmoregulation is observed in Escherichia coli, Rhodobacter sphaeroides or Erwinia chrysanthemi (Bohin, 2000;Cogez et al., 2001;Talaga et al., 2002).The results of our studies indicate that biosynthesis of cyclic glucans by M. huakuii is osmotic pressure dependent.The observation that the periplasmic pool of b-glucans is highly dependent on osmolarity of the environment suggests that the mesorhizobial cyclic b-glucan can function as an osmoprotectant during a free-state growth of this bacterium.
The hypothesis concerning the osmoprotective function of cyclic glucans does not work in the case of bacteroids (Breedveld &  Miller, 1998).The osmotic pressure within root nodules is moderately elevated, but the level of b-glucans synthesised by bacteroids is similar to that in a low osmolarity medium.There are two possible explanations: (i) the regulation of glucan biosynthesis differs from that in the free-living Rhizobium, or (ii) the main function of those oligosaccharides is not osmoprotection.Osmoregulated periplasmic glucans seem to be universal constituents of the envelope of Gram-negative bacteria.Various functions are ascribed in the literature to the particular bacterial b-glucans.Presumably, the biological function of primary importance of these molecules remains to be determined (Talaga et al., 2002) The similarity of the b-(1,2) glucan synthesised by M. huakuii IFO 15243 T and by a fast growing R. loti strain AYAC 1 BII; (Lepek et al., 1990) shows that these bacteria are closely related to the Rhizobium, Sinorhizobium and Agrobacterium cluster.Presumably, all these bacteria possess a very similar enzymatic pathway for b-glucan synthesis.Interestingly, Estrella and co-workers (Estrella et al., 2000) described the slow growing NZP 2309 strain of Rhizobium loti that produces a b-(1,3);(1,6) cyclic glucan that is representative for bradyrhizobia.NZP 2309 differs from Bradyrhizobium USDA 110 type strain but is phylogenetically closely related.Data are the averages from three independent experiments; S, standard deviation.

Figure 2 .
Figure 2. Positive-ion MALDI-TOF mass spectra of methylated glucan sample from M. huakuii IFO 15243 T .The m/z values are reported as the nominal mass of the pseudomolecular ion [M+Na] + .DP, degree of polymerisation.

Figure 3 .
Figure 3. A. DQF-COSY spectrum of M. huakuii glucan (*signal is attributed to Tris contamination).B. Partial TOCSY spectrum of the glucan from M. huakuii.The H 1 proton ring system is marked.C. Part of NOESY spectrum.The interresidue cross peak (H 1 /H 2 *) is marked with asterisk.
Figure 4. 13 C NMR analysis of cyclic b-(1,2) glucan preparation from M. huakuii.*signals at 60.2 and 62.1 p.p.m. are attributed to Tris contamination of the sample.