Structural characterization of neutral and anionic glucans from Mesorhizobium loti

doi:10.1016/j.carres.2008.07.007

Carbohydrate Research

Volume 343, Issue 14, 22 September 2008, Pages 2422-2427

https://doi.org/10.1016/j.carres.2008.07.007 Get rights and content

Abstract

The periplasmic glucans of Mesorhizobium loti were isolated and separated into fractions according to their acidity. NMR spectroscopy confirmed their backbone structure to be a cyclic β-(1→2)-d-glucan as in the case of other rhizobia, and revealed no non-glycosidic substituents in the neutral fraction, and glycerophosphoryl and succinyl residues as major and minor substituents, respectively, in the anionic fractions. MALDI-TOF mass spectrometry showed that the anionic glucans contain one, two, or three such substituents per molecule according to their acidity, and, in contrast, that all the anionic subfractions have a similar size distribution to that of the neutral glucans, where molecules composed of 20–24 glucosyl residues are predominant. These results clarify the periplasmic glucan composition in terms of charge-to-mass ratios in M. loti cells.

Graphical abstract

Introduction

Gram-negative bacteria generally contain low-molecular-weight glucose polymers in the periplasm. Despite their structural diversity, such periplasmic glucans are considered in common to play an essential role in the envelope organization, and mutants defective in the biosynthesis show a pleiotropic phenotype including a defect in hypo-osmotic adaptation.¹ Moreover, in the case of pathogenic or symbiotic bacteria, periplasmic glucans are known to be crucial for the interaction with their eukaryotic hosts; as an example of their roles, periplasmic glucans were reported to suppress the host defense/immune mechanisms for plant symbiont Bradyrhizobium japonicum, mammalian pathogen Brucella abortus, and plant pathogen Xanthomonas campestris pv. campestris.2, 3, 4 In the case of Brucella, it has been proposed that the function of the molecule is to extract cholesterol from eukaryotic membranes so as to disrupt cholesterol-rich lipid rafts present on phagosomal membranes.⁵ As for the family Rhizobiaceae, which includes various pathogenic or symbiotic species, cyclic β-(1→2)-d-glucans have been determined to be their periplasmic glucans from the structural analyses with various bacteria such as Agrobacterium, Brucella, Mesorhizobium, Rhizobium, and Sinorhizobium species.6, 7, 8, 9, 10, 11, 12, 13 The molecules consist of β-(1→2)-linked d-glucosyl residues with the degrees of polymerization (DPs) ranging between 17 and 28, and some residues are modified by non-glycosidic substituents; the substituents are phosphoglycerol in A. tumefaciens,¹⁴ succinic acid in B. abortus,¹⁵ both in S. meliloti,¹⁶ and both methylmalonic acid and succinic acid in R. radiobacter,¹⁷ but no substituents in M. huakuii.¹³ Such substituents confer negative charge on the glucans, which would lead to the change in cell-envelope characteristics. Whereas the anionic glucans are divided into more than one fraction according to the acidity within one organism, the charge-to-mass ratio has not been accurately characterized for each of the fractions.

Rhizobia are distinguished by the symbiotic nitrogen fixation with leguminous plants. S. meliloti is accepted as a model species for molecular genetics that establishes symbiosis with plants forming indeterminate-type nodules (e.g., alfalfa), the one of the major nodule types. The mutants for cyclic β-(1→2)-d-glucan synthesis were shown to be defective in the infection of host plants.¹⁸Mesorhizobium loti is another model species that establishes symbiosis with plants forming determinate-type nodules (e.g., Lotus japonicus), the other of the major nodule types. Its periplasmic glucans, however, have not been structurally characterized, whereas some M. loti studies dealt with the glucans through the chromatographic analyses.19, 20 We reported a novel M. loti mutant that shows defective infection of L. japonicus. It was suggested that the cep gene product, designated in that work, acts on a successful symbiosis by affecting the content of periplasmic glucans through the genetic and chromatographic analyses.²¹ Thus, we attempted in this work to determine the structure of the glucans, with special regard to the charge-to-mass ratios of the molecules.

Section snippets

M. loti contains cyclic β-(1→2)-d-glucans partially substituted with phosphoglycerol and succinic acid

M. loti cells were extracted with 70% ethanol, and the extract was subjected to gel-filtration chromatography. The glucan fractions, which occurred as a major peak having a K_av of 0.45–0.7 as previously shown,²¹ were pooled and then analyzed by anion-exchange chromatography. The chromatogram displayed five peaks, indicating one neutral (N) and four anionic (A1 to A4) subfractions (Fig. 1). This elution profile is similar to that reported previously except for an additional peak (A4).²¹ Each of

Bacterial strain and culture condition

M. loti ML001, a streptomycin-resistant derivative of M. loti wild-type strain MAFF303099,²¹ was grown at 30 °C with shaking in glutamic acid–d-mannitol–salts medium with a modification of the d-mannitol concentration to 1.0 g/L.⁷

Isolation of neutral and anionic glucans

M. loti cell pellets harvested from 8-L culture grown to an optical density at 660 nm of 0.7 were extracted with 240 mL of 70% (v/v) ethanol at 70 °C for 1 h. The extract was subjected to gel filtration on a HiPrep 16/60 Sephacryl S-100 HR column (1.6 × 60 cm; GE Healthcare).

Acknowledgments

We thank Mrs. Teiko Yamada (Tohoku University) for technical assistance on NMR spectroscopy. This work was supported in part by Grant-in-Aid for Scientific Research (No. 19580077) from Japan Society for the Promotion of Science, and by Grant-in-Aid for Scientific Research on Priority Areas ‘Comparative Genomics’ from the Ministry of Education, Culture, Sports, Science and Technology.

References (28)

J.-P. Bohin
FEMS Microbiol. Lett.
(2000)
J. Celli
Res. Microbiol.
(2006)
A. Dell et al.
Carbohydr. Res.
(1983)
L.P.T.M. Zevenhuizen et al.
Carbohydr. Res.
(1983)
M. Batley et al.
Biochim. Biophys. Acta
(1987)
M. Hisamatsu
Carbohydr. Res.
(1992)
K.J. Miller et al.
Biochim. Biophys. Acta
(1987)
Y. Jung et al.
Carbohydr. Res.
(2005)
A.A. Bhagwat et al.
Plant Physiol.
(1999)
B. Arellano-Reynoso et al.
Nat. Immunol.
(2005)

L.A. Rigano et al.

Plant Cell

(2007)

D.R. Bundle et al.

Infect. Immun.

(1988)

L.P.T.M. Zevenhuizen et al.

Anton. Leeuw.

(1990)

M.W. Breedveld et al.

J. Bacteriol.

(1993)

Cited by (15)

Signal molecules and cell-surface components involved in early stages of the legume-rhizobium interactions
2015, Applied Soil Ecology
Citation Excerpt :
The inhibition of CG biosynthesis during the growth in high-osmolarity media indicates a role of these oligomers in bacterial adaptation to low-osmolarity conditions. Similarly to S. meliloti, M. loti, M. huakuii, and Agrobacterium tumefaciens produce the β-glucan type, in which d-glucose monomers are linked by β-(1 → 2)-glycosidic bonds (Fraysse et al., 2003; Choma and Komaniecka, 2003; Kawaharada et al., 2008). On the other hand, Bradyrhizobium species such as B. japonicum, B. elkanii, B. liaoningense, and B. yuanmingense produce β-(1 → 3); β-(1 → 6) type CGs, which are substituted by phosphocholine and highly decorated with acetate and succinate (Choma and Komaniecka, 2011).
Legumes are a highly important source of food, feed, and biofuel crops. With a few exceptions, these plants can enter into a complex symbiotic relationship with specific soil bacteria collectively called rhizobia. This interaction leads to formation of a new, highly specialized root organ – the nodule, inside which bacteria, differentiated into bacteroids, reduce atmospheric dinitrogen into forms of nitrogen that are useable by the plant. The legume–rhizobium association is highly specific and tightly controlled mainly by the host plant, in such a way that a particular legume interacts with only a limited set of rhizobial species. The establishment of symbiosis is a complex process, which involves a coordinated exchange of multiple signals between the host plant and its microsymbiont; among them, flavonoids secreted by legume roots and rhizobial lipochitin oligosaccharides (Nod factors) play a crucial role. Also, other plant-derived and bacterial cell-surface components and low-molecular-weight metabolites are engaged in the signaling. Among these, there are several proteins derived from the host plants (Nod factor receptors, signal transduction cascade proteins, lectins, trifolins, remorins), non-flavonoid inducers of rhizobial nod genes, H₂O₂, NO, and phytohormones. Additionally, rhizobia are actively involved in extracellular signaling to their host legumes to initiate infection and nodule morphogenesis. These bacteria produce and secrete many different compounds including hopanoids, indole-3-acetic acid, quorum sensors, bradyoxetin, lumichrome, H₂O₂ and NO, several types of surface polysaccharides (extracellular polysaccharide, lipopolysaccharide, capsular polysaccharide, cyclic β-glucan, glucomannan, gel-forming-polysaccharide, cellulose), and proteins secreted via types I and III secretion systems (glycanases, rhicadhesins, NodO, and Nops – nodulation outer proteins). They all contribute to various stages of symbiotic interactions, e.g., attachment to roots, host recognition, infection thread formation, and invasion of nodules. This review summarizes many aspects of the roles of extracellular signals, proteins, and polysaccharides in the early stages of legume–rhizobium symbiosis, showing high complexity in this “molecular dialog” and its interconnection with other cellular processes.
Characterization of cyclic β-glucans of Bradyrhizobium by MALDI-TOF mass spectrometry
2011, Carbohydrate Research
Citation Excerpt :
As mentioned above, the 13C NMR spectra shown in the article by Miller et al.7 have indicated that the B. japonicum 32H1 cyclic β-glucan was highly substituted in comparison to B. japonicum USDA 110 glucans.7 A recently published work by Kawaharada et al.19 has proven that not only Bradyrhizobium but also Mesorhizobium loti can produce anionic glucans multisubstituted with succinyl and phosphoglycerol residues. Three genes coding the biosynthesis pathway of β-glucans have been identified in B. japonicum (ndvB, ndvC, and ndvD).1,9
Periplasmic, cyclic β-glucans isolated from Bradyrhizobium elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium yuanmingense strains have been investigated by means of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), 1D and 2D nuclear magnetic resonance (NMR), as well as standard chemical methods. These compounds are built of 10–13 d-glucose residues. The main fractions contain molecules assembled of 12 hexose units (M_w = 1945.363 Da). Glucose monomers are linked by β-(1→3) or β-(1→6) glycosidic bonds. The ratio of β-(1→3) to β-(1→6) linked glucose is approximately 1:2. Moreover, methylation analysis demonstrated the presence of terminal, non-reducing, as well as branched (i.e., 3- and 6-substituted) glucoses. Thus, the basic structure of the investigated compounds is similar to that of periplasmic oligosaccharides from Bradyrhizobium japonicum and Azorhizobium caulinodans strains. The analyzed cyclic β-glucans are substituted by phosphocholine (PC) (one or two residues per ring) and highly decorated with acetate and succinate. The substituents are arranged diversely in the population of cyclic β-glucan molecules. The concentrations of cyclic β-glucans in Bradyrhizobium periplasmic space are osmotically regulated and increase in response to a decrease of medium osmolarity.
Structure, Physicochemical Properties and Biological Activity of Lipopolysaccharide from the Rhizospheric Bacterium Ochrobactrum quorumnocens T1Kr02, Containing d-Fucose Residues
2024, International Journal of Molecular Sciences
New two-stage pH combined with dissolved oxygen control strategy for cyclic β-1,2 glucans synthesis
2023, Applied Microbiology and Biotechnology
Structural characterization of glycerophosphorylated and succinylated cyclic β-(1→2)-d-glucan produced by sinorhizobium mliloti 1021
2020, Polymers
Synthesis, characterization, and biological activity of aminated zymosan
2020, ACS Omega

View all citing articles on Scopus

View full text

Structural characterization of neutral and anionic glucans from Mesorhizobium loti

Abstract

Graphical abstract

Introduction

Section snippets

M. loti contains cyclic β-(1→2)-d-glucans partially substituted with phosphoglycerol and succinic acid

Bacterial strain and culture condition

Isolation of neutral and anionic glucans

Acknowledgments

FEMS Microbiol. Lett.

Res. Microbiol.

Carbohydr. Res.

Carbohydr. Res.

Biochim. Biophys. Acta

Carbohydr. Res.

Biochim. Biophys. Acta

Carbohydr. Res.

Plant Physiol.

Nat. Immunol.

Plant Cell

Infect. Immun.

Anton. Leeuw.

J. Bacteriol.