concentrations as high as 5 to 20% of the total cellular dry weight under certain culture conditions, the cyclic ,B-glucans are major cellular constituents. In Agrobacterium and Rhizobium species, these molecules contain glucose residues linked solely by ,B-(1,2) glycosidic bonds. However, in Bradyrhizobium species, the glucose residues are linked by both ,B-(1,3) and P-(1,6) glycosidic bonds. While these molecules contain glucose as the only hexose monomer, they may become highly substituted with nonsugar moieties such as sn-1-phosphoglycerol. The degree of polymerization (DP) of the cyclic ,B-glucans ranges from 10 to 13 in Bradyrhizobium japonicum to 17 to 40 in Rhizobium meliloti. The first report of cyclic P-glucans came in 1942 with their discovery in the extracellular media of Agrobacterium tumefaciens cultures (83). These glucans, originally referred to as crown gall polysaccharides, were described as low-molecularmass glucose polymers of around 3,600 Da. Since that report, cyclic ,B-glucans have been found in cultures of all Agrobacte

In standard liquid medium containing 5 g mamitoll-' and 1 g glutamic acid 1-l, Rhizobium mefifoti SU-47 cells accumulated 350 mg cyclic 1,2-Q-glucans (g protein)-'. The cyclic glucans were 36 % glycerol-1-phosphatesubstituted and 64% were uncharged. In the same medium with 10 g mamitoll-' , repeating units of succinoglycan (1110 mg 1-l) were found as extracellular carbohydrates, and only low amounts of the succinoglycan polymer (up to 300 mg 1-l) were excreted. By raising the osmotic pressure of the medium by the addition of NaCl or other ionic and non-ionic osmolytes, succinoglycan production could be stimulated: up to 2.4 g 1-1 at 0.2 M-NaCI was produced at the expense of the repeating units. Above 0.2 M-NaCl growth was slowed down, and succinoglycan excretion diminished. At 1 M-NaCl growth stopped completely. In standard medium containing 0.6 M-NaCl the amount of cellular cyclic l,2-Q-glucans was lowered to 150 mg (g protein)-l out of which the glycerol-1-phosphatesubstituted glucan fraction was reduced to 15%. Instead, high amounts of oligosaccharides were synthesized as osmoprotectants, with trehalose as the major component [up to 200 mg (g protein)-']. Glycogen synthesis was completely suppressed at this salt concentration, while p l y Q-hydroxybutyric acid synthesis was unaffected.

In this study, we characterized four Tn5 mutants derived from Rhizobium leguminosarum RBL5515 with respect to synthesis and secretion of cellulose fibrils, extracellular polysaccharides (EPS), capsular polysaccharides, and cyclic beta-(1,2)-glucans. One mutant, strain RBL5515 exo-344::Tn5, synthesizes residual amounts of EPS, the repeating unit of which lacks the terminal galactose molecule and the substituents attached to it. On basis of the polysaccharide production pattern of strain RBL5515 exo-344::Tn5, the structural features of the polysaccharides synthesized, and the results of an analysis of the enzyme activities involved, we hypothesize that this strain is affected in a galactose transferase involved in the synthesis of EPS only. All four mutants failed to nodulate plants belonging to the pea cross-inoculation group; on Vicia sativa they induced root hair deformation and rare abortive infection threads. All of the mutants appeared to be pleiotropic, since in addition to defects in the synthesis of EPS, lipopolysaccharide, and/or capsular polysaccharides significant increases in the synthesis and secretion of cyclic beta-(1,2)-glucans were observed. We concluded that it is impossible to correlate a defect in the synthesis of a particular polysaccharide with nodulation characteristics.

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