Oligo- and polysaccharide synthesis by Rhizobium leguminosarum and Rhizobium meliloti

Rhizobium and Agrobacterium species are capable of synthesizing a variety of extracellular and cellular oligo- and polysaccharides. Changes in environmental conditions may all affect the composition, physical properties, and relative amounts of oligo- and polysaccharides. Interest in the field of Rhizobium polys accharides has resulted from a development in two distinct areas, (i) the role of oligo- and polysaccharides in the microbe- plant interaction, and (ii) studies on the physico- chemical properties of microbial polysaccharides with a potential technical application (CHAPTER 1).In this thesis two model strains, Rhizobium leguminosarum bv trifolii TA-1 and Rhizobium meliloti SU-47, were used to study the extent of polysaccharide-production as influenced by cultural conditions. Although this study was aimed at polysaccharide-production in general, most research was focused on the regulation of cyclic (1,2)-β-glucan synthesis. Therefore, inCHAPTER2 a summary on the chemistry, occurrence, biological function and potential applications of these compounds is given.CHAPTER3 describes the enhanced excretion of cyclic (1,2)-β-glucan by Rhizobium leguminosarum bv trifolii TA-1 as the result of (i) incubation at superoptimal temperature for growth (30-33°C) and (ii) growth at high cell densities. At 33°C, EPS and CPS production was inhibited and up to 3.9 g of cyclic (1,2)-β-glucan/l was produced in the production medium (10 g mannitol and 1 g glutamic acid/l medium) with a rate of 400 mg glucans/g protein/day by a biomass of 540 mg protein/l. At 25°C, the optimal temperature for growth of strain TA-1, EPS and CPS were the main carbohydrate products synthesized, while hardly any glucans were detected in the medium. Other R. leguminosarum strains showed a comparable "temperature-effect". In a medium containing 50 g mannitol and 10 g glutamic acid per liter high cell densities of strain TA-I (3.95 g protein/l) were obtained and 10.9 g/l of cyclic (1,2)-β-glucan within 10 days at 25°C, were excreted, while CPS production was strongly suppressed. The cyclic (1,2)-β-glucans were neutral and had degrees of polymerization (DP) ranging from 17 to 25 with DP=19 as the major glucan cycle.In production processes, aimed at high volumetric yields, high concentrations of organic nutrients and mineral salts in liquid media may lead to a considerable high osmotic pressure. Therefore, and because synthesis of cyclic glucans were found to be osmo-regulated in some members of the family of Rhizobiaceae, the influence of the osmotic pressure of the medium on the growth of and polysaccharide synthesis by R. meliloti SU-47 and R. leguminosarum bv trifolii TA-1 was studied (CHAPTERS4 and 5). The ability of members of the Rhizobiaceae to grow at high osmotic pressures of the medium, and their tolerance towards several ionic and non-ionic osmolytes, depend strongly on the species and the type of osmolyte. Strains of R. meliloti and A. tumefaciens could grow well in media up to 1 M NaCl while R. leguminosarum could only tolerate up to 0.35 M NaCl. In standard- or production medium with low osmotic pressure cells of strain SU-47 accumulated up to 350 mg cyclic (1,2)-β-glucans/g protein, of which 36% were glycerol-1-phosphate substituted and 64% were neutral. By increasing 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/l at 0.2 M NaCl), at the expense of the repeating units. Furthermore, the amount of cellular cyclic (1,2)-β-glucans was lowered, to 150 mg/g protein at 0.6 M NaCl, of which the glycerol-1-phosphate substituted glucan fraction was reduced to 15%. Instead, oligosaccharides up to 250 mg/g protein were synthesized with trehalose as the major component. Glycogen synthesis was fully suppressed at this salt concentration. No cyclic glucans were found in the medium (CHAPTER4). By increasing the osmotic pressure of the medium, the synthesis of EPS and CPS by R . leguminosarum bv trifolii TA-1 was suppressed, and cyclic glucans were excreted instead (1500- 2000 mg of glucans/l). This proceeded with a rate of 220 mg glucans/g protein/day by a biomass of 520 mg protein/l. at 0.2 M NaCl. Intracellular cyclic (1,2)-β-glucan concentrations remained at 45-100 mg/g protein during the stationary phase, independent of the osmotic strength of the medium. Parallel to the increasing osmotic pressure of the medium, the disaccharide trehalose accumulated in the cells, up to 130 mg/g protein (CHAPTER5).The response to a NaCl-shock on cellular carbohydrates of NaCl-free grown cells of R. leguminosarum bv trifolii TA-1 and R. meliloti SU- 47 was investigated in non-growing cultures in a batch-fermenter and in cell suspensions using in vivo NMR (CHAPTER6). In a glutamic acid-free medium containing NaCl TA-1 cells but especially SU-47 cells responded immediately by synthesizing trehalose, while glycogen and the external substrate mannitol were metabolized. Without mannitol in the medium trehalose synthesis was slower and parallelled the breakdown of the reserve materials glycogen and PHB. 13C-NMR experiments with 25-fold concentrated cell-suspensions using 13C 1 -mannitol as substrate revealed that 20% of the trehalose synthesized was derived from the substrate, but 80% from other sources. Therefore, trehalose synthesis occurred from the internal pool of glycogen and/or PHB, whether mannitol was present or not. Cells of strains TA- 1 and SU-47 that had accumulated trehalose metabolized this compound again in a low osmolarity environment. As trehalose is a general occurring compound found in cells of Rhizobium at much lower concentrations the higher concentrations which were measured during the conditions of osmotic stress could be explained by the role of trehalose as osmo-protectant. The cellular phosphoglycerol-substituted and neutral cyclic (1,2)-β-glucans of NaCl-free grown SU-47 cells were not degraded nor excreted after the NaCl-shock. With in vivo31P-NMR the glycerol-1-phosphate substituted cyclic glucans in cell suspensions of strain SU-47 could well be observed.InCHAPTER7 the synthesis of cyclic (1,2)-β-glucans from UDP-[ 14C]-glucose by a crude membrane preparation and whole cells of Rhizobium leguminosarum by trifolii TA-1 was investigated. The observed enhanced excretion of cyclic (1,2)-β-glucans by strain TA-1 was due to an increased permeability of the outer membrane for cyclic glucans. This was concluded from the following observations: (i) Incubation of repeatedly frozen and thawed TA-1 cells with UDP- 14C-glucose resulted in the appearance of a labelled glucan in the medium. Cells grown in the presence of NaCl and cells cultured at 33°C showed the same effect, while cells cultured at 25°C in the absence of NaCl did excrete only little cyclic glucans; (ii) the presence of 0.1 mM EDTA, a complexing agent of divalent cations which results in a weakened LPS layer, also induced glucan excretion in growing cultures of strain TA- 1; (iii) R. leguminosarum bv viciae RBL5515, exoB8 ::Tn5 mutant cells excreted a labelled glucan fraction, while the parent strain RBL-5515 did not. The mutant was affected in the production of EPS, CPS, and LPS (CHAPTER9), which all might explain the greater permeability of the mutant cells towards cyclic (1,2)-β-glucans. Hardly any difference in biosynthetic activity was observed between membrane fractions of TA-1 cells grown in the presence (0.2 M) or absence of NaCl. Glucan formation in vitro and glucan excretion by whole cells was strongly inhibited in the presence of 50 mg/ml cyclic glucan, indicating biosynthesis of cyclic (1,2)-β-glucans in strain TA-1 to be controlled by end-product inhibition. Therefore, the constant loss of glucans from osmotically-stressed TA-1 cells prevented end- product inhibition and resulted in glucan accumulation in the medium of up to 1600 mg/l. Cyclic (1,2)-β-glucans from Rhizobium meliloti and Agrobacterium tumefaciens are thought to be involved in both infection behaviour and osmo- regulation at low osmotic pressure of the medium. Combining the results obtained on the synthesis of cyclic glucans, it is doubtful that cyclic glucans of R . leguminosarum play an important role in osmo-adaptation at low osmolarity, for the following reasons: (i) cellular glucan concentrations are much lower than in R. meliloti; (ii) the great majority of the glucans in R. leguminosarum are neutral molecules, and (iii) no repression of glucan synthesis at enhanced osmolarity of the medium occurs.InCHAPTER8 Rhizobium leguminosarum bv trifolii TA-1 was used a model organism to study the influence of growth rate and medium, composition on exopolymer production. Circumstances leading to a high CPS-yield also lead often to concomitantly high EPS-production, rendering the medium viscous, and making the process of recovery of CPS more difficult. In continuous cultures, EPS was at every dilution rate (between D=0.02-0.12 h -1) the most abundant polysaccharide present in production medium, while CPS synthesis occurred only at low specific growth rates. Only low amounts of cyclic glucans were excreted (10-30 mg/l). In production medium EPS was synthetized in the active phase of growth and continued in stationary phase (up to 1.6-2.1 g/l CPS-synthesis which takes place only in stationary phase and in the presence of excess mannitol, was produced up to 1.8 g/l during batch incubation for 14 days at 25°C. Maximal CPS production was 2.9 g CPS/l medium, with 1 g protein as biomass in a medium containing 20 g/l mannitol and 2 g/l glutamic acid. The maximum specific growth rate was μ max =0.133 h -1. To washed cells at which 10 g/l mannitol was added CPS synthesis reached 2.1 g/l, but EPS- synthesis was lower (0.8 g/l).InCHAPTER9 the polysaccharide production by Rhizobium leguminosarum RBL5515 and some Tn::5 generated mutants affected in polysaccharide synthesis was studied in production medium. The EPS of the wildtype strain was composed of a K-36 type octasaccharide repeating unit with sugar composition of glucose: glucuronic acid: galactose in the ratios of 5:2:1. The CPS from the R. leguminosarum strains investigated had all a constant sugar composition of glucose: galactose: mannose in the ratios of 1:4:1. The presence or absence of either the pRL1JI or pSym5, the Sym plasmids of R. leguminosarum bv viciae and trifolii, respectively, did not influence the quantities of these polysaccharides synthesized, being comparable to strain TA-1. The production patterns of exopolysaccharides of their Tn::5 generated mutants were clearly different from most wild-type production patterns by the synthesis of altered EPS-structures, low production of CPS and/or EPS, and enhanced excretion of cyclic (1,2)-β-glucans (all mutants; 400-1000 mg/l) as compared to strain RBL5515 (50 mg/l). The mutants RBL5515, exo4 ::Tn5, RBL5515, exoB8 ::Tn5 and RBL5515, exo344 ::Tn5 formed 5-20% of wildtype EPS-level. Both mutants RBL5515, exoB8 Tn::5 and RBL5515, exo344 ::Tn5 synthesized a truncated EPS with a heptasaccharide-repeating unit missing the terminal galactose in the side chain. While the RBL5515, exoB8 ::Tn5 mutant was shown to be devoid of UDP-glucose 4' epimerase activity, the activity of this enzyme and of other enzymes involved in the synthesis of UDP-galactose were comparable in RBL5515, exo344 ::Tn5 and the wildtype. It was concluded that RBL5515, exo344 ::Tn5 was affected in a galactose transferase activity. Since this mutant failed to nodulate plants belonging to the pea inoculation group, and the presence of the terminal galactose in the side chain of the EPS of R. leguminosarum bv viciae was not required for succesful nodulation, it was postulated that the amount of EPS produced by RBIL5515 exo344 ::Tn5 and the herewith related viscosity is insufficient for nodulation.Finally, CPS of R. leguminosarum is a polymer which forms gels already at 0.2 % w/ v which is even lower than for agar and therefore has considerable potential applications. Cyclic (1,2)-β-glucans are potentially useful molecules because of their relatively hydrophobic internal space, which make them suitable as an inclusion agent. By choosing the adequate cultural conditions and/or strains, R.leguminosarum was able to produce CPS (CHAPTERS8, 9) or cyclic glucans (CHAPTERS3,5) as the main polysaccharides. The aim of this thesis was to extend the knowledge on the production of Rhizobium polysaccharides as influenced by cultural conditions. The results described in this thesis work can help to stimulate the application-oriented research on hydrocolloïds (CPS, EPS) and inclusion agents (cyclic glucans), and may lead to a better understanding of the biological role of these compounds.