Osmoprotection mechanisms in rhizobia isolated from Vicia faba var. major and Cicer arietinum Fatiha Brhada, M. Poggi, G. van de Sype, Daniel Le Rudulier To cite this version: Fatiha Brhada, M. Poggi, G. van de Sype, Daniel Le Rudulier. Osmoprotection mechanisms in rhizobia isolated from Vicia faba var. major and Cicer arietinum. Agronomie, EDP Sciences, 2001, 21 (6-7), pp.583-590. 10.1051/agro:2001148. hal-00886135 HAL Id: hal-00886135 https://hal.archives-ouvertes.fr/hal-00886135 Submitted on 1 Jan 2001 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Agronomie 21 (2001) 583–590 583 © INRA, EDP Sciences, 2001 Original article Osmoprotection mechanisms in rhizobia isolated from Vicia faba var. major and Cicer arietinum Fatiha BRHADAa*, M.C. POGGIb, G. VAN DE SYPEb, Daniel LE RUDULIERb a Laboratoire de Microbiologie et Biologie moléculaire, Faculté des Sciences, Université Mohammed V, BP 1014, Rabat, Morocco b Laboratoire de Biologie Végétale et Microbiologie, Faculté des Sciences et Techniques, Université de Nice-Sophia Antipolis, France (Received 22 January 2001; revised 9 May 2001; accepted 13 July 2001) Abstract – Research of mechanisms involved in osmoprotection of two rhizobia strains isolated from nodules of Vicia faba var. major and one strain nodulating Cicer arietinum, showed that choline and glycine-betaine improved growth in salt stress conditions. Provided radioactive choline was converted into glycine betaine catabolized at low osmolarity and accumulated under osmotic stress. Enzyme activities involved in the synthesis of glycine betaine from choline were not modified by salt whereas addition of choline to the growth medium enhanced them. Exogenous radioactive glycine betaine was accumulated in salt stressed bacteria after one hour incubation but catabolized four hours later, suggesting a transient accumulation. Growth experiments indicated that betaine and its derivatives could be used as nitrogen and carbon sources. An investigation, by liquid phase chromatography, revealated accumulation of glutamate and alanine at different levels according to the strain and degree of stress. rhizobia / osmoprotection / choline / glycine betaine Résumé – Mécanismes d’osmoprotection chez les rhizobia isolés de Vicia faba var. major et de Cicer arietinum. La recherche des mécanismes impliqués dans l’osmoprotection chez deux souches de rhizobium isolées des nodosités de Vicia faba var. major et une souche nodulant Cicer arietinum, a montré que la glycine bétaïne et la choline améliorent la croissance bactérienne sous stress salin. Lors d’études métaboliques, la choline intracellulaire radioactive est transformée en glycine bétaïne et ce indépendamment de l’osmolarité du milieu. En effet, les activités choline déshydrogénase et glycine bétaïne aldéhyde déshydrogénase ne sont pas modi- fiées par le sel. Néanmoins, la présence de la choline dans le milieu augmente les deux activités enzymatiques. L’incubation en pré- sence de la glycine bétaïne durant une heure est suivie d’une accumulation de ce composé chez les cellules cultivées à 0,15 M NaCl. Cette accumulation n’a plus lieu quatre heures plus tard. Des expériences de croissance avec la glycine bétaïne ou ses métabolites montrent son utilisation comme source de carbone et d’azote. La recherche, par chromatographie en phase liquide a révélé l’accumu- lation du glutamate et de l’alanine à des niveaux variables en fonction de la souche et du degré de stress. rhizobia / choline / glycine betaine / osmoprotection 1. INTRODUCTION Due to their excessive salt concentrations and high pH values, saline and alkali soils are unsuitable for the Salinity is one of the major factors responsible for growth of the crop legume plants and their root-nodule deterioration of soil and making it unfit for agriculture. bacteria. The osmotic stress prevailing in these areas and Communicated by Jean-Jacques Drevon (Montpellier, France) * Correspondence and reprints [email protected] 584 F. Brhada et al. non-adaptability of both symbionts may be important Growth of this bacterium at elevated osmolarity results limiting factors. It has been shown in many reports that in inhibition of this catabolism [1, 25]. Although the soil salinity may have an adverse effect on the establish- choline-betaine pathway has been well characterized in ment of functional symbiosis [24]. Hence, it is necessary S. meliloti, there is little information concerning other to inoculate with strains selected for their osmotic toler- members of Rhizobiaceae. ance. rhizobia differ in their ability to tolerate salt [8, 12]. The upper limit of salt concentration tolerated by We have recently examined salt tolerance of two rhi- species of rhizobia depends on strains, varying from zobia, RlF12 and RlF16, isolated from nodules of Vicia 0.2% to 10% of NaCl [31]. El Sheikh and Wood [8] faba and one, Rch60, from Cicer arietinum cultivated in reported that fast-growing rhizobia were more salt-toler- arid and semi-arid areas of Morocco. Increasing the salt ant than the slow growers. concentration in the medium reduced both the growth rate and the final yield of all strains but less in the chick- An increase in extracellular osmolarity inhibits a pea strain, Rch60, more salt-tolerant [5]. Addition of number of physiological and biochemical activities in glycine betaine or choline at 1 mM in salt-added medium the bacteria, hence growth is generally reduced. had a beneficial role on the growth of the three strains. However, bacteria have involved a variety of adaptive However, the choline effect was delayed and less pro- mechanisms in order to restore the cell turgor pressure nounced. Both molecules were taken up by cells grown and then reduce the osmotic potential between the cell at low osmolarity, and whereas glycine betaine uptake and the environment [4]. Among these mechanisms, one activity was stimulated in cells grown in the presence of of the most clearly established is the accumulation of 0.15 M NaCl, choline uptake was strongly inhibited by ions such as potassium [30] and organic compatible salt in all tested strains. In cells grown with exogenous solutes like amino acids, sugars and betaines [16]. choline, the uptake inhibition exerted by salt was The most universally adopted compatible solute is relieved, mainly in the strain isolated from nodules of glycine betaine, which can be accumulated during stress Cicer arietinum. On the basis of kinetic determinations, by a large variety of bacteria [7]. This was reported for in control cells as well as in salt-stressed cells, only Sinorhizobium meliloti in 1986 by Le Rudulier and high-affinity uptake activities were observed for glycine Bernard [17]. Recently, Boncompagni et al. [2] reported betaine and choline. Periplasmic proteins that bound [ ] the utilization of glycine betaine as osmoprotectant by glycine betaine or choline were identified 5 . Rhizobium tropici, R. galegea, Sinorhizobium fredii, In this paper we provide information on the biosyn- Mesorhizobium loti and M. hualkuii. The accumulation thesis and catabolism of glycine betaine in strains RlF12, of glycine betaine, which enhances growth at high osmo- RlF16 and Rch60, and results of research into other larity, results either via transport from the environment osmo-protectants by liquid phase chromatography or via biosynthesis from precursors, choline or glycine (HPLC). betaine aldehyde [7]. In a few bacteria such as the halophilic Ectothiorhodospora, de novo biosynthesis is observed [11]. 2. MATERIALS AND METHODS Biosynthesis of betaine results from oxidation of choline via a two-step reaction with betaine aldehyde as 2.1. Bacteria and medium intermediate. In microorganisms two choline oxidase systems are possible. A soluble choline oxidase system Rhizobium strains RlF12 and RlF16, isolated from capable of catalysing both choline and betaine aldehyde Vicia faba, strain Rch60 nodulating Cicer arietinum and oxidations in vitro has been identified in Arthrobacter [ ] [ ] the Sinorhizobium meliloti strain 102F34 were main- globiformis 14 and Alcaligenes sp. 23 and a mem- tained on Yeast Extract Mannitol medium, pH 6.8 [28]. brane-bound choline dehydrogenase employed in conju- gation with a soluble betaine aldehyde dehydrogenase is In osmoprotection assays, strains RlF12, RlF16 and used in Pseudomonas aeruginosa [19], in E. coli [15] Rch60 were grown at 30 °C in a mannitol-aspartate-salts and also in Sinorhizobium meliloti [25]. Furthermore, the medium (MAS). Increased osmolarity was obtained by actual pathway of biosynthesis of glycine betaine from adding NaCl at 0.15 and 0.3 M. The osmolarity of the choline and its degradation by this species is well known different media was measured by using a microosmome- and evidence of their osmomodulation has been provided ter (H. Roebling) and corresponds to 52, 327 and [25]. Effectively, if glycine betaine is a metabolic end 600 miliosmoles/kg water respectively for MAS medium product in Escherichia coli and other enteric bacteria 0 M, 0.15 M and 0.3 M NaCl. When added, glycine [21] this