Glycine betaine and other structurally related compounds improve the salt tolerance of Rhizobium meliloti

Plant Science Letters, 31 (1983) 291--302 Elsevier Scientific Publishers Ireland Ltd.

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GLYCINE BETAINE AND OTHER S T R U C T U R A L L Y R E L A T E D COMPOUNDS IMPROVE THE SALT TOLERANCE OF R H I Z O B I U M MELILOTI D. SAUVAGEa, J. HAMELINb and F. LARHERa aLaboratoire de Biologie v~g$tale, U,E.R. Physique, Chimie, Biologie, Universit# de Nancy I, B.P. 239, 54506 Vandoeuvre-les-Nancy Cedex, and bGroupe de Recherche de Physicochimie strueturale, U.E.R. Structure et Propri@tds de la Mati@re, Universitd de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex (France) (Received December 10th, 1982) (Revision received March 9th, 1983) (Accepted March 9th, 1983) SUMMARY R h i z o b i u m meliloti Be 151, an effective strain of R h i z o b i u m for Medicago sativa, was f o u n d to be a facultative halotolerant bacterium. The presence of NaC1 at concentrations as high as 75 mM did not inhibit the bacterial growth. The strain tolerated salt concentrations up to 600 mM although a 40% inhibition in growth was observed at 400 mM NaC1. Addition of 10 mM glycine betaine helped the strain to overcome the diminution in growth in the presence of'sodium chloride. The extent of N-methylation of glycine is important in producing the anti-stress effect, since the N-trimethylated c o m p o u n d was more effective than the N-dimethylated which again was superior to the N-monomethylated derivative of glycine. On the contrary, glycine itself acted as a p o t e n t inhibitor of R. meliloti growth. Some kind of protection against the deleterious effects of saline conditions was also noted with some alicyclic betalnes such as ~-homobetaine and ~/-butyrobetaine as well as with cyclic betaines like stachydrin and homostachydrin. Glycine betaine and stachydrin which exhibited the most pronounced anti-stress effect were utilized by the bacteria as substrates in the absence of any conventional carbon source. The significance of these findings in relation to the Medicago s a t i v a - - R h i z o b i u m meliloti symbiosis is discussed. K e y words: N2 fixation -- R h i z o b i u m meliloti -- Salt tolerance -- Glycine betaine -- Stachydrin

INTRODUCTION Glycine betaine, which is found to accumulate under conditions of stress 0304-4211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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either in various halophytic species [1,2] or in a few glycophytic higher plants like spinach [3], barley [4,5], Euphorbia trigona [6] and various species of Beta [7] was assumed to be a compatible solute [8] involved in osmotic adjustment according to the model described by Flowers et al. [9]. A similar role has also been suggested for this compound in the extremely halophilic phototrophic bacterium, Ectothiorhodospira halochloris and in the salt resistant cyanobacterium Synechocystis D U N 52 [11]. Recently, the contribution of glycine betaine in regulating the cellular osmotic potential has been assessed in cells of both higher plants and bacteria which had been subjected to an increasing osmotic stress [10,12,13]. The results suggest that betaine accumulation m a y be an adaptive process. In addition to this function, a number of protective effects have recently been demonstrated for glycine betaine viz., (i) it m a y exert a stabilizing effect on the membranes either of bacteria subjected to high osmolarities (14) or of higher plants submitted to the deleterious effects of high temperatures as well as heat coupled with oxalates [15,16]; (ii)it inhibits the thermal denaturation of various enzymatic proteins [17,18] and (iii)it is an efficient osmoticum for chloroplast isolation [19]. Glycine betaine has also been found to improve the growth of the halotolerant bacterium Bal subjected to osmotic stress via a non-penetrating solute such as KCI [14]. Le Rudulier and BouiUard [20] have recently demonstrated a significant anti-osmotic stress action of glycine betaine for Klebsiellapneumoniae grown in presence of 650 m M NaCl. They also showed that the presence of glycine betaine in the medium could induce an effective protection of nitrogenase synthesis. It has been demonstrated that exogenous glycine betaine can be absorbed by a few leguminous plants via the root system and that it exerts a significant protection against polyethylene glycol (PEG) induced water stress in these species [21]. The hypothesis of an anti-stressproperty of exogenous glycine betaine in the Legume-Rhizobium symbiosis was thus put forward. Since the behaviour of Rhizobiaceae in the presence of glycine betalne is not known, especially in NaCl stress conditions, an attempt was made to determine a possible protective role for this exogenous organic solute and also its structurally related compounds on Rhizobium meliloti growing in media enriched with sodium chloride. MATERIALS AND METHODS An effective strain of Rhizobium meliloti (Be 151) obtained from N. Amarger (Laboratoire de Microbiologie des sols de I'INRA, Dijon, France) was used in this study. Yeast Extract (Difco)-Mannitol (YEM) broth culture of Wright [22] was used as the reference medium. In order to determine their respective effects, different concentrations of NaC1 (0--1 M), glycine betaine (0--100 mM) and its related compounds (10 mM each) were added to the medium. A 400 mM NaC1 concentration was found to be sufficient

293 to produce condition of stress. This concentration was therefore used in the following experiments. The pH was adjusted to 6.8--7.0 and 4-ml aliquots of each medium were dispensed into test tubes, autoclaved at 120°C for 20 min and inoc~ulated with 0.5 ml of an aqueous suspension of Rhizo. bium obtained from an agar slope. The tubes were incubated at 28°C on a rotary shaker for different periods of time. The extent of growth of the bacterium was measured with a Lumetron colorimeter at 420 nm by the difference in optical density ( 4 0 . D . ) from the time of inoculation to the time of measurement. Results presented here are the averages of t w o independent experiments; each of them performed in duplicate.

Chemicals Sodium chloride, glycine, N-methylglycine, N~iimethylglycine and Ntrimethylglycine were of commercial origin (Sigma). Stachydrin and homostachydrin were synthesized respectively from sodium salts of proline and pipecolic acid, following the m e t h o d o f Buchel and Korb [23] modified by Ikutani [24]. ~-Homobetaine was synthesized according to Le Berre and Delacroix [25] and 7-butyrobetaine was synthesized according to the procedure of Anderson et al. [26]. RESULTS

Salt tolerance of Rhizobium meliloti The growth curves obtained for R. meliloti in the presence of various NaC1 concentrations are presented in Fig. l a . The lag phases were 7 h for the cultures grown in a medium containing 0--250 mM NaC1; this was lengthened for the higher concentrations of salt and at a maximum NaCl (1 M) concentration, the lag phase was 30 h. It is evident from these curves that the beginning of the stationary phase of each NaC1 concentration was reached at approximately the same time. The relative growth of R. meliloti in various media supplemented with NaC1 is shown in Fig. l b . This data m a y suggest a slight stimulatory effect on growth up to 75 mM. An inhibitory effect was observed at a concentration as high as 150 mM. A complete inhibition of growth was obtained at a concentration of I M NaC1. Behaviour o f Rhizobium meliloti in presence o f glycine betaine In order to assess the influence of exogenous glycine betaine on the growth of Rhizobium meliloti, this solute was added to different kinds of media, viz.: Medium I: mineral c o m p o n e n t s of Wright medium + biotin (200 ~zg • 1-1) + mannitol (10 raM) Medium H: mineral c o m p o n e n t s of Wright medium + biotin + glycine betaine (1 or 10 raM)

294

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Fig. 1. Salt tolerance of R. meliloti Be 151. (a) Growth curves on Wright medium supplemented with various concentrations of NaCI: =, 3 mM; o, 25 mM; A, 75 raM; a, 150 mM; e, 200 m M ; v , 250 m M : ~ , 400 mM; ~, 500 mM; O------G, 800 raM; o------D, 1000 raM. (b) The relative growth of R. meliloti Be 151 in the premmce of different concentrations of NaCI (the results are expressed as percent of the control, after 57 h of incubation).

Medium III: mineral components of Wright m e d i u m + yeast extract (YE) + glycine betaine (1 or 10 mM) Medium I V : Wright m e d i u m (YEM) + glycine betaine from 0 to 50 mM The various growth curves obtained (Figs. 2a and 2b) show that glycine betaine (Fig. 2a, m e d i u m II) m a y induce a significant growth of R. meliloti at very low concentrations when it was used as a carbon source (compared to m e d i u m I). The growth was improved when YE was added to the m e d i u m (medium III). In contrast, when the m e d i u m was supplemented with 50 mM mannitol (Fig. 2b) the addition of glycine betaine had no effect on the bacterial growth. From our experiments it was apparent that glycine betaine present in a mannitol deficient m e d i u m m a y be used as a substrate for R h i z o b i u m growth. Fig. 2. Effect of glycine betaine (GB) on the growth of R. reel/lot/Be 151. (a) Growth curves o n various media: I, 10 mM mannitol plus biotin (200 ~g • 1-1); II, Glyeine betaine plus biotin: v , 1 mM GB; o, 10 mM GB; HI, Glycine betaine plus YE:V, 1 mM G B ; . , 10 mM GB; *, YE alone. (b) Growth curves on Wright medium supplemented with different concentrations of glycine betaine. (c) s, control; o, Wright medium supplemented with 400 mM NaCI + 10 mM GB; D, Wright medium supplemented with 400 mM NaCI.

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296 Improvement o f salt tolerance of Rhizobium meliloti in presence of glycine betaine R. meliloti was grown in YEM m e d i u m containing 400 m ~ NaCI and various concentrations of glycine betaine. The addition of this solute to the medium (Table I) significantly improved the growth of R. meliloti up to 20 mM glycine betaine, although m a x i m u m growth was obtained with 10 mM glycine betaine. These values are those recorded after 62 hours of incubation. In fact, it is evident from Fig. 2c that until an incubation period of at least 72 h the protective effect of betalne (10 raM) was not complete. If the growth curves of R. meliloti in the standard medium supplemented or not with NaC1 are coinpared to those obtained in the presence of glycine betaine it is clear that the protective effect of betaine is only observed after 24 h incubation. After this time, the growth is significantly increased with respect to the control. In fact, the growth rate reached almost the same value as that of bacteria growing in the absence of NaC1. A m a x i m u m growth rate was obtained at 72 h of incubation for the culture containing glycine betalne plus NaC1, which was exactly the same as the control (YEM). It is evident, then, that the stationary phase is only lengthened when 10 mM glycine betaine is present in the medium. Effects of glycine betaine or its structurally related precursors on the growth o f Rhizobium meliloti in the presence o f NaCl In this experiment, NaC1 was used at a concentration of 400 mM and glycine, N-methylglycine (sarcosine) N-dimethylglycine or N-trimethylglycine (glycine betaine) at a concentration of 10 mM. The kinetic curves (Fig. 3) show that glycine betaine may relieve the growth inhibition induced by sodium chloride as already shown in Fig. 2c. Among the other solutes used, only dimethylglycine exhibited some protective effect. The addition of sarcosine to the m e d i u m had no effect on the growth while glycine appeared to be an inhibitor of R. meliloti, especially when the bacteria were subjected to a high NaC1 salinity.

Effects of g.homobetaine and 7.butyrobetaine on the growth of Rhizobium melilotiin the presence of NaCl The growth curves (Figs. 4a and 4b) show a significant protective effect exerted by betaines, 7-butyrobetaine (Fig. 4a) being more effective than ~-homobetaine (Fig. 4b). However, complete recovery of growth as observed with glycine betaine was never attained. The protective effect of 7"butyrobetaine became apparent earlierthan that of glycine betaine. Effects of stachydrin and homostachydrin on the growth of Rhizobium meliloti in the presence of NaCl The experimental conditions are the same as those stated for the other solutes. To assess the relative effectiveness of these solutes with respect to glycine betaine, the latter was used as control (Fig. 4c). Glycine betaine

297 TABLE I IMPROVEMENT OF THE GROWTH OF R. MELILOTI Be 151 BY GLYCINE BETAINE IN THE PRESENCE OF 400 mM NaCI Glycine betaine conc. in the external medium (raM)

Total growth a expressed as ~ O.D. after 62 h incubation

Increase in bacterial growth (% of control)

0 1 7 10 20 100

0.99 0.97 1.11 1.17 1.12 0.88

---2.0 +12.1 +18.2 +13.1 --11.1

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Time in hr. Fig. 3. Effect of glycine betaine and its structurally related compounds on the growth of R. meliloti Be 151 in the presence of 400 mM NaCI. o, control; o, Wright medium + 400 mM NaC1; o, Wright medium + 400 mM NaC1 + 10 mM G B ; v , Wright medium + 400 mM NaC1 + 10 mM dimethylglycine; e, Wright medium + 400 mM NaCI + 10 mM N-methylglycine;v, Wright medium + 400 mM NaCI + 10 mM glycine.

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T i m e in hr. Fig. 4. The effect of different kinds of betaines on the growth of R. meliloti Be 151. Effect ofalicyclic betaines: (a) m, control; v, 400 mM NaC1; e, 400 mM NaC1 + 10 mM 7-butyrobetaine. (b) m, control; o, 400 mM NaCI; e, 400 mM NaCI + 10 mM E-homobetaine. Effect of cyclic betaines: (c) = control; o, 400 mM NaCI; o 400 mM NaCl + 10 mM GB; ~, 400 mM NaCI + 10 mM stachyd r i n ; , , 400 mM NaCI + 10 mM homostachydrin.

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was found to be the most effective of these three betaines. However, similar results were observed with stachydrin although the growth response was delayed by about 24 h. I-Iomostachydrin also exerted some protective effect against NaCI (the total growth was improved by 20%). In addition, the protective effect of stachydrin was apparent after 25 h of incubation while that of homostachydrin was only detected after an incubation period of 42 h. Betaines as growth substrates for Rhizobium meliloti Be 151 The effect of glycine betaine is one of protection in that this solute maintains growth of the bacteria. This observation prompted us to study the growth of R. meliloti in Wright's medium lacking mannitol and NaC1 but supplemented with a 10 mM concentration of ~-homobetaine, 7-butyrobetaine, stachydrin or homostachydrin. The growth curves (Fig. 5) on these media compared with those obtained (a)

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hr Fig. 5. The effect of different betaines as growth substrates for R. meliloti Be 151. (a) A 10 mM stachydrin + Y E ; o , YE alone as control ( b ) v , 10 mM homostachydrin + YE. (c) o, 10 mM 7-butyrobetaine + YE. (d) u, 10 mM ~-homobetaine + BE.

300 in a control medium containing only the mineral components of the Wright's medium and Y E show that growth was not modified in the presence of either ~-homobetaine, 7-butyrobetaine, or homostachydrin. O n the other hand, when stachydrin was added to the medium, growth was significantly better. Thus, as demonstrated for glycine betaine, the anti-stresseffect of stachydrin m a y be associated with some kind of property related to its nutritional value for R. meliloti. DISCUSSION In this study, Rhizobium meliloti Be 151 has been shown to tolerate relatively high NaC1 concentrations. Moreover, bacterial growth is even slightly stimulated by a 75 mM NaC1 concentration. The growth rate is only reduced at NaC1 concentrations higher than 150 raM. These results are in good agreement with those obtained by Subba Rao et al. [27] and Steinborn and Roughley [28] with other strains of R. meliloti. A stimulatory effect of NaC1 has also been shown for the JI strain of Rhizobiurn effective on Sesbania cannabina [29]. NaC1 is also tolerated in a wide range of concentrations by the P5 strain of R. japonicum [29] and by the 'Rhizobium mesquite' [30]. Thus, R. meliloti Be 151 may be considered as a facultative halotolerant bacterium along with a few other strains of Rhizobium species. ~uch behaviour is in contrast with that observed with R. trifolii (strain i'A 1 ), whose growth was completely inhibited by a 20 mM NaC1 concentration (Sauvage, unpublished results). Some other species of Rhizobium [ 31 ] are also NaCl-sensitive. Despite its basic salt tolerant .behaviour, R. meliloti can, however, be adversely affected by very high concentrations of sodium chloride. This inhibitory effect m a y b e alleviated by adding glycine betaine to the medium. The improvement in growth observed in the presence of both sodium chloride and glycine betaine may be related to an anti-stress effect since in the absence of NaC1, exogenous glycine betaine exerts no effect on bacterial growth when mannitol is present (Fig. 2b). Alternatively, the posi@jve effect of glycine betaine on growth may be sodium chloride dependent.~urthermore, in the absence of any carbon source in the liquid medium glycine betaine was found to be a substrate for growth. This indicates that R. meliloti possesses the enzymatic system for its utilization, presumably via a demethylation process. It was found that other structurally related solutes exerted very little (N-dimethylglycine) or no effect (N-methylglycine) in the presence of NaC1. This result suggests that the protective function is related to the extent of N-methylation of the amino acid precursor. Such a specific function for glycine betaine has also been reported for the bacterium Ba 1 where it exerted a protective function against the adverse effect of saline conditions on the respiratory system [32]. The authors susgested that glycine betaine may help in stabilizing the conformation of enzymes within

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the cell by its binding to negatively charged sites on the proteins. Structurally related solutes devoid of the typical quaternary methylated a m m o n i u m group failto do so. The observation of an inhibitory effect of glycine on R. meliloti grown under high concentrations of NaCl is in good agreement with that of Kurkdjian et al. [33]. They have shown that 10 m M glycine induced a significant decrease in membrane thickness in relation to a diminution in mucopeptide and protein contents of the cell wall. The slight improvement of salt tolerance of R. meliloti Be 151 as observed in the presence of ~-homobetaine and 7-butyrobetaine indicates that the structural requirements for the protective effect are very important. However, stachydrin, the proline betaine, m a y also exert a very significant protective effect on R. meliloti against the deleterious effects of NaCl. This finding m a y indicate that the typical linear dipole of alicyclic betaines is not an absolute requirement for an anti-stressfunction. A m o n g the five betaines used in this study, only glycine betaine and stachydrin behaved as substrates for the growth of R. meliloti on Wright's medium with and without yeast extract and lacking mannitol. This is noteworthy since Medicago sativa, the host of Rhizobium meliloti, accumulates significant amounts of both stachydrin and glycine betaine [34]. Antistress properties have to be predicted to occur in vivo for these two solutes especially in the nodules i n d u c e d b y R. meliloti on the roots of Medicago sativa. For glycine betaine and stachydrin to be substrates for the growth of R. meliloti they first have to be absorbed by the bacterial cells. This transport process may be mediated by specific permeases. If this occurs, the lag phase observed in the presence of betaines could correspond to the period of time needed for the biosynthesis of these perrneases. This absorption could provide the bacterial cells with either a substrate for growth in the absence of mannitol or a compatible solute allowing osmotic adjustment of R. meliloti when subjected to saline conditions. ACKNOWLEDGMENTS

Thanks are due to Professor Mangenot (Universitd de Nancy) for allowing the authors to use the facilitiesof his laboratory and S.M. Imamul H u q w h o helped in translating the manuscript. The authors wish to express their gratitude to Dr. S.F. McNally (University of Dublin) for her help in writing the revised form of the manuscript. Technical assistance of L. Patard is also acknowledged. REFERENCES 1 2 3 4 5

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