Soil Biology & Biochemistry 34 (2002) 593±597
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Intracellular Fe content in¯uences nodulation competitiveness of Sinorhizobium meliloti strains as inocula of alfalfa Federico Battistoni, RauÂl Platero, Francisco Noya, Alicia Arias, Elena Fabiano* Laboratorio de EcologõÂa Microbiana, Departamento de BioquõÂmica, IIBCE-MEC, Av. Italia 3318, Montevideo, CP 11600, Uruguay Received 17 April 2001; received in revised form 20 September 2001; accepted 3 November 2001
Abstract Rhizobia, as well as most soil bacteria, frequently face variable Fe conditions. The effects of Fe limitation or starvation upon rhizobia infectiveness are not fully understood. Our aim was to evaluate the effects of Fe limitation as well as the ability to acquire Fe in rhizobia competitiveness. Sinorhizobium meliloti 242 wild type strain and one of two-iron acquisition mutants (2.1 and 5.6) were co-inoculated at equal ratio onto alfalfa plants. Legumes were grown under gnotobiotic conditions in Fe-supplemented or Fe-chelated de®ned medium. Our results show that highly ef®cient Fe acquisition systems are involved in nodule competitiveness when Fe availability is low. Moreover, Fe-scarce inocula were out-competed by Fe-suf®cient inocula. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Iron starvation; Rhizobia; Competitiveness; Alfalfa; Sinorhizobium; Siderophores; Nodule occupancy
1. Introduction Soil bacteria generally inhabit in environments where the availability of some essential nutrients is scarce. Moreover, in habitats, such as the soil and rhizospheres, bacteria have to compete with other soil organisms to acquire their nutrients. For instance, it is well known that accessibility to nutritional iron could in¯uence microbial competition (Bossier et al., 1988). Most bacteria have evolved or acquired the ability to express high af®nity systems to internalize this metal under low available Fe conditions (Guerinot, 1994; Neilands, 1981a). One of such systems involves the production and utilization of low molecular weight organic compounds termed siderophores (Neilands, 1981b). Since production and utilization of siderophores could be affected by chemical, physical and biological factors, the ecological relevance of siderophores will depend upon the nature of soil and rhizosphere microenvironments (Buyer and Sikora, 1990; Loper and Buyer, 1991). Rhizobia are soil bacteria able to develop nitrogen-®xing symbiosis with legume plants. A prerequisite for their successful inoculation is the use of highly competitive strains. Amarger and Lobreau (1982) de®ned `nodulation competitiveness' as the relationship between the proportion of nodules occupied with a strain and the ratio of such strain * Corresponding author. Tel.: 1598-2-4871616; fax: 1598-2-4875548. E-mail address:
[email protected] (E. Fabiano).
in the inoculum mixture. Several phenotypes and genotypes of rhizobia have been described as involved in nodulation competitiveness: antibiosis, motility, lipopolysaccharide, exopolysaccharide production (Robleto et al., 1998; Triplett and Sadowsky, 1992; Lagares et al., 1992). In contrast to siderophores of pseudomonas, there are only few studies addressing the role of rhizobial siderophores in competitiveness and their symbiotic relevance is not clearly understood (Tang et al., 1990; Barran and Brom®eld, 1993; Guerinot, 1994). We have evaluated the in¯uence of Fe and siderophores upon nodulation competitiveness of rhizobia in competition experiments performed in alfalfa plants, between a native strain of Sinorhizobium meliloti and two isogenic mutants with de®cient siderophore-mediated Fe acquisition systems. 2. Materials and methods 2.1. Bacteria and media The strains of S. meliloti used in this study and their relevant characteristics are listed in Table 1. Bacterial cells were grown in TY liquid medium (Beringer, 1974). Fe-limited medium (TYE) was made by supplementation with ethylendiamine di (o-hydroxyphenylacetic acid) (EDDHA), a chelator virtually Fe 31 speci®c (Angerer et al., 1992). Antibiotics were added at the following ®nal concentrations: 100 mg streptomycin ml 21 (Sm) or 50 mg
0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(01)00215-2
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Table 1 S. meliloti strains used in this study Strain 242 2.1 5.6
Relevant characteristics
Reference
r
Spontaneous Sm mutant of 259, wild type strain Tn5-mob derivative of 242, presents an impaired growth on Fe-limited medium, is able to use ferrichrome and different heme-compounds but not rhizobactin 242-rich supernatant as Fe sources Tn5-mob derivative of 242, presents an impaired growth on Fe-limited medium, is not able to use rhizobactin 242-rich supernatant, ferrichrome and different heme-compounds as Fe sources
Fabiano et al. (1994) Noya et al. (1997) Noya et al. (1997)
neomycin ml 21 (Nm). EDDHA was purchased from SIGMA (St. Louis, Mo).
(Stevens et al., 1999). Fe-cell content was determined by the ferrozine method (Carter, 1971).
2.2. Plants
2.5. Nodule occupancy
Medicago sativa bv Creoula plants were used for competition assays. Seeds were surface disinfected by treatment with 95% ethanol for 30 s and 7 mM HgCl2, 50 mM HCl for 3 min, washed several times with distilled water and germinated in Petri dishes on sterile water±agar at 28 8C. One day later the seedlings were transferred aseptically to tubes containing 20 ml of N-free Jensen medium (JM) solidi®ed with 1.5% agar (Vincent, 1970). Fe-suf®cient medium or Fe-limited medium were obtained by the addition of 60 mM FeCl3 or 25 mM EDDHA, respectively. Plants were grown at 21 ^ 2 8C in a light controlled room with a photoperiod of 12 h. Plants inoculated with a singlestrain were used as reference.
The effect of EDDHA on cell growth was studied. Flasks with 50 ml of TY supplemented with increasing concentration of EDDHA (from 0 to 1 mM for the wild type strain and from 0 to 25 mM for mutants strains) were inoculated with washed cells taken from log-phase cultures. These studies were done at 40 h (early stationary phase) after inoculation. To study the effect of Fe addition on the growth of Fe-starved cells, tubes with 6 ml of medium were used. After 27 h of inoculation in TYE broth, FeCl3 was added at the same EDDHA molarity. Growth measurements were taken at different time-points over a period of 100 h. Flasks or tubes were incubated with shaking at 30 8C. Growth was determined by measuring optical density at 620 nm. At least three independent assay were performed for each treatment.
Bacteria were grown to early stationary phase on ¯asks with 50 ml of TY or TY supplemented with EDDHA at a ®nal concentration of 100 mM for the wild type, or 10 mM for the 2.1 mutant. Cultures with a ®nal OD620 nm of 0.7±1.2 were pelleted and washed cells were resuspended in sterile water to an OD620 nm of 0.10 (1 £ 10 8 cfu ml 21). These dilutions were used to make mixtures of wild type strain and mutant strains in a 1:1 ratio. Plants were inoculated with these mixtures at a ®nal concentration of 5 £ 10 5 cfu per tube. At least 12 tubes (two plants per tube) were used for each inoculation mixture. The inocula mixtures were prepared independently twice. Nodules were harvested from each plant 8 weeks after inoculation, and pooled. The pool of nodules from each treatment was surface sterilized with 7 mM HgCl2 in 50 mM HCl and washed several times with sterile deionized water (Fabiano and Arias, 1991). Nodules were individually placed in the wells of sterile polystyrene 96-well microtiter plate (NUNC w), containing 30 ml of 150 mM NaCl per well. A replica plater (SIGMA w) was used to macerate nodules and suspensions obtained were inoculated onto TY streptomycin supplemented media (TYS). Plates were incubated for 3 d at 30 8C and the colonies were transferred to the following solid media: TYS (where the parental and mutant strains can grow). TYS neomycin-supplemented media (TYSN, where only the mutants can grow), TYS supplemented with 500 mM EDDHA (TYSE, where only the parental strain can grow). Five intact nodules from each sterilized pool were placed onto TY solid media without antibiotic as a control for the sterilization process.
2.4. Iron assay
2.6. Data analysis
Flasks containing 2 l of TY or TYE were inoculated with about 5 £ 10 9 viable cells of 242, 2.1 and 5.6 strains. Final concentration of EDDHA is indicated in each case. When the culture reached stationary phase, cells were pelleted and washed twice with 0.1 mM Tris±HCl buffer, pH 7. Pellets were freeze-dried and the dry weights were determined. Dried-cells were digested overnight with the addition of concentrated HNO3 and heated for 4 h at 125 8C. Finally, samples were resuspended in 20 ml of 190 mM HNO3
P-values were calculated using a two-tailed binomial distribution with P equal to the initial composition of the mixture, i.e. 50% (Langley, 1971).
2.3. Effect of Fe-starvation on bacterial growth
3. Results 3.1. Effect of EDDHA on rhizobial growth Fig. 1 shows that an EDDHA concentration of 5 mM
F. Battistoni et al. / Soil Biology & Biochemistry 34 (2002) 593±597
Fig. 1. Effects of Fe starvation on 40 h culture of 242 (A) and mutants 2.1 and 5.6 (B). The data are representative of three independent experiments.
markedly affected the growth of the Fe-transport mutants 2.1 and 5.6. Particularly, the growth of mutant 5.6 was impaired at concentrations as low as 3 mM EDDHA. This extremely limited tolerance to EDDHA of mutant 5.6 could be related to the inability of this mutant to acquire Fe from siderophores or heme-compounds as compared to mutant 2.1 which can use different Fe sources (Table 1). In contrast, the wild type strain cultures could reach an OD620 nm of about 0.7 even in the presence of 500 mM EDDHA. The EDDHA limitation of bacterial growth was overcome by the addition of FeCl3 to the medium (Fig. 2), con®rming that cultures on TYE were being speci®cally Fe-starved. Based on results shown in Fig. 1 nodulation competition was assayed with Fe-starved cultures of wild type and mutant 2.1 strains obtained when the TY medium was supplemented with 100 or 10 mM EDDHA, respectively. 3.2. Intracellular Fe content Intracellular iron content was measured in bacteria grown under the same conditions used for nodule competition assays. Iron content of 242 wild type strain and the mutants strains is shown in Fig. 3. Results obtained indicate that when inocula were grown in Fe-chelated medium, cells present lower intracellular Fe content than for bacteria grown in TY medium.
595
Fig. 2. Effect of Fe addition on the growth of Fe-starved cultures of wild type strain 242 (A) or mutant 5.6 (B). Cells were cultured in Fe-suf®cient medium (TY) or Fe-limited medium (TY plus 250 mM EDDHA for the wild type strain or 50 mM EDDHA for the mutant); FeCl3 was added to cultures in Fe-limited medium at 27 h. The curves show the results from one of three independent experiments.
3.3. Nodulation competition assays Phenotypes of bacteria recovered from nodules of plants inoculated with a single-strain were tested on TYS, TYSN and TYSE plates, in addition to CAS-plates, which allow the detection of secreted siderophores (Schwyn and Neilands, 1987). The inoculant phenotype was retained on bacteria recovered from nodules, indicating that the Tn5mob insertion was stably maintained during the infection process. The methodology used for the determination of nodule occupancy enabled us to differentiate nodules occupied by the mutants from nodules occupied by the wild type strain. Values of dual-strain occupancy ranged from 0 to 15% of total nodules tested in each assay. No correlation between dual-strain occupancy and different assay conditions could be detected (data not shown). 3.3.1. Effects of siderophore-mediated Fe transport systems With the aim of evaluating the role of high af®nity Fe transport systems on the infective ability of rhizobia, alfalfa plants were co-inoculated with a mixture of the wild type strain and one of the Fe acquisition mutants (2.1 or 5.6). When the two strains were grown on Fe-suf®cient media prior to inoculation, the wild type and the mutants were recovered from nodules in the same proportion as used in the inocula mixture (Table 2). However, when experiments were performed with Fe-limited inocula, the wild type strain
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Fig. 3. Fe content of 242, 2.1 and 5.6 cells grown on Fe-rich or Fe-chelated medium.
occupied a signi®cantly greater (P-value ,0.01) proportion of the nodules (76 or 88% from plants grown on Fesupplemented or Fe-chelated medium, respectively). These data provide in planta evidence of the competitive advantage conferred by the siderophore-mediated Fe transport system when the Fe availability in the bacterial growth environment is low. 3.3.2. Effects of Fe bioavailability of the inoculum culture medium As shown in Table 2 and Fig. 3 when the experiments were performed with a mixture of strains 242 Fe-limited cells and mutant Fe-suf®cient cells, the infectivity of the wild type strain decreased signi®cantly. By growing the inoculum on Fe-suf®cient media instead of using Fechelated media increased remarkably the number of nodules occupied by the strain. This is shown by comparing I vs. III where the wild type growth condition was changed and II vs. III where the mutant growth condition was changed (Table 2). These results clearly indicate that intracellular Fe content of the inoculum has a signi®cant effect (P-value ,0.01) on the infective ability of rhizobia strain. The comparison of values obtained for I and IV, III and V assays indicate that the particular phenotype of both siderophore-transport mutants (Noya et al., 1997) did not affect the infectivity phenotype.
4. Discussion Reports about Fe regulated genes in rhizobia are scarce. In addition, very little is known about the role of this metal in the molecular regulation of the infectivity process. Bittinger et al. (1997), reported that rosR is involved in the competitive ability for nodulation in R. etli and Hussain and Johnston (1997), found that transcription of ros gene needed the presence of Fe in the growth medium of Agrobacterium radiobacter. Results presented in this work (Table 2) indicate that the intracellular Fe status of the cells used in the inoculant mixture affects the nodulation competitiveness of rhizobia. At present we are unable to explain the precise basis of the role of Fe in nodulation competitiveness but taking these observations into account, it is attractive to speculate that the intracellular Fe concentration affects the expression of a RosR-like protein, which would be required for a normal infectivity process in 242 strain. Certainly, exhaustive studies will be needed to con®rm this hypothesis. As we previously have shown high af®nity Fe acquisition systems in rhizobia not only involve its own siderophore, but also different compounds can be used as Fe sources as well, e.g. ferrichrome, hemin and leghemoglobin (Noya et al., 1997). Those microorganisms which possess diverse and highly ef®cient systems for Fe acquisition will be able to acquire this metal more effectively in competition with
Table 2 Nodulation competitiveness of 242 wild type strain and two Fe-acquisition mutants, 2.1 and 5.6. Plants were grown for 8 weeks on JM supplemented with 60 mM FeCl3 or 25 mM EDDHA. (*Proportion followed by an asterisk indicates a signi®cant (P-value ,0.01) difference between the proportion of wild type strain used in the inoculum (50%) and the proportion of the same strain recovered from nodules) Treatment
I II III IV V a b c d
Inoculation mixture a
242 (1):2.1 (1) c 242 (2):2.1 (2) 242 (2):2.1 (1) 242 (1):5.6 (1) 242 (2):5.6 (1)
Percent of 242-nodule occupancy b Plants grown on JM 1 60 mM FeCl3
Plants grown on JM 1 25 mM EDDHA
56 (120) 76* (78) 15* (53) 62 (50) 15* (41)
56 (52) 88* (40) 17* (65) ND d 19* (53)
The two strains were mixed in the inoculum at a 1:1 ratio. Number of nodules tested is indicated in parenthesis. (1) cells grown in Fe-suf®cient medium; (2) cells grown in Fe-depleted medium. ND, not determined.
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other soil microorganisms. Considering that Fe content of cell could in¯uence rhizobia infectiveness, we can presume that those strains that possess ef®cient ways to achieve appropriate intracellular Fe concentrations will have an advantage to nodulate their host plant. This characteristic would confer competitive advantages to these particular strains under soil conditions where bioavailability of Fe is limited. Studies with non-sterile soil would be helpful to elucidate the ecological relevance of this trait. In summary, results presented in this work provide evidences that the presence of intact high af®nity Fe acquisition systems assist nodule competitiveness and that an increase in Fe bioavailability of the inoculant medium led to an increase in nodule occupancy. Acknowledgements We are very grateful to P. Lemanceau and K. Carson for helpful discussion and G. Gualtieri for critical comments on the manuscript. This research was partially supported by grants from International Foundation for Sciences Sweden, Third World Academy of Sciences, Italy and PEDECIBAÐ Uruguay. References Angerer, A., Klupp, B., Braun, V., 1992. Iron transport systems of Serratia marcescens. Journal of Bacteriology 174, 1378±1387. Amarger, N., Lobreau, J.P., 1982. Quantitative study of nodulation competitiveness in Rhizobium strains. Applied and Environmental Microbiology 44, 583±588. Barran, L.R., Brom®eld, E.S.P., 1993. Does siderophore production in¯uence the relative abundance of Rhizobium meliloti in two-®eld populations? Canadian Journal of Microbiology 39, 348±351. Beringer, J.E., 1974. R factor transfer in Rhizobium leguminosarum. Journal of General Microbiology 84, 188±198. Bittinger, M.A., Milner, J.L., Saville, B.J., Handelsman, J., 1997. rosR, a determinant of nodulation competitiveness in Rhizobium etli. Molecular Plant±Microbe Interactions 10, 180±186. Bossier, P., Hofte, M., Verstraete, W., 1988. Ecological signi®cance of siderophores in soil. Advances in Microbial Ecology 10, 385±414.
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