INTRODUCTION

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Attempts to improve nitrogen fixation by introducing highly efficient Rhizobium strains often fail in soils that already contain indigenous rhizobial populations due to problems with competitiveness (6, 31, 32, 34). Despite being much less efficient at nitrogen fixation, the indigenous populations are generally better adapted and more persistent, with a higher level of infectivity, resulting in a higher level of occupancy of the nodules formed. Thus, to enhance nitrogen fixation, the strains used as inocula not only must fix nitrogen efficiently but also must be highly competitive. Genetic modifications can be used to increase the competitiveness of Rhizobium strains. An example of the successful use of this strategy is the improvement of Rhizobium etli competitiveness, even under agricultural conditions, achieved by introducing genes encoding trifolitoxin (23, 24).

Our laboratory has studied the genetics of competitiveness in Sinorhizobium meliloti. The putA gene, which encodes proline dehydrogenase, the enzyme catalyzing the oxidation of proline to glutamate, is one of the genetic loci thought to be involved in this symbiotic property. The ability of a PutA mutant to colonize the root surface was found to be impaired, as were nodulation efficiency and competitiveness (11, 12). The root exudates of the host plant of S. meliloti, alfalfa (Medicago sativa L.), contain proline and compounds such as betaines and stachydrine that release proline upon degradation (20). Mutants impaired in stachydrine utilization have also recently been shown to be affected in their ability to colonize alfalfa roots (20). These observations suggest that proline may be an important energy source for the bacteria during the first stages of the infection process. However, mutants with mutations in either the putA gene or the stachydrine utilization (stcD) gene continue to produce effective nodules (12, 20). Therefore, proline does not appear to be an important energy source for nitrogen fixation within bacteroids.

Complementation of the PutA mutant with a cosmid containing the wild-type putA gene did more than merely restore the competitiveness of the mutant to wild-type levels; it actually rendered the complemented mutant more competitive than the wild type (11). This suggested that manipulation of the expression of this gene could be used to improve the competitiveness of S. meliloti strains in field conditions. In S. meliloti, the putA gene is transcriptionally activated by proline and the resulting PutA protein serves as an autogenous repressor (30). In this work, we constructed a plasmid containing a putA gene that had high basal expression levels but was also inducible by proline. We evaluated the competitiveness of S. meliloti strains harboring this plasmid in microcosm and contained field release experiments. The increased competitiveness of S. meliloti containing the modified putA construct is discussed.

	MATERIALS AND METHODS

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Bacterial strains, media, growth conditions, plasmids, and DNA manipulation. The bacterial strains and plasmids used in this study are listed in Table 1. Plasmid DNA was routinely isolated and manipulated by following standard protocols (26). Escherichia coli was grown at 37°C in Luria-Bertani medium (26). S. meliloti strains were grown at 28°C in tryptone-yeast medium (1) or in defined minimal medium (MM) (22). Antibiotics, fungicide, and mercury chloride were used as required at the following concentrations: ampicillin, 200 µg/ml; spectinomycin, 100 µg/ml; streptomycin, 50 µg/ml for E. coli and 250 µg/ml for S. meliloti; kanamycin, 50 µg/ml for E. coli and 180 µg/ml for S. meliloti; cycloheximide, 200 µg/ml; and mercury chloride, 3.75 µg/ml for E. coli and 1.5 µg/ml for S. meliloti.

S. meliloti strain used for genetic modification. The M4 strain (Table 1) was chosen for genetic modification; this strain is tagged with the gusA gene in a nonessential region of the chromosome, between the recA and alaS genes, and is naturally resistant to streptomycin (29).

Plasmid stability. Stationary-phase cultures were diluted in fresh medium without selection to give an initial optical density of 0.03 at 620 nm and cultivated for 12 h (four generations). This process was repeated, and finally samples were serially diluted and plated onto solid media in the absence of selective drugs. One hundred colonies were chosen and picked onto plates with and without the selective antibiotics or mercury. The percentage of plasmid loss per generation was calculated with the formula [1  (Fr/Fi)/n] × 100, where Fr is the antibiotic resistance cell fraction after n generations and Fi is the initial fraction of resistant cells.

-Galactosidase assays. -Galactosidase activity was measured using the sodium dodecyl sulfate-chloroform method described by Miller (18), with S. meliloti cultures grown to exponential phase in MM broth with or without 0.2% (wt/vol) proline.

Microcosm studies. Ten alfalfa (Medicago sativa L. cv. Aragón) seedlings were grown in pots containing 125 g of soil, originating from Riego de la Vega (see "Field experiment plots and sites" below), mixed with 125 g of sterile sand. This soil is an eutric fluvisol (sand, 34%; silt, 44%; clay, 22%; pH 7.1; organic matter, 2.2%; total N, 0.18%; total organic carbon, 1.5%) with an indigenous S. meliloti population of 10² cells/g of dry soil. The pots were inoculated with 75 ml of water-inoculum mixture to obtain 10³ to 10⁴ CFU/g of soil mixture. Pots were covered with a layer of sterile perlite to avoid desiccation and placed in the growth chamber with a light-dark cycle consisting of 16 h of light and a temperature of 25°C followed by 8 h of darkness and a temperature of 18°C, with the humidity kept at 50%. Three replicates per treatment were performed. Roots were washed, and nodule occupancy was determined as described below.

Preparation of inoculum and seed coating for field release. To prepare the inoculum, cultures were grown to early stationary phase in tryptone-yeast medium supplemented with mercury chloride and appropriate antibiotics. The culture was washed and concentrated in MM to approximately 10¹¹ bacteria/ml. The inoculum was mixed with sterile peat at a 2:3 (vol/wt) ratio. Fifty grams of seeds (1 g = 440 seeds) was coated with 1 g of peat-inoculant mixture, 3 ml of sterile water, and 1 ml of adhesive (40% [wt/vol] gum arabic solution), resulting in a density of approximately 10⁵ bacteria/seed. The mixture was allowed to dry, and the seeds were then sown immediately, with 1.4 g of coated seeds per pot in each field plot.

Field experiment plots and sites. The field experiment was conducted at Riego de la Vega, León, Spain (42°24'N, 5°59'W). This agricultural field has a long history of alfalfa, sugarbeet, and barley crops. The indigenous S. meliloti population in this field site before the release experiment was 10² cells/g of dry soil (as determined using the most-probable-number method described by Brockwell [4]).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Map of the field site at Riego de la Vega, León, Spain. Three plots each were inoculated with M401 or M403 or were left untreated (NI).

Persistence. The persistence of the inoculum was determined on three different surfaces: bulk soil (soil not associated with alfalfa roots), rhizosphere soil (soil adhering to the root surface), and rhizoplane (the root surface). Samples from each surface type were serially diluted in sterile water and plated on MM supplemented with 20 µg of X-Gluc (5-bromo-4-chloro-3-indolyl--D-glucuronide) (Apollo Scientific Ltd., Stockport, United Kingdom) per ml, streptomycin, mercury chloride, and cycloheximide. Persistence was assessed by counting the blue S. meliloti colonies that developed. To obtain rhizosphere soil, alfalfa roots were washed in sterile phosphate-buffered saline (PBS) (140 mM NaCl, 3 mM KCl, 8 mM Na₂HPO₄, and 1.5 mM NaH₂PO₄) for 20 min. The buffer used for washing was then centrifuged for 5 min at 8,700 × g at 4°C. The pellet corresponded to rhizosphere soil, 1 g of which was used to produce serial 1:10 dilutions, which were then plated. To determine the persistence of bacteria on the rhizoplane, roots from which rhizosphere soil had been removed were washed three more times with 50 ml of sterile PBS for 10 min each. The nodules were removed from these roots, and nodule occupancy was assessed. We then ground 0.5 g (wet weight) of root in 4.5 ml of sterile PBS in a sterile mortar. The resulting suspension was serially diluted (1:10) in sterile water and plated.

Nodule occupancy. We incubated the nodules, overnight at 37°C in the dark, in 1 mM X-Gluc in 50 mM sodium phosphate buffer (pH 7.5)-1% (wt/vol) sodium dodecyl sulfate. Nodule occupancy by modified and wild-type organisms was assessed by counting blue (gusA-containing strains) and white nodules, respectively. Nodules from each of the three replicates per treatment were pooled prior to -glucuronidase activity assays. The total number of nodules analyzed at each time is indicated in Tables 3 and 5. The 95% confidence intervals were estimated by multiplying standard errors of the proportions by 1.96. For statistical analyses, z scores for proportions were determined and contrasted to confidence levels of 0.05.

	RESULTS AND DISCUSSION

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Modification of putA gene expression. Previous studies (11) have suggested that the competitiveness of S. meliloti strains could be increased by increasing expression of the putA gene. To test this hypothesis, we inserted a promoter that would ensure high basal putA gene expression. As we have previously shown (30), the promoter sequences required for expression of the putA gene are located between 190 and 290 nucleotides upstream from the translation start site. The 190-nucleotide upstream region present in pMH310 was insufficient to drive putA gene expression (Table 2). The constitutive promoter of the kanamycin resistance gene (nptII) was placed 190 nucleotides upstream from the translation start site of the putA gene, and a transcriptional fusion between this combination and lacZ was produced in pRG970 to give pMH210 (Table 1; Fig. 2). -Galactosidase activity in the presence and absence of proline was measured for S. meliloti strain GRM8, which harbored pMH210 (Table 2). These results were compared with those previously obtained in the laboratory (30). In the absence of proline, pMH210 gave six-times-higher levels of expression than the wild-type promoter region present in pJZ301. In the presence of proline, the level of expression of putA by pMH210 was four times higher than that in the absence of this amino acid.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   DNA fragments fused to the reporter gene lacZ of pRG970. Open boxes and arrows indicate DNA sequences of putA, the filled box indicates the promoter of the kanamycin resistance gene, and the filled circles indicate the proline regulation region. pJZ301 carries a putA::lacZ transcriptional fusion containing the wild-type promoter region (12). pMH210 carries a putA::lacZ transcriptional fusion containing the constitutive promoter of the kanamycin resistance gene 190 nucleotides upstream from the putA gene. pMH310 carries a putA::lacZ transcriptional fusion containing only the 190-nucleotide region upstream from the putA gene (30).

Microcosm experiments. Proline exuded by alfalfa plants may be an important energy and carbon source for S. meliloti during the colonization and infection process (11, 12, 20). Thus, an enhanced ability to metabolize this energy source may constitute a metabolic advantage, rendering the strain more competitive. The properties of the promoter region from pMH210 coupled with a functional putA gene may confer such a metabolic advantage on the bacteria. We therefore fused the promoter region of pMH210 to the remainder of the putA gene, to obtain the plasmid pMJV3 (Table 1). Soybean and alfalfa plants have been shown to accumulate proline in their nodules when grown under salt or drought stress (8, 13, 14). This may result in an increase in the availability of proline within root exudates. If this is indeed the case, then any metabolic advantage conferred by the higher level of putA gene expression from pMJV3 should be observed more clearly under drought conditions. We therefore performed microcosm studies under these conditions with an inoculum containing pMJV3.

Construction of the inoculum for field release. According to regulatory requirements, the kanamycin gene of pMJV3 was replaced with a mercury resistance cassette, giving pBBRHG3. As a control, the kanamycin gene of pBBR1MCS-2 was also replaced with the same cassette to give pBBRHG1 (Table 1). These two plasmids, pBBRHG1 and pBBRHG3, were introduced into S. meliloti strain M4 by conjugation, resulting in strains M401 (control strain) and M403 (carrying in the plasmid vector a putA gene under the control of the pKm promoter), respectively.

Environmental release. (i) Inoculation. The positive results obtained with the modified putA gene in the microcosm experiments conducted under simulated drought conditions led us to study the effect of this gene in a contained field release experiment. M401 and M403 were applied during the spring of 1999 to an agricultural field in Riego de la Vega, León, Spain. The soil of this field site was previously used for the microcosm experiments.

(ii) Persistence. The inocula used in our field experiment persisted throughout the experiment in rhizosphere soil and on the rhizoplane, with more than 10² bacteria per g (wet weight) of soil or root (Fig. 3). However, M403 was less persistent than M401 on these surfaces. This is probably because pBBRHG1 was more stable in M401 than pBBRHG3 was in M403 (the proportions of plasmid-containing cells after eight generations were 88.9 and 76.4%, respectively; the corresponding values for M4/pBB1MCS-2 and M4/pMJV3 were 100 and 95.8%, respectively). However, neither type of inoculum persisted well in bulk soil (Fig. 3A). Persistence assays performed with bulk soil surrounding the pots sown with seeds coated with M401 or M403 also showed that neither of these types of inoculum was present (data not shown). The higher persistence within the pots of the inoculum in rhizosphere soil and on the rhizoplane is not surprising, as the bacteria coating the seeds probably multiply preferentially along the growing root surface.

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Persistence of the control strain M401 () and the PutA-overproducing strain M403 () in bulk soil (A), in rhizosphere soil (B), and on the rhizoplane (C). The values shown for day 0 correspond to the CFU of each strain released per seed. Mean values and error values were calculated from three independent measurements. Error bars indicate standard errors, at a confidence level of 95%.

(iii) Nodule occupancy. The frequencies of nodule occupancy by M401 and M403 in the field experiment are indicated in Table 4. These results are very similar to those obtained with M4/pBBR1MCS-2 and M4/pMJV3 in the microcosm experiments performed under simulated drought conditions. In the field, M403, which contains the modified putA gene, occupied a larger proportion of the nodules than the control strain (M401) 1 month after inoculation. This result is especially interesting because the plants in the field were watered in the same way as crops would have been in an agricultural setting. In the following months, however, the proportion of nodules occupied by M403 did not change significantly, whereas that of nodules occupied by M401 increased, reaching levels similar to those for M403 2 months later. Once this level of nodule occupancy was attained by M401, it remained constant throughout the rest of the experiment. These results imply that an equilibrium in nodule occupancy was reached by both types of inoculum but that this equilibrium was reached more rapidly by M403. We cannot rule out that the transient advantage in competitiveness could be due to the reduced plasmid stability observed in M403 (see above). The integration of the modified putA gene into the chromosome would clarify this question.