INTRODUCTION

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The environments where most prokaryotic species are found in nature are often limited in nutrients, with a changing composition in both space and time. Many adaptations to these conditions are known, some of which result from high-affinity uptake systems, differentially expressed enzymes, and a variety of metabolic control mechanisms (27). Amidst bacterial processes that depend on the relative scarcity of one macronutrient, atmospheric N₂ fixation in N-poor environments has caused considerable interest for more than a century (11, 39).

Many species of the family Rhizobiaceae are outstanding in that they fix N₂ only in symbiosis with legume plants. This interaction starts in the soil with a specific plant-rhizobium molecular signal exchange involving plant flavonoids released into the root exudates, which induce the expression of the nod operons in the rhizobia. These genes encode the enzymes for the biosynthesis and release of a lipochitooligosaccharide known as Nod factor, which triggers nodule development in the inner root cortex with no requirement for the presence of active rhizobia (38). The nodule will provide the rhizobia with the nutrients and the microaerobic environment required for N₂ fixation (38). In parallel with plant nodule organogenesis, rhizobia are chemoattracted to the root surface (21), attach to it in nonspecific (48) as well as in bacterial lectin-mediated specific (22, 29) ways, penetrate the root hairs, forming characteristic structures known as infection threads, and finally invade the developing nodule (49), where they subsequently differentiate into bacteroids, a distinct rhizobial form which is the only one able to reduce atmospheric N₂ (38).

The process of infection and bacteroid differentiation is strongly dependent on the structure of the bacterial surface polysaccharide, which seems to play a role not only in recognition of rhizobia by the plant, but also as a signal to prevent the elicitation of plant defense activities arising against bacterial penetration (2, 24). In addition, some plant lectins, such as soybean lectin (SBL), are released into the rhizosphere (52, 55) and specifically stimulate rhizobial adsorption and infection (30, 55). Although the mechanism of plant lectin action remains obscure, its role in restricting rhizobium-host specificity range was demonstrated in studies with transgenic plants (15, 52).

Therefore, rhizobial adsorption, root hair infection, nodule formation, and nitrogen fixation are key steps of a complex process, each one contributing to a different level of symbiotic recognition and effectiveness. Throughout this process soil nitrogen sources like nitrate and ammonia (hereafter referred to as combined nitrogen) are required in limited amounts. It is well known that legumes possess a systemic regulatory control able to detect the presence of combined nitrogen in the rhizosphere and block nodulation in response (45), although the identity of the plant messenger that controls nodulation remains to be elucidated (47). Moreover, when combined nitrogen is administered to already nodulated plants, established nodules undergo senescence faster (33).

Other studies addressed the effect of changes in combined nitrogen availability from the rhizobial side. In Bradyrhizobium japonicum and Sinorhizobium meliloti the induction of nod genes is inhibited by high amounts of combined nitrogen (17, 56). On the other hand, induction of nod genes under N starvation conditions was weaker in mutants with mutations in the NtrBC two-component regulatory system of both species, although the extent to which this global regulatory system actually modulates nod gene expression was not fully established (56). Nodulation and nitrogen fixation were severely impaired in Rhizobium etli when bacterial N assimilation was artificially increased by introducing the Escherichia coli glutamate dehydrogenase gene (34). In addition, it was reported that N-limited R. etli formed more infection threads in common beans than either C-limited or exponentially growing cells (10). Indeed, the convenience of using a culture medium with a high C/N ratio instead of a low one to get better nodulation is well known in the industry of rhizobial inoculants for legume crops and was also related with an enhanced bacterial ability to initiate root hair infections (26). All these results indicate that, as for the plant counterpart, the presence of combined nitrogen in excess impairs key rhizobial symbiotic activities. On the other hand, limitation of combined nitrogen might stimulate the rhizobia for infection and nodulation (10). Although the latter could have important biotechnological implications, this has been less studied than the effects of N excess.

In B. japonicum, as in the other rhizobia, NH₄⁺ assimilation proceeds through the high-affinity glutamine synthetase (GS)-glutamate synthase (GOGAT) cycle (12). In addition, these rhizobacteria possess two different GSs. GSI, encoded by the glnA gene, is similar to other prokaryotic GSs except that its synthesis is not regulated by the two-component system NtrBC, its activity being controlled by adenylyl transferase-dependent adenylylation. GSII, encoded by the glnII gene, is similar to the chloroplastic GS, and its synthesis is under the control of NtrBC (12, 32). When N supply is sufficient, GSI is partially active, depending on the number of adenylylated (inactive) subunits per GSI dodecamer. As cell demand for N compounds increases, GSI deadenylylates, and GSII synthesis is induced.

Both control systems respond to the relative amounts of 2-oxoglutarate and glutamine inside the cell (12, 34, 40). A high 2-oxoglutarate/glutamine ratio signals a limitation of N precursors, and thus deadenylylation of GSI and synthesis of GSII are promoted in order to increase NH₄⁺ assimilation. These two control levels operating on different GS enzymes might help the rhizobia become highly efficient for scavenging, in their natural environments, the low levels of N sources that are needed to establish a productive nitrogen-fixing symbiosis, as already mentioned. On the other hand, when bacterial growth is N-limited, the relative C/N imbalance causing an abundance of C nutrients often results in changes of carbon fluxes, favoring the synthesis of polysaccharides and/or polyhydroxyalkanoates (31, 57). Since certain surface polysaccharides are involved in infection and nodulation efficiency, it is important to establish whether N-limiting conditions could favor their accumulation. In addition, this understanding could provide additional insight into the coordination of the C and N metabolic fluxes in rhizobia.

Here we present our study about the effects of N limitation on C accumulation in B. japonicum and its relation to the enhancement of its symbiotic association with soybean.

	MATERIALS AND METHODS

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Bacterial strains and culture conditions. B. japonicum LP 3001 and LP 3004 are Nod⁺ Fix⁺ spontaneous derivatives, spectinomycin (Sp) resistant and streptomycin (Sm) resistant, respectively, of strain E111 (29). Bj110-573 is a tetracycline (Tc)-resistant nodC::lacZ chromosomal fusion (16), kindly provided by Michael Göttfert (Dresden, Germany). Antibiotics were used at the following concentrations: Sp, 300 mg liter¹; Sm, 400 mg liter¹; Tc, 100 mg liter¹; cycloheximide (Ch), 50 mg liter¹. Bacteria were maintained on yeast extract-mannitol (YM) medium (54) with 30% (vol/vol) glycerol at 80°C.

Determination of GS activity. GSI and GSII activities were assayed with permeabilized cells (3). Cultures (300 ml) were halted by the addition of 3 ml of a 1% (wt/vol) solution of hexadecyltrimethylammonium bromide (CTAB). Cells were harvested by filtration through a 0.2-µm-pore membrane, washed once with 1% (wt/vol) KCl, and resuspended in 3 ml of 1% KCl. GS was measured by its -glutamyl transferase (-GT) activity (3). To discriminate between GSI and GSII, the activity was measured before and after a 1-h incubation at 50°C, which irreversibly inactivates GSII (14). The adenylylation state of GSI was determined by inhibiting the activity of adenylylated subunits in the presence of 60 mM MgCl₂, and the mean number of adenylylated subunits was calculated by comparison with the activity measured in the absence of MgCl₂ (46). Specific activity was expressed as nanomoles of -glutamyl hydroxamate produced per minute per milligram of protein (3).

Polysaccharide preparations. Cultures (300 ml) of B. japonicum 3001 were centrifuged at 12,000 × g for 40 min. The supernatant was used for exopolysaccharide (EPS) preparation, and the cell pellet was used for the capsular polysaccharide (CPS). The EPS was precipitated with 3 volumes of ethanol 96% at 20°C, and after resuspension in 0.5 M NaCl, it was stored at 20°C for further analysis. To prepare the CPS (36), pelleted bacteria were washed with 0.5 M NaCl and shaken in a Beckman Omnimixer for 30 s at mean power. This suspension was centrifuged for 15 min at 14,000 rpm in a microcentrifuge, the supernatant was stored at 20°C, and the cell pellet was reserved for cell protein determination. EPS and CPS were viewed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by Alcian blue-silver staining (43).

Analytical determinations. Protein concentration was determined as described (9) in supernatants from cells disrupted by ultrasonic treatment with a Sanyo Soniprep 150 using three 10-s pulses at mean power. Polysaccharides were determined with 0.2% (wt/vol) anthrone in 95% H₂SO₄, as described (51), using glucose as the standard. EPS was determined in the above-described culture supernatants, and CPS was quantified in the Omnimixer-shaken cell supernatants.

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nodC expression. Strain Bj110-573 was cultivated as described above, and at the appropriate times, the culture was divided into 2-ml aliquots. For induction, genistein was added at a final concentration of 2 µM (1), and incubation was continued at the same temperature and shaking conditions for an additional 12 to 14 h. Then the cells were permeabilized with 0.1% (wt/vol) SDS and chloroform (35), and the -galactosidase activity was determined using chlorophenol red galactopyranoside (CPRG) as the substrate (1). Activity is expressed in Miller units (35) except that CPRG hydrolysis was quantitated using absorbance at 574 nm.

SBL binding. SBL was isolated from soybean Promax seeds (kindly provided by A. Perticari, INTA, Argentina) by affinity column chromatography on -amino-caproyl-N-acetyl-D-galactosamine agarose; its purity was evaluated as described (30), and an aliquot from this preparation was labeled with fluorescein isothiocyanate (FITC) (5). Rhizobia were incubated with 60 µg of FITC-SBL ml¹ for 30 min at 28°C without shaking. The percentage of fluorescent cells was recorded by fluorescence/phase-contrast microscopy in a Neubauer chamber as described (5).

Plant experiments. Soybean Promax seeds were surface sterilized by immersion in 96% ethanol for 5 s and then in 20% (vol/vol) commercial bleach for 10 min, followed by six sterile distilled-water washes. Seeds were germinated on aqueous agar (1.5%, wt/vol) in darkness (29).

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	RESULTS

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Physiological and symbiotic characterization of B. japonicum LP 3001 grown in N-limited Götz media. Growth of strain LP 3001 in GN0, GN0.1, and GN1 in batch cultures was studied by recording the OD₅₀₀ and viable cell concentration every 24 h over a 20-day period. The resulting growth curves are shown in Fig. 1, being representative of three independent experiments with essentially the same results. During the first 5 days, all three cultures were in the exponential phase of growth despite the different N availability; afterwards the cultures entered the stationary phase: GN0 at day 6, GN 0.1 at day 10, and GN1 at day 13. After these time periods, the OD₅₀₀ remained constant, although at higher readings for GN1 (around 1.42) than for GN0.1 (0.51) and GN0 (0.28), reflecting the N limitation for growth (Fig. 1A).

View larger version (12K): [in this window] [in a new window]   FIG. 1.   Growth of B. japonicum LP 3001 in GN0 (squares), GN0.1 (triangles), and GN1 (circles). (A) Biomass, as estimated by optical density at 500 nm. (B) Viable cell counts.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Nodulation profiles produced in soybean primary roots by B. japonicum LP 3001 grown for the indicated times (days) in GN0 (A and D), GN0.1 (B and E), or GN1 (C and F). In A, B, and C, the distribution of the nodules along the primary roots of 42 plants is shown as the number of nodules within intervals of 1 RDU from the root tip (RT) mark made at inoculation. The zero value corresponds to the root tip position; negative values represent those parts of the roots which grew after inoculation. In D, E, and F, the RDU interval where most nodules appeared (most frequent interval, circles) and the mean number of nodules per primary root, including those with zero nodules (triangles), are shown as a function of the culture age in days.

Effects of N limitation on activity of GS and on carbon sink. While 14-day-old GN0 and GN0.1 cultures were clearly N limited, up to 5 days of growth the cultures in all three media were in exponential phase (Fig. 1). This suggests that whatever the amount of N added to the medium, even a small amount of NH₄⁺ carried over from the starter culture, estimated to be less than 40 µM final concentration in GN0, was enough to sustain growth for these first 5 days at a rate of 1.2 day¹ (Fig. 1). This growth rate agrees with previous reports of B. japonicum growth with mannitol as the carbon source (25). Rhizobia are able to satisfy their N needs in very scarce environments by means of their two GS enzymes (12), which together form a highly efficient N-assimilating system. Regulation is achieved by sensing the 2-oxoglutarate:glutamine ratio, which is indicative of the C/N relationship inside the cell. Therefore, similar growth rates of young cultures in GN0, GN0.1, and GN1 could be obtained through this metabolic control of N assimilation.

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View larger version (44K): [in this window] [in a new window]   FIG. 3.   FTIR spectra of 5-day-old (dashed lines) and 14-day-old (solid lines) B. japonicum LP 3001 grown in GN0 (A), GN0.1 (B), or GN1 (C).

Symbiotic performance in relation to N limitation. CPS contains the binding site for SBL (5), which stimulates adsorption of rhizobia to roots, as well as infection and competition for nodulation (30). Since N starvation strongly increased CPS production (Table 2), we tested whether, under those conditions, SBL binding to rhizobia and its further stimulation of adsorption were also enhanced by N starvation. To this end we incubated the different cultures of strain LP 3001 in the presence of FITC-labeled SBL for at least 30 min (5) and recorded the percentages of fluorescent cells. We also incubated cells for 12 h in the presence of unlabeled SBL and recorded the adsorption index to soybean roots. We observed that SBL binding was strongest to 5-day-old GN0 cells, intermediate to 14-day-old GN0 and GN0.1 cells, and lowest to the other cultures. The adsorption index increased by 276.0% in 5-day-old GN0 rhizobial cells and 58.3% in the 5-day-old GN0.1 culture, but was half-reduced in 14-day-old GN0 or GN0.1 cultures (Table 3). Cell clumping was not significant, as observed under the microscope.

View larger version (31K): [in this window] [in a new window]   FIG. 4.   Nodulation profiles produced in soybean primary roots by B. japonicum LP 3001 grown in GN0 (A and B), GN0.1 (C and D), or GN1 (E and F) for 5 days (A, C, and E) or 14 days (B, D, and F). The distribution of the nodules along the primary roots of 42 plants is shown as the number of nodules within intervals of 1 RDU from the root tip (RT) mark made at inoculation. The zero value corresponds to the root tip position; negative values represent those parts of the roots which grew after inoculation.

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	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

The ability of B. japonicum to survive and remain infective after months of incubation in deionized water is known (13), as well as its fitness under starvation conditions (23). Here we have shown that growth can be sustained for at least six generations with micromolar amounts of NH₄⁺ as the N source in minimal medium (Fig. 1). On the other hand, the Götz medium, with a 2 mM NH₄⁺ concentration and 28 mM mannitol as the carbon source (GN1), allowed the production of a considerable biomass and viable cells (Fig. 1) and did not produce any symptom of N starvation in B. japonicum. Thus, total biomass could not be increased in this medium by raising the NH₄⁺ concentration 10-fold. In addition, GS, one of the main N-assimilating activities in free-living B. japonicum, was downregulated in stationary-phase GN1 cultures, GSI activity being half-repressed and GSII not being induced (Table 1), indicating a low 2-oxoglutarate/glutamine intracellular ratio (34, 40). Furthermore, extracellular polysaccharides did not rise from exponential to stationary phase in GN1 (Fig. 3 and Table 2), indicating that inside the cell C was not in excess over N.

The state of N starvation in the other cultures was indicated by the control of N assimilation by the GS enzymes, which appeared to be regulated in two consecutive levels. When available N was more than enough to satisfy the cell's needs (as in the 14-day-old GN1 culture), GSI was partially inactivated by adenylylation, and GSII activity was negligible; as the level of available N decreased, the first increase in GS activity was achieved through GSI deadenylylation, and only when N needs were extreme (as in the 5-day-old GN0 culture) was GSII activity significantly induced in addition to GSI complete deadenylylation (Table 1). Thus, total GS activity was significantly higher for N-starved cultures.

When rhizobial cultures enter stationary phase, electron transport could drop, leading to accumulation of carbon and reducing power, which might inhibit 2-oxoglutarate dehydrogenase (44). However, in B. japonicum a bypass through 2-oxoglutarate decarboxylase and succinic semialdehyde dehydrogenase was recently identified (20), whereby under certain circumstances the tricarboxylic acid cycle would be less impaired in both the free-living and bacteroid states. In addition, N starvation precludes protein synthesis, leading to a rise in energy charge if an appropriate C source is present. Here we observed whether those possible excesses in C, reducing power, and/or energy charge were directed to the synthesis of polysaccharides and P(3HB).

In N-limited cultures, the rise in EPS and CPS production with respect to total cell protein was much higher than accumulation of P(3HB). On the other hand, when growth was limited by another nutrient, C was preferentially channeled to P(3HB) synthesis (Fig. 3 and Table 2). In agreement with our results, S. meliloti was also found to produce more EPS than P(3HB) when growth was N limited (50). Since P(3HB) is intracellular, its accumulation should be limited by cell size and also by the kinetics of P(3HB) granule formation (31). Therefore, as the C/N ratio increases, C excess over P(3HB) storage capacity could be channeled to extracellular structures such as CPS and excreted into EPS. Although somewhat obvious, this cell compartmentation of C polymers does not seem to be the only explanation for the difference in C channeling in response to the C/N ratio.

P(3HB) is more reduced than mannitol, CPS, or EPS, and polysaccharide synthesis by overflow metabolism might involve a recycling of carbon through dehydrogenating pathways (42), thus contributing much less to the disposal of excess reducing power than P(3HB) synthesis does. This could indicate that N-limited cultures accumulated less reducing power, probably as a consequence of their lower biomass. In addition, the high NH₄⁺ affinity of the GS enzymes, inferred from the ability of these rhizobia to grow at very low NH₄⁺ concentrations, might allow the glutamate resulting from 2-oxoglutarate transamination to be taken up by the GS-GOGAT cycle at a rate sufficient to continue with NAD(P)H consumption (e.g., for glutamine synthesis) and to prevent, to some extent, the accumulation of acetyl-coenzyme A at stationary phase in N-limited cultures. By contrast, GN1 cultures could accumulate enough glutamate to cause product inhibition of GOGAT-catalyzed transamination because of its high NH₄⁺ availability and low GS activity, which would lead to higher 2-oxoglutarate concentration and less NADPH consumption at this step.

Despite higher polysaccharide accumulation in N-starved cultures, we observed that the degree of EPS and CPS polymerization was the same in all six cultures, suggesting that the influence of N starvation on extracellular polysaccharide production was exerted at early biosynthetic steps.

Differences in EPS and CPS production might at least in part explain the different nodulation profiles produced by the stationary-phase cells (Fig. 2), if we assume that in either case only a subpopulation of the rhizobial cells in the culture are able to infect and nodulate the roots (53). When grown in N-starving conditions, the relative size of this subpopulation seemed to diminish as age increased, although individual cells retained their ability to nodulate fast: the reduction in the mean number of nodules per plant was 66% in GN0 and 87% in GN0.1, while most of the nodules appeared around the position occupied by the root tip at inoculation (Fig. 2D and E). On the contrary, the reduction in the mean number of nodules per plant with N-sufficient rhizobia was only 28% as age increased, but the most frequent position of these nodules was displaced 5 RDU towards the growing root apex (Fig. 2F), suggesting that individual bacterial cells were slower to nodulate. This indicates that these cells could require more time to initiate infections, perhaps because of changes in CPS (and EPS) content and composition that had to take place in the rhizosphere (4).

Cells with higher CPS content might bind more SBL, depending on the composition of these CPS (5). In contrast to previous findings employing N-sufficient cultures, we observed that under N-starving conditions the cultures possessed more EPS and CPS in stationary than in exponential phase (28, 36). Nevertheless, SBL binding was the highest in 5-day-old GN0 cultures (Table 3), suggesting that their CPS had a different composition than those of stationary-phase bacteria for the same media (5). Such a compositional change might have important symbiotic implications because (i) exoB mutants unable to synthesize UDP-galactose, the precursor of the SBL-binding monosaccharide unit, show delayed nodulation and impaired competitiveness (41) and (ii) preincubation of rhizobia in SBL preparations was shown to improve early preinfection and nodulation as well as competitiveness (30).

Stimulation of adsorption by the SBL was strongly enhanced by N starvation only in young cultures (Table 3), but there was some stimulation in the GN0.1 culture. The stimulation value obtained for 5-day-old GN0.1 cells approaches the values we obtained before with yeast extract-mannitol medium (30), which has an approximate total N concentration of 0.3 mM and 56 mM mannitol as the main carbon source. Therefore, we can suggest that SBL stimulation of rhizobial adsorption to soybean roots requires rhizobia grown in N-starving conditions (at least at an early starvation state), in partial correlation with the CPS rhizobial content. On the other hand, a contrary effect was observed in the 14-day-old cultures, where adsorption of the N-limited ones was partially inhibited by SBL binding (Tables 2 and 3). These results were not affected by differential cell clumping, as indicated by direct microscopic observation of the cells, in agreement with our previous results (29, 30). Although different incubation times of rhizobia with SBL were used to study SBL binding and stimulation of adsorption, this result might indicate that beyond or as a consequence of SBL binding, a certain mechanism(s) operative in exponential cells only (55) activated a higher adsorption. The existence of such a mechanism is suggested by the long incubation time required for an enhanced adsorption response (30).

In addition to plant lectin action on adsorption, another important preinfection event is the induction of the nod genes by root-exuded flavonoids. Induction of nodC gene expression by genistein was higher in the N-starved cultures (Table 4). This result adds to previous ones on ammonia inhibition of nodY and nodD gene expression in B. japonicum (56). On the other hand, the small nodC induction observed in the 14-day-old GN1 culture could be related to the inhibitory activity of a recently detected quorum-sensing signal molecule (G. Stacey, B. Zhang, Y. Chen, D. Xu, Y.-H. Lee, C. Bickley, D. Lohar, G. Liao, G. Copley, and J. Loh, Abstr. 13th Int. Congress Nitrogen Fixation, abstr. L36, 2001) which might not accumulate in the less dense GN0 or GN0.1 cultures.

Nodulation efficiency (Fig. 4) and competitiveness (Table 5) were also improved by N limitation. Nodulation profiles of young cultures correlated with SBL stimulation of adsorption, since the GN0 and GN0.1 cultures were the more effective and the only ones in which adhesiveness was stimulated by lectin pretreatment (Fig. 4 and Table 3). However, nodulation efficiency in 14-day-old N-limited cells was higher than in GN1-grown cells (Fig. 4), in contrast to stimulation of adsorption which was lacking in all three 14-day-old cultures (Table 3). The trend for nodulation profiles in stationary-phase cultures correlated better with EPS and CPS content (Table 2) and nodC induction (Table 4), both of which were higher in N-limited cultures. Competition for nodulation correlated well with the level of extracellular polysaccharide content and nodC induction (Tables 2, 4, and 5), the GN0 N-starved culture showing the highest response in these three traits, while the GN0.1 could compete against the GN1 culture only under a stringent competition represented by high inoculum doses. This range of competitive ability correlates with the effects of SBL in stimulating adsorption, in agreement with previous observations (30).

The results presented here indicate that rhizobial N starvation has a positive influence on the symbiosis of B. japonicum with soybean plants, through parallel effects on the EPS and CPS structure (41), nod gene induction (38), and SBL stimulation of adsorption (30), all of which resulted in increased nodulation efficiency and competitiveness.