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Rhizobial bacteria form symbiotic associations with leguminous plants that are manifested as root nodules comprised of bacteria within specialized plant cells. The bacterial partner fixes atmospheric nitrogen to ammonia, which can be assimilated by the plant host to fulfill its nitrogen requirement, and the endosymbiont receives carbon sources ultimately derived from plant photosynthesis.

Cooperative associations of bacteria with animal or plant hosts involve the exchange of organic nutrients (1, 2, 7, 9, 13-15). Bacterial auxotrophic mutants have been used to assess amino acids, vitamins, or other metabolites that the prokaryote is able to obtain from the eukaryote in symbiosis (7, 9, 13, 15). Numerous amino acid auxotrophs of Vibrio fischeri can successfully colonize the light organ of its host, the squid Euprymna scolopes, showing that host-derived amino acids support bacterial proliferation in symbiosis (7). Some Sinorhizobium meliloti amino acid auxotrophs can effectively infect alfalfa (Medicago sativa) whereas others cannot (9), indicating that some amino acids can be provided by the host.

A role for proline as an energy source for the symbiotic bacteria (bacteroids) Bradyrhizobium japonicum (10) and S. meliloti (8) has been described. Proline oxidation activity is present in bacteroids, and an S. meliloti mutant defective in the proline oxidation enzyme proline dehydrogenase is impaired in its ability to colonize alfalfa (8). It has been suggested that proline utilized by bacteroids in nodules can be supplied by the plant host and that exogenously supplied proline enhances nitrogen fixation (10, 18). Furthermore, it has been proposed that proline exuded into the rhizosphere by the plant may enhance bacterial proliferation and colonization (8). In the present study, we sought to determine whether B. japonicum obtains plant-derived proline by investigating whether a proline auxotroph can establish an effective symbiosis on soybean plants. We isolated the B. japonicum proC gene, encoding the proline biosynthesis enzyme -1-pyrroline-5-carboxylate (P5C) reductase and demonstrated that the proC gene is essential for symbiosis with soybean.

Complementation of an E. coli proC mutant with B. japonicum genomic DNA. Escherichia coli strain E1772 is a proline auxotroph due to a mutation in the proC gene (encoding P5C reductase) (12). Strain E1772 was transformed en masse with a B. japonicum expression library (3), and complementing transformant clones were identified as ampicillin-resistant colonies that grew on minimal media lacking exogenous proline. We obtained 180 colonies out of approximately 10⁶ clones screened, and plasmids were isolated from 10 of the larger colonies. Four of these contained plasmids with identical restriction enzyme digestion patterns, and one of them, p238, was chosen for further analysis. Reintroduction of p238 into strain E1772 conferred proline prototrophy on the mutant (Fig. 1), which confirms that the complemented phenotype of the transformants was due to the plasmid and not to a chromosomal reversion.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Complementation of E. coli proC strain E1772 with B. japonicum library clone. Cells harboring either p238 (squares), which contains a 1.3-kb genomic insert, or the pBluescript SK control (circles) were grown in minimal M9 media in the presence (open symbols) or absence (closed symbols) of 0.4 mM proline. Strain E1772(p238) grew essentially as well in the presence of proline as in its absence (data not shown).

The nucleotide sequence of the 1.3-kb insert of p238 insert was determined, and it contains an 873-bp open reading frame encoding a peptide 290 amino acids in length. Database searches revealed that the protein had the highest homology to P5C reductases from diverse organisms including humans (36% identity, 45.4% similarity) (6), soybeans (34.7% identity, 43.5% similarity) (4), and the bacteria Pseudomonas aeruginosa (37.6% identity, 44.4% similarity) (16) (Fig. 2) and E. coli (33.6% identity, 41.5% similarity) (5). In addition, the cyanobacterium Synechocystis PCC6802 genome project (GenBank accession no. P74572) identified a putative P5C reductase gene whose product had the greatest identity (42.5%) and overall similarity (51.3%) to the B. japonicum protein. This identity, along with the ability of p238 to complement E. coli strain E1772, strongly indicates that the B. japonicum proC gene was isolated.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Sequence alignment of the P5C reductases from B. japonicum and P. aeruginosa. Solid lines, identity between the sequences; dotted lines, conservative amino acid substitutions.

Construction of a B. japonicum proC mutant strain. A proC mutant strain was constructed with B. japonicum strain I110 by gene-directed mutagenesis. To do this, a 128-bp SacII fragment within the proC open reading frame was removed and replaced with a 2-kb omega () cassette encoding resistance to both spectinomycin and streptomycin (Fig. 3A). The disrupted gene borne on pLO1 (11) was introduced into B. japonicum, and homologous recombination with genomic DNA was screened for by growth of colonies in the presence of the antibiotics and sucrose, the latter of which selects against single recombinants. This procedure yielded proC mutant strain I110proC, which was chosen for further studies. Southern blot analysis of I110 DNA digested with SmaI or SalI using the SalI fragment containing proC as a probe yielded single fragments 2.5 and 1 kb in size, respectively, whereas genomic DNA from strain I110proC gave single bands of 4.5 and 3 kb, respectively, as predicted (data not shown). Furthermore, pLO1 was absent in strain I110proC as determined by Southern blotting using the plasmid as probe. Thus, B. japonicum I110 contains a single proC gene that was disrupted by the  cassette in the mutant.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Construction of B. japonicum proC strain I110proC and demonstration of its proline auxotrophy. (A) A restriction map of the 1.3-kb Sau3AI insert of complementing plasmid p238, showing restriction sites of NarI (N), SalI (S), PvuI (P), and SacII (Sc). The arrow denotes the open reading frame encoding P5C reductase. The internal 128-bp SacII fragment (solid portion of arrow) was removed and replaced with an  cassette. The disrupted gene was used to replace the wild-type proC gene by homologous recombination. (B) B. japonicum cells were grown in minimal medium containing no proline (closed symbols) or 1.7 mM proline (open symbols). Mutant strain I110proC showed no growth in medium lacking exogenous proline (closed circles). Growth of parent strain I110 in the absence of proline (squares) was essentially the same as in its presence (data not shown).

Strain I110proC was grown in liquid medium in the absence or presence of exogenous proline and was found to be a strict proline auxotroph (Fig. 3B). These observations are consistent with a single proC gene and suggest that there is not an alternative pathway for proline formation, at least under the conditions examined.

The proC gene is required for symbiosis with soybean. To examine whether the proC gene was necessary for symbiosis with soybeans, 2-day-old germinated seedlings were inoculated with parent strain I110 or mutant strain I110proC at the time of planting, and nodules from 26-day-old plants were analyzed (Table 1). Nodules elicited by the proC mutant were small and starchy in appearance and contained few viable bacteria. Furthermore, those nodules did not fix nitrogen or contain a detectable quantity of leghemoglobin, and thus they lacked conspicuous bacterial and plant markers, respectively, of a developed and functional nodule. Supplementation of the plant nutrient medium with 1.7 mM proline did not alter bacterial viability in nodules or nitrogen fixation activity (data not shown).

To verify that the symbiotic phenotype was the result of the disruption of proC in strain I110proC rather than of a possible polar effect of the  cassette, we tested the ability of a wild-type copy of proC to rescue the mutant strain. Plasmids are often unstable in symbiotic B. japonicum cells, making in trans complementation experiments difficult to interpret. Thus, we constructed a strain containing a normal copy of proC upstream of the mutant gene in the chromosome, which preserves any polar effects of the  cassette and specifically allows assessment of the effect of proC on complementation. Partial diploid strain I110ppd was constructed by single recombination of the 1.3-kb Sau3A fragment containing proC as the only open reading frame ligated into pLO1 as described above. The relative positions of the wild-type and mutant proC genes in the chromosome were determined by Southern blot analysis of PvuI-digested genomic DNA (data not shown). Strain I110ppd grew as well as the parent strain in liquid culture or on plates in the absence of proline (data not shown). Nodules elicited by strain I110ppd were similar to those elicited by the wild-type strain with respect to all parameters tested (Table 1), showing that proC complemented the mutant. We conclude that the B. japonicum proC gene is essential for symbiosis with soybeans and suggest that the soybean host is unable to rescue the proline auxotroph.

Conclusions. In the present work, we isolated B. japonicum proC and constructed a mutant strain defective in that gene. Addition of proline was both necessary and sufficient to compensate for the defective gene with respect to growth in culture. Strain I110proC was unable to elicit developed, nitrogen-fixing nodules on soybean, indicating that proC is required for symbiosis. Although proline is proposed to serve as an energy source for B. japonicum in nodules (10), a mutant defective in proline dehydrogenase, the enzyme which oxidizes proline to derive energy, forms an effective symbiosis (17). Based on this, it is likely that the symbiotic requirement for proC is primarily for protein synthesis.

Because some amino acid auxotrophs can be rescued symbiotically (9), and plant-derived proline is proposed to be available to rhizobia in the rhizosphere and in nodules (8, 10), the essentiality of the B. japonicum proC gene for symbiosis was somewhat surprising. The present work indicates that strain I110proC does not obtain an exogenous source of proline in its association with soybean that is sufficient to satisfy its auxotrophic requirement. This suggests that it is unlikely that plant-derived proline can serve as an energy source for B. japonicum throughout infection and subsequent nodule development and function. It is possible that the endosymbiont obtains proline from the host only after the nodule has fully formed and that the proline auxotrophy prevents early nodule development. Nevertheless, several amino acid auxotrophs can establish effective symbiosis, and therefore the plant can directly affect bacterial metabolism very early in the interaction in those cases.

Nucleotide sequence accession number. The Sau3AI partial digest fragment insert of p238 including the B. japonicum proC gene is in GenBank under accession number AF302126.