The Enhancement of Ammonium Assimilation inRhizobium etliPrevents Nodulation ofPhaseolus vulgaris

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Abstract
The modification of the ammonium assimilation pathway of Rhizobium etli (GS-GOGAT) by adding an additional ammonium assimilation enzyme, GDH, strongly affects its symbiotic interaction with beans. The plasmid pAM1a, based in the stable vector pTR101 (M. Weinstein, R. C. Roberts, and D. R. Helsinki, J. Bacteriol. 174,7486-7489, 1992), containing the Escherichia coli gdhA gene flanked by two transcription-translation terminators was constructed. The expression of GDH in both, the wild type (CFN42/pAM1a) and a ntrC- mutant (CFN2012/pAM1a) R. etli strains, gave a similar metabolic effect, i.e., high GDH and reduced GOGAT activities, and an increased synthesis and excretion of several amino acids. The total inhibition of bean nodulation was observed when the minimum optimal inoculum of R. etli CFN42/pAM1a was used; however, an effective symbiosis occurred with the CFN2012/pAM1a mutant strain. While a total inhibition of the induction of the nodA gene by bean root exudate or by naringenin was observed in the CFN42/pAM1a strain, at 10 mM ammonium, the CFN2012/pAM1a showed an optimal nodA gene induction. A correlation between nodA gene induction, Nod factor production, and nodulation was observed. We conclude that in R. etli, there is a down-regulation of nod gene expression and nodulation when a high internal nitrogen content is built up by the presence of a functional GDH and that NtrC is involved in such regulation. An instability of the plasmid harboring the gdhA gene was observed during symbiosis, indicating a strong selection against cells containing this plasmid.

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