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The symbiotic interaction between Rhizobium etli and Phaseolus vulgaris, the common bean plant, ultimately results in the formation of nitrogen-fixing nodules. Many aspects of the intermediate and late stages of this interaction are still poorly understood. The R. etli relA gene was identified through a genome-wide screening for R. etli symbiotic mutants. RelA has a pivotal role in cellular physiology, as it catalyzes the synthesis of (p)ppGpp, which mediates the stringent response in bacteria. The synthesis of ppGpp was abolished in an R. etli relA mutant strain under conditions of amino acid starvation. Plants nodulated by an R. etli relA mutant had a strongly reduced nitrogen fixation activity (75% reduction). Also, at the microscopic level, bacteroid morphology was altered, with the size of relA mutant bacteroids being increased compared to that of wild-type bacteroids. The expression of the N -dependent nitrogen fixation genes rpoN2 and iscN was considerably reduced in the relA mutant. In addition, the expression of the relA gene was negatively regulated by RpoN2, the symbiosis-specific N copy of R. etli. Therefore, an autoregulatory loop controlling the expression of relA and rpoN2 seems operative in bacteroids. The production of long-and short-chain acyl-homoserine-lactones by the cinIR and raiIR systems was decreased in an R. etli relA mutant. Our results suggest that relA may play an important role in the regulation of gene expression in R. etli bacteroids and in the adaptation of bacteroid physiology.

The Rhizobium etli rpoN1 gene, encoding the alternative sigma factor ς54 (RpoN), was recently characterized and shown to be involved in the assimilation of several nitrogen and carbon sources during free-living aerobic growth (J. Michiels, T. Van Soom, I. D’hooghe, B. Dombrecht, T. Benhassine, P. de Wilde, and J. Vanderleyden, J. Bacteriol. 180:1729–1740, 1998). We identified a second rpoN gene copy in R. etli,rpoN2, encoding a 54.0-kDa protein which displays 59% amino acid identity with the R. etli RpoN1 protein. TherpoN2 gene is cotranscribed with a short open reading frame, orf180, which codes for a protein with a size of 20.1 kDa that is homologous to several prokaryotic and eukaryotic proteins of similar size. In contrast to the R. etli rpoN1mutant strain, inactivation of the rpoN2 gene did not produce any phenotypic defects during free-living growth. However, symbiotic nitrogen fixation was reduced by approximately 90% in therpoN2 mutant, whereas wild-type levels of nitrogen fixation were observed in the rpoN1 mutant strain. Nitrogen fixation was completely abolished in the rpoN1 rpoN2 double mutant. Expression of rpoN1 was negatively autoregulated during aerobic growth and was reduced during microaerobiosis and symbiosis. In contrast, rpoN2-gusA and orf180-gusA fusions were not expressed aerobically but were strongly induced at low oxygen tensions or in bacteroids. Expression of rpoN2 andorf180 was abolished in R. etli rpoN1 rpoN2 andnifA mutants under all conditions tested. Under free-living microaerobic conditions, transcription of rpoN2 andorf180 required the RpoN1 protein. In symbiosis, expression of rpoN2 and orf180 occurred independently of the rpoN1 gene, suggesting the existence of an alternative symbiosis-specific mechanism of transcription activation.

The Rhizobium etli CNPAF512 fnrN gene was identified in the fixABCX rpoN 2 region. The corresponding protein contains the hallmark residues characteristic of proteins belonging to the class IB group of Fnr-related proteins. The expression of R. etli fnrN is highly induced under free-living microaerobic conditions and during symbiosis. This microaerobic and symbiotic induction of fnrN is not controlled by the sigma factor RpoN and the symbiotic regulator nifA or fixLJ, but it is due to positive autoregulation. Inoculation of Phaseolus vulgaris with an R. etli fnrN mutant strain resulted in a severe reduction in the bacteroid nitrogen fixation capacity compared to the wild-type capacity, confirming the importance of FnrN during symbiosis. The expression of the R. etli fixN, fixG, and arcA genes is strictly controlled by fnrN under free-living microaerobic conditions and in bacteroids during symbiosis with the host. However, there is an additional level of regulation of fixN and fixG under symbiotic conditions. A phylogenetic analysis of the available rhizobial FnrN and FixK proteins grouped the proteins in three different clusters.Soil bacteria belonging to the genera Rhizobium, Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, and Sinorhizobium (collectively referred to as rhizobia) elicit the formation of nodules on the roots of their leguminous hosts. In these specialized organs, the bacteria are released into the plant cells and differentiate into bacteroids that fix atmospheric nitrogen into ammonia that can be assimilated by the host plant. The nodules provide the microoxic conditions required for functioning of the oxygen-sensitive nitrogenase enzyme complex.In rhizobia, NifA activates transcription of several nitrogen fixation genes in conjunction with 54 RNA polymerase (12). In Rhizobium etli CNPAF512, NifA is strictly required for nitrogen fixation activity in nodules of Phaseolus vulgaris and controls the expression of several genes involved in nitrogen fixation, including nifH, iscN, and orf180-rpoN 2 (13,33,34