DIFFERENTIAL REGULATION OF RHIZOBIUM-ETLI RPON2 GENE-EXPRESSION DURING SYMBIOSIS AND FREE-LIVING GROWTH

The Rizobium etli rpoN1 gene, encoding the alternative sigma factor si gma(54) (RpoN), was recently characterized and shown to be involved in the assimilation of several nitrogen and carbon sources during free-l iving aerobic growth (J. Michiels, T. Van Soom, I. D'hooghe, B, Dombre cht, T, Benhassine, P, de Wilde, and J, Vanderleyden, J, Bacteriol. 18 0:1729-1740, 1998), We identified a second rpoN gene copy in R. etli, rpoN2, encoding a 54.0 kDa protein which displays 59% amino acid ident ity with the R. etli RpoN1 protein. The rpoN2 gene is cotranscribed wi th 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 eukar yotic proteins of similar size. In contrast to the R. etli rpoN1 mutan t strain, inactivation of the rpoN2 gene did not produce any phenotypi c defects during free-living growth. However, symbiotic nitrogen fixat ion was reduced by approximately 90% in the rpoN2 mutant, whereas wild -type levels of nitrogen fixation were observed in the rpoN1 mutant st rain, Nitrogen fi ration was completely abolished in the rpoN1 rpoN2 d ouble mutant. Expression of rpoN1 was negatively autoregulated during aerobic growth and was reduced during microaerobiosis and symbiosis. I n contrast, rpoN2-gusA and orf180-gusA fusions were not expressed aero bically but were strongly induced at low oxygen tensions or in bactero ids. Expression of rpoN2 and orf180 was abolished in R. etli rpoN1 rpo N2 and nifA mutants under all conditions tested. Under free-living mic roaerobic conditions, transcription of rpoN2 and orf180 required the R poN1 protein. In symbiosis, expression of rpoN2 and orf180 occurred in dependently of the rpoN1 gene, suggesting the existence of an alternat ive symbiosis-specific mechanism of transcription activation.