Analysis of the expression from Rhizobium meliloti fix-promoters in other Rhizobium backgrounds

Departamento de Microbiologíc y Parasitología, Facultad de Farmacia, Universidad de Sevilla, c/o Profesor García Gonzalez s/n, 41012 Sevilla, Spain 5 Author for correspondence: Antonio j. Palomares. Tel: +34 5 455 67 66. Fax: +34 5 462 81 62. ABSTRACT Using translational fusions to lacZ, we have measured expression from the promoters of Rhizobium meliloti regulatory genes, nifA and fixK , and structural genes, nifH and fixA , in other fast-growing rhizobia whose nitrogen fixation regulation is less known. Neither nifA nor fixK promoters were activated under both free-living microaerobic and symbiotic conditions, except in R. tropici , where clear symbiotic activation of either nifA or fixK expression could be observed. Both nifH and fixA promoters showed strong heterologous activation during symbiosis and weak activation under free-living nitrogen starvation conditions. Only when the nifH promoter was in R. tropici and R. leguminosarum bv. phaseoli, was clear induction observed in the microaerobic free-living state. Deletion analysis of these promoters suggested that a NifA binding site (UAS) was needed for full heterologous activation of nifHp , either in microaerobiosis or symbiosis. In contrast, the UAS region seemed to be unnecessary for fixA activation. However, a region containing a potential integration host factor (IHF) binding site was observed to be needed for complete heterologous symbiotic induction from fixAp. The moderate induction observed in nitrogen-free medium only required the 54 holoenzyme recognition sequence; this may be indicative of the existence of non-specific activation by NtrC-like proteins. Our results suggest possible common and different features in the control mechanisms of the nitrogen fixation gene expression among Rhizobium species. Keywords: Rhizobium meliloti, promoters, nitrogen fixation genes, gene expression, heterologous expression