Characterization of sequences downstream from transcriptional start site of Rhizobium meliloti nifHDK promoter

In free-living state, the nifHDK promoter P1 of Rhizobium meliloti is induced in response to microaerobiosis and expressed to a high level, while the fixABCX promoter P2 is not. The sequences upstream from both P1 and P2 share extended homology (about 85 %), which are about 160 bp in length, but the sequences downstream of the respective transcriptional start site are different. When the downstream sequence (DS) of P2 was replaced by the corresponding fragment from + 17 to + 61 of P1, the expression of P2 is greatly increased under free-living condition by lowering the oxygen tension, and the activity of P2 promoter can also be significantly enhanced in E. coli by the NifA protein. The difference between the DS regions of P1 and P2 promoter resulted in different expressions of P1 and P2 promoter under free-living microaerobic condition and in E. coli. The expression of P2 does not depend on the downstream sequences from the promoter element during symbiosis. Primer extension experiments identified the transcriptional start site of P2. Transcription from P2 was nor changed when P2 promoter region was inserted by P1 DS. Under symbiotic conditions, levels of expression of P2 were independent of the P1 DS region. It indicates that the regulations of P2 under symbiotic conditions are different from those under free-living conditions.