REGULATORY PROTEINS AND CIS-ACTING ELEMENTS INVOLVED IN THE TRANSCRIPTIONAL CONTROL OF RHIZOBIUM-ETLI REITERATED NIFH GENES

In Rhizobium etli the nitrogenase reductase genes are reiterated. Stra in CE3 has three copies; nifHa and nifHb form part of nifHDK operons w ith the nitrogenase structural genes, while nifHc is linked to a trunc ated nifD homolog. Their sequences are identical up to 6 residues upst ream from a sigma(54)-dependent promoter. A remarkable difference amon g them is the absence of canonical NifA binding sites upstream of nifH c while a canonical binding site is located 200 bp upstream of nifHa a nd nifHb. To evaluate the transcriptional regulation of the reiterated nifH genes, we constructed fusions of nifHa and nifHc with the lacZ g ene of Escherichia coli. Both genes were expressed at maximum levels u nder 1% oxygen in free-living cultures, and their expression declined as the oxygen concentration was increased. This expression was depende nt on the integrity of nifA, and nifHc was expressed at higher levels than nifHa. The same pattern was observed with root nodule bacteroids. Expression of both genes in E. coli required sigma(94) in addition to NifA bound to the upstream activator sequence. In vivo dimethyl sulfa te footprinting analyses showed that NifA binds to the canonical site upstream of nifHa and to a TGT half-site 6 nucleotides further upstrea m. NifA protected an imperfect binding site upstream of nifHc at posit ion 85 from the promoter. The integration host factor stimulated each gene differently, nifHa being more dependent on this protein. The abov e results correlate the asymmetric arrangement of cis-acting elements with a differential expression of the reiterated nifH genes, both in c ulture and during symbiosis with bean plants.