Azorhizobium caulinodansNitrogen Fixation(nif/fix)Gene Regulation: Mutagenesis of thenifA─24/─12 Promoter Element, Characterization of antrA(rpoN)Gene, and Derivation of a Model

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Abstract
Using site-directed mutagenesis, mutations were introduced in the -24/-12 promoter element of the Azorhizobium caulinodans nifA gene, and chimeric nifA-lacZ reporter gene fusions were constructed. Single base-pair mutations in the conserved -25 or -13 G residues were found to reduce or abolish nifA promoter activity, respectively, demonstrating that the -24/-12 promoter element is important for nifA gene expression and suggesting the involvement of a sigma 54 (NtrA/RpoN)-type transcription factor in nifA gene regulation. A 2-bp mutation at positions -25 and -16 was found to create a relatively nitrogen-control-independent, highly expressed nifA promoter. Using a heterologous ntrA(rpoN) gene probe, an A. caulinodans ntrA(rpoN)-like gene was cloned and the DNA sequence of this gene and flanking regions was determined. The presence of three open reading frames (ORF1-3) was demonstrated. ORF2 was found to contain regions sharing a high degree of homology with all characterized bacterial ntrA(rpoN) genes. ORF1 was found to share homology with ORFs found upstream of other bacterial ntrA(rpoN) genes, which have been postulated to encode members of a superfamily of ATP-binding proteins. Transposon Tn5 insertion mutations were introduced into the cloned ntrA(rpoN) gene, and chromosomal ntrA(rpoN)::Tn5 A. caulinodans mutants were created. The resulting mutants were found to be unable to fix nitrogen in the free-living state (Nif- in culture) or in stem or root nodules induced on Sesbania rostrata (Fix- in planta), and to be unable to grow aerobically in the presence of nitrate as sole nitrogen source (Ntr-). A nifH-lacZ gene fusion was found to be silent in ntrA(rpoN)::Tn5 mutant strains, but a nifA-lacZ gene fusion was found to be expressed at a wild-type level, suggesting that the ntrA(rpoN) gene identified here controls the expression of some of the A. caulinodans nif genes, but not the central nif regulatory gene nifA. Based on these results, a new model for the regulation of nif/fix gene expression in A. caulinodans is proposed.

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