Sequence analysis and expression patterns of two nifA genes from Frankia EuIK1

Abstract

Two nifA genes were cloned from Frankia EuIK1 strain, a symbiont of Elaeagnus umbellata Thunb. and analysed on the basis of their deduced amino acid sequences and expression patterns. The complete nucleotide sequence of 1926 bp of nifA1 and 1524 bp of nifA2 was determined, respectively. A putative NifA-binding site was found –95 to about –80 bp upstream of start codon for nifA1 ORF as TGT-N₁₀-ACA, but the clpB ORF was followed by nifA2 ORF with 15 bp of intergenic sequence. Deduced amino acid sequence showed that two nifA genes encode typical NifA, having three major domains and two linkers, and their central domains of NifA1 and NifA2 showed sequence similarity of 70–75% with those from other NifA proteins. However, entire NifA2 ORF is more similar to alternative NifA (60%) than to typical NifA (53%). Conserved amino acid sequence in helix-turn-helix motif of typical NifA was also found in NifA1, but it was not conserved in NifA2, which is also common in alternative NifA proteins. Moreover, the expression of nifA1 during nodule development was similar to that of Rhizobium meliloti in that it was expressed at low level constitutively, while that of nifA2 was similar to the pattern of nifH, structural gene for nitrogenase reductase, in that its transcripts level was changed in accordance with nitrogenase activity. These results indicate that nifA1 and nifA2 might be classified into typical nifA and alternative nifA, respectively. This is the first report on the presence of two nifA genes in Frankia.