MOLECULAR-STRUCTURE OF THE FRANKIA SPP NIFD-K INTERGENIC SPACER AND DESIGN OF FRANKIA GENUS COMPATIBLE PRIMER

The nifD-K intergenic spacer (IGS) of ArI3 and ACoN24d were found to h ave a length 265 and 199 nucleotides, respectively. They are markedly less conserved than the two neighbouring genes and have, in some insta nces, a repeated structure reminiscent of an insertion event. The repe ated sequence and the IGSs have no detectable homology with sequences in DNA databanks. The IGS has a stem-loop structure with a low folding energy,lower than that between nifH and nifD. No convincing alignment of IGS sequences could be obtained among Frankia strains. Only betwee n ACoN24d and ArI3, which belong to the same genomic species, was the alignment good enough to permit detection of a doubly repeated structu re. No promoter could be detected in the IGSs. The putative nifK open reading frame (ORF) in Frankia strain ArI3 has a length of 1587 nucleo tides, starting with a GTG codon, preceded by a ribosome binding site of a structure similar to that of nifH (GGAGGN(7)). The codon usage wa s similar to that of previously sequenced Frankia genes with a strong bias toward G- and C-ending codons except in the case of glycine where GGT is frequent. Alignment of the three Frankia nifK sequences (EUN1f ; ArI3 and ACoN24d) with those of other nitrogen-fixing bacteria permi tted detection of a sequence conserved among the three Frankia strains but absent in the other sequences. A primer targeted to that region i n combination with FGPD807-85 amplified the nifD-KIGS sequences of all Frankia strains (except the non-nitrogen-fixing Frankia strains CN3 a nd AgB1-9) and yet failed to amplify DNA of all other nitrogen-fixing bacteria. Conversely, the failure of primer FGPK700'-92 to amplify Aln us-infective strains could be explained by point mutations in the 3' p art of the primer.