Mutants in the nodFEL promoter of Rhizobium leguminosarum bv. viciae reveal a role of individual nucleotides in transcriptional activation and protein binding.

The highly conserved nod box sequence in the promoters of the inducible nodulation genes of rhizobia is required for transcription activation together with NodD, a LysR-type transcriptional regulator, and a flavonoid as a coinducer. DNA fragments containing nod box sequences form two binding complexes when crude preparations of Rhizobium leguminosarum bv. viciae are used: a NodD-dependent and an additional, NodD-independent complex. The role of individual nucleotides in the conserved nod box sequence in complex formation and in nodulation gene expression was investigated by introducing 13 individual base-pair substitutions in the nodF nod box of R. leguminosarum bv. viciae and studying their effect on promoter activity and protein-DNA complex formation. Two mutants showed decreased NodD binding and decreased promoter activity. Five mutants showed a NodD-dependent complex as with the wild-type nodF nod box, whereas their promoter activity was severely reduced after induction. This result is in agreement with earlier observations that NodD DNA binding also occurs in the absence of inducer. Four mutants were impaired in the formation of the NodD-independent retardation complex. Three of them showed no alterations in promoter activity, meaning that no specific role for the protein forming the NodD-independent complex could be established. The two mutants in the highly conserved LysR motif of the nod box were unable to direct coinducer-dependent promoter activity but, unexpectedly, their retardation patterns were not altered. The remaining two mutants showed constitutive promoter activity. The results are discussed in terms of the relevance of conserved nucleotides and motifs identified in the nod box.