Localization of functional regions of the Rhizobium nodD product using hybrid nodD genes

The flavonoid-inducible nod promoters of Rhizobium are positively regulated by the nodD gene which is highly conserved in various Rhizobium species. The nodD gene are functionally different in (i) their response to various exogenously added flavonoid inducers, (ii) the extent to which they mediate the activation of the flavonoid-inducible promoters, and (iii) the extent to which they repress their own constitutive transcription. In order to localize the regions of the nodD product which determine these differences, two series of nodD hybrid genes have been constructed. In one series the 5' moiety is derived from the R. meliloti nodD1 gene and the 3' moiety from the R. trifolii nodD gene. In the other series, the origins of the nodD moieties are reversed. Two regions of the nodD product appeared to be involved in autoregulation and it was also shown that the nodD promoters differ in their susceptibility to autoregulation. Many regions, dispersed over the entire nodD product, are involved in the specificity of activation by flavonoids. Several hybrid nodD genes were characterized which activate transcription with novel inducers. Furthermore, two classes of hybrid nodD genes were found from which the activation characteristics differ completely from those of the parental nodD genes. The first class activates the nodABCIJ promoter to the maximum level in the absence of flavonoid inducer. This level can no longer be influenced, positively or negatively, by the presence of (iso-)flavonoids. With the second class of hybrids, activation of the nodABCIJ promoter, even in the presence of flavonoid inducers, is no longer possible.