Single and multiple mutations affecting properties of the regulatory gene nodD of Rhizobium

nodD of Rhizobium leguminosarum has two regulatory properties: it autoregulates and, in cells grown with specific flavonoids, activates other nod genes. We isolated mutations in nodD affecting one or both properties. Those abolishing autoregulation and nod gene induction were at the 5' end of nodD, as were those which only affected autoregulation. Mutations affecting nod gene activation are at the 3' end of nodD. Eleven mutations in this region of nodD were isolated: some had little effect on the regulatory properties; other reduced activation of other nod genes. 265 bps were removed from the 3' end of nodD: this abolished nodD function. Doubly mutant derivatives of nodD were constructed by making nodD genes with a mutation that conferred the ability to activate transcription of nod genes in the absence of inducers (class IV) plus another that abolished autoregulation and/or flavonoid-dependent nod gene activation. The behaviour of such double mutants was complex; e.g. in one case, a doubly mutant nodD gene containing the class IV mutation, coupled to one of those that (alone) abolished autoregulation and nod gene induction, was similar in behaviour to the wild type. In other cases, double mutants were similar to one of the parentals.