Genetic and physical analysis of the nodD3 region of Rhizobium meliloti

Abstract

The nodulation (nod) genes of the symbiont Rhizobium meliloti are transcriptionally controlled by protein activators in the nodD gene family. While NodD1 and NodD2 act in concert with small molecular weight inducers provided by the host legume plant, NodD3 is an inducer-independent activator of the nod promoters. We determined the sequence of the nodD3 gene, confirmed the expression of a 35 kDa protein in vitro, and determined the insertion points of five Tn5 insertions in the region of the nodD3 gene. We found the NodD3 amino acid sequence to be markedly diverged from the sequences of NodD1 and NodD2, which were more similar to the inducer-dependent NodD of another species, Rhizobium leguminosarum biovar viciae. The expression of nodD3 is not well understood, but involves at least SyrM, another positive activator related to the LysR-NodD family. One of the phenotypically mutant Tn5 insertions used in genetic studies of NodD3-dependent nod regulation lacks NodD3 protein as determined by Western blots, but another expresses about 50-60% of the wild type level. The location of these Tn5 insertions substantially upstream of the open reading frame for NodD3 suggests importance of relatively distant regulatory sequences for nodD3 expression. An insertion that did not cause a NodD3- phenotype is located in the extreme C-terminus of the protein coding region.