TRANSCRIPTIONAL ORGANIZATION AND EXPRESSION OF NOIXWBTUV, A LOCUS THAT REGULATES CULTIVAR-SPECIFIC NODULATION OF SOYBEAN BY RHIZOBIUM-FREDII USDA257

Rhizobium fredii is a nitrogen-fixing bacterial symbiont of soybean and a number of other legume species. We have studied the transcriptional organization of a Sym plasmid focus that restricts the host range of R. fredii USDA257 at both the host species and cultivar level. The genes of this host-specificity locus, noIXWBTUV, are transcribed from three promoters. Two of these, which are upstream of nolW and nolBTUV, are oriented face to face and initiate transcription at sites that are 14 bp apart. The third lies upstream from nolX. The nolW promoter is constitutive, whereas the nolB and nolX promoters are inducible by flavonoid signals. We have attempted to express genes from this locus in Escherichia coli systems, both in vivo and in vitro. We detected the insert- and orientation-specific expression of two genes, nolX and nolW, but we were unable to obtain expression of nolBTUV. Antiserum raised against NolT nevertheless detected an abundantly expressed polypeptide of the predicted size in protein extracts of USDA257. This observation, as well as RNA dot blot data from a series of mutants, indicates that nolBTUV is expressed as a single transcriptional unit in R. fredii. Immunological detection of NolT, and of a second protein, NolX, was strictly dependent on flavonoid induction. The NolX protein was larger than the size predicted from the previously published nucleotide sequence, and this led to resequencing and revision of the open reading frame.