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 an d a number of other legume species. We have studied the transcriptiona l organization of a Sym plasmid focus that restricts the host range of R. fredii USDA257 at both the host species and cultivar level. The ge nes of this host-specificity locus, noIXWBTUV, are transcribed from th ree promoters. Two of these, which are upstream of nolW and nolBTUV, a re oriented face to face and initiate transcription at sites that are 14 bp apart. The third lies upstream from nolX. The nolW promoter is c onstitutive, whereas the nolB and nolX promoters are inducible by flav onoid signals. We have attempted to express genes from this locus in E scherichia coil systems, both in vivo and in vitro. We detected the in sert- 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 observatio n, as well as RNA dot blot data from a series of mutants, indicates th at nolBTUV is expressed as a single transcriptional unit in R. fredii. Immunological detection of NolT, and of a second protein, NolX, was s trictly dependent on flavonoid induction. The NolX protein was larger than the size predicted from the previously published nucleotide seque nce, and this led to resequencing and revision of the open reading fra me.