The Chromosomal Hxy Gene Cloning And Its Function Studies In Rhizobium Leguminosarum Bv.Viciae

Thirteen nod genes have been identified on the Sym plasmid (pRL1 JI) of Rhizobium leguminosarum bv. viciae, and arranged in five transcription units: nodO, nodMNT, nodFEL, nodD and nodABCIJ. Among them, nodD is the major regulatory gene of all the nod operon. NodD binds specifically to the promoter region of nod genes and upon induction by flavonoid, activates the expression of nod genes. During analyzing the interaction between NodD and nod-boxes in Rhizobium leguminosarum bv. viciae, a novel protein factor (HurL) on the bacterial chromosome has been identified to bind specifically with nod promoters for the first time in this lab .By constructing the hurL mutant, we found that inactivation of hurL gene led the R. leguminosarum bv. viciae to severe impairment in growth and nodulation. In addition to the hurL gene, we also found two partial ORFs were located on the cloned 3.1 kb Pstl fragment of genomic DNA corresponding to hurL region. In this work, one of the two partial ORFs , hypothesis protein of unknown function, have been studied .By southern blot and Genome Walking experiments we cloned the R. leguminosarum bv. viciae chromosomal hxy gene region and sequenced it. Sequence analysis and database search revealed that hxy gene is lowly homologous to any other gene that has been published and the hypothesis protein of hxy gene is also very lowly homologous to any other known protein. This result strongly suggests that the hxy gene is a new gene. To study its function, the R. leguminosarum bv. viciae hxy gene was mutated by insertion of a kanamycin resistance cassette through homologous recombination. The obtained hxy mutant strain mutDw6 exhibited no difference by comparison with the wild-type 8401 pRLUI in growth under free-living conditions. Further studies of NodD production and nod-gene-lacZ fusions expression in the hxy mutant strain mutDw6 revealed that inactivation of hxy gene had little influence up on nodD/A/F genes transcription. These results suggested that hxy is not required for the...