The Chromosomal HurL Gene Mutation In R. Leguminosarum Bv. Viciae Affects The Expression Of Nod Genes And Its Nodulation Phenotype

Thirteen nod genes have been identified on the Sym plasmid (pRL1JI) of Rhizobium leguminosarum bv. viciae, and lie 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. The expression of nodD is subject to negative autoregulation. During analyzing the interaction between NodD and nod-boxes in Rhizobium leguminosarum bv. viciae, a novel protein factor (HurL) has been identified to bind specifically with nod promoters and involve in in vitro nodD transcription, but its in vivo function remained unknown. In this work we proved that the gene coding for HurL protein is located on the bacterial chromosome, and cloned and sequenced the R. leguminosarum bv. viciae chromosomal hurL gene region. Sequence analysis and database search revealed that HurL is highly homologous to members of bacterial HU protein family. The R. leguminosarum bv. viciae hurL gene was mutated by insertion of a kanamycin resistance cassette. The obtained hurL mutant strain M704 exhibited poor growth under free-living conditions and showed higher sensitivity to UV irradiation. Further studies of NodD production and nod genes-lacZ fusions expression in the hurL mutant strain M704 revealed that inactivation of hurL gene led to severe impairment in NodD protein production and nodD gene transcription, repression of the inducible expression of nodA and nodF, and slight enhancement in the expression of px2, a gene identified earlier in this lab. These results suggested that hurL is required for maintaining the normal expression of nod genes in R. leguminosarum bv. viciae. By nodulation tests with Pisum sativum cv. Frisson and Vicia hirsute, we found that inactivation of the hurL gene caused failure in inducing nodulation, whereas the same mutant complemented with an intact R. leguminosarum bv. viciae hurL gene formed nodules in wild-type amounts, indicating that hurL gene is required for nodulation. Although the function of hurL gene in inducing nodulation remains unknown, the results of this work revealed that in addition to the nod and exo genes located on Sym plasmid, the R. leguminosarum chromosomal hurL gene is also involved in controlling its capacity in inducing nodulation on host plant.