Host regulation of nodulation genes in Rhizobium meliloti and Bradyrhizobium japonicum

Rhizobium melilotl and Bradvrhizobium japonicum were analyzed by biochemical and molecular genetic methods to identify genes which respond to signals from their host plants, B. japonicum was previously shown to respond to seed lectin from its soybean host to become "competent" to nodulate. B. jiaponicum strain USDAllO was thus treated with soybean lectin or with genistein which induces the known nodulation (nod) genes. The proteins from treated and untreated cells were separated by two-dimensional electrophoresis to identify proteins induced by these host molecules. Two relatively abundant proteins induced by soybean lectin (S55), and by genistein (G58) were identified.

In a genetic approach, cosmid pR32 which contains the common nod genes and surrounding DNA, was searched for host inducible genes by fusing the lacZ structural gene, encoding ^β-galactosidase, to promoters within pR32. Transposon Tn3-lac was allowed to transpose randomly within pR32, and - 100 such pR32::Tn3-lac constructs were restriction mapped and mated from E. coli into B. japonicum. By assaying the B. japonicum (pR32;;Tn3-lac) strains with and without soybean root exudate, two genistein-inducible operons, called HIL3 and HIL4, were newly identified. The B. iaponicum USDA 110 nodP gene was also subcloned and transferred into a Rhizobium meliloti nodD₁-mutant. This complementation experiment established the functionality of the B. iaponicum nodP gene and indicated its broad specificity towards flavonoids.

Rhizobium meliloti was previously known to carry three plant flavonoid-inducible operons made up of seven genes (nodABC, nodFEG and nodHl) involved in alfalfa nodulation. DNA sequence analysis by Dr. Kondorosi's group in Szeged, Hungary, identified a promoter sequence termed the "nod box" upstream of all three inducible operons. Three additional loci were identified which bore sequence homology to a synthetic nod box oligonucleotide, only one of which was associated with known nodulation genes. The unstudied two promoters, termed n4 and n5, were fused to the promoterless lac operon using transposon Mud-lacPR13. and found to be induced by the alfalfa-excreted flavone, luteolin. By constructing site-directed Tn5 mutants downstream of n4 and n5 it was found that n5 plays a significant role in nodulation of two R. meliloti hosts, alfalfa and sweetclover, whereas n4 was important only for alfalfa nodulation. Hybridization data suggested that the n5 locus is conserved among species of Rhizobium. whereas n4 is probably unique to R. meliloti strains, in agreement with the host-specific phenotype of n4 locus mutants.