Genetic and functional studies on novel nodulation genes in Bradyrhizobium japonicum

The DNA region downstream of the nodYABCSUIJ operon in Bradyrhizobium japonicum strain USDA110 was analysed. The DNA sequence of four novel genes (i.e., nolMNO and ORFA) and a previously identified genetic locus (i.e., nodZ) was determined and the function and transcriptional control of nolNO, ORFA and nodz were studied. nolMNO were shown to be co-transcribed with the common nodulation genes, nodABC, thus, establishing the nodYABCSUIJnolMNO operon. nolo showed similarity to nodu from various rhizobia on the amino acid level. The lipo-oligosaccharide nodulation signals produced by strains SL67 (nolO^-) and SL65 (nolNO^-) were purified and their chemical structures were determined (chemical analysis was done by Russell Carlson, The University of Georgia). In addition to the wild-type signal molecule, both mutants produced modified compounds that are not produced by the parent strain USDA110. The most prevalent difference observed was the absence of the 2-0-methylfucosyl residue from the mutant structures. In addition, metabolites were found in which the N-acetylglucosamine residue at the reducing end was glycosidically linked to glycerol. These alterations in the profiles of nodulation signals produced by strains SL67 and SL65 were accompanied by reduced nodulation efficiency on all hosts tested. The expression of nodz and ORFA was studied using lacz gene fusions. Both genes appear to be constitutively expressed under laboratory conditions. The transcriptional start site of ORFA was determined revealing a 128 nucleotide overlap between the transcripts of the divergently transcribed nodz and ORFA. Mutational inactivation of ORFA had no effect on nodulation. DNA Southern hybridization studies suggested. that nolO, ORFA and nodz may be linked in other rhizobial strains.