Identification of genes involved in the rhizobium-ligunlt symbiosis by mu-di (Kan, lao-generated transcription fusions

The process by which free-living Rhizobi-um forms nitrogen-fixing nodules on the roots of leguminous plants involves the induction and repression of a number of bacterial genes. To help identify these genes and to determine their mode of regulation, we have transposed the E. coli lac operon, minus its promoter, randomly around the chromosome of R. japonicum, a soybean nodulating bacterium. This was accomplished using the defective phage, mu-dl (Kan, lac) placed onto a self-transmissible, narrow host-range suicide vector, pGS6. Among the mu-dl insertion mutants examined, auxotrophs were detected at frequencies of about 0.3%, and nodulation and nitrogen fixation defective mutants at about 4%. Many of the symbi-otically defective mutants were found to have deletions in their indigenous plas-mids. Three mutants showed increased P-galactosidase levels when grown in the presence of soybean or kidney bean root-extracts but not in the presence of root-extracts from other legumes or a non-legume, indicating specific plant control of Rhizobium gene expression.