Cloning of hemA from Rhizobium sp. NGR234 and symbiotic phenotype of a gene-directed mutant in diverse legume genera

Summary The hemA gene which encodes -aminolaevulinic acid synthase (ALAS), was cloned and characterized from the broad host-range Rhizobium strain NGR234. A cosmid, identified by hybridization with the cloned gene of R. meliloti and complementation of an R. meliloti hemA mutant, was subcloned to yield a 5.5 kb fragment containing the entire NGR234 gene. A physical-genetic map was made and the interposon was introduced into a single EcoRI site which bisects the gene. The mutated gene was homogenotized into NGR234 to generate a hemA mutant, with a view to evaluating the role of rhizobial bacteroid ALAS activity for a wide variety of legume symbioses. The mutant strain formed an ineffective (Fix^–) symbiosis with all tested host plants. These included tropical legumes that produce either indeterminate (Leucaena) or determinate (Desmodium, Macroptilium, Lablab, Vigna) root nodules.Abbreviations ALA -aminolaevulinic acid - ALAS aminolaevulinic acid synthase - Lb leghaemoglobin - Lb-haem haem moiety of leghaemoglobin