A MUTANT BRADYRHIZOBIUM-JAPONICUM DELTA-AMINOLEVULINIC-ACID DEHYDRATASE WITH AN ALTERED METAL REQUIREMENT FUNCTIONS IN-SITU FOR TETRAPYRROLE SYNTHESIS IN SOYBEAN ROOT-NODULES

The tetrapyrrole synthesis enzyme S-aminolevulinic acid (ALA) dehydratase requires Mg2+ for catalytic activity in photosynthetic organisms and in Bradyrhizobium japonicum, a bacterium that can reside symbiotically within plant cells of soybean root nodules or as a free-living organism. ALA dehydratase from animals and other non-photosynthetic organisms is a Zn2+-dependent enzyme. A modified B. japonicum ALA dehydratase, ALAD, was constructed by site-directed mutagenesis of hemB in which three proximal amino acids conserved in plant dehydratases were changed to cysteine residues as is found in the Zn2+-dependent enzyme of animals. These substitutions resulted in an enzyme that required Zn2+ rather than Mg2+ for catalytic activity, and therefore a region of the ALA dehydratase from B. japonicum, and probably from plants, was identified that is involved in Mg2+ dependence. In addition, the data show that a change in only a few residues is sufficient to change a Mg2+-dependent ALA dehydratase to a Zn2+-dependent one. B. japonicum strains were constructed that contained a single copy of either hemB or the altered gene hemB integrated into the genome of a hemB(-) mutant. Cultures of the hemB strain KPZn3 had Zn2+-dependent ALA dehydratase activity that functioned in vivo as discerned by its heme prototrophy and expression of wild type levels of cellular hemes. Strain KPZn3 elicited root nodules on soybean that contained viable bacteria and exhibited traits of normally developed nodules, and the symbiotic bacteria expressed nearly wild type levels of cellular hemes. We conclude that the Zn2+-dependent ALAD can function and support bacterial tetrapyrrole synthesis within the plant milieu of root nodules.