CHARACTERIZATION OF A 5-AMINOLEVULINIC ACID SYNTHASE MUTANT OF AZORHIZOBIUM-CAULINODANS ORS571

Using a heterologous 5-aminolevulinic acid synthase (ALAS) gene probe from Rhizobium meliloti, we cloned an Azorhizobium caulinodans hemA-like locus and constructed a hemA::Tn5 insertion mutant via gene replacement. The resulting mutant strain was found to be a strict 5-aminolevulinic acid (ALA) auxotroph, to be nitrogen-fixation proficient in culture (Nif+) and to induce ineffective, nitrogen fixation-deficient (Fix-) root nodules, lacking leghemoglobin, on Sesbania rostrata. Moreover, the hemA::Tn5 A. caulinodans mutant (ALAS122) was found to be unable to induce stem-nodules on S. rostrata (Nod-). Second-site suppressor mutations, capable of growing on full medium (TY) in the absence of additional ALA, were isolated (ALAS122RT strains). hemA::Tn5 strains carrying these secondary mutations were found to be disturbed in free-living nitrogen fixation (Nif- to Nif+/-) and in some cases in nitrogen assimilation (Ntr-/+), but displayed the same symbiotic phenotype as the original hemA::Tn5 strain (Fix- on roots; Nod- on stems). The Ntr-/+ phenotype of the ALAS122RT strains could be complemented by plasmids carrying the ntrC and/or ntrYX loci of A. caulinodans, suggesting a role for the ntr system in controlling the activation of a (normally cryptic) secondary ALA biosynthesis pathway in strains carrying the second-site suppressor mutations. Growth of the ALAS122RT strains on TY medium lacking ALA was found to be inhibited significantly by inhibitors of the C5 ALA synthesis pathway, such as gabaculine and amino-oxyacetate (AOA), suggesting the possibility that both the Shemin and C5 ALA synthesis pathways may exist in A. caulinodans.