Characterization of a 5-Aminolevulinic Acid Synthase Mutant of Azorhizobium caulinodans ORS571. K. Pawlowski. Max-Planck-Institut fuer Zuechtungsforschung, Abteilung Genetische Grundlagen der Pflanzenzuechtung, D-5000 Cologne 30, Germany. S. P. Gough(2), C. G. Kannangara(2), and F.J. de Bruijn(1,3). (1)Max-Planck-Institut fuer Zuechtungsforschung, Abteilung Genetische Grundlagen der Pflanzenzuechtung, D-5000 Cologne 30, Germany; (2)Carlsberg Laboratory, Department of Physiology, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark; (3)MSU-DOE Plant Research Laboratory and Department of Microbiology, Michigan State University, East Lansing 48824 U.S.A. MPMI 6:35-44. Accepted 4 November 1992. Copyright 1993 The American Phytopathological Society.

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.