MOLECULAR-CLONING AND SEQUENCE-ANALYSIS OF AN AZOSPIRILLUM-BRASILENSEINDOLE-3-PYRUVATE DECARBOXYLASE GENE

Azospirillum brasilense isolated from the rhizosphere of different pla nts has the ability to excrete indole-3-acetic acid (IAA) into the cul ture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome l ibrary in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. bra silense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA productio n in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that th is fragment contains one complete open reading frame. The predicted pr otein sequence shows extensive homology with the indole-3-pyruvate dec arboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense muta nt Sp245a, constructed by homogenotization of a Tn5 insertion derivati ve of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 102 1 resulted in increased IAA production. Cell-free extracts prepared fr om R. meliloti and A. brasilense transconjugants harboring this gene c ould convert indole-3-pyruvic acid to indole-3-acetaldehyde and trypto phol. These results clearly demonstrate that IAA production in A. bras ilense is mediated by indole-3-pyruvate decarboxylase.