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Bacteria of the nitrogen-fixing genus Azospirillum live in association with roots of many plants. Bacterial phytohormone synthesis is proposed to influence the host plant root proliferation. Analysis of tryptophan (Trp), indole-3-acetamide (IAM), and indole-3-acetic acid (IAA) synthesis of the mutant Azospirillum brasilense strain SpM7918 showed an indoleacetamide accumulation concomitant with reduced indoleacetic acid synthesis. The IAA deficiency, and IAM accumulation could be reversed with a specific cosmid from an A. brasilense Sp245 library. The identity of the indoleacetic acid and indoleacetamide produced was confirmed by HPLC with on-line mass spectrometry. Specific radioactivities of tryptophan, indoleacetamide, and indoleacetic acid formed during" H-IAM and" H-Trp feeding experiments revealed multiple IAA biosynthetic pathways in Azospirillum: the indoleacetamide pathway, a second tryptophan …