An antimicrobial compound was isolated from Azospirillum brasilense culture extracts by high-performance liquid chromatography and further identified by gas chromatography-mass spectrometry as the auxin-like molecule, phenylacetic acid (PAA). PAA synthesis was found to be mediated by the indole-3-pyruvate decarboxylase, previously identified as a key enzyme in indole-3-acetic acid (IAA) production in A. brasilense. In minimal growth medium, PAA biosynthesis by A. brasilense was only observed in the presence of phenylalanine (or precursors thereof). This observation suggests deamination of phenylalanine, decarboxylation of phenylpyruvate, and subsequent oxidation of phenylacetaldehyde as the most likely pathway for PAA synthesis. Expression analysis revealed that transcription of the ipdC gene is upregulated by PAA, as was previously described for IAA and synthetic auxins, indicating a positive feedback regulation. The synthesis of PAA by A. brasilense is discussed in relation to previously reported biocontrol properties of A. brasilense.Azospirillum is a well-studied genus of plant growth-promoting bacteria (PGPB), which colonizes the rhizosphere of numerous crop plants in tropical and subtropical regions (5, 6). Different mechanisms, such as phytohormone production, nitrate reduction, and nitrogen fixation, have been proposed to explain improved plant growth following inoculation with Azospirillum (5,7,9,22,37,38,53). The production of phytohormones, and more specifically the auxin indole-3-acetic acid (IAA), has been recognized as an important factor in direct plant-growth-promoting abilities of A. brasilense (5,18,19,37).Azospirillum sp. are not typical biocontrol agents of soilborne plant pathogens (5). Apart from some reports on bacteriocins and siderophores, no other antibacterial substances in Azospirillum sp. have been identified so far (39,40,50,55,60). However, there have been reports of moderate biocontrol capabilities of Azospirillum brasilense against crown gall disease, bacterial leaf blight of mulberry, and bacterial leaf and/or vascular diseases of tomato (1,3,4,46,54). In addition, A. brasilense can restrict the proliferation of other nonpathogenic rhizosphere bacteria (21). Nevertheless, the exact mechanisms involved in Azospirillum acting as a putative biocontrol agent are not yet known. Some reports therefore indicate that the protective mechanism may be indirectly explained by the plant growth promotion effect or by outcompeting other bacteria hosted by the same plant (3,46).In the present study, we attempted to further screen A. brasilense supernatant (extracts) for the presence of metabolites (besides IAA), which may be involved in the persistence of Azospirillum in the rhizosphere. This screening led to the identification of phenylacetic acid (PAA), an auxin-like molecule with antimicrobial activity. MATERIALS AND METHODSStrains, plasmids, media, and culture conditions. Strains and plasmids used in this study are listed in Table 1. A. brasilense was grown at 30°C in Luria-Bertani (LB) med...

Expression of the Azospirillum brasilense ipdC gene, encoding an indole-3-pyruvate decarboxylase, a key enzyme in the production of indole-3-acetic acid (IAA) in this bacterium, is upregulated by IAA. Here, we demonstrate that the ipdC gene is the promoter proximal gene in a bicistronic operon. Database searches revealed that the second gene of this operon, named iaaC, is well conserved evolutionarily and that the encoded protein is homologous to the Escherichia coli protein SCRP-27A, the zebrafish protein ES1, and the human protein KNP-I/GT335 (HES1), all of unknown function and belonging to the DJ-1/PfpI superfamily. In addition to this operon structure, iaaC is also transcribed monocistronically. Mutation analysis of the latter gene indicated that the encoded protein is involved in controlling IAA biosynthesis but not ipdC expression. Besides being upregulated by IAA, expression of the ipdC-iaaC operon is pH dependent and maximal at acidic pH. The ipdC promoter was studied using a combination of deletion analyses and site-directed mutagenesis. A dyadic sequence (ATTGTTTC(GAAT)GAAACAAT), centered at -48 was demonstrated to be responsible for the IAA inducibility. This bacterial auxin-responsive element does not control the pH-dependent expression of ipdC-iaaC.