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...

N-Acyl homoserine lactones (AHLs) are molecules that are synthesized and detected by many gram-negative bacteria to monitor the population density, a phenomenon known as quorum sensing. Salmonella enterica serovar Typhimurium is an exceptional species since it does not synthesize its own AHLs, while it does encode a LuxR homologue, SdiA, which enables this bacterium to detect AHLs that are produced by other species. To obtain more information about the specificity of the ligand binding by SdiA, we synthesized and screened a limited library of AHL analogues. We identified two classes of analogues that are strong activators of SdiA: the N-(3-oxo-acyl)-homocysteine thiolactones (3O-AHTLs) and the N-(3-oxo-acyl)-trans-2-aminocyclohexanols. To our knowledge, this is the first report of compounds (the 3O-AHTLs) that are able to activate a LuxR homologue at concentrations that are lower than the concentrations of the most active AHLs. SdiA responds with greatest sensitivity to AHTLs that have a keto modification at the third carbon atom and an acyl chain that is seven or eight carbon atoms long. The N-(3-oxo-acyl)-trans-2-aminocyclohexanols were found to be less sensitive to deactivation by lactonase and alkaline pH than the 3O-AHTLs and the AHLs are. We also examined the activity of our library with LuxR of Vibrio fischeri and identified three new inhibitors of LuxR. Finally, we performed preliminary binding experiments which suggested that SdiA binds its activators reversibly. These results increase our understanding of the specificity of the SdiA-ligand interaction, which could have uses in the development of anti-quorum-sensing-based antimicrobials.

No abstract