University of Antwerp, Department of Biology, B-2610 Antwerp, Belgium

Background and objectives
Bacteria of the nitrogen-fixing genus Azospirillum live in association with roots of many plants. Like most rhizospere bacteria, Azospirillum produce phytohormones. Excretion of phytohormones by plant-associated bacteria may promote plant growth and improve crop yield. The increased plant growth observed after inoculation with Azospirillum was shown to be due to IAA and not to an enhanced nitrogen supply by the bacteria [1]. Little is known so far about the pathway(s) involved. Most of the studies on the metabolic pathways of IAA are based on plant pathogenic bacteria. Azospirillum is the best known non-pathogenic bacterium. Indole-3-pyruvic acid and indole-3-acetaldehyde are IAA intermediates in A. lipoferum. There is also evidence for the presence of the IAM pathway in A. brasilense, whereas the presence of indole-3-ethanol, indole-3-methanol and indole-3-lactate were demonstrated in A. brasilense cultures. Multiple IAA biosynthetic pathways were already demonstrated in Azospirillum: the IAM pathway, a second tryptophan-dependent and a tryptophan-independent pathway, the latter being predominant in case no tryptophan was supplied to the medium [2]. The indole-pyruvate decarboxylase gene was cloned from A. brasilense [3]. In the concomitant mutant SpM7918, IAA biosynthesis was reduced to 10% of the wild-type production [2]. The results presented here are part of a PhD project with the following objectives: (i) characterization of pathways involved in auxin metabolism in A. brasilense; (ii) characterization of precursors of the tryptophan-independent pathway; (iii) interregulation and interaction between different auxin metabolic pathways; (iv) analysis of the functional relevance of microbial phytohormone synthesis upon the interaction with the host.

Materials and methods
The strains were grown at 30C and 199 r.p.m. in MMAB for 90 h. At different time intervals, bacterial cells were seperated from the culture medium by centrifugation (10 min 2000 g). The concentrations of the different compounds were determined using micro LC/LC linked to electrospray tandem mass spectrometry [4]. In the feeding experiments, bacterial cultures are fed with H-labelled IAA precursors. The cultures are sampled, purified and measured by IP-RP-HPLC with online UV and fluorescence detection.

Results and conclusions
We analysed the IAA intermediates in growth media and bacterial cells of two different wild-type strains, Sp6 (WT) and Sp245 (WT) and the IPyA-decarboxylase mutant of Sp245 (Sp245b). Both wild-type strains analysed showed comparable concentrations of free IAA, while clear differences in IAA intermediates could be observed. Striking differences between the wild-type strain Sp245 and the IAA mutant were observed. These data can be explaned by an altered regulation mechanism in the mutant strain, due to feedback regulation. These results show that the interrelationship between the different IAA biosynthetic pathways is more complex than has been assumed.

1. Barbieri P, Zanelli T, Galli E, Zanetti G, 1986. FEMS Microbiology Letters 36, 87.
2. Costacurta A, Keijers V, Vanderleyden J, 1994. Molecular and General Genetics 243, 463.
3. Prinsen E, Costacurta A, Michiels K et al., 1993. Molecular Plant-Microbe Interactions 6, 609.
4. Prinsen E, Van Dongen W, Esmans E, Van Onckelen H, 1997. Journal of Mass Spectrometry 32, 12.