Targeted engineering of Azospirillum brasilense SM with indole acetamide pathway for indoleacetic acid over-expression Mandira Malhotra and Sheela Srivastava Abstract: Rhizospheric bacterial strains are known to produce indole-3-acetic acid (IAA) through different pathways, and such IAA may be beneficial to plants at low concentrations. IAA biosynthesis by a natural isolate of Azospirillum brasilense SM was studied and observed to be tryptophan-inducible and -dependent in nature. While our work demonstrated the operation of the indole pyruvic acid pathway, the biochemical and molecular evidence for the genes of the indole acetamide (IAM) pathway were lacking in A. brasilense SM. This led us to use the IAM pathway genes as targets for metabolic engineering, with the aim of providing an additional pathway of IAA biosynthesis and improving IAA levels in A. brasilense SM. The introduction of the heterologous IAM pathway, consisting of the iaaM and iaaH genes, not only increased the IAA levels by threefold but also allowed constitutive expression of the same genes along with efficient utilization of IAM as a substrate. Such an engineered strain showed a superior effect on the lateral branching of sorghum roots as well as the dry weight of the plants when compared with the wild-type strain. Such an improved bioinoculant could be demonstrated to enhance root proliferation and biomass productivity of treated plants compared with the parental strain. Key words: indole-3-acetic acid, tryptophan, indole-3-acetamide, iaaM-iaaH, metabolic engineering. Résumé : On sait que les souches bact ériennes de la rhizosph ère ont la capacité de produire de l’acide indole-3 acétique (AIA) par l’intermédiaire de différentes voies métaboliques et que cet AIA à faible concentration est bénéfique pour la plante. La biosynthèse de AIA par un isolat naturel de la souche SM de Azospirillum brasilense a été étudiée et s’est révélée inductible par le tryptophane et dépendant du tryptophane dans la nature. Bien que notre travail ait démontré l’implication de la voie métabolique de l’acide indole pyruvique, les preuves moléculaires et biochimiques de l’existence des gènes impliqués dans la voie de l’indole acétamide (IAM) sont absentes chez la souche A. brasilense SM. Ceci nous a amené à utiliser les gènes impliqués dans la voie de l’IAM comme cibles en ingénierie métabolique afin de générer un sentier de biosynthèse de AIA additionnel et améliorer les niveaux de AIA chez A. brasilense SM. L’introduction d’une voie IAM hétérologue comprenant les gènes iaaM et iaaH a non seulement augmenté les niveaux de IAA de trois fois, mais elle a aussi permis l’expression constitutive de ces gènes parallèlement à une utilisation efficace de IAM comme substrat. Une telle souche modifiée a démontré sa supériorité quant à la formation de ramifications secondaires des racines de sorgho ainsi que dans la production en poids sec de plants, comparativement à la souche sauvage. Cet inoculant biologique amélioré pourrait augmenter la prolifération des racines et la productivité des plants traités, comparativement à la souche parentale. Mots clés : acide indole 3-acétique, tryptophane, indole-3-acétamide, iaaM-iaaH, ingénierie métabolique. [Traduit par la Rédaction] Malhotra and Srivastava 1084 Introduction The rhizosphere, being a nutrient-rich zone, allows higher microbial growth than bulk soil. Rhizospheric bacteria can stimulate plant growth through the production of phyto- hormones, which may regulate certain physiological processes of plants, leading to consequent changes in root growth (Dobbelaere et al. 2003). It has long been known that Azo- spirilla produce phytohormones, i.e., auxins, cytokinins, and gibberellins (Tien et al. 1979), and that inoculation of such bacteria to seeds results in an improvement of root growth in terms of root hairs and lateral roots (Dobbelaere et al. 1999). Multiple pathways for indole-3-acetic acid (IAA) biosynthesis have been documented from the rhizospheric bacterium Azospirillum (Patten and Glick 1996). The indole pyruvic acid (IPyA) pathway operates in plants like Arabidopsis (Tam and Normanly 1998) as well as in plant-beneficial bacteria like Azospirillum and Rhizobium in an inducible manner and is subjected to extremely tight regulation (Costacurta and Vanderleyden 1995; Patten and Glick 1996). However, Rhizo- bium, Bradyrhizobium, and the plant pathogens Pseudomonas syringae and Agrobacterium tumifaciens synthesize IAA via a constitutive pathway involving the production of indole-3- acetamide (IAM) (Patten and Glick 1996). The IAM pathway has earlier been reported from Azospirillum brasilense Sp7 1078 Can. J. Microbiol. 52: 1078–1084 (2006) doi:10.1139/W06-071 © 2006 NRC Canada Received 10 April 2006. Revision received 29 June 2006. Accepted 10 July 2006. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 5 December 2006. M. Malhotra and S. Srivastava. 1 Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. 1 Corresponding author (e-mail: srivastava_sheela@yahoo.com).