Organization of the ipdC region regulates IAA levels in different Azospirillum brasilense strains: molecular and functional analysis of ipdC in strain SM.

Presence of the indole-3-pyruvic acid pathway (IPyA) of indole-3-acetic acid (IAA) biosynthesis was demonstrated by identifying the ipdC gene encoding indole-3-pyruvate decarboxylase (IPDC) in Azospirillum brasilense strain SM. Comparison with other A. brasilense strains, Sp7 and Sp245, revealed homology in the gene and its 5' regulatory region. The 3' region of strain SM carries a truncated iaaC gene implicated in controlling IAA biosynthesis in strain Sp245. While the ipdC transcript could be visualized by reverse transcription polymerase chain reaction (RT-PCR), truncated iaaC was non-functional. Strain SM derivatives carrying higher copy number of ipdC and P(ipdC) showed improved IAA biosynthesis. P(ipdC) showed sequence elements that are part of composite auxin-responsive promoters. Expression of ipdC was upregulated by IAA, other auxins, temperature and nutrient stress, and an increase in pH. Heterologous overexpression of a functional iaaC gene from strain Sp245 in strain SM confirmed its role in controlling IAA biosynthesis and lowering ipdC expression which may be effected by dissociating IAA-transcriptional regulator interactions in the 5' region. However, the effect of the introduced iaaC was overcome when both ipdC and iaaC were expressed from similar plasmid background. This analysis confirmed that strain-based differences in IAA biosynthesis could be explained by differential regulation of ipdC expression.

An ipdC gene knock-out of Azospirillum brasilense strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion.

The indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of IAA biosynthesis in Azospirillum brasilense SM was functionally disrupted in a site-specific manner. This disruption was brought about by group II intron-based Targetron gene knock-out system as other conventional methods were unsuccessful in generating an IAA-attenuated mutant. Intron insertion was targeted to position 568 on the sense strand of ipdC, resulting in the knock-out strain, SMIT568s10 which showed a significant (~50%) decrease in the levels of indole-3-acetic acid, indole-3-acetaldehyde and tryptophol compared to the wild type strain SM. In addition, a significant decrease in indole-3-pyruvate decarboxylase enzyme activity by approximately 50% was identified confirming a functional knock-out. Consequently, a reduction in the plant growth promoting response of strain SMIT568s10 was observed in terms of root length and lateral root proliferation as well as the total dry weight of the treated plants. Residual indole-3-pyruvate decarboxylase enzyme activity, and indole-3-acetic acid, tryptophol and indole-3-acetaldehyde formed along with the plant growth promoting response by strain SMIT568s10 in comparison with an untreated set suggest the presence of more than one copy of ipdC in the A. brasilense SM genome.

Targeted engineering of Azospirillum brasilense SM with indole acetamide pathway for indoleacetic acid over-expression.

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.