Cloning And Functional Analysis Of Genes Invovled In Indole-3-acetic Acid (IAA) Synthesis From Azospirillum Brasilense

Azospirillum brasilense is a nitrogen-fixing microorganism that has a potential use as an inoculant for promoting plant growth in grass and cereals. A. brasilense Yu62 which was isolated from the rhizosphere of maize in Beijing, China, is able to produce more than 50μg/ml IAA into the medium supplemented with exogenous Trp. To bring more information about synthesis and regulation of indole-3-acetic acid (IAA) from A. brasilense, a Tn5-insertion library of A. brasilense Yu62 was constructed and subjected to screening for IAA producing mutants. More than 5,000 Km transconjugants obtained were screened for mutants of IAA production. 5 mutants showing decreased IAA production were selected by colorimetric assays and HPLC method. Southern blot analysis with Tn5 fragment as a probe showed that each mutant contained only one hybridizing band, which suggests they are all single transposon insertion mutants.The five Tn5-inserted genes from these mutants were cloned and the DNA sequences flanking Tn5 were determined. These genes include atrA encoding GntR-family transcriptional regulator, ftsA encoding iron binding protein component of ABC-type Fe~3+ transport system, omaA encoding outer membrane protein, aldA encoding aldehyde dehydrogenase and trpE encoding anthranilate synthase component I . Complementation tests showed that atrA, ftsA and omaA were able to restore the IAA production of their mutants, respectively. At the same time, atrB encoding phosphotransferase and atrC encoding 4-aminobutyrate aminotransferase were cloned, and atrA, artB and artC were clustered in a cluster.atrB and atrC mutants were constructed by insertion of km cassettes, respectively. The results showed that the IAA level secreted by atrC mutant were decreased when compared to that of wild type strain, while the amount of IAA produced by atrB mutant was same as that of the wild type strain. The data suggest that atrC in the atrABC cluster was involved in IAA synthesis. Comparison of Fe~3+ concentrations in culture supernatants of the wild-type strain, the ftsA mutant and the complemented strain revealed that the iron-uptake ability of the ftsA mutant was highly reduced. This result also points to the necessity of iron as a metal ion in IAA synthesis. Statistical analysis showed no significant difference in the IAA accumulated in cells between the omaA mutant and the wild-type strain, suggesting the omaA might not affect IAA secretion but involve in IAA production in other unknown manners. Mutation of trpE gene led to decreased IAA Production and the IAA production could be restored by adding anthranilate into the medium. Different with A. brasilens Sp7, the trpE is not fused together with trpG and no trpL was found upstream the trpE sequence in Yu62. It suggests that there may be two copies of trpE genes exist in A. brasilense.The ipdC gene, encoding IPyA decarboxylase (IPDC) of the IPyA pathway, was cloned by PCR and cloned into the broad-host-range vector pLAFR3, then the ipdC multi-copy strain of A. brasilense Yu62 was obtained. Comparison of the IAA production between wild type and ipdC multi-copy strain showed that no obvious difference was existed.