Molecular cloning and sequence analysis of an Azospirillum brasilense indole-3-pyruvate decarboxylase gene.
Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading frame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.
Costacurta
A
A
F.A. Janssens Laboratory of Genetics, Catholic University of Leuven, Heverlee, Belgium.
Keijers
V
V
Vanderleyden
J
J
D000595:Amino Acid Sequence; D015806:Azospirillum brasilense / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004352:Drug Resistance, Microbial; D016972:Enterobacter cloacae / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial / Q000235:genetics*; D007210:Indoleacetic Acids / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011993:Recombinant Fusion Proteins; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000201:enzymology
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D007210:Indoleacetic Acids; D011993:Recombinant Fusion Proteins; C030737:indoleacetic acid; C068832:indolepyruvate decarboxylase; D002262:Carboxy-Lyases