Characterization of two trpE genes encoding anthranilate synthase alpha-subunit in Azospirillum brasilense.
The previous report from our laboratory has recently identified a new trpE gene (termed trpE2) which exists independently in Azospirillum brasilense Yu62. In this study, amplification of trpE(G) (termed trpE1(G) here) confirmed that there are two copies of trpE gene, one trpE being fused into trpG while the other trpE existed independently. This is the first report to suggest that two copies of the trpE gene exist in this bacterium. Comparison of the nucleotide sequence demonstrated that putative leader peptide, terminator, and anti-terminator were found upstream of trpE1(G) while these sequence features did not exist in front of trpE2. The beta-galactosidase activity of an A. brasilense strain carrying a trpE2-lacZ fusion remained constant at different tryptophan concentrations, but the beta-galactosidase activity of the same strain carrying a trpE1(G)-lacZ fusion decreased as the tryptophan concentration increased. These data suggest that the expression of trpE1(G) is regulated at the transcriptional level by attenuation while trpE2 is constantly expressed. The anthranilate synthase assays with trpE1(G)- and trpE2- mutants demonstrated that TrpE1(G) fusion protein is feedback inhibited by tryptophan while TrpE2 protein is not. We also found that both trpE1(G) and trpE2 gene products were involved in IAA synthesis.
Ge
Shi-Mei
SM
College of Biological Sciences and National Key Laboratory for Agrobiotechnology, Key Laboratory of Agro-Microbial and Application, China Agricultural University, Beijing 100094, PR China.
Xie
Bao-En
BE
Chen
San-Feng
SF
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000878:Anthranilate Synthase / Q000096:biosynthesis / Q000235:genetics*; D015806:Azospirillum brasilense / Q000201:enzymology*; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D015964:Gene Expression Regulation, Bacterial; D005819:Genetic Markers; D005838:Genotype; D007763:Lac Operon; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D021382:Protein Sorting Signals; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D005819:Genetic Markers; D010455:Peptides; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D000878:Anthranilate Synthase