Characterization of two trpE genes encoding anthranilate synthase α-subunit in Azospirillum brasilense

Abstract

The previous report from our laboratory has recently identified a new trpE gene (termed trpE 2) which exists independently in Azospirillum brasilense Yu62. In this study, amplification of trpE( G) (termed trpE 1( 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 trpE 1( G) while these sequence features did not exist in front of trpE 2. The β-galactosidase activity of an A. brasilense strain carrying a trpE 2- lacZ fusion remained constant at different tryptophan concentrations, but the β-galactosidase activity of the same strain carrying a trpE 1( G)- lacZ fusion decreased as the tryptophan concentration increased. These data suggest that the expression of trpE 1( G) is regulated at the transcriptional level by attenuation while trpE 2 is constantly expressed. The anthranilate synthase assays with trpE 1( G) − and trpE 2 − mutants demonstrated that TrpE 1(G) fusion protein is feedback inhibited by tryptophan while TrpE 2 protein is not. We also found that both trpE 1( G) and trpE 2 gene products were involved in IAA synthesis.