The two paralogue phoN (phosphinothricin acetyl transferase) genes of Pseudomonas putida encode functionally different proteins

Phosphinothricin (PPT) is a non-specific inhibitor of glutamine synthetase that has been employed as herbicide for selection of transgenic plants expressing cognate resistance genes. While the soil bacterium PseudomonasputidaKT2440 has been generally considered PPT-sensitive, inspection of its genome sequence reveals the presence of two highly similar open reading frames (PP_1924 and PP_4846) encoding acetylases with a potential to cause tolerance to the herbicide. To explore this possibility, each of these genes (named phoN1 and phoN2) was separately cloned and their activities examined in vivo and in vitro. Genetic and biochemical evidence indicated that phoN1 encodes a bona fidePPT-acetyl transferase, the expression of which suffices to make P.putida tolerant to high concentrations of the herbicide. In contrast, PhoN2 does not act on PPT but displays instead activity against methionine sulfoximine (MetSox), another glutamine synthetase inhibitor. When the geometry of the substrate-binding site of PhoN1 was grafted with the equivalent residues of the predicted PhoN2 structure, the resulting protein increased significantly MetSox resistance of the expression host concomitantly with the loss of activity on PPT. These observations uncover intricate biochemical and genetic interactions among soil microorganisms and how they can be perturbed by exposure to generic herbicides in soil.