Partial Cloning of the Ammonium Transporter Genes of Azospirillum Brasilense and Herbaspirillum seropedicae

Abstract

Ammonium transporter genes (ami) have been described in several organisms and they code for transmembrane proteins with high homology (Merrick, Edwards, 1995). High ammonium concentrations seem to inhibit this transport and, in some organisms, the ntr system is involved (Merrick, Edwards, 1995). Here we describe the partial cloning of the ammonium transporter gene (amtB) of A. brasilense and H. seropedicae. Degenerate primers related to homologous aminoacid sequences of the ammonium transporter proteins of E. coli, B. subtilis and M. tuberculosis were used to amplify DNA fragments of A. brasilense strain FP2 and H. seropedicae strain SmR1. These amplified DNA fragments were cloned and sequenced. A 150 bp DNA fragment amplified from the genome of H. seropedicae showed 68% identity with the E. coli AmtB at the aminoacid level and to other ammonium transporter proteins present in the database. This result indicates the presence of an amtB-like gene in H. seropedicae. A 510 bp DNA fragment amplified from the genome of A. brasilense strain FP2 was almost identical to the sequence published by Van Dommelen et al (1998) except for 2 positions (GenBank AF082303). This DNA fragment was used to generate an mutant of A. brasilense by insertion of a lacZ::Km cassette. This mutant showed a decrease in ammonium uptake of 43% compared with the wild type when tested at pH 6.8 and when grown in 5 mM glutamate. No significant difference was observed when cells were grown in 20 mM suggesting that the amtB gene is expressed under ammonium-limiting conditions. In both the wild type and the mutant strains, a decrease of about 50% in ammonium uptake was observed when the pH was decreased from 6.8 to 5.6. This effect of pH on ammonium uptake suggests, as proposed by Soupene et al (1998), that is the transported species.