Ammonia-dependent growth (Adg-) mutants of Rhodobacter capsulatus and Rhodobacter sphaeroides: comparison of mutant phenotypes and cloning of the wild-type (adg A) genes

Abstract

Mutants of Rhodobacter capsulatus deficient in growth on nitrogen sources other than NH+ 4 were compared with mutants of a similar phenotype isolated from Rhodobacter sphaeroides. In addition to N2 and some amino acids (glutamate, alanine, proline, arginine), mutants of R. sphaeroides and R. capsulatus strain AD2 were unable to utilize NO3 − as sole nitrogen source for growth. Under conditions of nitrogen starvation, mutants of both species lacked the methylammonium (ammonium) uptake system, which was found in the wild-type strains under these conditions. The wild-type (adgA) genes complementing these mutants were isolated from gene banks of the respective species and localized to a 2·9 kb BamHI-SalI fragment in R. sphaeroides and to a 1·7 kb SmaI fragment in R. capsulatus. These two fragments hybridized strongly with each other, showing that they contain homologous sequences. Furthermore, the adgA gene from R. capsulatus fully restored the wild-type phenotype to Adg− mutants of R. sphaeroides and vice versa. Inactivation of the chromosomal adgA gene by insertion of an antibiotic-resistance cassette resulted in a severe inhibition of growth in rich medium and in minimal medium containing NH+ 4. This suggests that the adg A gene is required for normal growth under all growth conditions.