Ammonium excretion by an L-methionine-DL-sulfoximine-resistant mutant of the rice field cyanobacterium Anabaena siamensis

Abstract

An ammonium-excreting mutant (SS1) of the rice field nitrogen-fixing cyanobacterium Anabaena siamensis was isolated after ethyl methanesulfonate mutagenesis by selection on 500 μM L-methionine-DL-sulfoximine. SS1 grew in the presence and absence of L-methionine-DL-sulfoximine at a rate comparable to that of the wild-type strain, with a doubling time of 5.6 h. The rate of ammonium release by SS1 depended on cell density; it peaked at the 12th hour of growth with 8.7 μmol mg of chlorophyll-1 h-1 (at a chlorophyll concentration of 5 μg ml-1) and slowed down to almost nil at the fourth day of growth. A similar pattern of release by immobilized SS1 was observed between 12 to 20 h after loading alginate beads in packed-bed reactors at the rate of 11.6 μmol mg of chlorophyll-1 h-1. The rate was later reduced significantly due to the fast growth of SS1 on the substrate. Prolonged release of ammonium at the peak level was achieved only by maintaining SS1 under continuous cultivation at low chlorophyll levels (5 to 7 μg ml-1). Under these conditions, nitrogen fixation in the mutant was 30% higher than that in its parent and glutamine synthetase activity was less by 50%. Immunoblot analysis revealed that SS1 and its parent have similar quantities of glutamine synthetase protein under ammonium excretion conditions. In addition, a protein with a molecular weight of about 30,000 seems to have been lost, as seen by electrophoretic separation of total proteins from SS1.