FUNCTIONAL AND GENETIC-CHARACTERIZATION OF THE (METHYL)AMMONIUM UPTAKE CARRIER OF CORYNEBACTERIUM-GLUTAMICUM

Under nitrogen starvation conditions, Corynebacterium glutamicum was f ound to take up methylammonium at a rate of 20 +/- 5 nmol . min(-1).(m g dry weight)(-1). The specific activity of this uptake was 10-fold lo wer when growing the cells under sufficient nitrogen supply, indicatin g a tight regulation on the expression level. The methylammonium uptak e showed Michaelis-Menten kinetics with an K-m of 44 +/- 7 mu M and wa s completely inhibited by the addition of 10 mu M ammonium, This findi ng and the fact that methylammonium was not metabolized by C. glutamic um strongly suggests that the uptake carrier actually represents an am monium uptake system, Methylammonium uptake was strictly dependent on the membrane potential, From the pH optimum and the accumulation of me thylammonium in equilibrium, it could be deduced that only one net cha rge is transported and, thus, that methylammonium is taken up in its p rotonated form via an uniport mechanism, The ant gene encoding the (me thyl)ammonium uptake system was isolated and characterized, The predic ted gene product of ant consists of 452 amino acids (M(r) = 47,699) an d shows 26-33% identity to ammonium transporter proteins from Saccharo myces cerevisiae and Arabidopsis thaliana. According to the hydrophobi city profile, it is an integral membrane protein containing 10 or 11 m embrane-spanning segments.