AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum

The uptake and assimilation of nitrogen sources is effectively regulated in bacteria. In the Gram-negative enterobacterium Escherichia coli, the NtrB/ C two-component system is responsible for the activation of transcription o f different enzymes and transporters, depending on the nitrogen status of t he cell. In this study, we investigated regulation of ammonium uptake in Co rynebacterium glutamicum, a Gram-positive soil bacterium closely related to Mycobacterium tuberculosis. As shown by Northern blot hybridizations, regu lation occurs on the level of transcription upon nitrogen starvation. In co ntrast to enterobacteria, a repressor protein is involved in regulation, as revealed by measurements of methylammonium uptake and beta-galactosidase a ctivity in reporter strains. The repressor-encoding gene, designated amtR, was isolated and sequenced. Deletion of amtR led to deregulation of transcr iption of amt coding for the C. glutamicum (methyl)ammonium uptake system. E. coli extracts from amtR-expressing cells were applied in gel retardation experiments, and binding of AmtR to the amt upstream region was observed. By deletion analyses, a target motif for AmtR binding was identified, and b inding of purified AmtR protein to this motif, ATCTATAGN(1-4)ATAG, was show n. Furthermore, the binding of AmtR to this sequence was proven in vivo usi ng a yeast one-hybrid system. Subsequent studies showed that AmtR not only regulates transcription of the amt gene but also of the amtB-glnK-glnD oper on encoding an amt paralogue, the signal transduction protein P-II and the uridylyltransferase/uridylyl-removing enzyme, key components of the nitroge n regulatory cascade. In summary, regulation of ammonium uptake and assimil ation in the high G+C content Gram-positive bacterium C. glutamicum differs significantly from the mechanism found in the low G+C content Gram-positiv e model organism Bacillus subtilis and from the paradigm of nitrogen contro l in the Gram-negative enterobacteria.