The response of Corynebacterium glutamicum to ammonium limitation was studied by transcriptional and proteome profiling of cells grown in a chemostat. Our results show that ammonium-limited growth of C. glutamicum results in a rearrangement of the cellular transport capacity, changes in metabolic pathways for nitrogen assimilation, amino acid biosynthesis, and carbon metabolism, as well as a decreased cell division. Since transcription at different growth rates was studied, it was possible to distinguish specific responses to ammonium limitation and more general, growth rate-dependent alterations in gene expression. The latter include a number of genes encoding ribosomal proteins and genes for F o F 1 -ATP synthase subunits.Corynebacterium glutamicum was isolated in 1957 by Kinoshita and coworkers in a screening program for L-glutamateproducing bacteria from a soil sample collected at Ueno Zoo in Tokyo, and at that time it was designated as Micrococcus glutamicus (10,21,33). Less than 50 years later, enormous amounts of L-glutamate (more than 1,500,000 tons per year) and L-lysine (more than 560,000 tons per year) are produced by use of different C. glutamicum strains, in addition to smaller amounts of some less industrially important amino acids (Lalanine, L-isoleucine, and L-proline) and in addition to different nucleotides (15). In contrast to closely related pathogenic species, such as Corynebacterium diphtheriae, Mycobacterium leprae, and Mycobacterium tuberculosis, C. glutamicum is generally recognized as a nonhazardous organism which is safe to handle. Furthermore, based on its extremely well-investigated central metabolism and well-established molecular biology tools, C. glutamicum is suitable as a model organism for high GϩC content gram-positive bacteria in general and mycolicacid-containing Actinomycetales in particular.The regulation of nitrogen metabolism in the Actinomycetales was the subject of research mainly in the last few years (for a review, see references 7 and 8). For C. glutamicum, detailed information of transport and assimilation of nitrogen sources as well as nitrogen regulation is available on a molecular level (for a review, see references 7 and 8). Uptake systems for ammonium, creatinine, and glutamate were studied, and assimilatory enzymes and pathways were investigated. Additionally, the key components of nitrogen control were identified; namely, AmtR, the master regulator of nitrogen control in C. glutamicum, GlnK, the sole P II -type signal transduction protein in this organism, and two modifying enzymes, a putative adenylyltransferase and the GlnD protein (32).While previous studies focused on specific genes or enzymes, here we present a global analysis of the C. glutamicum ammonium limitation response by transcriptional profiling and twodimensional gel electrophoresis. In this study these two global approaches were combined with the continuous cultivation of cells. In contrast to shaking flask experiments, this technique allows us to establish highly defined growth conditions o...