The complete genome sequence of Corynebacterium glutamicum strain R was determined to allow its comparative analysis with other corynebacteria. The biology of corynebacteria was explored by refining the definition of the subset of genes that constitutes the corynebacterial core as well as those characteristic of saprophytic and pathogenic ecological niches. In addition, the relative scarcity of corynebacterial sigma factors and the plasticity of their two-component system machinery reflect their relatively exacting nutritional requirements and reduced membrane-associated and secreted proteins. The conservation of key genes and pathways between corynebacteria, mycobacteria and Nocardia validates the use of C. glutamicum to study fundamental processes that are conserved in slow-growing mycobacteria, including pathogenesis-associated mechanisms. The discovery of 39 novel genes in C. glutamicum R that have not been previously reported in other corynebacteria supports the rationale for sequencing additional corynebacterial genomes to better define the corynebacterial pan-genome and identify previously undetected metabolic pathways in these organisms.