1 Institut für Genomforschung und Systembiologie, Centrum für Biotechnologie, Universität Bielefeld, Universitätsstrasse 27, 33615 Bielefeld, Germany 2 Institut für Biochemie, Universität zu Köln, Zülpicher Strasse 47, 50674 Köln, Germany Correspondence Jörn Kalinowski joern.kalinowski{at}cebitec.uni-bielefeld.de The gene products of the rbsRACBD ( rbs ) operon of C. glutamicum ( cg1410 – cg1414 ) encode a ribose-specific ATP-binding cassette (ABC) transport system and its corresponding regulatory protein (RbsR). Deletion of the structural genes rbsACBD prohibited ribose uptake. Deletion of the regulatory gene rbsR resulted in an increased mRNA level of the whole operon. Analysis of the promoter region of the rbs operon by electrophoretic mobility shift assays identified a catabolite-responsive element ( cre )-like sequence as the RbsR-binding site. Additional RbsR-binding sites were identified in front of the recently characterized uriR operon ( uriR - rbsK1 - uriT - uriH ) and the ribokinase gene rbsK2 . In vitro , the repressor RbsR bound to its targets in the absence of an effector. A probable negative effector of RbsR in vivo is ribose 5-phosphate or a derivative thereof, since in a ribokinase ( rbsK1 rbsK2 ) double mutant, no derepression of the rbs operon in the presence of ribose was observed. Analysis of the ribose stimulon in the C. glutamicum wild-type revealed transcriptional induction of the uriR and rbs operons as well as of the rbsK2 gene. The inconsistency between the existence of functional RbsR-binding sites upstream of the ribokinase genes, their transcriptional induction during growth on ribose, and the missing induction in the rbsR mutant suggested the involvement of a second transcriptional regulator. Simultaneous deletion of the regulatory genes rbsR and uriR finally demonstrated a transcriptional co-control of the rbs and uriR operons and the rbsK2 gene by both regulators, RbsR and UriR, which were furthermore shown to recognize the same cognate DNA sequences in the operators of their target genes. Abbreviations: ABC, ATP-binding cassette; CDS, coding sequence; EMSA, electrophoretic mobility shift assay; IMPACT, intein-mediated purification with an affinity chitin-binding tag; RACE, rapid amplification of cDNA ends Two supplementary tables, listing bacterial strains and plasmids, and oligonucleotides used in this study, with supplementary references, are available with the online version of this paper.