ABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Corynebacterium glutamicum/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Bacterial , Repressor Proteins/metabolism , Ribose/metabolism , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Binding Sites , Biological Transport , Corynebacterium glutamicum/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Gene Deletion , Molecular Sequence Data , Operon , Repressor Proteins/chemistry , Repressor Proteins/geneticsABSTRACT
The soil bacterium Corynebacterium glutamicum is a model organism in amino acid biotechnology. Here we present the identification of two different L-methionine uptake systems including the first characterization of a bacterial secondary methionine carrier. The primary carrier MetQNI is a high affinity ABC-type transporter specific for l-methionine. Its expression is under the control of the transcription factor McbR, the global regulator of sulfur metabolism in C. glutamicum. Besides MetQNI, a novel secondary methionine uptake system of the NSS (neurotransmitter:sodium symporter) family was identified and named MetP. The MetP system is characterized by a lower affinity for methionine and uses Na(+) ions for energetic coupling. It is also the main alanine transporter in C. glutamicum and is expressed constitutively. These observations are consistent with models of methionine, alanine, and leucine bound to MetP, derived from the X-ray crystal structure of the LeuT transporter from Aquifex aeolicus. Complementation studies show that MetP consists of two components, a large subunit with 12 predicted transmembrane segments and, surprisingly, an additional subunit with one predicted transmembrane segment only. Thus, this new member of the NSS transporter family adds a novel feature to this class of carriers, namely, the functional dependence on an additional small subunit.
