Characterization of the ROK-family transcriptional regulator RokA of Streptococcus pneumoniae D39.
The Gram-positive human pathogen Streptococcus pneumoniae possesses an unusually high number of gene clusters specific for carbohydrate utilization. This provides it with the ability to use a wide array of sugars, which may aid during infection and survival in different environmental conditions present in the host. In this study, the regulatory mechanism of transcription of a gene cluster, SPD0424-8, putatively encoding a cellobiose/lactose-specific phosphotransferase system is investigated. We demonstrate that this gene cluster is transcribed as one transcriptional unit directed by the promoter of the SPD0424 gene. Upstream of SPD0424, a gene was identified encoding a ROK-family transcriptional regulator (RokA: SPD0423). DNA microarray and transcriptional reporter analyses with a rokA mutant revealed that RokA acts as a transcriptional repressor of the SPD0424-8 operon. Furthermore, we identified a 25 bp AT-rich DNA operator site (5'-TATATTTAATTTATAAAAAATAAAA-3') in the promoter region of SPD0424, which was validated by promoter truncation studies, DNase I footprinting and electrophoretic mobility-shift assays. We tested a large range of different sugars for their effect on the expression of the SPD0424-8 operon, but only moderate variation in expression was observed in the conditions applied. Therefore, a co-factor for RokA-mediated transcriptional control could not be identified.
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
Sulman
S
Shafeeq
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
Tomas G
TG
Kloosterman
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
Vijayanand
V
Rajendran
Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG, Groningen, The Netherlands.
Oscar P
OP
Kuipers
D018983:DNA Footprinting; D004252:DNA Mutational Analysis; D020869:Gene Expression Profiling; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D046228:Microarray Analysis; D005810:Multigene Family; D009875:Operator Regions, Genetic; D010770:Phosphotransferases / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D013296:Streptococcus pneumoniae / Q000235:genetics*; D014158:Transcription, Genetic
D012097:Repressor Proteins; D010770:Phosphotransferases