Expression, autoregulation, and DNA binding properties of the Mycobacterium tuberculosis TrcR response regulator.
The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis. Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M. tuberculosis growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli. Upon induction of the TrcR protein, there was a >500-fold increase in beta-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR. This report indicates that the M. tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter.
Haydel
Shelley E
SE
Department of Biology, Washington University, St. Louis, Missouri 63130, USA. haydel@biology.wustl.edu
Benjamin
William H
WH
Dunlap
Nancy E
NE
Clark-Curtiss
Josephine E
JE
D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000494:pharmacology; D005809:Genes, Regulator; D006706:Homeostasis; D008264:Macrophages / Q000382:microbiology; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D004247:DNA; D003850:Deoxyribonuclease I