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.