Mycobacterium tuberculosis Origin of Replication and the Promoter for Immunodominant Secreted Antigen 85B Are the Targets of MtrA, the Essential Response Regulator

Abstract

Efficient proliferation of Mycobacterium tuberculosis (Mtb) inside macrophage requires that the essential response regulator MtrA be optimally phosphorylated. However, the genomic targets of MtrA have not been identified. We show by chromatin immunoprecipitation and DNase I footprinting that the chromosomal origin of replication, oriC, and the promoter for the major secreted immunodominant antigen Ag85B, encoded by fbpB, are MtrA targets. DNase I footprinting analysis revealed that MtrA recognizes two direct repeats of GTCACAgcg-like sequences and that MtrAϳP, the phosphorylated form of MtrA, binds preferentially to these targets. The oriC contains several MtrA motifs, and replacement of all motifs or of a single select motif with TATATA compromises the ability of oriC plasmids to maintain stable autonomous replication in wild type and MtrA-overproducing strains, indicating that the integrity of the MtrA motif is necessary for oriC replication. The expression of the fbpB gene is found to be down-regulated in Mtb cells upon infection when these cells overproduce wild type MtrA but not when they overproduce a nonphosphorylated MtrA, indicating that MtrAϳP regulates fbpB expression. We propose that MtrA is a regulator of oriC replication and that the ability of MtrA to affect apparently unrelated targets, i.e. oriC and fbpB, reflects its main role as a coordinator between the proliferative and pathogenic functions of Mtb.

Septal Localization of the Mycobacterium tuberculosis MtrB Sensor Kinase Promotes MtrA Regulon Expression

“…In this regard, a candidate of interest is MtrA D13A , which has a mutation in the signal receiver domain and is phosphorylationdefective but is proficient at binding to its target PfbpB and modulating its expression (14). We further confirmed this assumption by evaluating MtrA D13A binding to the oriC target by EMSA (9). Again, MtrA D13A bound oriC proficiently in the absence of ATP (supplemental Fig.…”

“…Electrophoretic Mobility Shift Assay (EMSA)-Interactions of MtrA and phosphorylated MtrA (MtrAϳP) with 5Ј-6-carboxyfluorescein-labeled PripA or oriC were assessed by EMSA as described (9). A 200-bp ripA upstream region was amplified using 6-carboxyfluorescein-labeled primers MVM782 and MVM783.…”

“…RNA Extraction, Reverse Transcription, and Quantitative Real-time (qRT) PCR-Total RNA was extracted from 7H9 broth-grown cultures of M. smegmatis as previously described (8,9,16,18,19). RNA was reverse-transcribed using random hexamers (Invitrogen) and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega), and the cDNA obtained was used to perform qRT-PCR in a Bio-Rad iCycler iQ5 TM Real-Time PCR detection system using 2ϫ iQ SYBR Supermix (Bio-Rad).…”

“…Chromatin Immunoprecipitation (ChIP) Assay-ChIP was carried with formaldehyde-cross-linked M. tuberculosis lysates and anti-MtrA and mock antibodies as described (9).…”

“…Earlier, we showed that the overproduction of a phosphorylation-competent wild type (WT) MtrA (MtrA WT ) compromised the ability of M. tuberculosis to replicate after infection, whereas the simultaneous overpro-duction of MtrA and the sensor kinase MtrB reversed the growth defect, suggesting that one role of the MtrB sensor kinase is to help regulate the phosphorylation status of MtrA (8). More recent data have identified oriC and the promoters (P) for dnaA and fbpB as MtrA targets (9). Other recent findings implicate MtrB in cell wall homeostasis in part through its association with the lipoprotein LpqB (10).…”