Identiﬁcation and Characterization of a Regulatory Sequence Recognized by Mycobacterium tuberculosis Persistence Regulator MprA

Establishment and maintenance of persistent, latent infection by Mycobacterium tuberculosis are dependent on expression of the mprA-mprB regulatory system. Previously, MprA and MprB were shown to participate in phosphotransfer reactions characteristic of two-component signaling systems. To begin identifying down- stream effector genes regulated by mprA-mprB during persistent stages of infection, a search for the regulatory sequence(s) recognized by response regulator MprA was carried out. Here, evidence is presented demonstrat- ing that MprA recognizes a 19-bp sequence comprising two loosely conserved 8-bp direct repeat subunits separated by 3 nucleotides. This motif, termed the MprA box, is found upstream of the mprA coding sequence and that of downstream gene pepD ( Rv0983 ). Protein phosphorylation was not required for binding to this DNA sequence by MprA in vitro; however, phosphorylation enhanced DNA binding by MprA and was required for the regulation of mprA and pepD by MprA in vivo. Binding of MprA to the MprA box was dependent on conserved nucleotides contained within repeat subunits and on the spacer length separating these repeats. In addition, recognition of this sequence proceeded via tandem binding of two monomers of MprA. Identiﬁcation of the genetic determinants regulated by MprA will ultimately enhance our understanding of the mechanisms utilized by M. tuberculosis to undergo latency. was added to phosphorylation buffer (50 mM Tris-HCl [pH 7.6], 50 mM KCl) supplemented with 20 mM MgCl 2 and 10 mM acetyl phosphate, and the mixtures were incubated at 37°C for 30 min. Previous studies have indicated that treatment with acetylphosphate under these conditions results in the phosphorylation of (cid:2) 25% of the His-MprA in the reaction (45). Electrophoretic mobility shift assays. For EMSAs, labeled probe DNA was incubated at room temperature for 10 min with various concentrations of re- combinant MprA protein in reaction buffer containing ﬁnal concentrations of 20 mM KCl, 5% glycerol, 10 ng of salmon sperm DNA per ml, 25 mM Tris-HCl, pH 8.0, 6 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM dithiothreitol, and 4.0 (cid:4) g of poly(dI-dC). Ten microliters from each binding reaction was then loaded onto a 4 to 6% nondenaturing polyacrylamide gel and electrophoresed for 2.5 to 3.0 h at 120 V and 4°C. Gels were dried and exposed to Biomax MR (Kodak) X-ray ﬁlm overnight. DNase I protection assays. DNA fragments comprising regions upstream of mprA or pepD were PCR ampliﬁed with primers mprAseq10up and mprAseq3 or mprBseq3up and Rv0983NotI and cloned into pCR2.1-TOPO. Inserts were removed from the clones by BamH1 and EcoRV digestion, resolved on a 1% agarose gel, recovered following gel extraction, and labeled at one end with [ (cid:3) - 32 P]dCTP and Klenow enzyme. Approximately 35 ng of labeled DNA was then incubated with 150 pmol of recombinant His-MprA protein in a reaction buffer containing 25 mM Tris-HCl, pH 8.0, 50 mM KCl, 6.25 mM MgCl 2 , 0.5 mM EDTA, 10% glycerol, and 0.5 be between 90 and 100%, and single PCR products were conﬁrmed by performing a postampliﬁcation melting curve analysis for each reaction. To compensate for potential variations in transcript numbers between treated and untreated cultures, mprA and pepD expression levels between strains were normalized to that of sigA because its expression is consti- tutive and unresponsive to stresses in M. tuberculosis (20). mprA and pepD induction levels following SDS treatment for each strain were calculated as follows: ([number of mprA or pepD SDS induced /[number sigA SDS induced ) ([number mprA pepD untreated sigA untreated Statistical analysis. All statistical analyses of and protected least-signiﬁcant difference) were performed ANOVA (version 1.11; Abicus