Characterization of Mycobacterium tuberculosis Rv3676 (CRPMt), a cyclic AMP receptor protein-like DNA binding protein

Abstract

Little is known about cyclic AMP (cAMP) function in Mycobacterium tuberculosis, despite its ability to encode 15 adenylate cyclases and 10 cNMP-binding proteins. M. tuberculosis Rv3676, which we have designated CRP(Mt), is predicted to be a cAMP-dependent transcription factor. In this study, we characterized CRP(Mt)'s interactions with DNA and cAMP, using experimental and computational approaches. We used Gibbs sampling to define a CRP(Mt) DNA motif that resembles the cAMP receptor protein (CRP) binding motif model for Escherichia coli. CRP(Mt) binding sites were identified in a total of 73 promoter regions regulating 114 genes in the M. tuberculosis genome, which are being explored as a regulon. Specific CRP(Mt) binding caused DNA bending, and the substitution of highly conserved nucleotides in the binding site resulted in a complete loss of binding to CRP(Mt). cAMP enhanced CRP(Mt)'s ability to bind DNA and caused allosteric alterations in CRP(Mt) conformation. These results provide the first direct evidence for cAMP binding to a transcription factor in M. tuberculosis, suggesting a role for cAMP signal transduction in M. tuberculosis and implicating CRP(Mt) as a cAMP-responsive global regulator.

FIG. 1.

Sequence logos of the E. coli CRP motif model (A) and the putative CRP_Mt motif model (B). The E. coli CRP motif model represents 87 experimentally identified (DNase I footprinted) binding sites; this motif model was used as prior information in the prediction of CRP_Mt binding sites (see Materials and Methods). The putative CRP_Mt motif model represents 58 predicted sites (see Table 2) from the set of intergenic regions from the M. tuberculosis genome. Sequence logos depict the relative frequency of each base at each position of the motif. The y axis indicates the information content measured in bits, and error bars represent standard deviations at each position due to the limited sample size (58).

FIG. 2.

EMSA experiment showing binding of CRP_Mt with motifs identified in captured DNA sequences. (A) The mobility of 28-bp synthesized probes containing motifs of 1B5 and 2D3 fragments was retarded by CRP_Mt, and the free probes reappeared in the presence of a 500-fold excess of unlabeled probe DNA, as specified. (B) The shift of the 2D3 28-bp motif probe by CRP_Mt could be competed by either 2D3 or 1B5 unlabeled motif DNA, but not by the 40-bp intergenic DNA upstream of Rv1624c (1624c) that was used as a negative control. This control Rv1624c DNA probe also failed to bind to CRP_Mt. The labeled DNA probe, unlabeled competitor DNA (cold DNA), and amount of CRP_Mt (nM) that was used are specified for each lane.

FIG. 3.

Sequence alignment of helix-turn-helix DNA recognition domains of CRP_Mt and E. coli CRP (Ec CRP) and FNR (Ec FNR) (Pfam 17.0, PF00325) (4). CRP and FNR amino acid residues that form hydrogen bonds with DNA bases are underlined.

FIG. 4.

EMSA showing CRP_Mt interactions with representative DNA binding sites identified by Gibbs sampling. DNA probes are as follows: A, the full-length Rv0884c-Rv0885 intergenic region, which contains a motif with a high probability of belonging to the CRP_Mt motif model; B, the 20-bp predicted binding site in the Rv0884c-Rv0885 intergenic region; and C, the 20-bp predicted binding site upstream of the Rv1230c open reading frame, which has a low probability of belonging to the motif model. For each lane, the CRP_Mt concentration (nM) is noted, and “cold DNA” specifies unlabeled competitor DNA fragments used at a 500-fold excess relative to the labeled probe DNA. The 40-bp Rv1624c nonspecific DNA was used as a negative control throughout.

FIG. 5.

Binding of CRP_Mt to native and modified sequences in the Rv0884c-Rv0885 intergenic sequence. (A) Sequences of the 20-bp native and modified sites are shown below the figure. Modified sites are marked with asterisks. The mobility of the native probe was retarded by CRP_Mt, and the reappearance of free probe was observed when a 500-fold excess of unlabeled native DNA or 1B5 or 2D3 DNA was present. The same concentration of unlabeled modified DNA and the Rv1624c 40-bp nonspecific DNA failed to compete the binding of the probe. Modified probes failed to bind CRP_Mt. (B) Binding of CRP_Mt to the Rv0884c-Rv0885 full-length intergenic DNA could be competed by a 500-fold excess of unlabeled 1B5 or 2D3 DNA, as well as by the native Rv0884c probe, but not the modified probes, as shown in panel A. “Cold DNA” refers to the competitor. The CRP_Mt concentration (nM) is noted for each lane in panel A and was 20 nM for panel B.

FIG. 6.

Effect of cAMP on affinity of CRP_Mt-DNA interaction, as measured by EMSA. (A) Binding of CRP_Mt to the 2D3 intergenic DNA probe in the presence of different amounts of cAMP in the binding buffer, as specified. (B and C) Comparison of CRP_Mt binding affinities in the presence and absence of 100 μM cAMP. (B) 2D3 probe; (C) Rv0884c-Rv0885 intergenic region probe. CRP_Mt concentrations are shown in nM.

FIG. 7.

Evidence of allosteric alteration of CRP_Mt by cAMP. (A) SDS-PAGE of CRP_Mt after limited proteolysis with trypsin in the presence or absence of cAMP or AMP. M, molecular weight marker (Molecular Probes). CRP_Mt treatment was as follows: lanes 1 to 3, trypsin digestion; lanes 4 to 6, undigested controls; lanes 2 and 5, addition of 100 μM cAMP; lanes 3 and 6, supplementation with 100 μM AMP. (B) EMSA of CRP_Mt using intergenic Rv0884c-Rv0885 DNA probe after limited proteolysis with trypsin in the presence or absence of 100 μM cAMP or 100 μM AMP. The CRP_Mt treatment is shown at the top, with the digested CRP_Mt concentration indicated in mM. An EMSA with all samples was performed with 100 μM cAMP in the binding reaction buffer. The figure is representative of three experimental repeats.

FIG. 8.

DNA bending by CRP_Mt. (A) Graphic showing the genetic structure of the Rv0884c-Rv0885 intergenic region. Five 156-bp subregions, designated F1 to F5, were amplified by PCR, with the binding site at a different location within each fragment, as shown. (B) Fragments F1 through F5 were labeled and used for EMSA with 35 nM of CRP_Mt. Unbound probes showed similar mobilities (left half of gel), while the mobility of each protein-DNA complex varied depending on the position of the CRP_Mt binding site within the fragment (right side of gel).