The atypical OmpR/PhoB response regulator ChxR from Chlamydia trachomatis forms homodimers in vivo and binds a direct repeat of nucleotide sequences

Abstract

Two-component signal transduction systems are widespread in bacteria and are essential regulatory mechanisms for many biological processes. These systems predominantly rely on a sensor kinase to phosphorylate a response regulator for controlling activity, which is frequently transcriptional regulation. In recent years, an increasing number of atypical response regulators have been discovered in phylogenetically diverse bacteria. These atypical response regulators are not controlled by phosphorylation and exhibit transcriptional activity in their wild-type form. Relatively little is known regarding the mechanisms utilized by these atypical response regulators and the conserved characteristics of these atypical response regulators. Chlamydia spp. are medically important bacteria and encode an atypical OmpR/PhoB subfamily response regulator termed ChxR. In this study, protein expression analysis supports that ChxR is likely exerting its effect during the middle and late stages of the chlamydial developmental cycle, stages that include the formation of infectious elementary bodies. In the absence of detectable phosphorylation, ChxR formed homodimers in vitro and in vivo, similar to a phosphorylated OmpR/PhoB subfamily response regulator. ChxR was demonstrated to bind to its own promoter in vivo, supporting the role of ChxR as an autoactivator. Detailed analysis of the ChxR binding sites within its own promoter revealed a conserved cis-acting motif that includes a tandem repeat sequence. ChxR binds specifically to each of the individual sites and exhibits a relatively large spectrum of differential affinity. Taken together, these observations support the conclusion that ChxR, in the absence of phosphorylation, exhibits many of the characteristics of a phosphorylated (active) OmpR/PhoB subfamily response regulator.

FIG. 1.

Recombinant ChxR purifies as a stable homodimer. Purified recombinant ChxR at 1, 10, or 100 μM was subjected to analytical size exclusion chromatography to determine the in vitro oligomeric state of the protein. A molecular mass standard curve was generated using bovine serum albumin (66 kDa), chicken ovalbumin (44 kDa), and horse myoglobin (17 kDa).

FIG. 2.

ChxR forms homodimers in vivo. Recombinant ChxR forms homodimers in vitro; however, the in vivo oligomeric state of ChxR was unknown. (A) To determine whether the primary amine chemical cross-linker DSS could capture ChxR homodimers, increasing concentrations (2.2, 3.5, 7, 14, and 21 μM) of purified, recombinant ChxR were incubated with 500 μM DSS. As a control, 21 μM ChxR was not incubated with DSS. Denatured samples were separated by SDS-PAGE and observed by Coomassie staining. (B) At 12, 24, and 36 hpi, C. trachomatis were enriched from infected L929 cells, and the relative amount of ChxR present was assayed by an immunoblot with polyclonal-monospecific antibodies against ChxR (αChxR). The alpha subunit of RNA polymerase (αRpoA) was used to normalize the amount of chlamydial protein (ChxR) each time point. (C) To test dimer formation in vivo, 10 mM DSS was added to uninfected host cells (Mock) or C. trachomatis-enriched lysates at 30 hpi (Infected). The samples were separated by SDS-PAGE, and an immunoblot was performed with antibodies against ChxR.

FIG. 3.

ChxR is associated with its own promoter in vivo. To determine whether ChxR recognizes the chxR promoter during a chlamydial infection, ChxR was cross-linked to DNA, using formaldehyde, at 36 hpi and immunoprecipitated from the lysates using antibodies that recognize ChxR (αChxR). PCR was then performed with primers specific for the chxR promoter. The lack of a PCR product with primers to the CT863 promoter supports that ChxR specifically recognizes the chxR promoter in vivo. The presence (+) or absence (−) of C. trachomatis genomic DNA (DNA) was used as PCR controls for both promoters.

FIG. 4.

chxR promoter region and putative ChxR binding motif. (A) The five putative binding sites (underlined and identified as DR5, DR4, DR3, DR2, and DR1) were derived from a previously reported DNase protection assay (28). A sixth recognition site (DR6) was later identified within the chxR promoter. Transcriptional start site and σ⁶⁶ holoenzyme promoter element are indicated by +1 and −35/−10, respectively (28) (B) Visual inspection of the six binding sites in the chxR promoter suggested a conserved direct repeat sequence. The two recognition motifs within the six DR sites were aligned. (C) A consensus sequence was generated using Weblogo (12) with each half site from the sequences in Fig. 4B. (D) The recognition sequence and linker length is listed (W = A/T, H = C/A/T, and N = G/C/A/T).

FIG. 5.

Binding of ChxR to individual DR sites. (A) To determine whether ChxR interacts with the six recognition sites within the chxR promoter individually, EMSAs were performed with 100 nM concentrations of each IR800-labeled binding site (DR1 to DR6) with (+) or without (−) 1 μM ChxR. A DNA sequence corresponding to the −120 to −95 region of the chxR promoter was used as a nonspecific DNA control (NC). The K_d for each binding site is given in Table 1.

FIG. 6.

Mutations within the DNA recognition motif significantly reduce ChxR-DNA interaction. (A) The DR2 nucleotide sequence from the chxR promoter is shown. The two consensus recognition sequences are underlined, and the boldface nucleotides indicate the sites of triple-cytosine mutations. (B) EMSAs were performed with increasing concentrations of ChxR (39 nM to 3.9 μM) and 39 nM concentrations of each DNA construct: wild-type sequence (WT), DR2-2 mutant, DR2-1 mutant, and DR2-1/2 double mutant.

FIG. 7.

Single-base-pair contributions to ChxR-DNA interactions within the DR2 binding site. EMSAs were performed with 5 μM ChxR and 50 nM DNA containing transversion mutations. The target DNA used in the experiment was the DR2 sequence from the chxR promoter, comprising the DR2 half-sites (underlined). The percentages of DNA shifted with each transversion mutation (n = 3) are shown in the graph relative to the DNA shifted with the wild-type sequence (n = 18). The amount of DNA shifted was quantified using the photon emission of the SYBR green at 520 nm. The mutations that resulted in a significant (P < 0.05) reduction of DNA-interaction are denoted by an asterisk.

FIG. 8.

ChxR^E49D retains homodimer formation and DNA binding activity. (A) To test homodimerization, 5 μM ChxR or 6 μM ChxR^E49D was incubated with 500 μM DSS. Proteins were separated by SDS-PAGE and visualized by Coomassie staining. (B) To determine the DNA binding activity of ChxR^E49D, EMSAs were performed in triplicate with 1.25 μM ChxR or 1.25 μM ChxR^E49D and 50 nM DNA from the DR2 site. The percentage of DNA shifted was quantified and normalized as described previously.