Stress response gene regulation in Chlamydia is dependent on HrcA-CIRCE interactions

Abstract

HrcA is a transcriptional repressor that regulates stress response genes in many bacteria by binding to the CIRCE operator. We have previously shown that HrcA regulates the promoter for the dnaK heat shock operon in Chlamydia. Here we demonstrate that HrcA represses a second heat shock promoter that controls the expression of groES and groEL, two other major chlamydial heat shock genes. The CIRCE element of C. trachomatis groEL is the most divergent of known bacterial CIRCE elements, and HrcA had a decreased ability to bind to this nonconsensus operator and repress transcription. We demonstrate that the CIRCE element is necessary and sufficient for transcriptional regulation by chlamydial HrcA and that the inverted repeats of CIRCE are the binding sites for HrcA. Addition of a CIRCE element upstream of a non-heat-shock promoter allowed this promoter to be repressed by HrcA, showing in principle that a chlamydial promoter can be genetically modified to be inducible. These results demonstrate that HrcA is the regulator of the major chlamydial heat shock operons, and we infer that the mechanism of the heat shock response in Chlamydia is derepression. However, derepression is likely to involve more than a direct effect of increased temperature as we found that HrcA binding to CIRCE and HrcA-mediated repression were not altered at temperatures that induce the heat shock response.

FIG. 1.

In vitro transcription to examine the effects of the CIRCE element and recombinant HrcA: structure of a pair of promoter constructs and results of an in vitro transcription reaction performed with C. trachomatis RNA polymerase in the absence (minus sign) or presence (plus sign) of 1,150 nM HrcA. (A) The wild-type dnaK promoter (upper line), containing a CIRCE element, was transcribed with the omcB promoter (lower line), and these promoters produced 153- and 125-nucleotide transcripts, respectively. w.t., wild type. (B) The dnaK promoter containing the hctA upstream region (upper line) and thus lacking the CIRCE element was transcribed with the hctA promoter containing the dnaK upstream region and its CIRCE element (lower line).

FIG. 2.

In vitro transcription with C. trachomatis and E. coli RNA polymerases. The upper band is the transcript from the dnaK promoter, and the lower band is the transcript from the omcB promoter. Lane 1, C. trachomatis RNA polymerase in the absence of HrcA; lane 2, C. trachomatis RNA polymerase and 1,150 nM HrcA; lane 3, E. coli RNA polymerase in the absence of HrcA; lane 4, E. coli RNA polymerase and 1,150 nM HrcA.

FIG. 3.

DNase I footprint of recombinant HrcA on the dnaK CIRCE region. (A) Each lane contained approximately 10,000 cpm of labeled dnaK probe. Lane 1, G sequencing ladder; lane 2, G-A sequencing ladder; lane 3, no HrcA; lane 4, 144 nM HrcA; lane 5, 288 nM HrcA. (B) Sequence of dnaK CIRCE region. Protected regions are underlined, and DNase-hypersensitive sites are indicated by vertical arrows.

FIG. 4.

Alignment of the consensus bacterial CIRCE sequence (29) and the sequences of CIRCE elements from C. trachomatis strain MoPn (51), C. trachomatis serovar D (47), C. pneumoniae strain CWL029 (17), and C. caviae strain GPIC (37). The heavy underlining indicates the CIRCE region; the lowercase letters indicate mismatches with the consensus CIRCE sequence; the light underlining indicates a region of extended complementarity; the arrows indicate the maximum extent of extended complementarity; and the asterisks indicate mismatches with the consensus sequence which are complemented in the inverted repeat.

FIG. 5.

Binding and transcription of the groE promoter by using recombinant HrcA. (A) EMSA with dnaK and groE promoter restriction fragments. The positions of bound and free probe are indicated on the right. Lanes 1 to 3, dnaK probe with no HrcA (lane 1), 100 nM HrcA (lane 2), or 400 nM HrcA (lane 3); lanes 4 to 6, groE probe with no HrcA (lane 4), 100 nM HrcA (lane 5), or 400 nM HrcA (lane 6); lanes 7 to 9, groE probe containing only one arm of the CIRCE inverted repeat with no HrcA (lane 7), 100 nM HrcA (lane 8), or 400 nM HrcA (lane 9). (B) In vitro transcription of wild-type groE promoter. Lanes 1 and 2, transcription of the dnaK promoter (upper band) and the lower omcB promoter (lower band) by using C. trachomatis RNA polymerase with no HrcA (lane 1) or 1,150 nM HrcA (lane 2); lanes 3 and 4, transcription of the groE promoter (upper band) and the omcB promoter (lower band) with no HrcA (lane 3) or 1,150 nM HrcA (lane 4). (C) In vitro transcription of hybrid groE CIRCE-dnaK core promoter. Lanes 1 and 2, transcription of the dnaK promoter (upper band) and the omcB promoter (lower band) by using C. trachomatis RNA polymerase with no HrcA (lane 1) or 1,150 nM HrcA (lane 2); lanes 3 and 4, transcription of the groE/dnaK hybrid promoter (upper band) and the omcB promoter (lower band) with no HrcA (lane 3) or 1,150 nM HrcA (lane 4).

FIG. 6.

Binding and transcription with recombinant HrcA at an elevated temperature. (A) EMSA reactions with 0.5 nM ³²P-labeled dnaK CIRCE probe and various concentrations of recombinant HrcA were performed in triplicate, and the results were quantified by phosphorimager analysis. The reaction mixtures were preincubated at room temperature for 15 min and then shifted to 37°C (▪), 42°C (▴), or 56°C (♦) for 5 min. The error bars indicate standard deviations from the mean. (B) In vitro transcription with E. coli RNA polymerase. The upper band was from the dnaK promoter, and the lower band was from the omcB promoter. The lanes show (from left to right) transcription at 37°C with no HrcA, transcription at 37°C with 1,150 nM HrcA, transcription at 42°C with no HrcA, and transcription at 42°C with 1,150 nM HrcA.