Cooperative Regulation of Campylobacter jejuni Heat-Shock Genes by HspR and HrcA

Abstract

The heat-shock response is defined by the transient gene-expression program that leads to the rapid accumulation of heat-shock proteins. This evolutionary conserved response aims at the preservation of the intracellular environment and represents a crucial pathway during the establishment of host–pathogen interaction. In the food-borne pathogen Campylobacter jejuni two transcriptional repressors, named HspR and HrcA, are involved in the regulation of the major heat-shock genes. However, the molecular mechanism underpinning HspR and HrcA regulatory function has not been defined yet. In the present work, we assayed and mapped the HspR and HrcA interactions on heat-shock promoters by high-resolution DNase I footprintings, defining their regulatory circuit, which governs C. jejuni heat-shock response. We found that, while DNA-binding of HrcA covers a compact region enclosing a single inverted repeat similar to the so-called Controlling Inverted Repeat of Chaperone Expression (CIRCE) sequence, HspR interacts with multiple high- and low-affinity binding sites, which contain HspR Associated Inverted Repeat (HAIR)-like sequences. We also explored the DNA-binding properties of the two repressors competitively on their common targets and observed, for the first time, that HrcA and HspR can directly interact and their binding on co-regulated promoters occurs in a cooperative manner. This mutual cooperative mechanism of DNA binding could explain the synergic repressive effect of HspR and HrcA observed in vivo on co-regulated promoters. Peculiarities of the molecular mechanisms exerted by HspR and HrcA in C. jejuni are compared to the closely related bacterium H. pylori that uses homologues of the two regulators.

Feeling the Heat: The Campylobacter jejuni HrcA Transcriptional Repressor Is an Intrinsic Protein Thermosensor

“…Recently, we determined the C. jejuni HrcA and HspR interactions on heat shock promoters by high-resolution DNase I footprints, showing that while DNA-binding of HrcA covers a compact region, HspR interacts with multiple high-and low-affinity binding sites [16]. In the present work, we show for the first time that in C. jejuni the HrcA repressor is the intrinsic heat-sensor of the heat-shock regulatory circuit.…”

“…C. jejuni recombinant His-tagged HrcA and HspR proteins were expressed in E. coli BL21(DE3) and purified through Ni 2+ -NTA affinity chromatography as previously described [16]. At the end of the purification, recombinant HrcA and HspR were stored in aliquots at −80 • C in Storage Buffer (for HrcA: 10 mM Tris-HCl, pH 8.0; 300 mM NaCl; 5 mM TCEP; 0.05% NP40; 10% glycerol; for HspR: 50 mM Tris-HCl, pH 8.0; 300 mM NaCl; 1 mM DTT; 0.05% NP40; 10% glycerol).…”

“…Following 75 s of incubation at room temperature, the reactions were stopped by adding 140 µL of DNase I Stop Buffer (192 mM NaOAc, pH 5.2; 32 mM EDTA; 0.1% SDS; 64 µg/µL sonicated salmon sperm DNA). Samples containing the labeled DNA fragments were then purified and loaded on 8M urea-6% polyacrylamide (19:1) gels as previously described [16]. The gel was blotted onto a 3 MM Whatman paper sheet (Thermo Scientific, Waltham, MA, USA), dried and autoradiographed.…”

“…First of all, we carried out EMSA (Electrophoretic Mobility Shift Assay) experiments using Pgro promoter probe and the purified HrcA protein. In order to investigate if temperature is able to influence HrcA activity, DNA-binding of the protein was promoted by incubating the reactions at 25 • C for 10 min (a condition previously used to study HrcA and HspR interaction with the DNA in [16]), then samples were shifted at 25, 37 or 42 • C for an additional period of time of 10 min, before separation of the complexes through native polyacrylamide gel electrophoresis. As reported in Figure 2A, at 25 • C the inclusion of increasing amounts of HrcA provoked the appearance of a single retarded band in a dose-dependent manner (Figure 2A, lanes 1-4), indicative of HrcA binding to Pgro, which harbors a single DNAbinding site [16].…”

“…Conversely, on the promoters upstream of the groES-groEL and hrcA-grpE-dnaK operons, HspR combines with HrcA and together exert transcriptional repression. In addition, it has recently been demonstrated a direct protein-protein interaction between HrcA and HspR and it has been shown that their binding on co-regulated targets occurs in a cooperative manner [16]. This negative regulatory circuit implies, at 37 • C which represents the normal temperature for the human host, a transcriptional repression of HSP-encoding genes operated by HspR and HrcA repressors bound to their operators within the heat-shock promoters.…”