Identification of a helix-turn-helix motif of Bacillus thermoglucosidasius HrcA essential for binding to the CIRCE element and thermostability of the HrcA-CIRCE complex, indicating a role as a thermosensor

Abstract

In the heat shock response of bacillary cells, HrcA repressor proteins negatively control the expression of the major heat shock genes, the groE and dnaK operons, by binding the CIRCE (controlling inverted repeat of chaperone expression) element. Studies on two critical but yet unresolved issues related to the structure and function of HrcA were performed using mainly the HrcA from the obligate thermophile Bacillus thermoglucosidasius KP1006. These two critical issues are (i) identifying the region at which HrcA binds to the CIRCE element and (ii) determining whether HrcA can play the role of a thermosensor. We identified the position of a helix-turn-helix (HTH) motif in B. thermoglucosidasius HrcA, which is typical of DNA-binding proteins, and indicated that two residues in the HTH motif are crucial for the binding of HrcA to the CIRCE element. Furthermore, we compared the thermostabilities of the HrcA-CIRCE complexes derived from Bacillus subtilis and B. thermoglucosidasius, which grow at vastly different ranges of temperature. The thermostability profiles of their HrcA-CIRCE complexes were quite consistent with the difference in the growth temperatures of B. thermoglucosidasius and B. subtilis and, thus, suggested that HrcA can function as a thermosensor to detect temperature changes in cells.

FIG. 1.

(A) Alignment of amino acid sequences of various HrcAs with the repressors of carbohydrate catabolic systems. The symbols indicate identical or conserved residues (▴), conserved substitutions (•), and semiconserved substitutions (○). HrcAs are from B. thermoglucosidasius (Bth) (29), Bacillus stearothermophilus (Bst) (16), B. subtilis (Bsu) (22), Bacillus halodurans (Bha) (26), Clostridium acetobutylicum (Cac) (18), and Staphylococcus aureus (Sau) (2). Eco GlpR, Eco GutR, Eco FucR, and Bsu IolR are the repressors of the E. coli glp (34), E. coli gut (31), E. coli fuc (13), and B. subtilis iol (32) genes, respectively. Boxes indicate the tentative helices assigned based on the HTH of E. coli GlpR. Numbers represent amino acid residue positions. (B) Different secondary-structure predictions for B. thermoglucosidasius HrcA. The software used for secondary predictions was DSC (12) for prediction 1, GORIII (8) for prediction 2, PHD (20) for prediction 3, Predator (7) for prediction 4, and Jnet alignment prediction (6) for prediction 5. The symbols for the results from these predictions are as follows: H, α helix; e, strand; and −, others. The putative helices presumed in this study are indicated with boxes. The two mutated residues (Lys31 and Arg43) are shown with arrows.

FIG. 2.

Binding curve of wild-type and mutant (K31P and R43D) B. thermoglucosidasius HrcA proteins to the CIRCE element-containing probe. The curves indicating the results for wild-type HrcA (○), K31P mutant HrcA (□), and R43D mutant HrcA (⋄) are shown. The protein concentration at which 50% of the target DNA remained free for the apparent equilibrium dissociation constants of the HrcA-CIRCE complex is also shown (dotted line).

FIG. 3.

Thermal denaturation profiles of B. subtilis (A) and B. thermoglucosidasius (B) HrcA by light-scattering assays in the presence of the plasmid pTY-1 containing the CIRCE element. Twenty-five micrograms of B. thermoglucosidasius HrcA or B. subtilis HrcA in 50 μl of 20 mM Tris-HCl-5 mM EDTA-6 M urea (pH 7.5) was added to the solution (2.95 ml of 20 mM Tris-HCl-5 mM EDTA [pH 7.5]) at temperatures of 20 (□), 30 (▵), 40 (♦), 50 (⋄), 60 (▿), and 70°C (○).