Renaturation of Bacillus thermoglucosidasius HrcA repressor by DNA and thermostability of the HrcA-DNA complex in vitro

Abstract

HrcA, a negative control repressor for chaperone expression from the obligate thermophile Bacillus thermoglucosidasius KP1006, was purified in a His-tagged form in the presence of 6 M urea but hardly renatured to an intact state due to extreme insolubility. Renaturation trials revealed that the addition of DNA to purified B. thermoglucosidasius HrcA can result in solubilization of HrcA free from the denaturing agent urea. Results from band shift and light scattering assays provided three new findings: (i) any species of DNA can serve to solubilize B. thermoglucosidasius HrcA, but DNA containing the CIRCE (controlling inverted repeat of chaperone expression) element is far more effective than other nonspecific DNA; (ii) B. thermoglucosidasius HrcA renatured with nonspecific DNA bound the CIRCE element in the molecular ratio of 2.6:1; and (iii) B. thermoglucosidasius HrcA binding to the CIRCE element was stable at below 50 degrees C whereas the complex was rapidly denatured at 70 degrees C, suggesting that the breakdown of HrcA is induced by heat stress and HrcA may act as a thermosensor to affect the expression of heat shock regulatory genes. These results will help to determine the nature of HrcA protein molecules.

FIG. 1

(A) Schematic representation and physical map of the hrcA-dnaK operon. The CIRCE element and the hrcA gene are indicated with a hairpin and thick arrow, respectively. A 1.1-kbp PCR product obtained from primers HF and HR, the EcoRI fragment cloned in pTY-1, and the BamHI fragment in pETTY are shown as bars, as are pTI-2 (26) containing the C terminus of the hrcA gene with the grpE, dnaK, and dnaJ genes. (B) DNA sequence of the N terminus of the hrcA gene and its 5′-flanking region. The vegetative promoter (−35 and −10 consensus sequence) is boxed. The CIRCE element is indicated with thick arrows. A Shine-Dalgarno sequence (SD) for ribosome binding is underlined.

FIG. 2

(A) SDS-PAGE pattern of B. thermoglucosidasius HrcA purified by metal chromatography; (B) native-PAGE pattern of B. thermoglucosidasius HrcA renatured with nonspecific DNA (pUC119). The gel concentrations used were 10% for SDS-PAGE and 7.5% for native PAGE. Each lane contained 2 μg of B. thermoglucosidasius HrcA protein. Molecular masses of the protein markers (M) in panel A are indicated in kilodaltons.

FIG. 3

(A) Band shift assays of the CIRCE probe with B. thermoglucosidasius HrcA renatured with nonspecific DNA (pUC119). Positions of the radiolabeled free DNA probe (F) and HrcA-probe complex (C) are marked. B. thermoglucosidasius HrcA samples contained no urea. Lane 1, labeled CIRCE probe (0.2 μg) alone; lane 2, labeled CIRCE probe (0.2 μg) with renatured HrcA (0.1 μg); lane 3, labeled nonspecific probe (0.2 μg) alone; lane 4, labeled nonspecific probe (0.2 μg) with renatured HrcA (0.1 μg); lane 5, same as lane 2 with nonlabeled CIRCE probe (1 μg); lane 6, same as lane 2 with nonlabeled nonspecific probe (1 μg). (B) Thermostability analyses of HrcA and probe complex by band shift assays. The binding reaction of B. thermoglucosidasius HrcA to the CIRCE probe was done at room temperature for 10 min; then each mixture was incubated at the indicated temperature for 1 min. Immediately after heat shock treatment, the samples were loaded into wells of a polyacrylamide gel. Lane 1, labeled CIRCE probe (0.2 μg) alone; lanes 2 to 7, the labeled CIRCE probe (0.2 μg) with renatured HrcA (0.1 μg); lane 2, no heat shock treatment after the binding reaction (control); lanes 3 to 7, heat shock treatment at 40, 50, 60, 65, and 70°C, respectively. Positions of the radiolabeled free CIRCE probe (F) and HrcA-probe complex (C) are marked.

FIG. 4

Light scattering patterns of B. thermoglucosidasius HrcA in the presence of DNA (A) and other proteins (B) and effect of temperature on light scattering patterns (C). Incubation was at 15°C throughout the experiments in panels A and B. (A) 25 μg of B. thermoglucosidasius HrcA (final concentration, 0.2 μM) in 0.1 ml of 20 mM Tris-HCl–5 mM EDTA–3 M urea (pH 7.5) was added to the solution (2.9 ml) as follows: no DNA (□), 50 μg of plasmid DNA pUC119 (◊), 100 μg of pUC119 (○), 20 μg of plasmid DNA pTY-1 containing the CIRCE element (▵), and 50 μg of pTY-1 (■). (B) The solution contained HrcA (final concentration, 0.2 μM) alone (◊) or with bovine serum albumin (0.2 μM; □); B. thermoglucosidasius DnaK (0.2 μM; ○) or B. thermoglucosidasius GroEL (0.2 μM; ▵). (C) The solution (3.0 ml) containing HrcA (25 μg) and plasmid DNA pTY-1 (20 μg) was preincubated with stirring at 15°C for 3 min; then the cuvette was transferred into the cell holder of a fluorescence spectrophotometer set at 15°C (⧫), 40°C (■), 50°C (▵), 60°C (○), 65°C (◊), or 70°C (□). In the experiment to investigate the effect of B. thermoglucosidasius GroEL (▾), the GroEL protein (final concentration, 0.2 μM) was added to the solution mixture in the cuvette, which was then transferred into the cell holder set at 70°C. Data collection always started when the cuvette was transferred into the cell holder.