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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.
Highlights
In the heat shock response of gram-positive bacteria and ␣-proteobacteria, the CIRCE-HrcA systems play an important role in the mechanism for regulation of expression of heat shock genes (14, 15, 29)
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 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°C whereas the complex was rapidly denatured at 70°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
Since the dnaK, grpE, and dnaJ genes were cloned from B. thermoglucosidasius KP1006 together with the C terminus of the hrcA gene in the same operon (26), we tried to clone the N terminus of the hrcA gene and its 5Ј-flanking region, using colony hybridization on the genomic library of various restriction enzyme-digested fragments
Summary
In the heat shock response of gram-positive bacteria and ␣-proteobacteria, the CIRCE (controlling inverted repeat of chaperone expression)-HrcA systems play an important role in the mechanism for regulation of expression of heat shock genes (14, 15, 29). The many examples of cooperation between HrcA and these chaperones for proper function of HrcA in diverse bacteria (1) are consistent with the common and stable characteristics that HrcA is hardly soluble and forms aggregates when expressed and purified from Escherichia coli. This unfavorable characteristic was reported for B. subtilis HrcA (19), B. stearothermophilus HrcA (12), Staphylococcus aureus HrcA (16), and C. acetobutylicum HrcA (17). On the basis of results of our in vitro studies, we discuss the possibility that HrcA binding to the CIRCE element may act as a thermosensor for regulation of the two chaperone operons in B. thermoglucosidasius KP1006
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