MTH1745, a protein disulfide isomerase-like protein from thermophilic archaea, Methanothermobacter thermoautotrophicum involving in stress response

Abstract

MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermoautotrophicum. The potential functions of MTH1745 are not clear. In the present study, we show a crucial role of MTH1745 in protecting cells against stress which may be related to its functions as a disulfide isomerase and its chaperone properties. Using real-time polymerase chain reaction analyses, the level of MTH1745 messenger RNA (mRNA) in the thermophilic archaea M. thermoautotrophicum was found to be stress-induced in that it was significantly higher under low (50 degrees C) and high (70 degrees C) growth temperatures than under the optimal growth temperature for the organism (65 degrees C). Additionally, the expression of MTH1745 mRNA was up-regulated by cold shock (4 degrees C). Furthermore, the survival of MTH1745 expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock (51.0 degrees C). These results indicated that MTH1745 plays an important role in the resistance of stress. By assay of enzyme activities in vitro, MTH1745 also exhibited a chaperone function by promoting the functional folding of citrate synthase after thermodenaturation. On the other hand, MTH1745 was also shown to function as a disulfide isomerase on the refolding of denatured and reduced ribonuclease A. On the basis of its single thioredoxin domain, function as a disulfide isomerase, and its chaperone activity, we suggest that MTH1745 may be an ancient protein disulfide isomerase. These studies may provide clues to the understanding of the function of protein disulfide isomerase in archaea.

Fig. 1

MTH1745 mRNA expression in M. thermoautotrophicum analyzed by real-time PCR. The level of MTH1745 mRNA was normalized by 16S rRNA levels and indicated in the respective growth temperature (a) or cold shock at 4°C for 2 h (b). The value of 65°C was set to 1 and the rest of the value normalized. The results were expressed as means ± SEM

Fig. 2

Survival versus time at 51.0°C. Two transformed E. coli, MTCON (with pET-28a) and MTDX01 (with pET-28a-MTH1745), were cultured as described and subjected to 51.0°C treatment for up to 60 min. After heat treatment, samples were taken at the time indicated, then diluted, and immediately plated on LB plus kanamycin plates. Survival (%) of MTDX01 cells (open triangle) and MTCON cells (open square) are shown. Mean survival is expressed as a percentage of the survival obtained in cultures

Fig. 3

MTH1745 prevents heat-induced denaturation of citrate synthase. Purified MTH1745 was incubated at 43°C with 150 nM citrate synthase, and the solution turbidity was measured at 360 nm. MTH1745 concentrations were: 0.0 nM (open square); 150 nM (ex mark). BSA concentrations were 150 nM (open triangle)

Fig. 4

In vivo restoration of DsbA-dependent alkaline phosphatase activity with periplasmic expressed MTH1745 in E. coli. Alkaline phosphatase activity is expressed as ΔA420_nm min⁻¹ΔA. E. coli strains, JCB570, JCB571, JCB573 (with MTH1745), JCB579 (with MTH1745, pSJS1240), JCB580 (with APss-MTH1745), JCB581 (with APss-MTH1745, pSJS1240) were used