Isolation Of Methanothermobacter Thermoautotrophicus And Molecular And Function Characterization Of Preotein Disulfide Isomerase (MTH1745)

Comparing with many other microorganisms,growth of Methanothermobacter thermoautotrophicus is characterized by some particularities.To study the stress related enzyme can help us to understand better the anti-stress molecular machaism of archaea.A new strain of Methanothermobacter thermoautotrophicus,named DX01,was isolated from a hot springs of China.The effect of growth temperature on the morphology,cell color,carbon source utilization and growth rate of M. thermoautotrophicus DX01 was investigated.The phenotypic characteristics of strain DX01 pellet presented quite distinctive,including brick red colouration and growth as short rods during culturing at 60℃.In order to isolatea up-regulated expression proteins at high temperature,comparative analysis was performed using protein fingerprint profiles at different cultured temperature.Methyl-coenzyme M reductase I, a key enzyme at methanogenesis,was up-regulated expression at 70℃.Isolation of this strain expands the known range of the M.thermoautotrophicus species.Although MTH1745 is a putative protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active site from the anaerobic archaea Methanothermobacter thermoautotrophicus△H~T,the potential functions of MTH 1745 are not clear.In the present study,we show a crucial role of MTH 1745 in protecting cells against stress which may be related to its functions as disulfide isomerase and chaperone properties.Using real-time PCR analyses,the level of MTH1745 mRNA in the thermophilic archaea M.thermoautotrophicus△H~T was found to be temperature-dependent,which was extremely higher at low(50℃)and high(70℃) growth temperature than at its optimal growth temperature(65℃).Additionally,the expression of MTH 1745 mRNA was up-regulated by cold shock(4℃).Furthermore, the survival rate of MTH1745 expressing E.coli cells was markedly higher than control cells in response to heat shock(51.0℃).These results indicated that MTH1745 play important roles in resistance of temperature stress.By assay of enzyme activities in vitro,MTH1745 exhibited chaperone activity by promoting the functional folding of citrate synthase after thermodenaturation.On the other hand, MTH1745 was also shown to function as disulfide isomerase with assays of the refolding of denatured and reduced ribonuclease A.These studies may provide clues to the better understanding of the function of protein disulfide isomerase in archaea.