The whole-genome sequence of Thermoanaerobacter tengcongensis, an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China, was completed in 2002. However, in vivo studies on the genes of this ...The whole-genome sequence of Thermoanaerobacter tengcongensis, an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China, was completed in 2002. However, in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system. In order to establish such a system, the plasmid pBOL01 containing the replication origin of the T. tengcongensis chromosome and a kanamycin resistance cassette, in which kanamycin resistance gene expression was controlled by the tte1482 promoter from T. tengcongensis, was constructed and introduced into T. tengcongensis via electroporation. Subsequently, the high transformation efficiency occurred when using freshly cultured T. tengcongensis cells without electroporation treatment, suggesting that T. tengcongensis is naturally competent under appropriate growth stage. A genetic transformation system for this strain was then established based on these important components, and this system was proved to be available for studying physiological characters of T. tengcongensis in vivo by means of hisG gene disruption and complementation.展开更多
Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms,and therefore enables these organisms to survive at extreme temperatures.Although it is well-known that thermostable p...Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms,and therefore enables these organisms to survive at extreme temperatures.Although it is well-known that thermostable proteins are critical for the growth of thermophilic organisms,the structural basis of protein thermostability is not yet fully understood.The histidine-containing phosphocarrier (HPr) protein,a phosphate shuttle protein in the phosphoenolpyruvate-dependent sugar transport system (PTS) of bacterial species,is an ideal model for investigating protein thermostability with respect to its small size and deficiency in disulphide bonds or cofactors.In this study,the HPr protein from Thermoanaerobacter tengcongensis (TtHPr) is cloned and purified.Crystal structure with good quality has been determined at 2.3 resolution,which provides a firm foundation for exploring the thermostable mechanism.However,it shows that the crystal structure is conserved and no clue can be obtained from this single structure.Furthermore,detailed comparison of sequence and structure with the homologs from mesoor thermophilic bacteria shows no obvious rule for thermostability,but the extra salt-bridge existing only in thermophilic bacteria might be a better explanation for thermostability of HPr.Thus,mutations are performed to interrupt the salt-bridge in HPrs in thermophilic bacteria.Using site-directed mutations and the circular dichroism method,thermostability is evaluated,and the mutational variations are shown to have a faster denaturing rate than for wild-type viruses,indicating that mutations cause instability in the HPrs.Understanding the higher-temperature resistance of thermophilic and hyperthermophilic proteins is essential to studies on protein folding and stability,and is critical in engineering efficient enzymes that can work at a high temperature.展开更多
基金supported by the grants from the National Natural Science Foundation of China(Grant Nos.30621005 and 31030003)the Ministry of Science and Technology of China(Grant No.2009CB118905)
文摘The whole-genome sequence of Thermoanaerobacter tengcongensis, an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China, was completed in 2002. However, in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system. In order to establish such a system, the plasmid pBOL01 containing the replication origin of the T. tengcongensis chromosome and a kanamycin resistance cassette, in which kanamycin resistance gene expression was controlled by the tte1482 promoter from T. tengcongensis, was constructed and introduced into T. tengcongensis via electroporation. Subsequently, the high transformation efficiency occurred when using freshly cultured T. tengcongensis cells without electroporation treatment, suggesting that T. tengcongensis is naturally competent under appropriate growth stage. A genetic transformation system for this strain was then established based on these important components, and this system was proved to be available for studying physiological characters of T. tengcongensis in vivo by means of hisG gene disruption and complementation.
基金supported by the National High Technology Research and Development Program of China (Grant No. 2006BAD06A04) from Ministry of Science and Technology of ChinaGeorge F. Gao is a leading principal investigator of the Innovative Research Group of the National Natural Science Foundation of China (Grant No. 80121003)
文摘Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms,and therefore enables these organisms to survive at extreme temperatures.Although it is well-known that thermostable proteins are critical for the growth of thermophilic organisms,the structural basis of protein thermostability is not yet fully understood.The histidine-containing phosphocarrier (HPr) protein,a phosphate shuttle protein in the phosphoenolpyruvate-dependent sugar transport system (PTS) of bacterial species,is an ideal model for investigating protein thermostability with respect to its small size and deficiency in disulphide bonds or cofactors.In this study,the HPr protein from Thermoanaerobacter tengcongensis (TtHPr) is cloned and purified.Crystal structure with good quality has been determined at 2.3 resolution,which provides a firm foundation for exploring the thermostable mechanism.However,it shows that the crystal structure is conserved and no clue can be obtained from this single structure.Furthermore,detailed comparison of sequence and structure with the homologs from mesoor thermophilic bacteria shows no obvious rule for thermostability,but the extra salt-bridge existing only in thermophilic bacteria might be a better explanation for thermostability of HPr.Thus,mutations are performed to interrupt the salt-bridge in HPrs in thermophilic bacteria.Using site-directed mutations and the circular dichroism method,thermostability is evaluated,and the mutational variations are shown to have a faster denaturing rate than for wild-type viruses,indicating that mutations cause instability in the HPrs.Understanding the higher-temperature resistance of thermophilic and hyperthermophilic proteins is essential to studies on protein folding and stability,and is critical in engineering efficient enzymes that can work at a high temperature.
