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
A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from α-glucose-1-phosphate (α-Glc-1-P) and glucose, was cloned from Thermoanaerobacter tengcongensis...A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from α-glucose-1-phosphate (α-Glc-1-P) and glucose, was cloned from Thermoanaerobacter tengcongensis and successfully expressed in Escherichia coli. The overexpressed TreP, with a molecular mass of approximately 90 kDa, was determined by SDS-PAGE. It catalyzes trehalose synthesis and degradation optimally at 70℃ (for 30 min), with the optimum pHs at 6.0 and 7.0, respectively. It is highly thermostable, with a 77% residual ac- tivity after incubation at 50℃ for 7 h. Under the optimum reaction conditions, 50 μg crude en- zyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/L α-Glc-1-P and 125 mmol/L glucose within 30 min, while only 1.5 mmol/L out of 250 mmol/L tre- halose is degraded within the same time period. Dot blotting revealed that the treP gene in T. tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied. Both results indicate that the dominant function of the T. tengcongensis TreP is catalyzing trehalose synthesis but not degradation. Thus it might provide a novel route for indus- trial production of trehalose.展开更多
The transition metal cobalt,an essential cofactor for many enzymes in prokaryotes,is taken up by several specifi c transport systems.The CbiMNQO protein complex be-longs to type-1 energy-coupling factor(ECF)transporte...The transition metal cobalt,an essential cofactor for many enzymes in prokaryotes,is taken up by several specifi c transport systems.The CbiMNQO protein complex be-longs to type-1 energy-coupling factor(ECF)transporters and is a widespread group of microbial cobalt transport-ers.CbiO is the ATPase subunit(A-component)of the cobalt transporting system in the gram-negative thermo-philic bacterium Thermoanaerobacter tengcongensis.Here we report the crystal structure of a nucleotide-free CbiO at a resolution of 2.3Å.CbiO contains an N-terminal canonical nucleotide-binding domain(NBD)and C-termi-nal helical domain.Structural and biochemical data show that CbiO forms a homodimer mediated by the NBD and the C-terminal domain.Interactions mainly via conserved hydrophobic amino acids between the two C-terminal do-mains result in formation of a four-helix bundle.Structural comparison with other ECF transporters suggests that non-conserved residues outside the T-component bind-ing groove in the A component likely act as a specifi city determinant for T components.Together,our data provide information on understanding of the structural organiza-tion and interaction of the CbiMNQO system.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This work was supported by the Chinese Acad-emy of Sciences(Grant No.KSCX2-SW-112)the National Natural Science Foundation of China(Grant No.30170015).
文摘A gene encoding the trehalose phosphorylase (TreP), which reversibly catalyzes trehalose degradation and synthesis from α-glucose-1-phosphate (α-Glc-1-P) and glucose, was cloned from Thermoanaerobacter tengcongensis and successfully expressed in Escherichia coli. The overexpressed TreP, with a molecular mass of approximately 90 kDa, was determined by SDS-PAGE. It catalyzes trehalose synthesis and degradation optimally at 70℃ (for 30 min), with the optimum pHs at 6.0 and 7.0, respectively. It is highly thermostable, with a 77% residual ac- tivity after incubation at 50℃ for 7 h. Under the optimum reaction conditions, 50 μg crude en- zyme of the TreP is able to catalyze the synthesis of trehalose up to 11.6 mmol/L from 25 mmol/L α-Glc-1-P and 125 mmol/L glucose within 30 min, while only 1.5 mmol/L out of 250 mmol/L tre- halose is degraded within the same time period. Dot blotting revealed that the treP gene in T. tengcongensis was upregulated in response to salt stress but downregulated when trehalose was supplied. Both results indicate that the dominant function of the T. tengcongensis TreP is catalyzing trehalose synthesis but not degradation. Thus it might provide a novel route for indus- trial production of trehalose.
基金the National Basic Research Program(973 Program)(Nos.2011CB910502 and 2012CB911101)the National Natural Science Foundation of China(Grant Nos.31030020 and 31170679).
文摘The transition metal cobalt,an essential cofactor for many enzymes in prokaryotes,is taken up by several specifi c transport systems.The CbiMNQO protein complex be-longs to type-1 energy-coupling factor(ECF)transporters and is a widespread group of microbial cobalt transport-ers.CbiO is the ATPase subunit(A-component)of the cobalt transporting system in the gram-negative thermo-philic bacterium Thermoanaerobacter tengcongensis.Here we report the crystal structure of a nucleotide-free CbiO at a resolution of 2.3Å.CbiO contains an N-terminal canonical nucleotide-binding domain(NBD)and C-termi-nal helical domain.Structural and biochemical data show that CbiO forms a homodimer mediated by the NBD and the C-terminal domain.Interactions mainly via conserved hydrophobic amino acids between the two C-terminal do-mains result in formation of a four-helix bundle.Structural comparison with other ECF transporters suggests that non-conserved residues outside the T-component bind-ing groove in the A component likely act as a specifi city determinant for T components.Together,our data provide information on understanding of the structural organiza-tion and interaction of the CbiMNQO system.
