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
In archaea, the HEL308 homolog Hel308a（or Hjm） is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is ...In archaea, the HEL308 homolog Hel308a（or Hjm） is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is limited. Herein, a structure-based functional analysis of Hjm was performed in the genetically tractable hyperthermophilic archaeon, Sulfolobus islandicus. Results showed that domain V and residues within it, which affect Hjm activity and regulation, are essential and that the domain V-truncated mutants and sitedirected mutants within domain V cannot complement hjm chromosomal deletion. Chromosomal hjm deletion can be complemented by ectopic expression of hjm under the control of its native promoter but not an artificial arabinose promoter. Cellular Hjm levels are kept constant under ultraviolet（UV） and methyl methanesulfonate（MMS） treatment conditions in a strain carrying a plasmid to induce Hjm overexpression. These results suggest that Hjm expression and activity are tightly controlled, probably at the translational level.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 grants from the National Natural Science Foundation of China (Nos. 3093002, 31170072 and 31470184 to Y.S.)
文摘In archaea, the HEL308 homolog Hel308a（or Hjm） is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is limited. Herein, a structure-based functional analysis of Hjm was performed in the genetically tractable hyperthermophilic archaeon, Sulfolobus islandicus. Results showed that domain V and residues within it, which affect Hjm activity and regulation, are essential and that the domain V-truncated mutants and sitedirected mutants within domain V cannot complement hjm chromosomal deletion. Chromosomal hjm deletion can be complemented by ectopic expression of hjm under the control of its native promoter but not an artificial arabinose promoter. Cellular Hjm levels are kept constant under ultraviolet（UV） and methyl methanesulfonate（MMS） treatment conditions in a strain carrying a plasmid to induce Hjm overexpression. These results suggest that Hjm expression and activity are tightly controlled, probably at the translational level.
