Genetic tools for the piezophilic hyperthermophilic archaeon Pyrococcus yayanosii.
The hyperthermophile Pyrococcus yayanosii CH1 is the only high-pressure-requiring microorganism obtained thus far within the archaea domain or among all non-psychrophiles in any domain. In this study, we developed a genetic manipulation system for P. yayanosii after first isolating a facultatively piezophilic derivative strain, designated P. yayanosii A1. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene was overexpressed in strain P. yayanosii A1 and was demonstrated to confer host cell resistance against simvastatin. Furthermore, using simvastatin as a selection marker, the endogenous pyrF of P. yayanosii A1 was disrupted through homologous recombination, thus generating the additional host strain P. yayanosii A2 (ΔpyrF). A markerless gene disruption vector was constructed by incorporating a pyrF-sim (R) cassette that enables the combined use of simvastatin resistance for positive selection and 5-FOA for counter selection. The utility of this versatile disruption system was demonstrated by deleting the carbon-nitrogen hydrolase of P. yayanosii strain A1. These results demonstrate that a variety of genetic tools are now in place to study unknown gene function and the molecular mechanisms of piezophilic adaptation in P. yayanosii.
Institute of Oceanology, Shanghai Jiao Tong University, Minhang Dongchuan Road 800, Shanghai, 200240, China.
Xuegong
X
Li
Ling
L
Fu
Zhen
Z
Li
Xiaopan
X
Ma
Xiang
X
Xiao
Jun
J
Xu
D017931:DNA Primers / Q000235:genetics; D004352:Drug Resistance, Microbial; D005821:Genetic Techniques; D005822:Genetic Vectors; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics; D009154:Mutation; D011312:Pressure; D019711:Pyrococcus / Q000235:genetics*; D019821:Simvastatin / Q000737:chemistry
D017931:DNA Primers; D019821:Simvastatin; D006903:Hydroxymethylglutaryl CoA Reductases