The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon （apyrF） as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding apyrF host. After positive selection of the single-crossover integration recombinants （pop-in） on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants （pop-out） with 5-fluoroorotic acid （5-FOA）, the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out the crtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea.
Hailong Liu Jing Han Xiaoqing Liu Jian Zhou Hua Xiang
Halophilic archaea (haloarchaea) inhabit hypersaline environments,tolerating extreme salinity,low oxygen and nutrient availability,and in some cases,high pH (soda lakes) and irradiation (saltern ponds).Membrane-associated proteins of haloarchaea,such as surface layer (S-layer) proteins,transporters,retinal proteins,and internal organellar membrane proteins including intracellular gas vesicle proteins and those associated with polyhydroxyalkanoate (PHA) granules,contribute greatly to their environmental adaptations.This review focuses on these haloarchaeal cellular and organellar membrane-associated proteins,and provides insight into their physiological significance and biotechnological potential.
CAI LeiZHAO DaHeHOU JingWU JinHuaCAI ShuangFengDASSARMA PriyaXIANG Hua
