Microbial rhodopsins of Halorubrum species isolated from Ejinoor salt lake in Inner Mongolia of China

Abstract

Microbial rhodopsins are photoactive proteins that use a retinal molecule as the photoactive center. Because of structural simplicity and functional diversity, microbial rhodopsins have been an excellent model system for structural biology. In this study, a halophilic archaea that has three microbial rhodopsin-type genes in its genome was isolated from Ejinoor salt lake in Inner Mongolia of China. A sequence of 16S rRNA showed that the strain belongs to Halorubrum genus and named Halorubrum sp. ejinoor (He). The translated amino acid sequences of its microbial rhodopsin-type genes suggest that they are homologs of archaerhodopsin (HeAR), halorhodopsin (HeHR) and sensory rhodopsin II (HeSRII). The mRNAs of three types of genes were detected by RT-PCR and their amounts were investigated by Real-Time RT-PCR. The amount of mRNA of HeSRII was the smallest and the amounts of of HeAR and HeHR were 30 times and 10 times greater than that of HeSRII. The results of light-induced pH changes suggested the presence of a light-driven proton pump and a light-driven chloride ion pump in the membrane vesicles of He. Flash induced absorbance changes of the He membrane fraction indicated that HeAR and HeHR are photoactive and undergo their own photocycles. This study revealed that three microbial rhodopsin-type genes are all expressed in the strain and at least two of them, HeAR and HeHR, are photochemically and physiologically active like BR and HR of Halobacterium salinarum, respectively. To our knowledge, this is the first report of physiological activity of HR-homolog of Halorubrum species.

Two Consecutive Polar Amino Acids at the End of Helix E are Important for Fast Turnover of the Archaerhodopsin Photocycle

“…The PCR product was separated by agarose gel electrophoresis and cloned into the pT7blue vector, which is designed for simplified cloning of PCR products. The obtained DNA sequences were determined by ABI 3130/3130xl Genetic Analyzer (Applied Biosystems) . To construct a high‐expression vector of He AR, a NdeI/XhoI fragment (containing the He AR‐coding region) from pT7blue‐ He AR was inserted into suitable sites on pET21c(+) (Novagen, Madison, WI) .…”

“…The amino acid sequence identities of He AR with AR1 and BR are 94% and 57%, respectively. All key functional amino acid residues of BR are conserved in He AR, including the transmembrane Asp, Glu and Arg residues associated with proton transport .…”

Functional Mechanism of Proton Pump-Type Rhodopsins Found in Various Microorganisms as a Potential Effective Tool in Optogenetics

“…aus-1 and aus-2 isolated from a lake in Western Australia by Mukohata et al [49]. Several aR homologous proteins, including aR-3 described above, have been discovered in different haloarchaeal species [50][51][52][53]. In addition, Mukohata et al successively identified two other H + -pump-like proteins belonging to a different clade from BR and aR: cruxrhodopsin-1 (cR-1) from Haloarcula argentinensis [54] and deltarhodopsin-1 (dR-1) from Haloterrigena sp.…”