Over-expression of a new photo-active halorhodopsin in Halobacterium salinarium

Abstract

The gene of haloopsin ( hop) from halobacterial strain shark was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of shark halorhodopsin (HR) showed that its homology with halobium HR was 62%. The gene product seems to be HR having several positively charged residues that are conserved in all known HRs. The gene encoding shark hop as well as that encoding halobium hop were successfully expressed in Halobacterium salinarium ( halobium) by using a plasmid shuttle vector containing the bacterioopsin ( bop) promoter. The expression level of shark HR is almost the same as that for halobium HR with the same shuttle vector containing the bop promoter. Under the physiological conditions, the anion pumping activity of the shark HR expressed in H. salinarium was almost the same as that for halobium HR; however, the anion selectivity and half-maximal anion transport were different. Furthermore, its absorption maximum in the absence of chloride shifted to approx. 596 nm in contrast to that for halobium HR. The half-lifetimes of HR 520 formation for shark HR and halobium HR were almost the same; however, the half-lifetime of its decay was approx. 6-times faster for shark HR than it was for halobium HR at a high chloride concentration (1000 mM). Even at a low chloride concentration (50 mM), HR 520 and HR 640 intermediates could be detected for shark HR, and the half-lifetime of HR 640 decay was found to be approx. 25 ms. In the presence of nitrate, the half-lifetime of HR 565 recovery for shark HR was approx. 10-times slower than that for halobium HR. Some of amino acid substitutions between shark HR and halobium HR may affect the anion selectivity and the photoreaction of HR.