SYNTHESIS OF A GENE FOR SENSORY RHODOPSIN-I AND ITS FUNCTIONAL EXPRESSION IN HALOBACTERIUM-HALOBIUM

We have designed, synthesized, and expressed in Halobacterium halobium a gene encoding sensory rhodopsin I (SR-I). The gene has been optimiz ed for cassette mutagenesis by incorporating 30 unique restriction sit es with uniform spacing throughout the 720-bp coding region. For expre ssion, the coding region was placed downstream of the promoter and tra nslation initiation region of the bacterioopsin gene on a selectable v ector. This construct encodes SR-I with an extended N terminus that in cludes the 13-amino acid leader sequence and the 8-amino acid N termin us of bacterioopsin. To obtain a SR-I- H. halobium strain for expressi ng the synthetic gene, we used homologous recombination to delete the chromosomal gene encoding SR-I, sopI. The deletion strain was transfor med with the synthetic sopI expression vector. Using antibody directed against the C-terminal region of SR-I, we detected in transformant me mbranes a protein with the electrophoretic mobility expected for SR-I with a processed N-terminal extension. The synthetic gene product was functionally identical to SR-I. Its flash-induced absorption differenc e spectrum and photochemical reaction cycle in membrane envelope vesic les were characteristic of SR-I. The protein fully restored phototaxis responses in the deletion strain.