A C-TERMINAL TRUNCATION RESULTS IN HIGH-LEVEL EXPRESSION OF THE FUNCTIONAL PHOTORECEPTOR SENSORY RHODOPSIN-I IN THE ARCHAEON HALOBACTERIUM-SALINARIUM

Expression of the gene encoding the halobacterial photoreceptor sensor y rhodopsin I (SRI), sopI, was studied by means of homologous gene tar geting. A sopI- Halobacterium salinarium mutant strain was constructed by homologous replacement of sopI with a novobiocin-resistant gyrB fr om Haloferax Aa 2.2. Cells bearing gyrB were resistant to novobiocin, indicating that the Haloferax gene is functional in H. salinarium. Com plementation of this deletion strain with sopI fused to the bacterio-o psin promoter resulted in the recovery of all phenotypical attributes of SRI. This establishes the first direct correlation between sopI and the function of its gene product. In the complemented deletion strain , functional expression of sopI occurred from the bop locus, where sop I had integrated by homologous recombination. This shows that cotransc ription of sopI and the gene encoding the SRI signal transducer, htrI, which is found in the wild type, is not a prerequisite for photosenso ry activity. Deletion of the last 43 bp at the 3' end of sopI resulted in a 10-fold increase in the amount of SRI, without affecting the act ivity of the pigment. The mRNA level of the truncated gene was not aff ected as compared to that of the wild type. We propose that regulation occurs at the protein level, probably through a negative determinant of protein stability located in the C-terminus of SRI. Replacement of the last 28 amino acids of bacteriorhodopsin by the last 29 amino acid s of SRI results in a decrease of the bacteriorhodopsin, supporting ou r observations. The C-terminus of SRI is the first domain with a downr egulating influence on protein levels thus far identified in H. salina rium. The system for SRI overexpression we present here greatly facili tates biochemical and biophysical studies on the photoreceptor and all ows investigation of the molecular interactions underlying the signal transduction chain of halobacterial photo-taxis.