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 sensory rhodopsin I (SRI), sopI, was studied by means of homologous gene targeting. A sopI- Halobacterium salinarium mutant strain was constructed by homologous replacement of sopI with a novobiocin-resistant gyrB from Haloferax Aa 2.2. Cells bearing gyrB were resistant to novobiocin, indicating that the Haloferax gene is functional in H. salinarium. Complementation of this deletion strain with sopI fused to the bacterio-opsin 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 sopI had integrated by homologous recombination. This shows that cotranscription of sopI and the gene encoding the SRI signal transducer, htrI, which is found in the wild type, is not a prerequisite for photosensory 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 activity of the pigment. The mRNA level of the truncated gene was not affected 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 acids of SRI results in a decrease of the bacteriorhodopsin, supporting our observations. The C-terminus of SRI is the first domain with a downregulating influence on protein levels thus far identified in H. salinarium. The system for SRI overexpression we present here greatly facilitates biochemical and biophysical studies on the photoreceptor and allows investigation of the molecular interactions underlying the signal transduction chain of halobacterial phototaxis.
Ferrando-May
E
E
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Brustmann
B
B
Oesterhelt
D
D
D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D006216:Halobacteriaceae / Q000235:genetics; D006217:Halobacterium / Q000235:genetics / Q000378:metabolism*; D025602:Halorhodopsins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009675:Novobiocin / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D025603:Sensory Rhodopsins; D017384:Sequence Deletion
D004269:DNA, Bacterial; D025602:Halorhodopsins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C061497:SRI protein, Halobacterium; D025603:Sensory Rhodopsins; D009675:Novobiocin; D001436:Bacteriorhodopsins