Properties of a second sensory receptor protein in Halobacterium halobium phototaxis.

A second slow-cycling retinylidene protein, in addition to slow-cycling (sensory) rhodopsin (SR), can be bleached with hydroxylamine and regenerated with all-trans retinal in photosensory signaling Halobacterium halobium membranes. Flash photolysis shows this protein undergoes a photochemical reaction cycle characterized by photoconversion of its ground state (lambda max 480 nm) to a species with lambda max less than or equal to 360 nm, which thermally regenerates the 480-nm species with a t1/2 of 260 msec at 25 degrees C, under conditions in which SR photocycles at 650 msec in the same membranes. Mutants characterized with respect to their phototaxis behavior are identified which contain SR and the 480-nm pigment, the latter ranging from undetectable to a concentration equal to that of SR. Receptor mutants lacking all phototaxis sensitivity lack both of the photochemically reactive proteins. The mutant properties contribute to an accumulation of behavioral and spectroscopic evidence that the 480-nm pigment is a second sensory photoreceptor in H. halobium. NaDodSO4-polyacrylamide gel electrophoresis of [3H]retinal-labeled membrane proteins from the mutants indicates SR and the 480-nm pigment contain distinct chromophoric polypeptides differing in their migration rates. The data implicate polypeptides of 25,000 Mr and 23,000 Mr as retinal-binding polypeptides of SR and the 480-nm protein, respectively.

Selection and properties of phototaxis-deficient mutants of Halobacterium halobium.

A method for isolating phototaxis-deficient (Pho-) mutants of Halobacterium halobium was developed. The procedure makes use of a flashing repellent light to induce frequent reversals of swimming direction by responsive cells, thereby impeding their migration along a small capillary and resulting in a spatial separation of the parent population and a population enriched for Pho- cells. Two classes of Pho- mutants were obtained by this selection scheme: those which have lost the chemotactic response (Che-) as well as phototaxis sensitivity (general taxis mutants), and those which are defective in steps specific to phototaxis (photosignaling mutants). In the latter class, several retinal synthesis mutants were isolated, as well as a strain which fit the expected properties of a mutant lacking a functional photoreceptor protein. On the basis of spectroscopic and swimming behavior studies, the retinal-containing protein, slow-cycling or sensory rhodopsin (SR), was previously proposed to be a dual-function sensory receptor mediating both attractant and repellent photosensing. The receptor mutant Pho81 fulfills two predictions which provide direct genetic evidence for this proposal. The mutant has lost SR photoactivity as determined by spectroscopic measurements, and it has simultaneously lost both attractant and repellent phototaxis sensitivity. Comparison of [3H]retinal-labeled membrane proteins from the mutant and its SR-containing parent implicated a 25,000 Mr polypeptide as the chromophoric polypeptide of SR.