We will study the relationship of protein methylation to receptor excitation in the photosensory and chemosensory signal transduction pathways of Halobacterium halobium. A major finding from studies of bacterial chemotaxis is that reversible methylation of integral membrane """"""""signal transducer"""""""" proteins plays a critical role in adapatation to chemotaxis stimuli. Recently a phototaxis system using a rhodopsin-like photoreceptor (the slow-cycling bacterial rhodopsin, """"""""SR"""""""") has been shown to govern excitation and adaptation motility responses in H. halobium analogous to those of bacterial chemotaxis. The sR protein undergoes two different photochemical reaction cycles, depending on excitation wavelength. One cycle generates an attractant signal, and the other generates a repellent signal. The generation of these signals can be controlled by controlling the spectral composition of the excitation light. Initial work from several laboratories indicates protein methylation reactions participate in the sR-mediated phototaxis as well as in chemotaxis behavior in these cells. Our objectives are: (1) to characterize the components of the protein methylation system in H. halobium phototaxis; (2) to assess their relationship of photoexcitation of the two sR photochemical reaction cycles to activation of the methylation system. Protein methylation will be monitored by in vivo labeling with (3H)methyl, protein separation by polyacrylamide gel electrophoresis, and radiolabel detection by autofluorgraphy and scintillation counting. In the same in vivo preparations in which the photostimulus-induced methylation is monitored, the photochemical reactions of sR will be analyzed by time-resolved flash photolysis. Our rationale is tht this combination of detailed receptor information and protein methylation data will clarify the relationship of receptor excitation to the methylation/demethylation reactions. The ability to use light to control sR signaling will permit us to probe some properties of the phototaxis methylation system's response to stimuli with a time resolution and control difficult to obtain with stimulation of chemotaxis systems by chemical gradients.
