Identification of Methylation Sites and Effects of Phototaxis Stimuli on Transducer Methylation in Halobacterium salinarum

Abstract

The two transducers in the phototaxis system of the archaeonHalobacterium salinarum, HtrI and HtrII, are methyl-accepting proteins homologous to the chemotaxis transducers in eubacteria. Consensus sequences predict three glutamate pairs containing potential methylation sites in HtrI and one in HtrII. Mutagenic substitution of an alanine pair for one of these, Glu265-Glu266, in HtrI and for the homologous Glu513-Glu514 in HtrII eliminated methylation of these two transducers, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autofluorography. Photostimulation of the repellent receptor sensory rhodopsin II (SRII) induced reversible demethylation of HtrII, while no detectable change in the extent of methylation of HtrI was observed in response to stimulation of its cognate sensory rhodopsin, the attractant receptor SRI. Cells containing HtrI or HtrII with all consensus sites replaced by alanine still exhibited phototaxis responses and behavioral adaptation, and methanol release assays showed that methyl group turnover was still induced in response to photostimulation of SRI or SRII. By pulse-chase experiments with in vivol-[methyl-3H]methionine-labeled cells, we found that repetitive photostimulation of SRI complexed with wild-type (or nonmethylatable) HtrI induced methyl group turnover in transducers other than HtrI to the same extent as in wild-type HtrI. Both attractant and repellent stimuli cause a transient increase in the turnover rate of methyl groups in wild-type H. salinarum cells. This result is unlike that obtained withEscherichia coli, in which attractant stimuli decrease and repellent stimuli increase turnover rate, and is similar to that obtained with Bacillus subtilis, which also shows turnover rate increases regardless of the nature of the stimulus. We found that a CheY deletion mutant of H. salinarum exhibited theE. coli-like asymmetric pattern, as has recently also been observed in B. subtilis. Further, we demonstrate that the CheY-dependent feedback effect does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell.

Evidence That Both Ligand Binding and Covalent Adaptation Drive a Two-State Equilibrium in the Aspartate Receptor Signaling Complex

“…The aspartate receptor of Escherichia coli and Salmonella typhimurium is an extensively studied example of the transmembrane receptors that typically regulate such pathways. The aspartate receptor and its closest prokaryotic relatives initiate taxis to a range of different stimuli including chemicals, heat, osmotic pressure, and light (Bibikov et al 1997; Falke et al 1997; Nishiyama et al 1999; Perazzona and Spudich 1999). The cytoplasmic domains of these taxis receptors are highly conserved, suggesting that they share similar molecular signaling mechanisms (Le Moual and Kirkland 1996), as confirmed by the construction of functional chimeric proteins formed by fusing different domains from closely related chemoreceptors (Krikos et al 1985; Slocum et al 1987; Tatsuno et al 1994; Weerasuriya et al 1998).…”

Equal-quantum Action Spectra Indicate Fluence-rate–selective Action of Multiple Photoreceptors for Photomovement of the Thermophilic Cyanobacterium Synechococcus elongatus¶

“…In this connection, sensory rhodopsin I (max, 587 nm), the photoreceptor for photoattractant response in Halobacterium salinarum (17), might be one of the possible candidates, considering that ''rhodopsins'' are suggested as the photoreceptor for phototaxis in the green flagellate algae Chlamydomonas reinhardtii (18)(19)(20)(21) and Volvox carteri (22). In the photoattractant system of H. salinarum, the existence of an adaptation system correlated with the methylation of a membrane protein associated with sensory rhodopsin is demonstrated and is important for the response to continuous light stimulus (23)(24)(25)(26). In the phototactic system of S. elongatus, green and yellow light were more effective than red light at high fluence rate, thus suggesting the possible existence of an adaptation system, which could possibly be correlated with methylation of photosystems for green and yellow lights.…”

Light‐induced switching of HAMP domain conformation and dynamics revealed by time‐resolved EPR spectroscopy

“…Similarly, for the other spin label positions a slight mutant-dependent variation is observed in the photocycle kinetics ( Table 1). The influence of spin labeling of the HAMP domain on the receptor, which is situated upstream, suggests that changes of the transducer state such as methylation [37] may also affect the receptor, possibly extending the regulatory role of the methylation sites to the receptor. Consistently, this variation in the kinetics is observed here for both optical as well as for EPR transients (cf.…”