Sensory rhodopsins I and II modulate a methylation/demethylation system in Halobacterium halobium phototaxis.

Abstract

This work demonstrates that phototaxis stimuli in the archaebacterium Halobacterium halobium control a methylation/demethylation system in vivo through photoactivation of sensory rhodopsin I (SR-I) in either its attractant or repellent signaling form as well as through the repellent receptor sensory rhodopsin II (SR-Il, also called phoborhodopsin). (14) and phototaxis behavior (15). A clue to the mechanism of information transfer from the SR-I chromophoric protein was the finding of a methylaccepting protein of =94 kDa covariant in a series of mutants with the SR-I chromophoric protein of 25 kDa (16). The 94-kDa protein resembles the chemotaxis signal generators ("transducers") of eubacteria (e.g., Escherichia colt), which transmit chemoreceptor signals from the membrane to a cytoplasmic sensory pathway. In eubacteria methylation of the transducers occurs by carboxylmethyl esterification of glutamate residues in the signal-transmitting domain and mediates adaptation to chemostimuli (17)(18)(19)(20).Reversible protein methylation was implicated in phototaxis and chemotaxis adaptation in H. halobium before the identification of specific taxis receptors (21-25). Methylaccepting membrane proteins in the 90-to 150-kDa range can be visualized by autofluorography of protein gels and are lost in taxis-mutants (16,26), are regained upon reversion to taxis' (16), and exhibit changes in extent of methylation induced by chemostimuli (26). The lability of the methyl linkages to mild base indicates these proteins are carboxylmethylated (16, 26). Hydrolysis of the carboxylmethyl ester bonds in E. coli results in methanol production (27, 28), which has been used to monitor methylesterase activity in vivo (29) and to detect changes in the rate of carboxylmethyl hydrolysis induced by chemostimuli (30). Applying this assay to H. halobium, Alam et al. (26) observed increases in the rate of evolution of volatile methyl groups after stimulation with chemotaxis effectors. Light-induced increases were also reported (26). These observations provided evidence for methylation involvement in phototaxis, but demonstration of control of methylation by photoactivation of the known photoreceptors was not available.Computerized cell-tracking methods have been implemented to study swimming behavior of H. halobium in response to selective photoactivation of 32), and spectroscopic procedures have been developed to monitor SR-I and SR-II photochemical reactions (5, [8][9][10]12). In the work presented here, we have applied these methods to characterize receptor mutants and to investigate Abbreviations: SR-I and -II, sensory rhodopsin I and II, respectively; BR, bacteriorhodopsin; HR, halorhodopsin.

Primary structure and functional analysis of the soluble transducer protein HtrXI in the archaeon Halobacterium salinarium

“…Halobacterial cells continually release methyl groups in volatile chemical form, and the rates of release are altered transiently by chemostimuli and photostimuli (1,19). We demonstrated that the rates of release of methanol by sensory stimuli also do not exhibit the same symmetry as those of E. coli: for halobacteria, both positive and negative stimuli result in an increased rate of methanol release (1).…”

Effect of loss of CheC and other adaptational proteins on chemotactic behaviour in Bacillus subtilis

“…Many archaea and some bacteria have CheC or the closely related CheX (Kirby et al, 2001). Some of these have polar flagella and, on negative stimuli, undergo repeated reversals of motion, such as the spirochaete Spirochaeta aurantia (Cercignani et al, 1998;Fosnaugh & Greenberg, 1988) and the archaeon Halobacterium salinarum (Spudich et al, 1989). It is clear that the main output of the receptor/CheA complex is to regulate CheY-P levels in these as in all chemotactic bacteria/ archaea but how CheY-P levels can regulate switching frequency (it primarily affects bias) has never been understood.…”

“…Backward swimming and reversals between directions, however, occur in wild-type. The increased reversals on negative stimulus might be due to changing CheY-P levels affecting affinity of CheC (or CheX) for the switch and possibly also to changing affinity for CheC (or CheX) at the receptors (due to methylation changes that occur there: Nordmann et al, 1994;Spudich et al, 1989). As a further parallel with B. subtilis, we note that a cheB mutant of H. salinarum shows increased frequency of reversals, with no effect on the ratio of CW and CCW rotation of the flagella, compared with wild-type (Rudolph & Oesterhelt, 1996).…”

Identification of distinct domains for signaling and receptor interaction of the sensory rhodopsin I transducer, HtrI

“…A second protein was implicated by mutant analysis to be required for SR-I control of cell motility responses (18). On the basis of its reversible carboxylmethylation and other biochemical similarities to eubacterial chemotaxis transducers, the protein was proposed to function as a halobacterial transducer for SR-I, relaying signals from the receptor to cytoplasmic components controlling the flagellar motor (21). The protein, now called HtrI, was isolated, and its gene was cloned and found to reside immediately upstream of sopI (27).…”