Identiﬁcation of Methylation Sites and Effects of ...the htrI promoter as described previously (26). PCR mutagenesis. Mutations introduced into the sequences of HtrI and HtrII were

JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Sept. 1999, p. 5676–5683 Vol. 181, No. 18

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Identification of Methylation Sites and Effects of PhototaxisStimuli on Transducer Methylation in

Halobacterium salinarumBASTIANELLA PERAZZONA AND JOHN L. SPUDICH*

Department of Microbiology and Molecular Genetics, The University ofTexas Medical School, Houston, Texas 77030

Received 6 May 1999/Accepted 14 July 1999

The two transducers in the phototaxis system of the archaeon Halobacterium salinarum, HtrI and HtrII, aremethyl-accepting proteins homologous to the chemotaxis transducers in eubacteria. Consensus sequencespredict three glutamate pairs containing potential methylation sites in HtrI and one in HtrII. Mutagenicsubstitution of an alanine pair for one of these, Glu265-Glu266, in HtrI and for the homologous Glu513-Glu514in HtrII eliminated methylation of these two transducers, as demonstrated by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis autofluorography. Photostimulation of the repellent receptor sensory rhodopsinII (SRII) induced reversible demethylation of HtrII, while no detectable change in the extent of methylation ofHtrI 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 responsesand behavioral adaptation, and methanol release assays showed that methyl group turnover was still inducedin response to photostimulation of SRI or SRII. By pulse-chase experiments with in vivo L-[methyl-3H]methi-onine-labeled cells, we found that repetitive photostimulation of SRI complexed with wild-type (or nonmeth-ylatable) HtrI induced methyl group turnover in transducers other than HtrI to the same extent as in wild-typeHtrI. Both attractant and repellent stimuli cause a transient increase in the turnover rate of methyl groups inwild-type H. salinarum cells. This result is unlike that obtained with Escherichia coli, in which attractant stimulidecrease 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 CheYdeletion mutant of H. salinarum exhibited the E. coli-like asymmetric pattern, as has recently also beenobserved in B. subtilis. Further, we demonstrate that the CheY-dependent feedback effect does not require thestimulated transducer to be methylatable and operates globally on other transducers present in the cell.

In the archaeon Halobacterium salinarum, phototaxis is me-diated by two photoreceptive protein complexes, sensory rho-dopsin I (SRI)-HtrI and sensory rhodopsin II (SRII)-HtrII.SRI is a photoreceptor for attractant (orange) and repellent(near-UV) stimuli, and SRII is a photoreceptor for repellent(blue-green) stimuli (8). HtrI (35) and HtrII (37) are integralmembrane proteins bound to SRI and SRII, respectively. TheHtr proteins belong to a family of transducer proteins thatare highly homologous in their cytoplasmic domains. The beststudied are the methyl-accepting chemotaxis receptor-trans-ducers (MCPs) of Escherichia coli and Salmonella typhimurium(5, 30). As in the enteric eubacteria, the conserved cytoplasmicdomains control a two-component regulatory system with thehistidine kinase CheA and its protein substrate, the responseregulator CheY (20).

In the chemotaxis system of E. coli, behavioral adaptation iscorrelated with the reversible methylation of the carboxyl sidechains of glutamyl residues (carboxylmethylesterification) (10,11, 19, 29, 32). A positive stimulus (i.e., addition of an attract-ant or removal of a repellent) causes an increase in the levelof methylation, and a negative stimulus causes a reductionin the number of methylated residues on the MCP proteinthrough which the stimulus signal is transmitted. Changes inthe steady-state methylation of MCPs upon stimulation have

been detected as changes in their migration rate determinedby sodium dodecyl sulfate-polyacrylamide gel electrophore-sis (SDS-PAGE) (2, 6). The rate of methyl group turnover invivo has been measured as the release of volatile radioactivemethyl groups by cells since a product of carboxylmethyl glu-tamate hydrolysis is methanol (10). Genes encoding a methylesterase, CheB (20), and a methyltransferase, CheR (9a), havebeen identified in H. salinarum.

The methylation system in H. salinarum is similar in somerespects to that of enteric bacteria and in others to the gram-positive B. subtilis. As in E. coli, methionine starvation elimi-nates swimming direction reorientation (tumbling in E. coliand swimming reversals in H. salinarum) and tactic responses(27). A cheB gene deletion mutant in H. salinarum has beenshown to exhibit a phenotype (frequent swimming reversals)similar to that of the corresponding mutant in E. coli (21), andloss of methylation attributed to a methyltransferase (CheR)mutation results in a smooth swimming phenotype as in E. coli(24, 31). Unlike E. coli, but as for B. subtilis (12), H. salinarumcells undergo increased turnover of methyl groups after both apositive and a negative stimulus when exposed to both chem-ical and light stimuli (1, 25). Increases in the extent of meth-ylation on gels following chemostimulation with histidine andleucine (attractants) and decreases after chemostimulationwith phenol (repellent) in cells labeled to steady state havebeen observed in H. salinarum (1). Changes in methylationupon photostimulation were detected in HtrII in the workreported here.

In this study, we have investigated the nature of methylation

* Corresponding author. Mailing address: Dept. of Microbiologyand Molecular Genetics, University of Texas Medical School, 6431Fannin St., Houston, TX 77030. Phone: (713) 500-5458. Fax: (713)500-5499. E-mail: [email protected].

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changes in the response to light stimuli in H. salinarum. Weidentified the sites of methylation in HtrI and HtrII and foundsimilarities to and differences from the methylation system inE. coli chemotaxis.

MATERIALS AND METHODS

Recipient strain and plasmids. The recipient strain for plasmid transformationwas Pho81Wr2 (bacteriorhodopsin2 halorhodopsin2 SRI2 SRII2 HtrI2

HtrII2; carotenoid deficient and lacking a restriction activity) (36). The plasmidpKJ306 is a shuttle vector able to replicate in both H. salinarum and E. coli (9,15), and genes encoding SRI apoprotein and HtrI were expressed from theirnative promoter. A modified form of the same plasmid, the expression vectorpPR5, was used to express genes encoding SRII and HtrII under the control ofthe htrI promoter as described previously (26).

PCR mutagenesis. Mutations introduced into the sequences of HtrI and HtrIIwere created by PCR site-directed mutagenesis (3). A 759-bp SacI/XhoI frag-ment from the native htrI gene containing the sites to be mutated was cloned inpBluescript KS2 (Stratagene, La Jolla, Calif.), and the resulting plasmid was

used as a template for PCR. Similarly, a 659-bp NotI/SmaI fragment from thenative htrII gene cloned in pBluescript KS2 was used to make the mutations inthis gene. Mutagenized fragments were reintroduced in the respective originalexpression plasmids. The T3 and T7 promoters and synthetic oligonucleotides(Bioserve, Laurel, Md.) were used to introduce the following pairs of mutations:Q258A-Q259A, E265A-E266A, E315A-Q316A, E336A-Q337A, E485A-E486A,E265A, and E266A in HtrI and E513A-E514A in HtrII. The triple mutant inHtrI contains the mutation pairs Q258A-Q259A, E265A-E266A, and E485A-E486A. It was made by mutating each pair in three different PCR mutagenesiscycles. Pfu DNA polymerase (Stratagene) was used for PCR amplification. Themutations introduced in HtrI and HtrII were confirmed by DNA sequencing.

Motion analysis. A computerized cell-tracking system (Motion Analysis, SantaRosa, Calif.) was used to monitor the swimming behavior of wild-type andmutant H. salinarum strains (28). Cells were grown to early stationary phase,diluted 1:10 in fresh complete medium (CM), and incubated for 1 h at 37°C withshaking. Cells containing SRI-HtrI were stimulated with 600-nm attractant lightin an infrared background and white light for repellent stimulation. Swimmingreversals in response to a 4-s step down in this light were recorded and analyzedon an SPARC-IPC workstation (Sun Microsystems, Mountain View, Calif.). TheSRII-HtrII photostimulus consisted of 9 s of illumination with a 500-nm light.Photostimuli were delivered from a Nikon 100-W Hg-Xe lamp beam with ap-propriate 40-nm band pass interference filters (Corion, Holliston, Mass.).

Autofluorography and immunoblotting. Cells were labeled in vivo withL-[methyl-3H]methionine in the presence of puromycin as described previously(24). The absolute amount of L-[methyl-3H]methionine uptake varied in differentcultures, but the relative amounts of label of specific bands corresponding tospecific methyl-accepting proteins was reproducible. Cells were precipitated inacetone by the protocol described by Spudich and Spudich (23) except that thedrying of the samples was done with a Speed-Vac (Savant Instruments, Inc.,Farmingdale, N.Y.). Acetone-precipitated cells or membrane proteins were sep-arated by SDS-PAGE (16) and analyzed by autofluorography of dried gels.Immunoblot analysis was performed by using a polyclonal antibody to the sig-naling domain of halobacterial transducers (HC23 antibody) as described previ-ously (38).

FIG. 1. Autofluorograms of SDS–8% polyacrylamide gels of protein fromL-[methyl-3H]methionine-labeled H. salinarum cells. (A) HtrI-containing cells;(B) HtrII-containing cells. Cells were exposed to dark and light cycles as shownat the top of the fluorograms. Light at 600 and 500 nm was used as stimuli forHtrI and HtrII, respectively. Acetone-precipitated cells were processed for SDS-polyacrylamide gels as described previously (23). Arrows on the left of the gelspoint to HtrI and HtrII migration positions.

FIG. 2. Predicted methylation sites. At the top of the figure in bold letters isthe consensus sequence for the methyltransferase CheR reported for entericbacteria (33). In HtrI and HtrII, the potential methylation sites are underlinedand the residues that are common to the consensus are in bold. Residues E265and E266 in HtrI and their flanking sequences perfectly match the consensussequence. Sequences flanking residue pairs Q258-Q259 and E485-E486 in HtrIand E513-E514 in HtrII differ from the consensus sequence by only one residue.MH1 and MH2 are the first and second methylation helices from the N terminus.

FIG. 3. Autofluorograms of L-[methyl-3H]methionine-labeled membraneproteins and immunoblot. (A) Methylation pattern of membrane proteins fromthe following strains: Pho81Wr2 (lane 1), Pho81Wr2/pKJ306WT (lane 2),Pho81Wr2/pKJ306Q258A-Q259A (lane 3), Pho81Wr2/pKJ306E265A-E266A(lane 4), Pho81Wr2/pKJ306E315A-Q316A (lane 5), Pho81Wr2/pKJ306E336A-Q337A (lane 6), Pho81Wr2/pKJ306E485A-E486A (lane 7). (B) Methylationpattern of membrane proteins. Lanes 1, 2, and 3 contain the same strains as lanes1, 2, and 4 in panel A, respectively. Lane 4 contains the Pho81Wr2/pKJ306 triplemutant (see Materials and Methods). (C) Immunoblot with HC23 antibody ofmembrane proteins. Lanes 1, 2, 3, and 4 contain the same strains as those lanesin panel B.

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Assay for volatile [3H]methyl group production. Cells radiolabeled as de-scribed above and immobilized on a 0.45-mm-pore-size nitrocellulose filter (Nal-gene, Rochester, N.Y.) were used in accordance with the same protocol asdescribed previously (25), except a flow rate of 1 ml/min was used and the voidvolume of the filter and outlet tubing was 0.4 ml.

Pulse-chase methyl turnover experiments. Cells were radiolabeled in vivo asdescribed above. The radioactivity was chased with a 103 excess of nonradioac-tive L-methionine (130 mM). Two-milliliter volumes of the cell suspensions werethen transferred to a disposable cuvette (1 cm by 1 cm by 4.5 cm) held in aremovable sample chamber taken from an SLM Aminco DW-2000 spectropho-tometer (SLM Instruments, Urbana, Ill.) and maintained at a constant temper-ature of 37°C with stirring throughout the experiment. Three different conditionswere used in three separate experiments of 75 min each: constant dark, repetitivecycles of 1 min of light and 1 min of dark, and constant light. Cells wereilluminated with light from a 100-W tungsten-halogen lamp passed through4-mm-thick heat-absorbing glass (Edmund Scientific, Barrington, N.J.) and a600-nm interference filter. Two-hundred-microliter samples were withdrawn ateach time point, precipitated in cold acetone, and processed for SDS-PAGE.Quantitation of the remaining label on autofluorographs of SDS-PAGE wasdone with SigmaScan (Jandel Scientific, San Rafael, Calif.).

RESULTS

Light induces large methylation changes in HtrII but not inHtrI transducers. Cells containing either SRI-HtrI or SRII-

HtrII complexes were labeled to a steady-state level in vivowith L-[methyl-3H]methionine. Fluorography of SDS-PAGEwas used to detect changes in methylation of both HtrI andHtrII upon specific light stimulation of the respective coupledreceptors. SRI stimulation with 600-nm light did not producedetectable changes in the level of methylation of HtrI proteinor other methylated proteins in the gel (Fig. 1A). SRII stimu-lation with 500-nm light caused a reduction of label in the bandcorresponding to the HtrII protein evident after 1 min ofillumination and further decreased after 5 min. Remethylationof HtrII protein was observed after 2 min in the dark, and itwas almost completed after 4 min. Exposing the cells to asecond cycle of illumination reproduced the demethylationresponse observed in the first cycle (Fig. 1B).

HtrI and HtrII contain putative methylation sites. Sequencecomparison of HtrI, HtrII, and other Htr proteins with the eu-bacterial chemoreceptors identifies glutamates or glutaminesat positions Q258-Q259, E265-E266, E315-Q316, E336-Q337in MH1 (methylation helix 1) (30) and E485-E486 in MH2 ofHtrI. HtrII shows in its MH1 glutamate residues E513-E514

FIG. 4. (A) Reversal frequency responses to photostimuli. Phototaxis responses of wild-type cells and HtrI mutants in response to a 4-s step down (dark bar undereach graph) in orange (600-nm) light. Stimuli were delivered at 26-s intervals and three or more sets of 16 stimuli each were averaged to produce the final data. (B)Autofluorograms of L-[methyl-3H]methionine labeled membrane proteins. Lane 1, Pho81Wr2/pKJ306E265A; lane 2, Pho81Wr2/pKJ306E266A.

FIG. 5. Volatile [3H]methyl group release upon SRI-HtrI photostimulation. (A) Pho81Wr2 (SRI2 HtrI2); (B) Pho81Wr2/pKJ306WT; (C) Pho81Wr2/pKJ306triple mutant. Each point corresponds to volatile counts per minute contained in a 0.5-ml fraction collected in 30 s. The variation in the ordinate scale is due to thevariation in extent of L-[methyl-3H]methionine uptake in different strains and on different days.

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that correspond to E265-E266 of HtrI. In HtrI, Q258-Q259and E265-E266 are homologous to Q297-Q298 and E303-E304of Tsr, respectively. Residues E315-Q316 and E336-Q337 areconserved among all the H. salinarum Htr proteins whose se-quences are available to date. A recognition sequence (con-sensus in Fig. 2) for the methyltransferase enzyme, CheR, asreported for the eubacterial chemotransducers (33), is pres-ent in the region flanking residues Q258-Q259, E265-E266,and E485-E486 of HtrI and E513-E514 of HtrII. Both HtrIand HtrII are missing at their C terminus the pentapeptidesequence (NWET/SF), which is conserved among the majorchemotransducers Tsr, Tar, and Tcp and found to be the site ofbinding of CheR (34).

Methylation of HtrI is lost when residues at positions 265and 266 are mutated to alanines. Single-residue pairs on HtrIwere mutated to alanines by PCR site-directed mutagenesis.Mutant cells were labeled in vivo with L-[methyl-3H]methio-nine, membrane proteins were isolated, and the pattern ofmethylated bands was analyzed by SDS-PAGE fluorography(Fig. 3A). Pho81Wr2, an HtrI2 SRI2 strain, did not show anymethylated band corresponding to the HtrI protein (Fig. 3A,lane 1). A distinct methyl-labeled band corresponding to HtrIwas observed in the wild-type strain (Fig. 3A, lane 2). Alaninesubstitutions of glutamates 265 and 266 caused loss of thismethylated band (Fig. 3A, lane 4). The other mutants did notshow detectable band differences from that of the wild-type(Fig. 3A, lane 3, 5, 6, and 7). Mutations at the three sites

containing the methyltransferase recognition sequence werecombined in a single construct (the triple mutant). In Fig. 3B,the triple mutant in lane 4, like the one with the mutationsE265A and E266A in lane 3, did not show any methylationcorresponding to the HtrI band. The amounts of HtrI proteinsin these mutants detected by immunoblot analysis were similarto that of the wild-type strain (Fig. 3C).

Behavioral analysis of methylation mutants of HtrI. Lightstimuli control the motility behavior of the cells by modulating

FIG. 6. Pulse-chase experiment with L-[methyl3H]methionine-labeled Pho81Wr2/pKJ306WT and triple mutant cells. (A) Autofluorogram. Radioactivity was chasedby adding a 103 excess of nonradioactive L-methionine to cells labeled in vivo as described in Materials and Methods. Wild-type cells were exposed to the dark, to thelight, and to alternating light and dark as described in Materials and Methods, and acetone-precipitated samples from each time point were processed for SDS-PAGE.(B) Amount of labeling corresponding to the HtrI band was quantified by using SigmaScan for each condition used. For the triple mutant, the amount of labelingcorresponding to the second band from the top (Fig. 3) was plotted. The values at 25 min were maximal and were therefore defined as 100% labeling.

FIG. 7. Autofluorograms of L-[methyl-3H]methionine-labeled membrane pro-teins and corresponding immunoblot. (A) Methylation patterns of membrane pro-teins from the following strains: Pho81Wr2 (lane 1), Pho81Wr2/pPR5E513AE514A(lane 2), Pho81Wr2/pPR5WT (lane 3). (B) Immunoblot. Samples were loaded inthe same order as in panel A, and the blot was labeled with HC23 antibody.



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their frequency of reversal of swimming direction. An increasein the intensity of orange (attractant) light suppresses reversalprobability, inducing the cells to swim smoothly towards higherintensities of the light. A step down in intensity of the samelight presents a negative stimulus to which the cells react byincreasing their reversal frequency and is commonly used as ameasure of function of the attractant signaling system. Wild-type cells respond to an orange step down stimulus by increas-ing their reversal frequency (excitation). Within the time thelight remains off (4 s), their reversal frequency returns to nearthe prestimulus level (adaptation) (Fig. 4). All the mutantsanalyzed, as well as the wild type, adapted to a step down inorange light. The mutation pair E265A-E266A, which causedloss of methylation of HtrI on SDS gels, produced a reducedresponse compared to that of the wild type. Single alaninesubstitution at positions E265 and E266 established that glu-tamate 265 is responsible for the behavioral phenotype seen inthe double mutant (Fig. 4A). In vivo labeling of the samemutants showed complete loss of methylation by the mutationE265A but not by E266A (Fig. 4B). The triple mutant in whichall the methylation sites with the methyl transferase recogni-tion sequence were changed to alanines exhibited the samebehavior as did the wild type (Fig. 4). Analysis of responses ofpairs of methylation site mutants showed that specificallyE485A-E486A restores the E265A-E266A reduced response(data not shown). The reversal frequencies of each of these

mutants, in the dark and after 2 to 5 min of light, was indis-tinguishable from that of the wild-type strain (1.3 min21).

Stimuli through nonmethylatable HtrI induce volatile meth-yl group release from cells. A vapor phase transfer assay wasused to measure stimulus-induced SRI-HtrI-dependent releaseof methyl groups. The light-induced peaks have been shown tobe due to [3H]methanol (18) consistent with carboxylmethylester hydrolysis. Transient increases in the rate of release ofvolatile [3H]methyl groups were detected after both attractantand repellent photostimuli in both wild-type and triple-mutantcells (Fig. 5B and C). Light-induced methyl release occursfrom the triple mutant even though no steady-state labeling ofHtrI was observed on gels. Therefore, photoactivated SRI-induced turnover of methyl groups in the cell does not dependon the methylated sites on the HtrI transducer.

SRI-HtrI photostimulation causes global methylation turn-over. The methanol release assay showed that there must beanother source of methyl groups different from the stimulatedtransducer HtrI. To identify this source, wild-type and triple-mutant cells were radiolabeled with L-[methyl-3H]methionine,excess nonradioactive methionine was added to the cells, andstimulus-induced chase of the methylation on gels was moni-tored. Cells were exposed to three different conditions: con-stant dark, cycles of 1 min of dark and 1 min of light, andconstant light in three separate assays. The rate of turnover ofmethyl groups on HtrI and on other transducer proteins wasaccelerated by cycles of stimulation in comparison to those ofthe constant-dark or constant-light conditions, showing thatthe stimulus and not the light per se accelerates the turnover(Fig. 6). Quantitation of the HtrI band in the three differentexperimental conditions showed that labeled methyl groups inthe protein in the dark and in the light are chased to 60 to 80%of the starting level of radioactivity in 75 min. Repetitive lightand dark stimuli induced a higher level of turnover that re-sulted in an almost complete chase of radioactivity to 10 to20% in the same time period (Fig. 6B).

The residue pair E513-E514 of HtrII is responsible for mostof the methylation seen in this protein. In HtrII, the residuepair E513-E514 is homologous to E265-E266 in HtrI and con-tains the only putative methylation site in HtrII with the trans-ferase recognition consensus sequence (Fig. 2). When theseresidues in HtrII were mutated to a pair of alanines, nearly allmethylation was lost (Fig. 7A, lane 2), even though the proteinwas expressed at the same level as the wild type (Fig. 7B, lanes2 and 3, respectively). However, motility analysis showed thatalanine substitutions at these sites did not impair the ability ofthe cells to respond to a repellent stimulus (a step-up in500-nm light for 9 s) and to adapt to it. Furthermore, thereversal frequencies of the mutant in the dark and after 2 to 5min in the light were indistinguishable from those of the wildtype (3 min21 in this particular experiment). As in SRI-HtrI,release of methyl groups upon SRII-HtrII-specific stimulationis not affected by the mutations at these sites (Fig. 8B).

Methyl group turnover is regulated by a CheY-dependentpathway. The response regulator gene cheY was deleted in thestrain Pho81Wr2 by using a gene replacement technique (14).The deletion mutant did not form a swarm ring in soft agarplates, as was observed previously for a similar cheY deletionmutant (21). The phenotype of the DcheY strain was smoothswimming both in the dark and in the light, and the reversalfrequency of this strain was restored to wild-type levels byplasmid expression of CheY. Expression of the SRI-HtrI orSRII-HtrII complexes in the cheY deletion mutant, as expect-ed, did not restore spontaneous reversals but did restore light-stimulated changes in methyl turnover rates. Volatile methanolrelease assay of the DcheY strains expressing the SRI-HtrI and

FIG. 8. Volatile [3H]methyl group release upon SRII-HtrII photostimula-tion. (A) Pho81Wr/pPR5WT; (B) Pho81Wr2/pPR5E513AE514A.



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the SRII-HtrII complexes showed in both cases reduction ofturnover of methyl groups upon positive stimuli (Fig. 9). Thus,the deletion of the cheY gene converted the pattern of stimu-lus-induced methanol release from symmetric to asymmetric(Fig. 9).

DISCUSSION

In the generally accepted model of bacterial chemotaxis,adaptation to chemostimuli is brought about by methylationand demethylation of glutamate residues on the receptor-transducer (MCP) protein (4, 5, 30). A change in the level ofmethylation on the stimulated MCP cancels the effect of ligandoccupancy and therefore resets the flagellar motor switch to itsprestimulus bias. We found that H. salinarum cells carryingtransducers mutated at their methylation sites, which conse-quently cannot be methylated, are still able to adapt to lightstimuli. Our results prove that adaptation to phototaxis stimuliin H. salinarum, defined as a return to the prestimulus flagellarbias within the time the stimulus persists, does not requirechanges in methylation of the transducer through which thestimulus is sent. This finding may seem initially to be funda-mentally different from the E. coli chemotaxis paradigm, butupon deeper analysis the difference may be a kinetic ratherthan a mechanistic one. For large and abrupt temporal gradi-ent stimuli with chemotaxis effectors, the time required tochange the level of methylation of the MCPs is, under manyexperimental conditions, the rate-limiting step for adaptation

to occur (30). Evidently, this is not the case for the analogoustemporal gradient photostimuli used here since the eliminationof Htr methylation sites does not alter the kinetics of recoveryto near the prestimulus reversal frequency. In their analysis,Stock and Surette (30) point out that in natural spatial gradi-ents much faster responses and faster adaptation mechanismsare necessary than would be possible from the relatively slowmethylation system in E. coli. Therefore, methylation is likelyto be one of several processes resetting flagellar motor switchbias in the fluctuating natural environment of the cells. Theother processes may dominate the behavior of H. salinarum inthe short time window.

Methylation changes on the stimulated transducer are clear-ly not required in H. salinarum in the short time in which mostof the recovery from a step up or step down in light intensityoccurs. However, methylation extent does bias the flagellarmotor switch in a similar manner as in E. coli. Demethylationby methionine starvation or cheR mutation causes smoothswimming, and overmethylation by cheB deletion causes a highswimming reversal frequency. Furthermore, the transducerHtrII undergoes demethylation in response to a repellent stim-ulus as do MCPs in E. coli. We did not observe a methylationincrease in response to attractant stimulation of HtrI, but smallchanges may have been below our level of detection. Giventhat methylation regulates the flagellar motor switch bias, it islikely to play a role in the adaptation of the cells over longertime scales, but transducer-specific methylation is not required.

We observed photostimulus-induced turnover of methyl

FIG. 9. Volatile [3H]methyl release group of SRI-HtrI and SRII-HtrII-containing cells in Pho81Wr2 (top panels) and Pho81Wr2DcheY background (bottompanels).



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groups monitored by methanol evolution, and pulse-chase mea-surements demonstrated that transducers other than that stim-ulated undergo methylation-demethylation reactions. Similar-ly, it has been shown in E. coli that attractant stimulation ofone MCP transiently increases the methylation level of anotherand that the inhibition of the methyl esterase is responsible forthis effect (22). A global methylation process was observed inour studies in all cases, including stimulation of a nonmethyl-atable transducer, and may be needed for altering the confor-mation of a cluster of transducers to contribute to resetting thekinase activity of the CheA protein. Such “adaptational cross-talk” has been suggested for E. coli, in which chemotaxis me-diated by the ribose and galactose transducer Trg lacking itsmethylation sites was restored by expressing the mutated trans-ducer in the presence of other chemoreceptors (7). Also, trans-ducer protein clustering, which would provide a mechanism forconformational coupling of stimulated and unstimulated trans-ducers, has been reported (17).

E. coli, B. subtilis, and H. salinarum all exhibit an enhancedrate of methanol evolution caused by stimuli that activate thehistidine kinase activity of CheA (repellent stimuli in H. sali-narum) (20). This is attributable to the activation of the methylesterase activity of CheB by phosphorylation in each case.E. coli shows a reduced rate of methanol evolution from stim-uli that inhibit CheA activity, as would be expected from thedephosphorylation of CheB. On the other hand, CheA kinase-inhibiting stimuli in B. subtilis and H. salinarum cause an in-crease in methanol evolution rate even though the activity ofCheB is predicted to be decreased (30). A clue to the basis ofthis anomalous effect has been discovered by Kirby et al. (13),who recently reported that deletion of CheY eliminates theunexpected enhancement of turnover rate in response to re-moval of the chemoeffector asparagine in B. subtilis. Theirexplanation is that addition and removal of asparagine eachcauses transient demethylation of its MCP and that the inabil-ity of the CheY null mutant to remethylate the ligand-boundMCP accounts for the lack of methanol production in responseto asparagine removal. The authors suggest a mechanism forthe CheY-P feedback effect, namely, that the CheY-P inter-action with the ligand-bound MCP affects the topology of theC terminus of the MCP so that the esterase has increasedaccess to an otherwise-less-accessible methylated residue (13).

Our results show that as in B. subtilis, a CheY-dependentprocess is responsible for the increased methanol evolutionrate following CheA-inhibiting stimuli in H. salinarum. In aCheY deletion mutant of H. salinarum, repellent and attract-ant stimuli result in increases and decreases, respectively, inthe methanol evolution (methyl group turnover) rate, as ex-pected from the respective phospho-activation and -deactiva-tion of CheB, and as is observed in wild-type E. coli. However,the explanation offered for B. subtilis does not seem to apply toH. salinarum. First, we do not observe remethylation of HtrIIin the continuous presence of light after the protein is demeth-ylated in response to blue light. The methylation changes ofHtrII in response to addition and removal of repellent bluelight are not transient, but methylation levels are shifted in amanner closely similar to those of E. coli MCPs in response toaddition and removal of repellent substances. Second, wefound with the triple mutant that activation of a nonmethylat-able HtrI still produces a symmetric pattern in release ofradiolabeled methanol. Therefore, the feedback effect of CheYis not localized to an effect on the stimulated transducer butmodulates methyl group turnover of other transducers in thecell. One possibility is that CheY increases the rate of meth-ylation, either by making methylation sites more accessible or

by activation of CheR activity, thereby increasing the turnoverrate.

ACKNOWLEDGMENTS

We thank Elena Spudich and Kwang-Hwan Jung for critically read-ing the manuscript and John R. Kirby for helpful discussions.

This work was supported by National Institutes of Health grantR01-GM27750 (to J.L.S.).

REFERENCES

1. Alam, M., M. Lebert, D. Oesterhelt, and G. L. Hazelbauer. 1989. Methyl-accepting taxis proteins in Halobacterium halobium. EMBO J. 8:631–639.

2. Boyd, A., and M. I. Simon. 1980. Multiple electrophoretic forms of methyl-accepting chemotaxis proteins generated by stimulus-elicited methylation inEscherichia coli. J. Bacteriol. 143:809–815.

3. Chen, B., and A. E. Przybyla. 1994. An efficient site-directed mutagenesismethod based on PCR. BioTechniques 17:161–162.

4. Eisenbach, M. 1996. Control of bacterial chemotaxis. Mol. Microbiol. 20:903–910.

5. Falke, J. J., R. B. Bass, S. L. Butler, S. A. Chervitz, and M. A. Danielson.1997. The two-component signaling pathway of bacterial chemotaxis: a mo-lecular view of signal transduction by receptors, kinases, and adaptationenzymes. Annu. Rev. Cell Dev. Biol. 13:457–512.

6. Hazelbauer, G. L., and P. Engstrom. 1981. Multiple forms of methyl-accept-ing chemotaxis proteins distinguished by a factor in addition to multiplemethylation. J. Bacteriol. 145:35–42.

7. Hazelbauer, G. L., C. Park, and D. M. Nowlin. 1989. Adaptational“crosstalk” and the crucial role of methylation in chemotactic migration byEscherichia coli. Proc. Natl. Acad. Sci. USA 86:1448–1452.

8. Hoff, W. D., K.-H. Jung, and J. L. Spudich. 1997. Molecular mechanism ofphotosignaling by archaeal sensory rhodopsins. Annu. Rev. Biophys. Biomol.Struct. 26:223–258.

9. Jung, K.-H., and J. L. Spudich. 1998. Suppressor mutation analysis of thesensory rhodopsin I-transducer complex: insights into the color-sensingmechanism. J. Bacteriol. 180:2033–2042.

9a.Jung, K.-H., and J. L. Spudich. Unpublished data.10. Kehry, M. R., and F. W. Dahlquist. 1982. The methyl-accepting chemotaxis

proteins of Escherichia coli. Identification of the multiple methylation siteson methyl-accepting chemotaxis protein I. J. Biol Chem. 257:10378–10386.

11. Kleene, S. J., M. L. Toews, and J. Adler. 1977. Isolation of glutamic acidmethyl ester from an Escherichia coli membrane protein involved in chemo-taxis. J. Biol Chem. 252:3214–3218.

12. Kirby, J. R., C. J. Kristich, S. L. Feinberg, and G. W. Ordal. 1997. Methanolproduction during chemotaxis to amino acids in Bacillus subtilis. Mol. Mi-crobiol. 24:869–878.

13. Kirby, J. R., M. M. Saulmon, C. J. Kristich, and G. W. Ordal. 1999. CheY-dependent methylation of the asparagine receptor, McpB, during chemotaxisin Bacillus subtilis. J. Biol. Chem. 274:11092–11100.

14. Krebs, M. P., R. Mollaaghababa, and H. G. Khorana. 1993. Gene replace-ment in Halobacterium halobium and expression of bacteriorhodopsin mu-tants. Proc. Natl. Acad. Sci. USA 90:1987–1991.

15. Krebs, M. P., E. N. Spudich, H. G. Khorana, and J. L. Spudich. 1993.Synthesis of a gene for sensory rhodopsin I and its functional expression inHalobacterium halobium. Proc. Natl. Acad. Sci. USA 90:3486–3490.

16. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature (London) 227:680–685.

17. Maddock, J. R., and L. Shapiro. 1993. Polar location of the chemoreceptorcomplex in the Escherichia coli cell. Science 259:1717–1723.

18. Nordmann, B., M. R. Lebert, M. Alam, S. Nitz, H. Kollmannsberger, D.Oesterhelt, and G. L. Hazelbauer. 1994. Identification of volatile forms ofmethyl groups released by Halobacterium salinarum. J. Biol. Chem. 269:16449–16454.

19. Nowlin, D. M., J. Bollinger, and G. L. Hazelbauer. 1987. Sites of covalentmodification in Trg, a sensory transducer of Escherichia coli. J. Biol. Chem.262:6039–6045.

20. Rudolph, J., N. Tolliday, C. Schmitt, S. C. Schuster, and D. Oesterhelt. 1995.Phosphorylation in halobacterial signal transduction. EMBO J. 14:4249–4257.

21. Rudolph, J., and D. Oesterhelt. 1996. Deletion analysis of the che operon inthe archaeon Halobacterium salinarium. J. Mol. Biol. 258:548–554.

22. Sanders, D. A., and D. E. Koshland, Jr. 1988. Receptor interactions throughphosphorylation and methylation pathways in bacterial chemotaxis. Proc.Natl. Acad. Sci. USA 85:8425–8429.

23. Spudich, E. N., and J. L. Spudich. 1982. Measurement of light regulatedphosphoproteins of Halobacterium halobium. Methods Enzymol. 26:213–216.

24. Spudich, E. N., C. A. Hasselbacher, and J. L. Spudich. 1988. Methyl-accept-ing protein associated with bacterial sensory rhodopsin I. J. Bacteriol. 170:4280–4285.

25. Spudich, E. N., T. Takahashi, and J. L. Spudich. 1989. Sensory rhodopsins



http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

I and II modulate a methylation/demethylation system in Halobacteriumhalobium phototaxis. Proc. Natl. Acad. Sci. USA 86:7746–7750.

26. Spudich, E. N., W. Zhang, M. Alam, and J. L. Spudich. 1997. Constitutivesignaling by the phototaxis receptor sensory rhodopsin II from disruption ofits protonated Schiff base-Asp-73 interhelical salt bridge. Proc. Natl. Acad.Sci. USA 94:4960–4965.

27. Spudich, J. L., and R. A. Bogomolni. 1988. Sensory rhodopsins of halobac-teria. Annu. Rev. Biophys. Biophys. Chem. 17:193–215.

28. Spudich, J. L., and E. N. Spudich. 1995. Selection and screening methods forhalophilic archael rhodopsin mutants, p. 23–28. In F. T. Robb et al. (ed.),Archaea: a laboratory manual. Cold Spring Harbor Laboratory Press, Pla-inview, N.Y.

29. Stock, J. B., and D. E. Koshland, Jr. 1978. A protein methylesterase involvedin bacterial sensing. Proc. Natl. Acad. Sci. USA 75:3659–3663.

30. Stock, J. B., and M. Surette. 1996. Chemotaxis, p. 123–145. In F. C. Neid-hardt, R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik,W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.), Esch-erichia coli and Salmonella: cellular and molecular biology. American Societyfor Microbiology, Washington, D.C.

31. Sundberg, S. A., R. A. Bogomolni, and J. L. Spudich. 1985. Selection andproperties of phototaxis-deficient mutants of Halobacterium halobium. J. Bac-teriol. 164:282–287.

32. Terwilliger, T. C., and D. E. Koshland, Jr. 1984. Sites of methyl esterification

and deamination on the aspartate receptor involved in chemotaxis. J. Biol.Chem. 259:7719–7725.

33. Terwilliger, T. C., J. Y. Wang, and D. E. Koshland, Jr. 1986. Surface struc-ture recognized for covalent modification of the aspartate receptor in che-motaxis. Proc. Natl. Acad. Sci. USA 83:6707–6710.

34. Wu, J., J. Li, G. Li, D. G. Long, and R. M. Weis. 1996. The receptor bindingsite for the methyltransferase of bacterial chemotaxis is distinct from the sitesof methylation. Biochemistry 35:4984–4993.

35. Yao, V. J., and J. L. Spudich. 1992. Primary structure of an archaebacterialtransducer, a methyl-accepting protein associated with sensory rhodopsin I.Proc. Natl. Acad. Sci. USA 89:11915–11919.

36. Yao, V. J., E. N. Spudich, and J. L. Spudich. 1994. Identification of distinctdomains for signaling and receptor interaction of the sensory rhodopsin Itransducer, HtrI. J. Bacteriol. 176:6931–6935.

37. Zhang, W., A. Brooun, M. M. Mueller, and M. Alam. 1996. The primarystructures of the Archaeon Halobacterium salinarium blue light receptorsensory rhodopsin II and its transducer, a methyl-accepting protein. Proc.Natl. Acad. Sci. USA 93:8230–8235.

38. Zhang, W., A. Brooun, J. McCandless, P. Banda, and M. Alam. 1996. Signaltransduction in the archaeon Halobacterium salinarium is processed throughthree subfamilies of 13 soluble and membrane-bound transducer proteins.Proc. Natl. Acad. Sci. USA 93:4649–4654.



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Documents

Identiﬁcation of Methylation Sites and Effects of ...the htrI promoter as described previously (26). PCR mutagenesis. Mutations introduced into the sequences of HtrI and HtrII were