Constitutive signaling by the phototaxis receptor sensory rhodopsin II from disruption of its protonated Schiff base–Asp-73 interhelical salt bridge

Rescue of Blue-Green Phototaxis Mutants by Plasmid-Mediated Expression of the htrII–sopII Gene Pair.

The htrII–sopII gene pair has been identified on a 6.5-kbp PstI fragment (11). Southern hybridization with an htrII-specific and a sopI -specific probe reveal a shift to a 7-kbp PstI fragment in the blind mutant Pho81Wr⁻ and the presence of a BglII site not present in the wild-type PstI fragment. Sequencing analysis (J. Zhu, E.N.S., M.A., and J.L.S., unpublished work) reveals that these changes are due to insertion of the 552-bp ISH2 transposable element, which is also responsible for disruption of the htrI–sopI pair in Pho81Wr⁻ (15).

SRII production from its native promoter is at low levels and is maximal during exponential-phase growth (2). To increase expression level in stationary-phase cultures, the htrII–sopII pair was placed under control of the htrI–sopI promoter on the expression vector pPR5 and introduced by transformation into Pho81Wr⁻ cells as well as into mutant strain Δ35, a derivative of Pho81 in which the htrII–sopII region has been excised. Southern hybridization of the transformants with a sopII-specific probe shows the inactivated copy of sopII on the 7-kbp PstI chromosomal fragment, as in untransformed Pho81Wr⁻, and the plasmid copy on a 6.5-kbp fragment (data not shown). No evidence of recombination, which would produce additional bands, was observed.

Expression of both htrII and sopII genes was evident by the restoration of (i) the absorption spectrum (see Fig. 1a) and flash-induced absorption changes characteristic of SRII (see Figs. 2 and 3), (ii) SRII-mediated phototaxis responses (see Fig. 4), and (iii) the HtrII methylation and immuno band (see Fig. 5). These data confirm that the putative htrII–sopII gene pair, assigned on the basis of sequence homologies, encode the phototaxis receptor SRII and its transducer HtrII.

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Figure 1

Absorption spectra of wild-type and D73N SRII in membranes and purified in lauryl maltoside. (a and c) Spectra generated by all-trans-retinal addition to hydroxylamine-bleached membranes from Pho81Wr⁻ containing wild-type and D73N SRII, respectively, in the presence of HtrII. (b and d) Spectra of purified His-tagged wild-type and D73N SRII, respectively, in lauryl maltoside.

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Figure 2

Laser flash-induced absorption transients of wild-type and D73N SRII in membranes. Membranes as in Fig. 1 containing wild-type (Left) and D73N (Right) SRII were subjected to a 532-nm laser flash at time 0, and absorption changes were monitored at 500 μs per point at the indicated wavelengths.

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Figure 3

Flash-induced absorption difference spectra of SRII and D73N in membranes. Maximum amplitudes of absorption changes monitored at various wavelengths as in Fig. 2 are shown for membranes containing wild-type and D73N SRII. The ordinate values are those of D73N, and SRII values were multiplied by 3 for plotting.

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Figure 4

Phototaxis responses of cells containing wild-type or D73N SRII. Reversal frequency transients from computerized cell-tracking and motion analysis for cells carrying SRII or D73N in the presence of HtrII are shown for a 10-ms 500-nm stimulus (arrow) at 100% stimulus intensity. (Inset) Fluence/response curves. Photoinduced reversals were assessed as the increased frequency of swimming reorientations in the 1-s period following the 10-ms stimulus. Stimuli were delivered at 30-s intervals and three sets of 16 stimuli were averaged to produce the reversal transients. Values in the fluence/response curves are each from one set of 16 stimuli.

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Figure 5

Immunoblot and methylation pattern of Pho81Wr⁻, Δ35, and transformant membranes. Shown are immunoblot with HC23 antibody (A) and methylation (B) fluorograms of membrane proteins from the following: a, Pho81Wr⁻; b, Pho81Wr⁻ × pPR73N; c, Pho81Wr⁻ × pPR5; d, Δ35; e, Δ35 × pPR73N; and f, Δ35 × pPR5.

Absorption Spectra of Wild-Type and D73N SRII.

Asp-73 in SRII is in the position homologous to that of the Schiff base counterions Asp-85 in BR and Asp-76 in the purple form of SRI. The known counterions have well characterized effects on the absorption spectra in BR and SRI. Accordingly, we replaced Asp-73 with Asn in SRII to test by absorption spectroscopy whether Asp-73 serves the analogous function.

Transformant membranes containing either wild-type or D73N SRII in the presence of HtrII were bleached with hydroxylamine as described (20) and reconstituted with all-trans-retinal to determine their visible absorption spectra. Wild-type SRII exhibits structured absorption with maxima at 487 nm and 460 nm (Fig. 1a), similar to the spectrum reported previously for hydroxylamine bleaching/reconstitution of Flx3b membranes (21). Purified His-tagged SRII in lauryl maltoside shows a nearly identical, but better resolved, spectrum (λ_max 487 nm), with an evident vibrational fine structure, as previously reported for native SRII (21).

The D73N mutation shifts the absorption maximum to longer wavelengths by ≈30 nm both in membranes and in purified pigment in lauryl maltoside (Fig. 1 c and d). A loss of vibrational fine structure is especially evident in the purified pigment spectra, which have higher signal-to-noise ratios. Using the λ_max values, we calculate a shift of 1078 cm⁻¹ caused by substitution of Asn for Asp-73 (Table 1). This value is identical to that observed from the replacement of the homologous residues Asp-85 in BR (22) and Asp-76 in the purple form of SRI (9). In BR and in HtrI-free SRI the Asn-induced red shift is known to be due to the neutralization of the anionic form of the aspartyl residues Asp-85 and Asp-76, respectively, which function as counterions to the protonated Schiff base in each of the two pigments. The homologous position of Asp-73 and the identical red shift strongly indicate that Asp-73 is anionic in SRII₄₈₇, as depicted in Table l, and functions similarly as an important counterion component in SRII. Further support for this interpretation is that acidic pH also induces a transition (pK_a 3.0) of the wild-type SRII absorption maximum to 514 nm, analogous to formation of the 605-nm form of BR due to Asp-85 protonation, that D73E exhibits a similar transition shifted to pK_a 5.1, and D73N does not exhibit a shift in absorption maximum in the range pH 1 to pH 6 (J. Zhu, E.N.S., M.A., and J.L.S., unpublished results).

Flash-Induced Absorption Changes in Membranes Containing Wild-Type and D73N SRII.

The main Schiff base counterion of BR, of purple SRI, and of human rod rhodopsin serves as the primary proton acceptor from the Schiff base after photoisomerization. Accordingly, we assessed whether photoinduced deprotonation of the Schiff base is blocked in D73N SRII.

Transformant membranes containing either wild-type or D73N SRII in the presence of HtrII were analyzed by laser flash kinetic UV–visible spectroscopy. Absorption transients were monitored at 450 nm, at which in wild-type SRII the depletion and return of SRII₄₈₇ are evident. The formation and decay of the two photointermediates SII₃₆₀ and SII₅₄₀ after a 532-nm laser flash were monitored at 370 nm and 550 nm, respectively. Transformant membranes containing wild-type SRII exhibited transients that fit the expected intermediates with formation of SII₃₆₀ in <1 ms, decay of this intermediate into SII₅₄₀ with a first-order half-time of 90 ms, and return of the initial state with a first-order half-time of 500 ms (Fig. 2). These values and the flash-induced absorption difference spectrum shown in Fig. 3 are similar to those reported previously (2).

D73N SRII shows no detected formation of a deprotonated intermediate as evidenced by the lack of a signal at 370 nm (Fig. 2). The flash-induced absorption difference spectrum shows a depletion with no evident increases in absorption. A similar difference spectrum is observed for the BR D85N mutant, in which Schiff base deprotonation is almost completely blocked (23), and for the light-driven chloride pump halorhodopsin, which does not deprotonate the Schiff base during its photocycle (24). The lack of positive bands indicates the photoproduct(s) of the D73N pigment mostly overlaps the absorption spectrum of the initial species. A blue-shifted photointermediate is indicated, since the depletion maximum of the difference spectrum at 530 nm is red-shifted from the 514-nm λ_max of D73N. The photocycle of D73N is slower than that of wild type (Fig. 2).

Phototaxis Responses of Cells Containing Wild-Type and D73N SRII.

To assess the importance of Schiff base deprotonation to phototaxis signaling, fluence/response curves of cells containing wild-type or D73N SRII were produced by motility analysis using 10-ms stimuli with 500 ± 20 nm light for which the 487-nm SRII and 514-nm D73N receptors have comparable absorption (Fig. 1 a and c). At a stimulus strength sufficient to saturate the repellent response in wild-type SRII, cells carrying the D73N receptor exhibit 16% of the response (Fig. 4 Inset). The D73N mutant receptor and wild-type SRII are present at similar concentrations in the cells as assessed by absorption measurement (Fig. 1 a and c), and therefore the efficiency of signal generation by SRII is reduced, but not eliminated, by the mutation.

Cells carrying D73N exhibit higher reversal frequencies in the dark than wild type, as is evident in the prestimulus computerized motion analysis data (Fig. 4). Visual single cell tracking confirms that the D73N receptor increases the spontaneous reversal frequency of the cells observed with nonactinic infrared background illumination. The mean ± SEM for four independent measurements was 3.5 ± 0.2 min⁻¹ and 10.8 ± 1.8 min⁻¹ for transformants carrying wild-type SRII or D73N, respectively. This finding implies that the excitation signal that induces reversals is constitutively generated by the D73N receptor and is incompletely compensated by the adaptation (methylation) system. However the D73N receptor is not fully activated, since photostimuli still elicit a partial response from cells carrying the mutant SRII.

D73N Causes HtrII Demethylation in the Dark.

According to the E. coli chemotaxis paradigm, repellent signals result in transducer demethylation in the adaptation process. Light-induced methyl group turnover measurements (25, 26) and effects of mutations on swimming behavior (27, 28) strongly support that this paradigm applies, although light-induced changes of methylation extent of the Htr proteins have not been directly demonstrated. In analogy to E. coli transducers, if the D73N receptor is in a constitutively active signaling state, the HtrII protein should demethylate as an adaptation response (6). In vivo l-[methyl-³H]methionine labeling confirms this expectation. The amounts of HtrII protein expressed with SRII or D73N are identical according to immunoblotting (Fig. 5A) but methylation of HtrII is greatly reduced in the dark in the presence of the D73N receptor (Fig. 5B). Moreover, HtrII migrates slightly slower in the D73N background, consistent with demethylation, which slows the migration of E. coli transducers (29).