A novel six-rhodopsin system in a single archaeon

Abstract

Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered six-rhodopsin system in a single archaeon, Haloarcula marismortui, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in H. marismortui, and these were then overexpressed in Escherichia coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in H. marismortui.

FIG. 1.

Gene candidate identification for cognate transducers. (A) Three htr open reading frames (ORFs) (light shading) conjugated either upstream or downstream of a corresponding opsin gene (dark shading). The gene locus tag is indicated for each htr. *, no annotation was made for this gene from the original genome project. (B) The translated protein sequences from the htr ORFs were analyzed using SMART (http://smart.embl-heidelberg.de). reHmHtrI represents restoration of the missing two-transmembrane region of HmHtrI (see the text for details). The scale bar shows the length of the amino acid sequence. The large open vertical rectangle indicates that this section of the protein sequence was analyzed as a transmembrane region. The small gray rectangle indicates the amino acid sequence predicted to be a low-complexity region. The pentagons illustrate protein-signaling domains. HAMP, histidine kinases, adenylyl cyclases, methyl binding proteins and phosphatases; MA (MCP), methyl-accepting chemotaxis protein.

FIG. 2.

Configurations, gene names, and protein names for the six rhodopsins and their cognate transducers from H. marismortui. HmBRI (bop) is a light-driven proton transporter; HmHR (hop) is a light-driven chloride transporter; HmBRII (xop1), originally predicted to be a BR precursor, is, like HmBRI, a light-driven proton transporter; and HmSRM (xop2), which was annotated as an “opsin of unknown function,” associates with HmHtrM (htrM), and its function is yet to be determined. HmSRI (sop1) associates with reHmHtrI (htr1), and HmSRII (sop2) associates with HmHtrII (htr2) to mediate photoattractant and photorepellent responses, respectively. The blue box indicates the two rhodopsins that are in addition to the currently known four-rhodopsin system identified from H. salinarum.

FIG. 3.

RT-PCR analysis of the rhodopsins and signal transducers. The mRNAs coding for all rhodopsins (bop, hop, sop1, sop2, xop1, and xop2) and transducers (htr1, htr2, and htrM) were detectable in the cDNA of H. marismortui grown with and without white-light illumination. The expression profiles of these genes in early-, mid-, and late-log-phase cultures (i.e., OD₆₀₀s of 0.5, 1.0, and 1.5, respectively) were analyzed by RT-PCR with gene-specific primers. The 16S rRNA transcript was used as an internal reference for relative quantification (see Fig. SA1 in the supplemental material).

FIG. 4.

Spectrum determination of the six individual purified rhodopsins from H. marismortui. (A) Group photo of all six rhodopsins purified from E. coli dissolved in MES buffer containing 0.05% n-dodecyl-β-d-maltoside, pH 5.8. From left to right, HmBRI, HmHR, HmSRI, HmSRII, HmBRII, and HmSRM. (B) Absorbance spectrum and absorbance peak of each rhodopsin. AU, arbitrary units.

FIG. 5.

Flash-induced absorption changes for the six purified H. marismortui rhodopsins. The purified proteins were dissolved in MES buffer containing 0.05% n-dodecyl-β-d-maltoside, pH 5.8, and transient absorbance changes were recorded at their corresponding ground state λ_max. (A) Transients were measured for HmBRI at 550 nm (top), HmBRII at 550 nm (middle), and HmHR at 580 nm (bottom), and all photocycles were completed within a millisecond. (B) Transients were measured for HmSRI at 570 nm (top), HmSRII at 480 nm (middle), and HmSRM at 500 nm (bottom), and photocycles were completed with a second. The curves represent the change of absorbance corresponding to the λ_max for each rhodopsin upon green-laser (532-nm) excitation.

FIG. 6.

Measurement of light-driven transport of ions. (A) A suspension of E. coli cells expressing HmBRI, HmBRII, or HmHR alone was used to measure light-induced pH changes. The beginning and end of illumination using a 10-mW, 532-nm continuous green laser are indicated by the arrows labeled “on” and “off,” respectively. (B) Chloride binding affinity determination for HmHR. The inset shows the spectral shifts of HmHR in different chloride concentrations. The direction of shift from high- to low-salt concentration is marked by an arrow.

FIG. 7.

Reversal kinetics upon 0.5-s pulse stimulation of HmSRII (A) and HmSRM (B) with 465- ± 15-nm and 540- ± 10-nm lights, respectively. Light-gray, dark-gray, and black circles represent 25%, 50%, and 75% light intensity, respectively. The estimated 75% light intensities for the 465- ± 15-nm and 540- ± 10-nm lights were 3.7 × 10⁶ and 2.9 × 10⁶ photons·mm⁻²·s⁻¹, respectively. Each panel was derived from 720 s of video from a single set of experiments. Error bars indicate 95% confidence intervals, assuming a binomial distribution; the number of trajectories ranged from 117 to 235.

FIG. 8.

Laser flash-induced absorption changes for HmSRM and HmSRM-HmHtrM fusion proteins. (A) Dark-state depletion and recovery of HmSRM and the HmSRM-HmHtrM fusion protein. Laser flash photolysis (laser pulse time, 10 ms, 1 W) of HmSRM and the HmSRM-HmHtrM fusion protein in 50 mM Tris-HCl, 4 M NaCl, pH 6.8, 0.05% DDM at room temperature. The absorption change (ΔAbs) of HmSRM (dark gray) and the HmSRM-HmHtrM fusion (light gray) were monitored at 503 nm and 507 nm, respectively, and the flash was set to a time of 1 s. (B) Duration of the M-intermediate in HmSRM and the HmSRM-HmHtrM fusion protein. Laser-induced photolysis (laser pulse time, 10 ms, 1 W) of HmSRM and the HmSRM-HmHtrM fusion protein in 50 mM Tris-HCl, 4 M NaCl, pH 6.8, 0.05% DDM at room temperature. The absorption change of HmSRM (dark gray) and the HmSRM-HmHtrM fusion (light gray) were monitored at 378 nm and 379 nm, respectively, and the flash was set to a time of 1 s. Data fitting (black) based on a one-phase exponential-decay model and the duration of the signaling state, M-intermediate, was 164 ms in HmSRM and 622 ms in the HmSRM-HmHtrM fusion form.