Haloarchaeal opsin sequence acquisition, alignment, and phylogenetic inference

During automated annotation of 80 haloarchaeal genomes with the Rapid Annotation Using Subsystem Technology (RAST) server [38], five sequences were annotated as opsin homologs which did not show significant sequence similarity to canonical haloarchaeal opsins by BLASTp search (e-value cutoff of 10⁻⁵) and which were missing the Schiff base lysine required for binding of the retinal chromophore in all experimentally characterized opsins (K216 in the haloarchaeal model opsin Halobacterium sp. NRC-1 bacteriorhodopsin). These sequences were also annotated as opsins in the NCBI genomic database. A BLASTp search (e-value cutoff of 10⁻⁵) against the 80 haloarchaeal genomes using these five opsin sequences as queries, recovered 43 additional K216-lacking, putative opsin homologs for a total of 48 homologs in 28 genomes. These were combined with 122 canonical haloarchaeal opsins recovered by BLASTp search (e-value cutoff of 10⁻⁵) of the 80 haloarchaeal genomes using all Haloarcula marismortui ATCC 43049 opsins as query sequences. This set of queries was chosen because Har. marismortui possesses homologs for each of the six previously categorized classes of haloarchaeal opsins [11,12]. A total of 170 haloarchaeal opsin homologs were included in subsequent analyses (sequences available at the Dryad Digital Repository (http://dx.doi.org/10.5061/dryad.963hr)).

Multiple sequence alignments were created separately for canonical and non-canonical haloarchaeal opsins using MUSCLE 3.8 [39,40], and checked for accuracy against a previously published haloarchaeal opsin alignment [30]. Miscalled start sites for 56 sequences were manually corrected based upon alignment with sequences of experimentally characterized opsins. Individual alignments were combined using the profile-profile alignment option in MUSCLE 3.8 and manually trimmed in the alignment editor Jalview [41]. A total of 204 positions were used to infer phylogeny using the Bayesian tree-building software MrBayes [42,43] with 1.2 million rounds of Markov chain Monte Carlo iteration. The final potential scale reduction factor was 1.000 and final standard deviation of split frequencies was 0.010270. The tree was visualized using FigTree [44]. The tree file and alignment used for tree inference are available from the Dryad Digital Repository (http://dx.doi.org/10.5061/dryad.963hr). For a fully expanded tree, see S5 Fig.

Distribution of opsin classes and retinal biosynthesis genes in the Haloarchaea

The presence/absence pattern of haloarchaeal opsin subclasses and the retinal biosynthesis genes crtY, crtE, crtB, crtI, and brp were superimposed on a previously published multi-marker phylogeny of the haloarchaea [20] using iTOL [45,46]. Subclass membership for each opsin homolog was determined based on clade affiliation in haloarchaeal opsins tree. Sequences for crtY, crtE, crtB, crtI, and brp homologs were retrieved via BLASTp searches against the local haloarchaeal database, using query sequences from six haloarchaeal species (sequences available at the Dryad Digital Repository (http://dx.doi.org/10.5061/dryad.963hr)) and an e-value cutoff of 10⁻²⁰. As all species with crtY and brp also had a full complement of crtE, crtB, and crtI, presence or absence of crtY and brp was used to represent retinal biosynthesis ability.

Genome context of ORPs

Genomic context of haloarchaeal ORPs was investigated using JContextExplorer [47]. Haloarchaeal genomes can be loaded using the “Retrieve Popular Genome Set” function. Annotations associated with this genome set were done using the RAST annotation service [38] (see [20]). To view ORP contexts, search by Cluster Number for “4077;15722” (group A) or “2453;13537” (group B). Cluster numbers represent homology families as defined in [20].

Structural modeling of ORP proteins

We used x-ray structure of Natronomonas pharaonis SRII (PDB ID: 3QAP) [48] as a template for generating structural models for three ORP proteins (Nab. magadii WP_004267173, Hrr. distributum ELZ45759, and Nbt. gregoryi WP_005575895) using the Rosetta-Membrane method [49–51]. We used Nmn. pharaonis SRII because it was identified by the HHpred server [52,53] as the closest structural homolog to the ORPs (21–25% sequence identity). Due to a two residue deletion in the loop between TM6 and TM7 in the Hrr. distributum ORP compared with Nmn. pharaonis SRII, we predicted the structure of this region de novo using Rosetta cyclic coordinate descent (CCD) and kinematic (KIC) closure loop modeling, developed to model loop structures with sub-angstrom accuracy [54,55]. Several rounds of CCD and KIC loop modeling were perforrmed with at least 10,000 models generated during each round. Models were ranked based on total Rosetta energy after each loop modeling round [49,51,56]. Ten percent of the lowest energy models were clustered [57] using a root mean square deviation (RMSD) threshold that placed 1–2% of all models in at least one of the largest clusters. Models representing centers of the top 20 clusters (early rounds) and/or the best 10 models by total energy (later rounds) were used as input for the next round of loop modeling. Rosetta's full atom relaxation protocol [51,56] was used to explore potential differences in backbone and side chain conformations of ORPs compared with the SRII template. Selection of the best ORP models was guided by clustering of the lowest energy models to generate the most frequently sampled conformations.

Intramolecular pathway and ligand-binding pocket predictions

Comparative structural models described above were used for prediction of ligand binding sites, internal cavities and tunnels using CAVER 3.0 [58]. Analysis was performed individually for each of the top five models generated by Rosetta for each ORP, which had a mean pair-wise RMSD across all models ranging from 0.610 to 0.694 Å. CAVER settings used were minimum probe radius of 0.9 Å, shell depth of 4 Å, shell radius of 3 Å, clustering threshold of 3.5 Å, 12 approximating balls, and maximum distance and desired radius for starting point optimization of 3 Å and 5 Å, respectively. Starting point coordinates were optimized to enable use of homologous starting positions for the majority of models. The starting point for all but two of the 15 ORP models was G193 (Nab. magadii) / A185 (Hrr. distributum) / G196 (Nbt. gregoryi). For two Hrr. distributum models, this starting position resulted in no predicted cavities, however, cavities similar to those predicted in other models were discovered with a starting position of Q175-I176. This starting position also resulted in no predicted cavities for SRII, as expected. For comparative visualization purposes, the starting position of P183 was used for SRII, resulting in prediction of a small, likely artifactual ligand binding pocket. For comparison, cavities were also predicted for bacteriorhodopsin (PMID: 4MD2) [59] and halorhodopsin (PMID: 1E12) [60] homologs. Starting points for these predictions were P4 and N3, respectively. Binding pockets and tunnels were visualized with MacPyMOL [61]. For comparison of cavity predictions between ORPs and canonical opsins, see Fig 3C. For comparison of all ORP models, see S2 Fig.

Virtual ligand screening methods

Autodock VINA [62] (1.1.2 May 11, 2011), downloaded from http://vina.scripps.edu/, was used for fast protein-ligand screening. For fast multi-threaded ligand screening the program idock [33] (v2.1.3) was downloaded from https://github.com/HongjianLi/idock. For additional docking experiments we used Autodock (v4.2.6) and Autodock tools [63], the PyRx [64] virtual screening software (v0.8) from http://pyrx.sourceforge.net/ as well as UCSF Chimera [65] for ligand visualization and protein modifications. For SDF file and metadata handling we used the freely available OSIRIS DataWarrior [66] www.openmolecules.org/datawarrior/. The statistical analysis of docking results was performed with StatSoft Statistica (v12) and computational compound clustering of results including 3D mapping was performed with CheS-Mapper [67]. Venn diagrams for compound overlap based on compound ID were calculated on the web page http://www.bioinformatics.lu/venn.php.

Protein data included three canonical opsins (PDB ID: 1E12, 3QAP, and 4MD2) and three ORPs (Nab. magadii WP_004267173, Hrr. distributum ELZ45759, and Nbt. gregoryi WP_005575895). Receptor preparation included removal of water and unwanted ligands as well as pdbqt conversion with AutoDock tools and PyRx software.

A total of 229,358 ligands were sourced from the Universal Natural Products Database (UNPD, pkuxxj.pku.edu.cn/UNPD/) [68]. The 3D-conformers were created using the ChemAxon molconvert software (ChemAxon molconvert; For 3D conformer generation; Molecule File Converter, version 6.0.5, 2013 ChemAxon Ltd. ChemAxon), utilizing the MMFF94 force field optimization with the lowest energy conformer option and explicit hydrogens. The SDF file was then transformed with OpenBabel [69] (v2.3.2) into a ligand pdbqt file and subsequently split into multiple single files with VINA_split.exe. Idock configurations files were created for each receptor with a box size of 15 Å on each axis, including the following docking parameters, (x:16.560 y:40.852 z:-4.072) for Nab. magadii WP_004267173, (x:14.244 y:40.502 z:-1.779) for Hrr. distributum ELZ45759 and (x:14.527 y:41.251 z:-2.290) for Nbt. gregoryi WP_005575895. All 229,358 ligands were screened for each protein. Because the scoring functions of VINA and idock are non-deterministic we repeated the scoring for the best candidates in order to screen for outliers. For the creation of a small decoy library [70], known ligands from the canonical opsins were submitted to http://dude.docking.org/generate. The decoy library with 750 compounds was then used to generate energy cutoff values for the idock-based screening.

The hardware for virtual screening included a Dual Xeon High Performance Workstation with two Intel E5-2687W processors (16 cores, 32 threads), 8 Tb RAID10 disks, 2 Tb SSDs and 196 Gb RAM under Windows 7 64-bit. All modeling was performed on an 80 Gb SoftPerfect RamDisk allowing sequential read/write speeds of up to 4 Gb/second. For additional discussion of ligand screening, see S1 File.

Type 1 opsin sequence acquisition, alignment, and phylogenetic inference

Sequences for bacterial, archaeal, and microbial eukaryotic type 1 opsins were obtained by BLASTp search of the NCBI nr and env_nr databases (as of October 30^th, 2012) with an e-value cutoff of 10⁻⁵ and maximum target sequences set to 100,000 using all Har. marismortui opsins as query sequences as described above. A total of 1,430 sequences were recovered. After removing mutants, synthetic constructs, highly fragmentary sequences and sequences lacking a predicted Bac_rhodopsin domain (Pfam clan CL0192), 907 opsin homologs remained. Sequences acquired from each database were independently aligned using MUSCLE 3.8.31 [39,40], manually trimmed to remove poorly aligned regions and to account for the fragmentary nature of metagenomic sequencing, and alignments combined using the profile-profile alignment option. After adding the 170 haloarchaeal sequences from our dataset, a total of 147 positions for 1,077 opsin sequences were used in tree inference. For these sequences, an initial guide tree was constructed using FastTree [71,72], then 1000 re-sampled alignments were generated using the Phylip package SEQBOOT [73] and used to determine bootstrap support values for clades in the initial tree with CompareToBootstrap.pl [74]. The resulting tree was visualized with FigTree [44]. The tree file is available from the Dryad Digital Repository (http://dx.doi.org/10.5061/dryad.963hr). For fully expanded tree, see S3 Fig. (red leaf labels represent sequences lacking the Schiff base lysine).

Phylogenetic distribution of haloarchaeal-type ORPs

All protein and peptide databases in CAMERA and the NCBI nr and env_nr databases (as of December 18^th, 2012) were interrogated for non-canonical opsin homologs using BLASTp searches with all 48 haloarchaeal ORPs as queries. BLASTp parameters and database details are listed in S2 Table. Redundant sequences were removed using Jalview [41] and unique sequences searched against the Pfam database [75] for presence of a Bac_rhodopsin domain (CL0192). Sequences with a Bac_rhodopsin domain were aligned using MUSCLE 3.8 [39,40] and presence or absence of K216 was recorded. To assess the evolutionary origin of the only non-haloarchaeal ORP recovered from this search (Trametes versicolor EIW60452), a phylogeny was inferred for all 170 haloarchaeal opsins and the T. versicolor ORP. Sequences were aligned using Muscle 3.8.31 [39,40] and alignment manually trimmed in Jalview [41]. After trimming, 220 positions were used in phylogenetic inference using FastTree [71,72] and the resulting tree was visualized in FigTree [44]. For tree see S4 Fig.

Confirmation of native transcription of ORPs

Liquid cultures of Hrr. litoreum JCM 13561, Hrr. distributum JCM 9910, Nab. magadii DSM 3394 and Nbt. gregoryi SP2 were grown to mid-log phase in JCM 168 (Hrr. litoreum and distributum) or DSM 371 (Nab. magadii and Nbt. gregoryi) media. Cell pellets were collected from 2 mL of mid-log phase cultures and RNA harvested using 1.0 mL TRIzol^® Reagent with standard extraction protocol followed by a DNase digestion step and a second TRIzol^® extraction with 0.5 mL Trizol^® Reagent. DNase digestion was with NEB DNase I (M0303S) using standard protocol. Following second Trizol^® extraction, RNA was tested for gDNA contamination using PCR with haloarchaeal 16S primers (S3 Table) prior to reverse-transcription. Reverse transcription was carried out with SuperScript^® III Reverse Transcriptase from Life Technologies^™ using standard protocols, and random hexamers (Qiagen, 79236). Presence of transcript was confirmed via PCR with primers listed in S3 Table. Cross-reactivity of primers for species with multiple ORP homologs was tested and primer sets found to be gene-specific. For Hrr. distributum, additional cultures were grown under the following conditions, and transcript presence verified using methods described above: a) 350 rpm, 3.42 M NaCl, b) 350 rpm, 5.0 M NaCl, c) 50 rpm, 3.42 M NaCl. Conditions (a) and (b) were incubated using a G-53 gyratory tier shaker (New Brunswick Scientific, M1074), condition (c) in an Innova 44R incubator (New Brunswick Scientific, M1282). For each condition, samples were collected at mid-log and stationary phase.

Growth of Halobacterium salinarum clones

Unless otherwise specified, Halobacterium salinarum NRC-1 and derivatives were grown in CM medium (250 g/L NaCl, 20 g/L MgSO₄ • 7H₂O, 2 g/L KCl, 3 g/L Na₃Citrate, 10 g/L Oxoid Neutralized Peptone (Oxoid, LP0037)) at 37°C in an Innova 44R incubator (New Brunswick Scientific, M1282) shaken at 175 rpm.

Cloning

In order to heterologously express His-tagged ORP proteins, genes encoding Nab. magadii WP_004267173 and Hrr. distributum ELZ45759 were PCR amplified from genomic DNA using primers listed in S3 Table in 50 μL PCR reactions: 5 μL 10X buffer, 10 μL Q solution, 2.5 μL 10 μM forward primer, 2.5 μL 10 μM reverse primer, 1.25 μL 10 mM dNTPs, 0.3 μL TAQ polymerase (Qiagen, Q201203), 0.1 μL Pfu Polymerase (Stratagene, 600153), 28.35 μL MilliQ H₂O using the following PCR cycle: 98°C 10 min, (98°C 30 sec, 55°C 1 min, 72°C 1 min 35 sec) x 25 cycles, 72°C 5 min in a Dyad Peltier Thermocycler (BioRad). Additionally, genes encoding a canonical BR (Har. marismortui YP_137573) and a colorless transmembrane protein (Hbt. sp. NRC-1 pstC2 VNG0455G), were amplified as controls from genomic DNA as described above. Amplified genes were then ligated into two expression vectors, one under the control of the NRC-1 bop promoter (pDJLCHIS) and the other under the control of the NRC-1 ferredoxin promoter (pMTFCHIS2) using the restriction enzymes NdeI (NEB, R0111S) and BamHI (NEB, R0136S for pMTFCHIS2) or HindIII (NEB, R0104S for pDJLCHIS). Plasmid sequences available from the Dryad Digital Repository (http://dx.doi.org/10.5061/dryad.963hr). Clones were transformed into chemically competent DH5α cells and plated on LB agar plates containing 100 μg/mL carbenicillin (Fisher Scientific, BP26485). Success of cloning was verified through Sanger sequencing using plasmid specific sequencing primers (S3 Table). Constructed pDJLCHIS and pMTFCHIS2 expression vectors (S6 Fig) were transformed into Hbt. sp. NRC-1 SD23 and Hbt. sp. NRC-1 SD20 strains, respectively.

Similarly, for heterologous expression in Escherichia coli, genes encoding Nab. magadii WP_004267173, Hrr. distributum ELZ45759, and Har. marismortui BR2 YP_137573 were PCR amplified from genomic DNA and cloned into pET29b+ (Novagen) using NdeI and HindIII restriction sites as described above. All cloned constructs, including an empty pET29b+ vector, were transformed into E. coli BLR(DE3) cells (Merck Millipore) for expression.

Protein-level expression of ORPs in heterologous hosts

To express ORP proteins in E. coli, all clones were grown in Luria Broth (LB) under 50 μg/mL kanamycin resistance (kan) in an Innova 44R Shaking Incubator (New Brunswick) with shaking at 175 rpm. The pET29b+/ORP, pET29b+/BR2 and pET29b+/empty BLR(DE3) clones were revived from freezer stock overnight in 4 mL of LB + kan at 37°C. Revived cells were re-cultured in 25 mL of LB + kan with a starting OD₆₀₀ of 0.1 and grown at 37°C to mid-log (OD₆₀₀ = 0.4). The mid-log subcultures were then used to inoculate 1 L LB + kan with a starting OD₆₀₀ of 0.01 and placed in the 37°C shaking incubator. Once the cultures returned to mid-log (OD₆₀₀ = 0.4), all cultures were induced with 0.5 mM IPTG (Fisher Scientific, BP1755), supplemented with 10 μM all-trans retinal (Sigma-Aldrich, R2500), and incubated at 18°C for 18 hours with shaking. Cells were harvested by centrifuging at 8000 rpm for 10 min and stored at -80°C until use.

To purify, cell pellets were thawed and re-suspended in 15 mL of solubilization buffer (100 mM Na/K phosphate pH 7.4, 2% Triton-X100, 10 μM all-trans retinal) with one cOmplete^® Mini EDTA free protease inhibitor cocktail tablet (Roche, 04693159001). Samples were sonicated three times with a Model 120 Sonic Dismembrator (Fisher Scientific, FB120) fitted with a Model CL-18 probe at 65% power alternatively for 2 seconds ON and 2 seconds OFF; for a total of 2 minutes ON. Sonicated samples were incubated on a rotisserie for 3 hours at 4°C. Cell debris was spun down by centrifugation at 8000 rpm for 10 minutes, and the supernatant was mixed with an equal volume of equilibration/wash buffer (50 mM Na⁺ phosphate, 300 mM NaCl, 10 mM imidazole; pH 7.4, 0.5% Triton-X100, 10 μM all-trans retinal). 250 μL of HisPur^™ Cobalt Resin (Thermo Scientific, 89964) was washed with 500 μL of equilibration/wash buffer and mixed with equilibrated lysate for 45 minutes in a rotisserie at 4°C. Conjugated resin was washed three times with 500 μL equilibration/wash buffer and eluted twice with 250 μL of elution buffer (50 mM Na⁺ phosphate, 300 mM NaCl, 150 mM imidazole; pH 7.4, 0.5% Triton-X100, 10 μM all-trans retinal). 250 μL of the first eluent was concentrated to ~100 μL using a Microcon 30 kDa MWCO centrifugal filter column (Millipore, 42410) and aliquoted into a Greiner Half Area UV-Star^® microplate (Greiner Bio-One, 675801). Absorbance was measured from 250–800 nm in 2 nm intervals with an Infinite M200 plate reader (Tecan, 30016056). Background (elution buffer) was subtracted from all spectra and spectra were normalized to the 280 nm absorbance value of the Har. marismortui BR2 positive control. For spectra see Fig 4C.

To verify expression of ORP protein, we performed a Western blot on HisPur^™ purified lysate from cloned ORPs in E. coli BLR(DE3) background using the following protocol: SDS-PAGE was run as described in SI Methods and the gel was soaked in 15 mL of transfer buffer (25 mM Tris base, 192 mM glycine, 10% methanol, pH 8.4) for 15 min. A 0.2 μm pore diameter nitrocellulose membrane (Biorad, 162–0112), foam pads, and 3 mm Whatman filters were soaked in transfer buffer before assembling the sandwich for transfer. Proteins were transferred from the PAGE gel to the nitrocellulose membrane at 30V for 1 hr on ice. His-tagged proteins were probed using the SuperSignal^® West HisProbe^™ Kit (Thermo Scientific^™, 15168) and signal was detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, RPN2232). For Western blot, see Fig 4B.

Protein-level expression of ORPs in native hosts

Several methods were used to investigate protein-level expression of ORP homologs in both native and heterologous hosts. First, we attempted identification of ORPs by LC-MS/MS in the native host Nbt. gregoryi SP2, which has four unique ORP genes. Cell pellets collected from mid-log phase cultures were lysed with 200 μL lysis buffer containing 100 mg SDS, 10 mL diH₂O, 60 μL DNase I (GoldBio D-300-1), 0.25 mg RNase A (Roche, 10109169001), and 1 cOmplete^® Mini EDTA free protease inhibitor cocktail tablet (Roche, 04693159001) per 15 mL buffer. Lysate was sonicated for 20 minutes (30 s on/off) on a Biorupter^® UCD-200 (Diagenode). Cellular debris was removed by centrifugation, and supernatant denatured for 10 min at 100°C with 6x SDS buffer. Approximately 40 μg protein was loaded into pre-poured 4–20% SDS-PAGE gel (Bionexus Inc., 2BNPC420) and run approximately 1 cm into gel. Total protein band was excised and subjected to in-gel trypsin digest according to the following protocol: gel was cut into 1 mm³ pieces, washed with 50 mM Ammonium Bicarbonate (AmBic), shrunk with acetonitrile (ACN), reduced with 10 mM DTT/50 mM AmBic, shrunk again with ACN, incubated in 55 mM iodoacetamide/50mM AmBic 20 min in the dark, washed with 50 mM AmBic, shrunk with ACN and partially dried in a vacuum concentrator (Labconco). Overnight digestion was carried out at 37°C with 250 ng of trypsin (Promega, V5117) in 50 mM AmBic (pH 8). The supernatant was sonicated in 60% ACN and 0.1% trifluoroacetic acid for 10 min, then dried in the vacuum concentrator. Digested peptides were analyzed by LC-MS/MS on a Thermo Q-Exactive mass spectrometer with Michrom Paradigm LC and CTC Pal autosampler. Peptides were directly loaded onto an Agilent ZORBAX 300SB C₁₈ reversed phase trap cartridge, which, after loading, was switched in-line with a Michrom Magic C₁₈ AQ 200 um x 150 mm C₁₈ column connected to a Thermo-Finnigan LTQ iontrap mass spectrometer through a Michrom Advance Plug and Play nano-spray source. The nano-LC column (Michrom 3μ 200Å MAGIC C18AQ 200μ x 150 mm) was used with a 90 min-long gradient (1–10% buffer B in 5 min, 10–35% buffer B in 65 min, 35–70% buffer B in 5 min, 70% buffer in 1 min, 1% buffer B in 14 min) at a flow rate of 2 uL min^-1 for the maximum separation of tryptic peptides. A top 15 method was used with Xcalibur software to collect Q-Exactive data, with a scan range of 300–1600 m/z. Results were searched against a Nbt. gregoryi database with cRAP proteins and reversed sequences (5402 proteins total) in X!Tandem [76] with a fragment ion mass tolerance of 20 PPM and a parent ion tolerance of 20 PPM. Carbamidomethyl of cysteine was specified in X!Tandem as a fixed modification. Glu->pyro-Glu of the N-terminus, ammonia-loss of the N-terminus, gln->pyro-Glu of the N-terminus, deamidation of asparagine and glutamine, oxidation of methionine and tryptophan, dioxidation of methionine and tryptophan and acetylation of the N-terminus were specified in X!Tandem as variable modifications. Scaffold v.4.0.7 [77] was used to validate peptide and protein identifications. Peptide identifications were accepted if they could be identified with confidence of greater than or equal to 95% and protein identifications were accepted if they could be identified with confidence of greater than or equal to 95% and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [78]. Proteins that contained similar peptides and could not be differentiated were grouped. Proteins sharing significant peptide evidence were grouped into clusters. No ORP proteins were identified from Nbt. gregoryi SP2 lysate.

After failing to detect ORP protein in a native host, we purified His-tagged cloned proteins from a Hbt. sp. NRC-1 SD23 bop(-) background using HisPur^® Cobalt resin (Thermo Scientific^™, 89965). Exudate was loaded onto a 4–20% SDS-PAGE gel as described above, and size separated. Based upon presence of band in the three experimental samples and absence in Hbt. sp. NRC-1 SD23 control lacking expression vector, the region of the gel corresponding to 50 kDa MW proteins was excised and prepared for proteomics as described above. No ORP proteins were detected.

We next performed Western blot on lysate from cloned ORPs in Hbt. sp. NRC-1 SD23 bop(-) background using the following protocol. SDS-PAGE was run as described above and gel soaked in 15 mL of transfer buffer (25 mM Tris base, 192 mM glycine, 10% methanol, pH 8.4) for 15 min. PVDF membrane (Novex, LC2002) was successively soaked in 100% methanol, MilliQ^™ H₂O, and transfer buffer. Foam pads and 3mm Whatman filters were also soaked in transfer buffer. Western gel was run at 30V for 1 hr. His-tagged proteins were probed using the SuperSignal^® West HisProbe^™ Kit (Thermo Scientific^™, 15168). Based upon presence of two bands at ~15 and ~20 kDa in the three experimental samples, the region of the gel corresponding to 13–25 kDa was extracted and prepared for proteomics as described above. No ORPs were detected, however, the three opsin proteins present in the host (HR, SRI, and SRII) were observed, indicating the region of the gel used was the correct MW range for opsins. Over 900 proteins were identified with a protein false discovery rate of 1.7% and peptide false discovery rate of 0.36%, using a Hbt. sp. NRC-1 database with ORPs added.