Spectral properties of bacteriophytochrome AM1_5894 in the chlorophyll d-containing cyanobacterium Acaryochloris marina

Acaryochloris marina, a unicellular oxygenic photosynthetic cyanobacterium, has uniquely adapted to far-red light-enriched environments using red-shifted chlorophyll d. To understand red-light use in Acaryochloris, the genome of this cyanobacterium was searched for red/far-red light photoreceptors from the phytochrome family, resulting in identification of a putative bacteriophytochrome AM1_5894. AM1_5894 contains three standard domains of photosensory components as well as a putative C-terminal signal transduction component consisting of a histidine kinase and receiver domain. The photosensory domains of AM1_5894 autocatalytically assemble with biliverdin in a covalent fashion. This assembled AM1_5894 shows the typical photoreversible conversion of bacterial phytochromes with a ground-state red-light absorbing (Pr) form with λBV max[Pr] 705 nm, and a red-light inducible far-red light absorbing (Pfr) form with λBV max[Pfr] 758 nm. Surprisingly, AM1_5894 also autocatalytically assembles with phycocyanobilin, involving photoreversible conversion of λPCB max[Pr] 682 nm and λPCB max[Pfr] 734 nm, respectively. Our results suggest phycocyanobilin is also covalently bound to AM1_5894, while mutation of a cysteine residue (Cys11Ser) abolishes this covalent binding. The physiological function of AM1_5894 in cyanobacteria containing red-shifted chlorophylls is discussed.


Results
Protein domain comparison. The AM1_5894 gene comprises 2544 nucleotides and encodes an 847-amino acid protein product, having a predicted molecular weight of 94.2 kDa. The overall domain structure of AM1_5894 is typical of other bacteriophytochromes, with a photosensory core module of PAS-GAF-PHY as well as a C-terminal signal transduction domain, consisting of a histidine kinase; and a receiver domain (Fig. 1). The PAS domain contains a Cys residue at its eleventh position that aligns with the conserved BV-binding Cys of typical BphPs. It also has a His residue (H249) in its GAF domain that is conserved (Fig. 1). The adjacent PCB/ PΦ B-binding Cys found in the GAF domain of cyanobacterial and plant phytochromes is absent in AM1_5894 (Fig. 1). AM1_5894 is more closely related to BphPs from non-photosynthetic and anoxygenic photosynthetic bacteria than cyanobacterial phytochromes (Cph), based on alignment of their GAF domains 40 . Extending this alignment to include both the GAF and PHY domains confirms that AM1_5894 is phylogenetically separate from other Cphs and cBphPs, apart from the BphPs from another Acaryochloris ecotype, CCMEE 5410, and the filamentous cyanobacterium Leptolyngbya PCC 7375 ( Figure S1). Unusually, the signal output domain of this cluster of three BphPs consists of a HWE (histidine-tyrosine-glutamate)-type histidine kinase and a receiver domain, which is a typical domain structure of Bathy BphPs. Bathy BphPs are most commonly found in Rhizobiales 10,41 . Although Acaryochloris MBIC 11017 does not contain any Cph-like phytochromes, a number of putative CBCRs, including the recently characterized AM1_1186, AM1_1870 and AM1_1557, are present [37][38][39] .
Scientific RepoRts | 6:27547 | DOI: 10.1038/srep27547 Expression of recombinant AM1_5894. Initial attempts to clone and express the full-length AM1_5894 were largely unsuccessful. A clone with a point mutation in its histidine kinase domain causing a Gly639 → Asp substitution was obtained, although only a small amount of soluble protein showing typical bacteriophytochrome spectral characteristics could be expressed in Escherichia coli ( Figure S2). Instead, a truncated construct was expressed in E. coli. This truncated protein (AM1_5894∆ HK) is 508-amino acids in length, compared with the full-length protein of 847 amino acids. AM1_5894∆ HK contains PAS, GAF and PHY domains that are essential for chromophore attachment and photochromism [42][43][44][45] , but lacks the output module, including the histidine kinase and receiver domain (Fig. 1). AM1_5894∆ HK was His-tag purified almost to homogeneity and used to examine the chromospectral properties of the AM1_5894 holoprotein.
Chromophore-protein Reconstitution. Based on sequence analysis, AM1_5894 was predicted to covalently bind BV, but not PCB because of a conserved Cys residue in its PAS domain, and lack of a conserved PCB/PΦ B-binding Cys residue in its GAF domain (Fig. 1). To confirm this prediction, overexpressed full-length AM1_5894D639G and truncated AM1_5894∆ HK were incubated in the absence or presence of either BV or PCB to facilitate autocatalytic attachment of the chromophore. Surprisingly, both reconstituted BV-and PCB-AM1_5894 protein complexes exhibited Zn-induced fluorescence on SDS-polyacrylamide gels (Fig. 2a). This demonstrates that both BV and PCB are covalently bound to AM1_5894 in vitro. Due to the low yields and point mutation of our full-length AM1_5894 clone, the truncated construct (AM1_5894∆ HK) was used for chromospectral analyses of AM1_5894.
In order to avoid any bias that may be generated by self-aggregated chromophores, either BV or PCB was added directly to lysed E. coli containing overexpressed AM1_5894∆ HK before His-tag purification. Further purification was carried out using non-denaturing size exclusion chromatography (Fig. 2b). Online spectra of the triangle; named after period circadian protein, aryl hydrocarbon receptor nuclear translocator protein and single-minded protein) that covalently binds BV in bacteriophytochrome (BphP) and cyanobacterial BV-binding bacteriophytochrome (cBphP) and the conserved Cys residue in the GAF domain (closed triangle; named after cGMP-specific phosphodiesterases, adenylyl cyclases and FhlA proteins) that binds PCB or phytochromobilin (PΦ B) in proteins in cyanobacterial phytochromes (Cphs) and plant phytochromes (Phys), respectively. The his residue adjacent this position which is conserved in most phytochromes is also marked (closed circle). HWE (histidine-tyrosine-glutamate -type histidine kinase(HK); HA_c (Histidine kinase-like ATPase c-terminal region); REC (cheY-like receiver domain). For other abbreviations refer to the text. Additional sequence information is given in Supplementary  chromophore-binding protein complexes confirmed the elution peak at 7.8 min corresponded to the assembled holoproteins, with the same spectra observed in vitro (Figs 2c and 3).
Spectral characterization. The absorption spectrum of dark-assembled BV-AM1_5894Δ HK resembles that of a typical phytochrome in its Pr state (Fig. 3a). It has two main absorption peaks centred at 380 and 705 nm, with a shoulder at approximately 650 nm (Fig. 3a). Upon illumination with red light (665 nm), there is a reduction in the 705 nm peak, accompanied by the appearance of a longer wavelength peak at 758 nm, which is indicative of the Pfr form (dashed line, Fig. 3a). Illumination of the Pfr form with far-red light (777 nm) or dark incubation causes AM1_5894 to switch back to its Pr state. The difference spectrum of BV-AM1_5894Δ HK Pfr minus Pr demonstrates a 378 nm minimum and 422 nm maximum peaks in the blue region, and 705 nm minimum and 758 nm maximum peaks in the far-red region (Fig. 3a). We found it was not possible to completely switch BV-AM1_5894Δ HK to its Pfr state, and the ratio between Pr and Pfr states remained stable after 15 min (Fig. 4a). The Pfr-state protein rapidly reverted back to its Pr state when exposed to far-red light (777 nm) (Fig. 4b) or incubated in the dark.
After dark assembly with the chromophore PCB, the AM1_5894Δ HK spectrum shares a similar, but blue-shifted peak structure to that of BV-AM1_5894Δ HK (Fig. 3). PCB-AM1_5894Δ HK has major peaks at 370 nm and 682 nm and a shoulder at approximately 625 nm (Fig. 3b). Illumination with red light initiates a partial switch to the Pfr state of PCB-AM1_5894Δ HK, with a maximum at 734 nm. The difference spectrum of Pfr minus Pr shows similar profiles and 368 nm minimum and 412 nm maximum peaks in the blue light region and 682 nm minimum and 734 nm maximum peaks in the red/far-red light region (Fig. 3b). By normalising at an absorbance of 280 nm, the absorbance intensity at 705 nm for BV-AM1_5894Δ HK is higher than that at 682 nm for PCB-AM1_5894Δ HK (Fig. 2c), indicative of a lower binding affinity of AM1_5894Δ HK for PCB compared with BV.
Like the BV-holoprotein, the PCB-holoprotein also could not be entirely switched to its Pfr state (Fig. 5). However, the switching kinetics between Pr-Pfr and Pfr-Pr were very different between BV-AM1_5894Δ HK and the PCB-AM1_5894Δ HK (Figs 4 and 5). The Pr-Pfr switching of BV-AM1_5894Δ HK was slow with a halflife of ~3.5 minutes while the Pfr-Pr switching was fast with a halflife of less than 5 sec (Fig. 4). Unlike the BV-AM1_5894Δ HK, the switching kinetics of PCB-AM1_5894Δ HK was approximately symmetric with one another, whether switching from Pr to Pfr or vice versa with a halflife of approximately 10 sec for the conversion (Fig. 5).

Differences in dark relaxation and photoactive light range.
Dark relaxation and the effects of different light wavelengths on the switching of the Pfr form of AM1_5894 to its Pr state were examined in detail for BV-AM1_5894Δ HK and PCB-AM1_5894Δ HK using different wavelength light emitting diodes (LEDs) (Fig. 6a,b, Figure S3). Maximal Pfr to Pr switching for both BV-and PCB-bound AM1_5894Δ HK was observed when exposed to far-red light (777 nm), with a similar rate of switching observed for PCB-AM1_5894Δ HK exposed to 735 nm light. Dark relaxation was rapid (approximately 85% complete after 30 s) for BV-AM1_5894Δ HK, with rates similar to those observed when it was exposed to 777 nm light, while exposure to other light wavelengths inhibited this switching (Fig. 6a). In contrast, there was only minimal dark relaxation observed for PCB-AM1_5894Δ HK after 60 s. The dark reversion of BV-AM1_5894Δ HK is faster than PCB-AM1_5894Δ HK. The low ratio of bound PCB in reconstituted PCB-AM1_5894Δ HK complexes (Fig. 2c) and the slower dark reversion (Fig. 6) support the fact that, as predicted, the BV-binding form is of the common chromophore-binding structure seen for BphPs, although it may also facilitate PCB-binding (Fig. 1).
The switching of PCB-AM1_5894Δ HK from its Pr to its Pfr state was more sensitive to light wavelength than BV-AM1_5894Δ HK. For both, 665 nm light was most efficient at inducing Pr to switch to Pfr (Fig. 6c,d). However, 710 nm and 638 nm incident light were similarly effective for BV-AM1_5894Δ HK (Fig. 6c). Unexpectedly, 450 nm light partially switches BV-AM1_5894Δ HK from Pr to Pfr states, as illustrated by both this   switching and the slight reduction in the Pfr to Pr switching when comparing 450 nm-exposed BV-AM1_5894Δ HK to that left in the dark (Fig. 6a,c).
It is known that a conserved Cys residue in the PAS domain of BphPs (Cys11 for AM1_5894Δ HK, Fig. 1) is the position for covalent binding of BV 5 . To determine whether Cys11 was involved in binding PCB, as well as BV, we constructed a C11S-AM1_5894Δ HK mutant, then BV or PCB was added to lysed E. coli containing overexpressed C11S-AM1_5894∆ HK before His-tag purification. Under non-denaturing conditions, both BV and PCB seemed to elute with protein together during size exclusion chromatographic purification. However, the BV-associated complex appears much weaker than for WT AM1_5894Δ HK with a very low chromophore ratio (Fig. 7c). Although there were protein/chromophore complexes with a similar retention time to that of the WT for PCB, the spectrum of PCB-C11S-AM1_5894Δ HK was different, with an extra peak at around 555 nm, likely to be "unspecified" protein-chromophore complexes (Fig. 7b). However, no Zn-induced fluorescence was observed on the SDS-polyacrylamide gel for the C11S-AM1_5894Δ HK mutant incubated with either BV or PCB (Fig. 7a,b). Thus, our data demonstrate that Cys11 of AM1_5894 is essential for covalent binding of both BV and PCB. Both chromophores showed non-covalently associated with the C11S-AM1_5894Δ HK mutant (Fig. 7b,c), but this association was unstable. We were unable to detect any photoconversion in this mutant, associated with either BV or PCB ( Figure S4).

Discussion
Acaryochloris uses red-shifted Chl d to capture light for photosynthesis in red/far-red light enriched environments 22 . To optimize the efficiency of light capture, Acaryochloris dynamically regulates chlorophyll-and phycobiliprotein-based light-harvesting complexes in a manner dependent upon light intensity and quality 30,36 . Here, we characterized the spectral properties of AM1_5894, a newly described BphP in Acaryochloris. The domain architecture of AM1_5894 resembles that of other canonical BphPs, having an N-terminal photosensory core module of PAS-GAF-PHY and a C-terminal signal transduction domain (Fig. 1). However, GAF/ PHY domains are phylogenetically similar to Bathy BphPs from soil dwelling bacteria ( Figure S1). Almost all these Bathy BphPs have a Pfr ground state, suggesting that these soil dwelling diazotrophs use these BphPs for a non-standard response to the perception of red light 10 . Although AM1_5894 appears related to Bathy Bphs, it has a photocycle that resembles the majority of characterized phytochromes, having a Pr ground state that switches to the Pfr light-activated state when exposed to red light. AM1_5894 and Bathy BphPs also share the same signal output domain structure, with an HWE-type histidine kinase and a C-terminal receiver domain (Fig. 1). These characteristics place AM1_5894 in a cluster of BphPs termed type 2 ( Figure S1) 10 and as only type 2 BphPs share an HWE-type histidine kinase domain they are thought to share a common evolutionary origin, which is   12 . The ability to use different chromophores of AM1_5894Δ HK in combination with recently identified CBCRs in Acaryochloris provides the molecular basis for Acaryochloris to thrive in different ecological niches, especially in diverse light environments.
Based on sequence alignments of the GAF and PHY domains of AM1_5894 with other bacterial and plant phytochromes, we initially hypothesized that AM1_5894 would covalently bind BV, but not necessarily PCB (Fig. 1). To our surprise, Zn-induced fluorescence on SDS-polyacrylamide gels suggests that AM1_5894 forms a covalent attachment to PCB as well as BV. Our experiments with a C11S-AM1_5894Δ HK mutant suggested that PCB is covalently bound to Cys 11 of AM1_5894, although the binding mechanism is unclear (Fig. 2). The GAF domains from two CBCRs from Acaryochloris have also been suggested to covalently bind PCB and BV based on Zn-induced fluorescence on SDS-polyacrylamide gels [37][38][39] . Other BphPs from non-photosynthetic bacteria, such as Deinococcus radiodurans, Pseudomonas syringae and P. aeruginosa, also bind PCB (and PΦ B) covalently, as well as their natural chromophore BV 37,38 . But since they do not produce PCB or PΦ B, the covalent binding of PCB to these Bphs is not physiologically relevant.
When bound to BV, their spectra and Pr/Pfr switching spectral properties resemble those of BphPs, under more red-shifted sensory light wavelengths. The reconstituted BV-AM1_5894Δ HK protein complexes have Pr/Pfr absorption maxima of 705 and 758 nm, respectively. These are ~20 nm-red-shifted compared to the PCB-AM1_5894Δ HK Pr/Pfr absorption maxima of 682 and 734 nm. The cBphP from Fremyella diplosiphone has similar Pr/Pfr spectral profiles, with a protein domain structure resembling that of AM1_5894, i.e., a Cys residue in its PAS domain and no canonical Cys residue in the GAF domain that covalently binds BV but not PCB 39 . Mutational analysis suggests that the BV-binding Cys residue in the PAS domain (C24) was not the site of attachment 46 . In fact, mutation of the His residue (H267) located adjacent to the canonical PCB/PΦ B binding site markedly affected BV-binding and spectral properties 5,47 . These data contrast with mutational analysis of BphPs in Pseudomonas aeruginosa, where mutation of the Cys residue in the PAS domain prevents covalent attachment of BV, while mutation of the corresponding conserved His residue does not 46 . The non-covalent attachment of PCB maintains a more conjugated chromophore, which could explain the red-shifted spectral properties in PCB-binding in cBphPs from Fremyella diplosiphone, compared with other PCB-phytochrome holoproteins. However, the abolition of the covalent attachment of PCB to the C11SAM1_5894Δ HK mutant indicates that this cysteine residue is the site of the PCB covalent binding (Fig. 7).
Acaryochloris is able to dynamically regulate its light-harvesting mechanism in response to different light conditions, reducing the level of phycobiliproteins (which absorb light at around 640 nm) in response to high levels of white light or under far-red (720 nm) light 30,36 . Under such conditions, phycobiliproteins are either unnecessary for light capture or unable to capture far-red light, respectively. We have shown that BV-AM1_5894 can switch from its Pr ground state to its Pfr light-activated state using light sources between 625-708 nm. It efficiently switches back to its ground state using light sources between 730-780 nm. The light wavelength range between 710-730 nm is physiologically relevant to Acaryochloris, since Chl d-protein complexes absorb maximally within this range in vivo. The presence of PCB-AM1_5894 in addition to CBCRs found in Acaryochloris provide it with an effective method of measuring changes in the light spectrum relevant to its photosynthetic absorption range, allowing it to regulate target gene expression. The ability of AM1_5894 to covalently bind both BV and PCB in vitro and possibly in vivo (both being present in Acaryochloris) offers much broader light detection compared with other phytochromes, giving an adaptive advantage to Acaryochloris in its niche environments.
Extending the search for GAF domains beyond typical Cphs and BphPs in the Acaryochloris genome identified at least 11 genes that encode CBCR-like GAF domains. To date, GAF domains from AM1_1186, AM1_1870 and AM1_1557, have been characterized [37][38][39] . The GAF domain of AM1_1186 binds PCB and acts as a so-called dual-Cys CBCR with a red/blue light photocycle 38 . A GAF domain from AM1_1557 (GAF domain 2) and AM1_1870 (GAF domain 3) both covalently bind PCB and BV. For both AM1_1552g2 and AM1_1870g3 bound to PCB, a red-green photocycle was observed, however this shifted to a far red-orange photocycle when PCB was replaced with BV 37,39 . The BV-bound recombinants have red-shifted absorption peaks at 697 nm for BV-AM1_1557g2 39 and 706 nm for BV-AM1_1870g3 37 . Although the spectral range in which CBCRs function is considerably greater than for other phytochromes, including BphPs, it is likely that the red/far-red photocycle of AM1_5894 best overlaps with the photosynthetically-relevant spectral range for Acaryochloris, making it a better candidate for regulating the photoacclimation exhibited by Acaryochloris.

Experimental Procedures
Expression and purification of recombinant AmrBphP. One canonical phytochrome, AM1_5894, was identified from the Acaryochloris marina MBIC11017 genome. The 2544 bp AM1_5894 open reading frame was amplified from genomic DNA of Acaryochloris using the following primers to allow In-Fusion ® (Clontech, CA, USA) cloning (forward: 5′ -CGCGCGGCAGCCATATGGAAATTAGAGAACTAGCGATTT-3′ , reverse: 5′ -GTTAGCAGCCGGATCCTTAGGCTAAGAGTTGATGAATG-3′ ). The underlined sequences are 5′ overhangs specific to the E. coli expression vector pET15b, which introduces a hexa-histidine tag to the expressed protein. A clone harbouring a full length AM1_5894 gene with a single amino acid substitution of Gly639 → Asp is designated as full-length AM1_5894D639G was obtained. To improve the protein overexpression yield, the 1524 bp N-terminal photosensory domain (AM1_5894Δ HK, lacking its histidine kinase and receiver domains) was amplified from Acaryochloris genomic DNA, using Phusion polymerase (New England Biolabs Ltd., UK) and the primer pair AmBph_ITA_FW (GCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGAAATTAGAGAACTAGCGATTTCT) and AmBph_ ITA_RV (CCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGTTATTCCTGCTGCTGGTTTTCTCC). The underlined 3′ sequences correspond to pET15b sequences, which allowed assembly of the PCR product into pET15b, using a Gibson Assembly ® master mix (New England Biolabs). A Cys11Ser mutant of AM1_5894Δ HK (C11S-AM1_5894Δ HK) was synthesized as a gBlock (Integrated DNA Technologies, Corlaville, IA, USA), with the overlapping pET15b sequence. It was also assembled into pET15b using a Gibson Assembly ® master mix (New England Biolabs). The resulting three constructs were used to transform DH5α E. coli cells. The fidelity of all clones (including the point mutation that caused the Gly639Asp mutation in the full length clone) was confirmed before being used to transform Rosetta E. coli cells.
Rosetta E. coli (BL21, DE3) cultures harbouring the pET15b constructs were grown at 37 °C for 1 h and then at 21 °C until OD 600 = 0.5 in LB media, supplemented with ampicillin and chloramphenicol at 100 μ g/ml and 35 μ g/ml, respectively. Expression was induced by the addition of 330 μ M Isopropyl β -D-1-thiogalactopyranoside (IPTG) and cultures were grown at 10 °C for 48 h to limit aggregation of the expressed protein. The cells were harvested by centrifugation and the cell pellet resuspended in binding buffer (50 mM potassium phosphate buffer, pH 8.0, 10% (v/v) glycerol,) prior to cell lysis. Cells were lysed using a pre-chilled French Pressure Cell (GlenMills, NJ, USA) at approximately 1500 psi. His-tagged AM1_5894 protein was purified from the clarified crude protein extract using Scientific RepoRts | 6:27547 | DOI: 10.1038/srep27547 a His Gravitrap column (GE Healthcare, Sweden) and eluted with 500 mM imidazole. In some instances, BV or PCB was added to the crude protein extract prior to purification. Eluted protein was concentrated using a 3 kDa cut-off protein concentrator (Merck Millipore, MA, USA) and rinsed in 50 mM potassium phosphate buffer in order to dilute imidazole concentration to the final of < 50 mM.
Chromophore reconstitution. Overexpressed AM1_5894Δ HK or full-length AM1_5894 (30 μ M concentration of crude extract) was reconstituted in vitro with 100 μ M of either BV (Frontier Scientific, USA) or PCB. PCB was isolated from powdered Spirulina (Lifestream International, NZ) by a modified methanolysis method 48 . PCB concentrations were determined spectroscopically using an extinction coefficient (ε 690nm ) of 37,900 M −1 cm −1 in methanol/5%HCl 49,50 . For confirmation of covalent binding between the chromophore and AM1_5894 or AM1_5894Δ HK, reconstituted chromophore-protein complexes were analyzed by size exclusion chromatography using a BioSec2000 column (300 × 4.6 mm, Phenomenex) on a Shimadzu HPLC (25 mM phosphate buffer with pH 8.0 as the mobile phase, with a flow rate of 0.2 ml/min). The assembled BV-AM1_5894Δ HK or PCB-AM1_5894Δ HK was separated by SDS-polyacrylamide gel and Zn-induced fluorescence was monitored to detect protein-bound chromophores, after incubating the gel in 1 mM ZnCl 2 for 10 min. Total protein composition for both was visualized with Coomassie Brilliant Blue staining.
Spectral characterization for photoconversion in vitro. All UV/visible absorption spectra were recorded with a UV2550 spectrophotometer (Shimadzu) at room temperature. For spectral analysis, approximately 30 μ M His-tag purified AM1_5894Δ HK (as determined by its absorbance at 280 nm) was mixed with BV or PCB at a final concentration of 20 μ M and the assembly was monitored by measuring the increase in absorbance at 705 nm or 682 nm, respectively. The kinetics of switching between the Pr (a red-light absorbing form) and Pfr (a far red-light absorbing form) states of BV-AM1_5894Δ HK or PCB-AM1_5894Δ HK were monitored over time by measuring spectral changes on exposure to either 665 nm (Pfr to Pr switch) or 777 nm (Pr to Pfr) monowavelength LEDs ( Figure S3). All LED applied in this experiment are dual in-line LEDs (DIP LED) powered by 1.5 V DC and 10-20 mA. The light intensity of the 665 nm LED is in the range between 3 and 8 μ mol photons m −2 s −1 monitored by LI-COR light meter.
The optimal photoactive wavelengths for AM1_5894 were examined by exposure of the Pfr AM1_5894Δ HK bound to BV or PCB to either darkness or a series of narrow bandwidth LEDs (450, 522, 710, 735 and 777 nm). To determine the optimal wavelengths for Pr to Pfr conversion, the Pr state of BV-AM1_5894Δ HK or PCB-AM1_5894Δ HK also was illuminated with a series of narrow bandwidth LEDs (450, 522, 597, 638, 665, 710, and 735 nm). Emission spectra for these LEDs are shown in Figure S3. The times of exposure for switching are based on the kinetics of switching, corresponding to just prior to 'saturation' of switching. To study reversion back to its initial state, purified AM1_5894Δ HK protein were exposed to either 665 nm or 777 nm light for at least 1 min, with reversion experiments conducted 5 min apart. BV-AM1_5894Δ HK reversion from its Pr to Pfr state was activated by exposures of at least 15 min.