Bacteriophytochromes: Phytochrome-Like Photoreceptors from Nonphotosynthetic Eubacteria

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Science  24 Dec 1999:
Vol. 286, Issue 5449, pp. 2517-2520
DOI: 10.1126/science.286.5449.2517


Phytochromes are a family of photoreceptors used by green plants to entrain their development to the light environment. The distribution of these chromoproteins has been expanded beyond photoautotrophs with the discovery of phytochrome-like proteins in the nonphotosynthetic eubacteria Deinococcus radiodurans andPseudomonas aeruginosa. Like plant phytochromes, theD. radiodurans receptor covalently binds linear tetrapyrroles autocatalytically to generate a photochromic holoprotein. However, the attachment site is distinct, using a histidine to potentially form a Schiff base linkage. Sequence homology and mutational analysis suggest that D. radioduransbacteriophytochrome functions as a light-regulated histidine kinase, which helps protect the bacterium from visible light.

The phytochrome family of dimeric photoreceptors regulates growth and development by sensing ambient light through the photointerconversion between an inactive red-light (R)–absorbing form and an active far-red-light (FR)–absorbing form (1). Although previously thought to be restricted to higher plants, the recent detection of phytochrome-like proteins in lower plants, algae, cyanobacteria, and purple bacteria suggests that all photosynthetic organisms contain phytochrome (2–4). The mechanism of action of the higher-plant phytochromes is still unclear, but recent studies suggest that they act as light-regulated protein kinases (5). This possibility has been strongly supported by sequence comparisons of the phytochrome-like proteins RcaE and Cph1 from the cyanobacteriaFremyella diplosiphon and Synechocystis sp. 6803, respectively (4, 6, 7), and Ppr from the purple bacterium Rhodospirillum centenum(3). These proteins contain a domain homologous to the chromophore-binding pocket of higher-plant phytochromes attached to a domain common among two-component histidine kinases (8). Based on the view that a photosynthetic bacterium is the progenitor of plant chloroplasts, it has been speculated that these prokaryotic genes represent the evolutionary origins of plant phytochromes (2,3).

Here, we show that phytochrome-like receptors are also present within several nonphotosynthetic organisms, with the discovery of related sequences in the heterotrophic eubacteria Deinococcus radiodurans and Pseudomonas aeruginosa. These genes, designated BphP for bacteriophytochrome photoreceptor, were discovered by scanning genomic databases for coding regions similar to those of phytochromes (9). The encoded D. radiodurans BphP (DrBphP) and P. aeruginosaBphP (PaBphP) proteins are 755 and 728 amino acids, respectively, with an overall amino acid sequence identity of 37% and a similarity of 48% to each other (10). Their NH2-terminal ∼500 amino acids are similar to the chromophore-binding region of Cph1, RcaE, Ppr, and plant phytochromes (Fig. 1, A and B). One important distinction is that both BphPs, like Ppr and possibly RcaE, do not contain the positionally conserved cysteine that is considered essential for autocatalytically linking the linear tetrapyrrole chromophore through a thioether bond (1, 11). Like the photosynthetic-bacteria sequences, the COOH-terminal ∼250 amino acids of DrBphP and PaBphP are related to histidine kinase domains present in environmental sensors that function as two-component regulators (8) (Fig. 1, A and B). This region contains the four conserved motifs that compose the catalytic center, including a positionally conserved histidine that serves as the phosphorylation site (Fig. 1A).

Figure 1

Relations among phytochromes and phytochrome-like proteins. (A) Amino acid sequence alignments of domains conserved among the phytochrome-like proteins ofDrBphP, PaBphP, SynechocystisCph1 (GenBank accession number AB001339), R. centenum Ppr (GenBank accession number AF064527), F. diplosiphon RcaE (GenBank accession number U59741), and the plant phytochrome Arabidopsis thaliana phyB (GenBank accession number X17342) (9). Boxes H, N, D/F, and G identify catalytic motifs that are common among two-component histidine kinases (8); the star identifies the conserved histidine residue essential for phosphate donation. Solid and open triangles identify the positionally conserved His260 and Cys259 that serve as a chromophore-binding site in DrBphP and in higher-plant phytochromes, respectively. The open diamond locates a second conserved cysteine in DrBphP (Cys289) that is important for spectral integrity but not essential for chromophore attachment. Numbering of the amino acids is based on DrBphP. Reverse type and gray boxes denote identical and similar amino acids, respectively. (B) DOTPLOT illustrating three conserved regions in DrBphP as compared to those from Synechocystis Cph1. I, an NH2-terminal domain of unknown function; II, the chromophore-binding pocket; and III, the histidine kinase motif.

Given the NH2-terminal sequence homology, we predicted that these BphPs would associate with linear tetrapyrroles to generate R/FR photoreversible chromoproteins. Despite the absence of the conserved cysteine, recombinant DrBphP apoprotein covalently bound phytochromobilin (PΦB) or phycocyanobilin (PCB) in vitro, as demonstrated by zinc-induced autofluorescence of the complex following SDS–polyacrylamide gel electrophoresis (PAGE) (Fig. 2A). In fact, the fluorescence intensity of these adducts rivaled that from a similar amount of oat phytochrome A (phyA) assembled in planta (12) (Fig. 2A), implying thatDrBphP binds chromophore as efficiently as plant phytochromes. Analysis of the PCB-DrBphP holoprotein by electrospray-ionization mass spectrometry (MS) following reversed-phase liquid chromatography (LC) showed that it, like plant phytochromes, bound only one chromophore per polypeptide (13). The assembled PCB-DrBphP complex had a mass ∼580 daltons greater than that of the apoprotein, a mass difference equal to that of a single PCB chromophore (586 daltons) (Fig. 3, A and B).

Figure 2

In vitro assembly and spectral properties of wild-type and mutated versions of DrBphP assembled with PΦB or PCB. (A and C) The reaction products subjected to SDS-PAGE and stained with Coomassie brilliant blue or analyzed for zinc-induced fluorescence of the bilin chromophore. Oat phyA represents phytochrome assembled in planta and purified from etiolated seedlings (12), and Apo representsDrBphP without chromophore addition. (B and D) Difference spectroscopy of the reaction products following saturating R and FR light. Difference maxima and minima are indicated. All holoproteins in (D) were assembled with PCB.

Figure 3

Characterization of the DrBphP holoprotein assembled with PCB. Molecular-mass determination of (A) Holo-DrBphP and (B) Apo-DrBphP by LC/MS. The masses indicated with arrowheads were calculated from multiple ion species of the corresponding proteins. (C) The LC absorption profile at 374 nm of theDrBphP-PCB holoprotein after cleavage with CNBr. The peaks containing the chromopeptide and free PCB released from the peptide during CNBr treatment are indicated. The peaks under the square bracket are contaminants present in the PCB preparation used to assemble Holo-DrBphP (10). Covalent binding of PCB toDrBphP was selective and specific, because there was no apparent binding of other ultraviolet-absorbing contaminants (10). (D) The MS spectrum showing the chromopeptide ion (arrowhead) identified it as a PCB-histidyl-homoserine lactone predicted to be released from the shown amino acid sequence (S, Ser; P, Pro; M, Met; H, His; Q, Gln; Y, Tyr; and L, Leu). (E) Mutation of His260 blocks chromophore attachment. Wild-type (WT) and H260A versions of the DrBphP apoprotein were incubated with PCB and analyzed as in Fig. 2.

Once assembled with either PΦB or PCB, the DrBphP holoprotein became photochromic, capable of repeated photointerconversions between a R- and a FR-absorbing form (12) (Fig. 2B). The absorbance-difference spectrum of the holoprotein resembled that of Cph1 (4, 6) and plant phytochromes (1, 11), but with a slight blue shift for the FR maximum [698 nm for DrBphP versus 709 nm for plant phytochrome (11) assembled with PCB] (Fig. 2B). A sizable photoreversible difference between 500 and 590 nm (Fig. 2B) was apparent, suggesting that the FR-absorbing form ofDrBphP holoprotein also absorbs green light.

The ability of DrBphP to covalently attach chromophore despite the absence of a cysteine residue at position 259 suggested that DrBphP (and likely PaBphP as well) binds chromophore by a different linkage. To test whether Met259 binds chromophore, using its sulfonyl moiety, we substituted this residue with alanine, which would block a thioether linkage (1, 11), or with cysteine, the consensus ligation site (1), and examined whether these mutant proteins could bind chromophore (14). The Met259 → Ala259 (M259A) and the Met259 → Cys259 (M259C) mutants covalently bound PCB to generate R/FR photoreversible chromoproteins, eliminating Met259 as the ligation site (Fig. 2, C and D). However, the difference spectra obtained with these mutants were altered from that of wild-type DrBphP, indicating that Met259 is necessary for chromoprotein integrity. Another potential chromophore-binding site was Cys289, the only other cysteine conserved in the NH2-terminal region of most phytochrome and phytochrome-like sequences (Fig. 1A). The Cys289 → Ala289 (C289A) mutant (14) also bound PCB covalently to generate a photoreversible chromoprotein but had aberrant spectral properties (Fig. 2, C and D). Collectively, these results show thatDrBphP does not use the typical thioether linkage to bind linear tetrapyrroles.

To locate the chromophore-binding site, we subjected the PCB-DrBphP holoprotein to chemical digestion with cyanogen bromide (CNBr) (13) and analyzed the resulting fragments by LC/MS. Several molecules with absorbance at 374 nm were resolved by LC (Fig. 3C). Subsequent MS analysis identified these compounds as free PCB, several contaminants, and a single PCB-bound peptide (10). The chromopeptide was a singly charged ion with a mass-to-charge ratio (m/z) of 805.6 (Fig. 3D), consistent with the expected mass of a CNBr product, PCB-histidyl homoserine lactone, formed from the release of a His-Met chromodipeptide from the holoprotein. Its mass predicted that PCB was bound to histidine through a Schiff base linkage by a dehydration reaction; this linkage would produce an unprotonated ion with a positive charge at the quaternary amine and a calculatedm/z of 805.5. The Met-His-Met sequence required to generate such a CNBr product appears only once in DrBphP (residues 259 through 261), locating His260 as the ligation site.

To confirm that His260 is the bilin linkage site, we substituted it with alanine (14) and tested whether this mutant protein could bind PCB. Compared to wild-typeDrBphP, the chromophore-binding activity of the His260 → Ala260 (H260A) mutant was reduced at least 100-fold (Fig. 3E). His260 is positionally conserved in all phytochromes and phytochrome-like proteins (Fig. 1A) and appears to be important for chromophore attachment in plant phytochromes (11). For other phytochrome-like proteins that do not contain the adjacent cysteine (PaBphP, Ppr, and RcaE), this histidine residue may serve as the ligation site. For those that have the adjacent cysteine (higher-plant phytochromes and Cph1), this histidine could form a transient Schiff base intermediate with the chromophore, which is then attacked by the sulfur nucleophile of the proximal cysteine to link the chromophore by a thioether bond.

Immediately downstream of BphP in an operon within theD. radiodurans genome is a coding region for a 126–amino acid protein with substantial homology to response regulators (8) (designated BphR for bacteriophytochrome regulator). Its closest sequence relative (62% similar over the entire length of the polypeptide) is Synechocystis Rcp1, the proposed phosphate acceptor of activated Cph1 (6). Given its linkage toDrBphP and the homology to Rcp1, we predict that D. radiodurans BphR (DrBphR) functions as a phosphate acceptor for the light-activated histidine kinase activity ofDrBphP, which in turn initiates its associated light-signaling pathway.

Because phytochromes and phytochrome-like proteins regulate pigmentation (3, 7, 15), it was likely that the BphP/BphR system also regulates pigment synthesis in D. radiodurans, the most abundant of which is the carotenoid deinoxanthin (16). To examine this, we generated null mutations in theBphP/BphR operon by homologous-gene replacement and examined the photoresponses of the cultures (17). Wild-type bacteria accumulated higher amounts of deinoxanthin when grown under increasing fluence rates of white light. However, this induction was severely repressed in all three mutant strains, ΔbphPΔbphR, ΔbphP, and ΔbphR (Fig. 4A). Red light increased pigmentation in wild-type cells, but far-red light had no observable effect in comparison to darkness (10), implying that the Pfr form was the biologically active conformation. The light-induced accumulation of carotenoid appears to help protect D. radiodurans from intense visible light. Whereas the wild type and the ΔbphPmutant grew at similar rates in the dark, colony growth of ΔbphP was markedly reduced when grown under intense light (Fig. 4B).

Figure 4

Role of BphP and BphR in D. radiodurans carotenoid accumulation and growth. (A) Deinoxanthin accumulation in wild-type, ΔbphPΔbphR, ΔbphP, or ΔbphR strains of D. radiodurans kept in the dark or subjected to various fluences of continuous white light for 4 days. Deinoxanthin content was expressed in relation to total protein. The data represent the mean of four experiments (error bars indicate ±SD). (B) Growth of individual colonies of wild-type and ΔbphP strains in the dark or in ∼350 μmol m−2 s−1 of white light. After 4 days, the area (error bars indicate ±SD) of ∼50 representative colonies was measured.

The presence of phytochrome-related proteins in D. radiodurans and P. aeruginosa expands the known types of signaling used by heterotrophic bacteria to respond to changing light. Although PaBphP was not directly tested here, we presume that its photochemical properties and mechanism of action are similar to that of DrBphP, given their extensive sequence homology within both the chromophore-binding pocket and the histidine kinase domain (Fig. 1A). The DrBphP photoreceptor is similar but photochemically distinct from plant phytochromes, having blue-shifted absorbance spectra and using a different linkage to bind chromophore, which involves a histidine rather than a cysteine to potentially form a Schiff base–type bond. The identity of the naturalDrBphP chromophore is unknown, but its similarity to PCB is likely.

Given that D. radiodurans and P. aeruginosa are distantly related to each other and to photosynthetic bacteria (10), these BphPs broaden our current view of phytochrome evolution. Because the other nonphotosynthetic bacteria whose genome sequences are available do not contain phytochrome-related genes (10), bacteriophytochromes are not universally distributed, thus raising the question as to how these eubacteria evolved or obtained this receptor. One possibility forD. radiodurans is horizontal gene transfer, likely from a cyanobacterium. Consistent with this possibility is that numerousD. radiodurans genes have a substantial sequence similarity to those within cyanobacteria (18). Whatever its origin, theD. radiodurans BphP pathway should provide a useful paradigm for studying the mechanism or mechanisms of phytochrome action because it offers for the first time a simplified phytochrome-like response in an organism that is naturally devoid of photosynthesis. In this case, the signal transduction chain could involve as few as three components: DrBphP, DrBphR, and the target genes that regulate deinoxanthin biosynthesis.

  • * To whom correspondence should be addressed. E-mail: vierstra{at}


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