Bacteriophytochromes: new tools for understanding phytochrome signal transduction

The recent discovery of phytochrome-like photoreceptors, collectively called bacteriophytochromes, in a number of bacteria has greatly expanded our understanding of the origins and modes of action of phytochromes in higher plants. These primitive receptors contain an N-terminal domain homologous to the chromophore-binding pocket of phytochromes, and like phytochromes, they bind a variety of bilins to generate photochromic holoproteins. Following the chromophore pocket is a domain similar to two-component histidine kinases, suggesting that these bacterial photoreceptors function in phosphorelay cascades that respond to the light environment. Their organization and distribution support the views that higher-plant phytochromes evolved from a cyanobacterial precursor and that they act as light-regulated kinases. With the ability to exploit bacterial genetics, these bacteriophytochromes now offer simple models to help unravel the biochemical and biophysical events that initiate phytochrome signal transmission.     

The impact of the phytochromes on photosynthetic processes

This review describes the phytochrome system in higher plants and cyanobacteria and its role in regulation of photosynthetic processes and stress protection of the photosynthetic apparatus. A relationship between the content of the different phytochromes, the changes in the ratios of the physiologically active forms of phytochromes to their total pool and the resulting influence on photosynthetic processes is reviewed. The role of the phytochromes in the regulation of the expression of genes encoding key photosynthetic proteins, antioxidant enzymes and other components involved in stress signaling is elucidated.     

High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution

Phytochromes are red/far red light photochromic photoreceptors that direct many photosensory behaviors in the bacterial, fungal, and plant kingdoms. They consist of an N-terminal domain that covalently binds a bilin chromophore and a C-terminal region that transmits the light signal, often through a histidine kinase relay. Using x-ray crystallography, we recently solved the first three-dimensional structure of a phytochrome, using the chromophore-binding domain of Deinococcus radiodurans bacterial phytochrome assembled with its chromophore, biliverdin IXalpha. Now, by engineering the crystallization interface, we have achieved a significantly higher resolution model. This 1.45A resolution structure helps identify an extensive buried surface between crystal symmetry mates that may promote dimerization in vivo. It also reveals that upon ligation of the C3(2) carbon of biliverdin to Cys(24), the chromophore A-ring assumes a chiral center at C2, thus becoming 2(R),3(E)-phytochromobilin, a chemistry more similar to that proposed for the attached chromophores of cyanobacterial and plant phytochromes than previously appreciated. The evolution of bacterial phytochromes to those found in cyanobacteria and higher plants must have involved greater fitness using more reduced bilins, such as phycocyanobilin, combined with a switch of the attachment site from a cysteine near the N terminus to one conserved within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. From analysis of site-directed mutants in the D. radiodurans phytochrome, we show that this bilin preference was partially driven by the change in binding site, which ultimately may have helped photosynthetic organisms optimize shade detection. Collectively, these three-dimensional structural results better clarify bilin/protein interactions and help explain how higher plant phytochromes evolved from prokaryotic progenitors.     

Phytochromes and photomorphogenesis in Arabidopsis.

Plants have evolved exquisite sensory systems for monitoring their light environment. The intensity, quality, direction and duration of light are continuously monitored by the plant and the information gained is used to modulate all aspects of plant development. Several classes of distinct photoreceptors, sensitive to different regions of the light spectrum, mediate the developmental responses of plants to light signals. The red-far-red light-absorbing, reversibly photochromic phytochromes are perhaps the best characterized of these. Higher plants possess a family of phytochromes, the apoproteins of which are encoded by a small, divergent gene family. Arabidopsis has five apophytochrome-encoding genes, PHYA-PHYE. Different phytochromes have discrete biochemical and physiological properties, are differentially expressed and are involved in the perception of different light signals. Photoreceptor and signal transduction mutants of Arabidopsis are proving to be valuable tools in the molecular dissection of photomorphogenesis. Mutants deficient in four of the five phytochromes have now been isolated. Their analysis indicates considerable overlap in the physiological functions of different phytochromes. In addition, mutants defining components acting downstream of the phytochromes have provided evidence that different members of the family use different signalling pathways.     

The Aspergillus nidulans phytochrome FphA represses sexual development in red light

Phytochrome photoreceptors sense red and far-red light through photointerconversion between two stable conformations, a process mediated by a linear tetrapyrrole chromophore. Originally, phytochromes were thought to be confined to photosynthetic organisms including cyanobacteria, but they have been recently discovered in heterotrophic bacteria and fungi, where little is known about their functions. It was shown previously in the ascomycetous fungus Aspergillus nidulans that asexual sporulation is stimulated and sexual development repressed by red light. The effect was reminiscent of a phytochrome response, and indeed phytochrome-like proteins were detected in several fungal genomes. All fungal homologs are more similar to bacterial than plant phytochromes and have multifunctional domains where the phytochrome region and histidine kinase domain are combined in a single protein with a C-terminal response-regulator domain. Here, we show that the A. nidulans phytochrome FphA binds a biliverdin chromophore, acts as a red-light sensor, and represses sexual development under red-light conditions. FphA-GFP is cytoplasmic and excluded from the nuclei, suggesting that red-light photoperception occurs in the cytoplasm. This is the first phytochrome experimentally characterized outside the plant and bacterial kingdoms and the second type of fungal protein identified that functions in photoperception.     

A New Appraisal of the Prokaryotic Origin of Eukaryotic Phytochromes

The evolutionary origin of the phytochromes of eukaryotes is controversial. Three cyanobacterial proteins have been described as ``phytochrome-like' and have been suggested to be potential ancestors of these essential photoreceptors: Cph1 from Synechocystis PCC 6803, showing homology to phytochromes along its entire length and known to attach a chromophore; and PlpA from Synechocystis PCC 6803 and RcaE from Fremyella diplosiphon, both showing homology to phytochromes most strongly only in the C-terminal region and not known to bind a chromophore. We have reexamined the evolution of the photoreceptors using for PCR amplification a highly conserved region encoding the chromophore-binding domain in both Cph1 and phytochromes of plants and have identified genes for phytochrome-like proteins (PLP) in 11 very diverse cyanobacteria. The predicted gene products contain either a Cys, Arg, Ile, or Leu residue at the putative chromophore binding site. In 10 of the strains examined only a single gene was found, but in Calothrix PCC 7601 two genes (cphA and cphB) were identified. Phylogenetic analysis revealed that genes encoding PLP are homologues that share a common ancestor with the phytochromes of eukaryotes and diverged before the latter. In contrast, the putative sensory/regulatory proteins, including PlpA and RcaE, that lack a part of the chromophore lyase domain essential for chromophore attachment on the apophytochrome, are only distantly related to phytochromes. The Ppr protein of the anoxygenic photosynthetic bacterium Rhodospirillum centenum and the bacterial phytochrome-like proteins (BphP) of Deinococcus radiodurans and Pseudomonas aeruginosa fall within the cluster of cyanobacterial phytochromes. Received: 9 December 1999 / Accepted: 10 May 2000     

A computational approach to discovering the functions of bacterial phytochromes by analysis of homolog distributions

Background  

Phytochromes are photoreceptors, discovered in plants, that control a wide variety of developmental processes. They have also been found in bacteria and fungi, but for many species their biological role remains obscure. This work concentrates on the phytochrome system of Agrobacterium tumefaciens, a non-photosynthetic soil bacterium with two phytochromes. To identify proteins that might share common functions with phytochromes, a co-distribution analysis was performed on the basis of protein sequences from 138 bacteria.     

An Evolutionarily Conserved Signaling Mechanism Mediates Far-Red Light Responses in Land Plants

Phytochromes are plant photoreceptors important for development and adaptation to the environment. Phytochrome A (PHYA) is essential for the far-red (FR) high-irradiance responses (HIRs), which are of particular ecological relevance as they enable plants to establish under shade conditions. PHYA and HIRs have been considered unique to seed plants because the divergence of seed plants and cryptogams (e.g., ferns and mosses) preceded the evolution of PHYA. Seed plant phytochromes translocate into the nucleus and regulate gene expression. By contrast, there has been little evidence of a nuclear localization and function of cryptogam phytochromes. Here, we identified responses to FR light in cryptogams, which are highly reminiscent of PHYA signaling in seed plants. In the moss Physcomitrella patens and the fern Adiantum capillus-veneris, phytochromes accumulate in the nucleus in response to light. Although P. patens phytochromes evolved independently of PHYA, we have found that one clade of P. patens phytochromes exhibits the molecular properties of PHYA. We suggest that HIR-like responses had evolved in the last common ancestor of modern seed plants and cryptogams and that HIR signaling is more ancient than PHYA. Thus, other phytochromes in seed plants may have lost the capacity to mediate HIRs during evolution, rather than that PHYA acquired it.     

Phytochrome evolution: Phytochrome genes in ferns and mosses

We have isolated phytochrome genes from the moss Physcomitrella , the fern Psilotum and PCR-generated phytochrome sequences from a few other ferns. The phytochrome gene of the moss Physcomitrella turned out not to contain the aberrant C-terminal third of the phytochrome from the moss Ceratodon , but the transmitter module-like sequences found in other phytochromes. A series of different phytochrome genes was detected in Psilotum . Differences between the amino acid sequences derived from them ranged from about 5 to more than 22%. Some of these genes are likely pseudogenes. Analysis by phylogenetic tree constructions revealed that higher and lower plant phytochromes evolved with different velocities. Lower plant phytochromes form a separate family characterized by a high degree of similarity. The amino acid differences between phytochrome types detected in a single species of higher plants are about two-fold higher than the differences between phytochromes of species of lower plants belonging to different divisions ( Physcomitrella and Selaginella ). Future studies on phytochrome sequences may eventually also throw light on the significance of Psilotum in the evolution of vascular plants.     

Chromatin,And Gene Regulation In Eukaryotic Cells At The Transcriptional Leve

Phytochromes are environmental sensors, historically thought of as red/far-red photoreceptors in plants. Their photoperception occurs through a covalently linked tetrapyrrole chromophore, which undergoes a light-dependent conformational change propagated through the protein to a variable output domain. The phytochrome composition is modular, typically consisting of a PAS-GAF-PHY architecture for the N-terminal photosensory core. A collection of three-dimensional structures has uncovered key features, including an unusual figure-of-eight knot, an extension reaching from the PHY domain to the chromophore-binding GAF domain, and a centrally located, long α-helix hypothesized to be crucial for intramolecular signaling. Continuing identification of phytochromes in microbial systems has expanded the assigned sensory abilities of this family out of the red and into the yellow, green, blue, and violet portions of the spectrum. Furthermore, phytochromes acting not as photoreceptors but as redox sensors have been recognized. In addition, architectures other than PAS-GAF-PHY are known, thus revealing phytochromes to be a varied group of sensory receptors evolved to utilize their modular design to perceive a signal and respond accordingly. This review focuses on the structures of bacterial phytochromes and implications for signal transmission. We also discuss the small but growing set of bacterial phytochromes for which a physiological function has been ascertained.     

Bacterial phytochromes: more than meets the light

Phytochromes are environmental sensors, historically thought of as red/far-red photoreceptors in plants. Their photoperception occurs through a covalently linked tetrapyrrole chromophore, which undergoes a light-dependent conformational change propagated through the protein to a variable output domain. The phytochrome composition is modular, typically consisting of a PAS-GAF-PHY architecture for the N-terminal photosensory core. A collection of three-dimensional structures has uncovered key features, including an unusual figure-of-eight knot, an extension reaching from the PHY domain to the chromophore-binding GAF domain, and a centrally located, long α-helix hypothesized to be crucial for intramolecular signaling. Continuing identification of phytochromes in microbial systems has expanded the assigned sensory abilities of this family out of the red and into the yellow, green, blue, and violet portions of the spectrum. Furthermore, phytochromes acting not as photoreceptors but as redox sensors have been recognized. In addition, architectures other than PAS-GAF-PHY are known, thus revealing phytochromes to be a varied group of sensory receptors evolved to utilize their modular design to perceive a signal and respond accordingly. This review focuses on the structures of bacterial phytochromes and implications for signal transmission. We also discuss the small but growing set of bacterial phytochromes for which a physiological function has been ascertained.     

Evolutionary relationships among photosynthetic prokaryotes (Heliobacterium chlorum, Chloroflexus aurantiacus, cyanobacteria, Chlorobium tepidum and proteobacteria): implications regarding the origin of photosynthesis

The presence of shared conserved insertions or deletions in proteins (referred to as signature sequences) provides a powerful means to deduce the evolutionary relationships among prokaryotic organisms. This approach was used in the present work to deduce the branching orders of various eubacterial taxa consisting of photosynthetic organisms. For this purpose, portions of the Hsp60 and Hsp70 genes, covering known signature sequence regions, were PCR-amplified and sequenced from Heliobacterium chlorum, Chloroflexus aurantiacus and Chlorobium tepidum. This information was integrated with sequence data for several other proteins from numerous species to deduce the branching orders of different photosynthetic taxa. Based on signature sequences that are present in different proteins, it is possible to infer that the various eubacterial phyla evolved from a common ancestor in the following order: low G+C Gram-positive (H. chlorum) --> high G+C Gram-positive --> Deinococcus-Thermus --> green non-sulphur bacteria (Cf. aurantiacus ) --> cyanobacteria --> spirochaetes --> Chlamydia-Cytophaga-Aquifex-flavobacteria-green sulphur bacteria (Cb. tepidum) --> proteobacteria (alpha, delta and epsilon) and --> proteobacteria (beta and gamma). The members of the Heliobacteriaceae family that contain a Fe-S type of reaction centre (RC-1) and represent the sole photosynthetic phylum from the Gram-positive or monoderm group of prokaryotes are indicated to be the most ancestral of the photosynthetic lineages. Among the Gram-negative bacteria or diderm prokaryotes, green non-sulphur bacteria such as Cf. aurantiacus, which contains a pheophytin-quinone type of reaction centre (RC-2), are indicated to have evolved very early. Thus, the organisms containing either RC-1 or RC-2 existed before the evolution of cyanobacteria, which contain both these reaction centres to carry out oxygenic photosynthesis. The eubacterial divisions consisting of green sulphur bacteria and proteobacteria are indicated to have diverged after cyanobacteria. Some implications of these results concerning the origin of photosynthesis and the earliest prokaryotic fossils are discussed.     

A singular bacteriophytochrome acquired by lateral gene transfer

Bacteriophytochromes are phytochrome-like proteins that mediate photosensory responses in various bacteria according to their light environment. The genome of the photosynthetic and plant-symbiotic Bradyrhizobium sp. strain ORS278 revealed the presence of a genomic island acquired by lateral transfer harboring a bacteriophytochrome gene, BrBphP3.ORS278, and genes involved in the synthesis of phycocyanobilin and gas vesicles. The corresponding protein BrBphP3.ORS278 is phylogenetically distant from the other (bacterio)phytochromes described thus far and displays a series of unusual properties. It binds phycocyanobilin as a chromophore, a unique feature for a bacteriophytochrome. Moreover, its C-terminal region is short and displays no homology with any known functional domain. Its dark-adapted state absorbs maximally around 610 nm, an unusually short wavelength for (bacterio)phytochromes. This form is designated as Po for orange-absorbing form. Upon illumination, a photo-reversible switch occurs between the Po form and a red (670 nm)-absorbing form (Pr), which rapidly backreacts in the dark. Because of this instability, illumination results in a mixture of the Po and Pr states in proportions that depend on the intensity. These uncommon features suggest that BrBphP3.ORS278 could be fitted to measure light intensity rather than color.     

Evolution of photosynthetic reaction centers

The common ancestor of all photosynthetic prokaryotes and organelles contained chlorophyll (Chl) a. All green and purple photosynthetic bacteria descended from a common bacteriochlorophyll (Bchl) a-containing ancestor which diverged from the Chl a line. Separate PS-I and PS-II reaction centers may have evolved before the appearance of Bchl a. When the transition to Bchl a occurred, the resultant organism contained two types of reaction center, “PS-I” and “PS-II.” One line of development eliminated “PS-II” and evolved into the green bacteria. The other line eliminated “PS-I” and became the purple bacteria. In the Chl a-containing organisms the evolution of PS-II continued until oxygen evolution was achieved.     

Evolution of bacterial denitrification and denitrifier diversity

Little is known about the role of nitrate in evolution of bacterial energy-generating mechanisms. Denitrifying bacteria are commonly regarded to have evolved from nitrate-respiring bacteria. Some researchers regard denitrification to be the precursor of aerobic respiration; others feel the opposite is true. Currently recognized denitrifying bacteria such as Hyphomicrobium, Paracoccus, Pseudomonas and Thiobacillus form a very diverse group. However, inadequate testing procedures and uncertain taxonomic identification of many isolates may have overstated the number of genera with species capable of denitrification. Nitrate reductases are structurally similar among denitrifying bacteria, but distinct from the enzymes in other nitrate-reducing organisms. Denitryfying bacteria have one of two types of nitrite reductase, either a copper-containing enzyme or an enzyme containing a cytochrome cd moiety. Both types are distinct from other nitrate reductases. Organisms capable of dissimilatory nitrate reduction are widely distributed among eubacterial groups defined by 16S ribosomal RNA phylogeny. Indeed, nitrate reduction is an almost universal property of actinomycetes and enteric organisms. However, denitrification is restricted to genera within the purple photosynthetic group. Denitrification within the genus Pseudomonas is distributed in accordance with DNA and RNA homology complexes. Denitrifiers seem to have evolved from a common ancestor within the purple photosynthetic bacterial group, but not from a nitrate-reducing organism such as those found today. Although denitrification seems to have arisen at the same time as aerobic respiration, the evolutionary relationship between the two cannot be determined at this time.     

From seed to seed: the role of photoreceptors in Arabidopsis development

As sessile organisms, plants have evolved a multitude of developmental responses to cope with the ever-changing environmental conditions that challenge the plant throughout its life cycle. Of the many environmental cues that regulate plant development, light is probably the most important. From determining the developmental pattern of the emerging seedling, to influencing the organization of organelles to best maximize energy available for photosynthesis, light has dramatic effects on development during all stages of plant life. In plants, three classes of photoreceptors that mediate light perception have been characterized at the molecular level. The phytochromes recognize light in the red portion of the spectrum, while cryptochromes and phototropins perceive blue and UVA light. In this review, we discuss the different aspects of development that are regulated by these photoreceptors in the model plant species Arabidopsis thaliana and how the phytochromes, cryptochromes, and phototropins bring about changes in development seen in the growing plant.     

RuBisCO-like proteins as the enolase enzyme in the methionine salvage pathway: functional and evolutionary relationships between RuBisCO-like proteins and photosynthetic RuBisCO

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key enzyme in the fixation of CO(2) in the Calvin cycle of plants. Many genome projects have revealed that bacteria, including Bacillus subtilis, possess genes for proteins that are similar to the large subunit of RuBisCO. These RuBisCO homologues are called RuBisCO-like proteins (RLPs) because they are not able to catalyse the carboxylase or the oxygenase reactions that are catalysed by photosynthetic RuBisCO. It has been demonstrated that B. subtilis RLP catalyses the 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) enolase reaction in the methionine salvage pathway. The structure of DK-MTP-1-P is very similar to that of ribulose-1,5-bisphosphate (RuBP) and the enolase reaction is a part of the reaction catalysed by photosynthetic RuBisCO. In this review, functional and evolutionary relationships between B. subtilis RLP of the methionine salvage pathway, other RLPs, and photosynthetic RuBisCO are discussed. In addition, the fundamental question, 'How has RuBisCO evolved?' is also considered, and evidence is presented that RuBisCOs evolved from RLPs.     

Evolution of bacterial denitrification and denitrifier diversity

Little is known about the role of nitrate in evolution of bacterial energy-generating mechanisms. Denitrifying bacteria are commonly regarded to have evolved from nitrate-respiring bacteria. Some researchers regard denitrification to be the precursor of aerobic respiration; others feel the opposite is true. Currently recognized denitrifying bacteria such as Hyphomicrobium, Paracoccus, Pseudomonas and Thiobacillus form a very diverse group. However, inadequate testing procedures and uncertain taxonomic identification of many isolates may have overstated the number of genera with species capable of denitrification. Nitrate reductases are structurally similar among denitrifying bacteria, but distinct from the enzymes in other nitrate-reducing organisms. Denitryfying bacteria have one of two types of nitrite reductase, either a copper-containing enzyme or an enzyme containing a cytochrome cd moiety. Both types are distinct from other nitrate reductases. Organisms capable of dissimilatory nitrate reduction are widely distributed among eubacterial groups defined by 16S ribosomal RNA phylogeny. Indeed, nitrate reduction is an almost universal property of actinomycetes and enteric organisms. However, denitrification is restricted to genera within the purple photosynthetic group. Denitrification within the genus Pseudomonas is distributed in accordance with DNA and RNA homology complexes. Denitrifiers seem to have evolved from a common ancestor within the purple photosynthetic bacterial group, but not from a nitrate-reducing organism such as those found today. Although denitrification seems to have arisen at the same time as aerobic respiration, the evolutionary relationship between the two cannot be determined at this time.     

Phytochrome structure and photochemistry: recent advances toward a complete molecular picture

Phytochromes are nature's primary photoreceptors dedicated to detecting the red and far-red regions of the visible light spectrum, a region also essential for photosynthesis and thus crucial to the survival of plants and other photosynthetic organisms. Given their roles in measuring competition and diurnal/seasonal light fluctuations, understanding how phytochromes work at the molecular level would greatly aid in engineering crop plants better suited to specific agricultural settings. Recently, scientists have determined the three-dimensional structures of prokaryotic phytochromes, which now provide clues as to how these modular photoreceptors might work at the atomic level. The models point toward a largely unifying mechanism whereby novel knot, hairpin, and dimeric interfaces transduce photoreversible bilin isomerization into protein conformational changes that alter signal output.     

Bathy Phytochromes in Rhizobial Soil Bacteria

Phytochromes are biliprotein photoreceptors that are found in plants, bacteria, and fungi. Prototypical phytochromes have a Pr ground state that absorbs in the red spectral range and is converted by light into the Pfr form, which absorbs longer-wavelength, far-red light. Recently, some bacterial phytochromes have been described that undergo dark conversion of Pr to Pfr and thus have a Pfr ground state. We show here that such so-called bathy phytochromes are widely distributed among bacteria that belong to the order Rhizobiales. We measured in vivo spectral properties and the direction of dark conversion for species which have either one or two phytochrome genes. Agrobacterium tumefaciens C58 contains one bathy phytochrome and a second phytochrome which undergoes dark conversion of Pfr to Pr in vivo. The related species Agrobacterium vitis S4 contains also one bathy phytochrome and another phytochrome with novel spectral properties. Rhizobium leguminosarum 3841, Rhizobium etli CIAT652, and Azorhizobium caulinodans ORS571 contain a single phytochrome of the bathy type, whereas Xanthobacter autotrophicus Py2 contains a single phytochrome with dark conversion of Pfr to Pr. We propose that bathy phytochromes are adaptations to the light regime in the soil. Most bacterial phytochromes are light-regulated histidine kinases, some of which have a C-terminal response regulator subunit on the same protein. According to our phylogenetic studies, the group of phytochromes with this domain arrangement has evolved from a bathy phytochrome progenitor.Phytochromes are biological photoreceptors that were discovered in plants, where they control development throughout the life cycle in manifold ways (21, 33). Today, a large number of homologs are known also from cyanobacteria, other bacteria, and fungi, which are termed cyanobacterial phytochromes (Cphs), bacteriophytochromes (BphPs), and fungal phytochromes (Fphs), respectively (20, 24). The chromophore is autocatalytically assembled within the N-terminal part of the protein, the photosensory core module (PCM), which contains the PAS, GAF, and PHY domains (30). Typically, phytochromes are converted by light between two spectrally different forms, the red-absorbing Pr and the far-red-absorbing Pfr forms. Photoconversion is initiated by an isomerization of the covalently bound bilin chromophore (32).Plant and cyanobacterial phytochromes incorporate phytochromobilin (PΦB) and phycocyanobilin (PCB) as natural chromophores, respectively, which are covalently bound to Cys residues in the GAF domains. All characterized phytochromes that belong to these groups have a Pr ground state. Plant phytochromes can undergo dark conversion of Pfr to Pr (5), whereas the Pfr form of typical cyanobacterial phytochromes is stable in darkness (26).Bacteriophytochromes utilize biliverdin (BV) instead as a natural chromophore (1), which is covalently attached to a Cys residue in the N terminus of the PAS domain (26). Since the conjugated system of BV is longer than that of PΦB or PCB, the absorption maxima of bacteriophytochromes are found at higher wavelengths than those of cyanobacterial or plant homologs.With the discovery of a bacterial phytochrome from Bradyrhizobium sp. strain ORS278, termed BrBphP1, the first phytochrome with a Pfr ground state and dark conversion from Pr to Pfr was found (10). Thereafter, five more phytochromes with dark conversion of Pr to Pfr were described: Rhodopseudomonas palustris BphP1 (RpBphP1) from strain CEA001, RpBphP5, and RpBphP6 from strain CGA009 (11); Agrobacterium tumefaciens Agp2 (or AtBphP2) from strain C58 (18); and Pseudomonas aeruginosa BphP1 (PaBphP1) (40). These phytochromes are now termed bathy phytochromes because the absorption maxima of their ground states are bathochromically (to longer wavelengths) shifted compared to those of all other phytochromes.Moreover, some other bacterial phytochromes with unusual properties have been described. In the Ppr from Rhodospirillum centenum, a photoactive yellow protein (PYP) domain is fused to the N terminus of a phytochrome homolog. The phytochrome part of Ppr assembles with BV to form a Pr adduct. However, irradiation does not result in the formation of Pfr but in a bleaching of the Pr spectrum (23). The BV adduct of RpBphP3 from R. palustris, which has a Pr ground state, photoconverts to the so-called Pnr form with a blue-shifted absorption maximum (12). RpBphP4 from R. palustris strains Ha2 and BisB5 and Bradyrhizobium BphP3 (BrBphP3) from Bradyrhizobium BTAi1, both with a Pr ground state, photoconvert into a long-lived MetaR form (8, 42). MetaRa and MetaRc are intermediates in the photoconversion from Pr to Pfr of prototypical phytochromes (3). BphP3 from the Bradyrhizobium strain ORS 278 is an exception among bacteriophytochromes as it binds PCB as a natural chromophore. This phytochrome adopts a so-called Po (P-orange) ground state with an absorbance maximum in the orange range (11, 15). Upon irradiation, this phytochrome converts into the Pr form. RpBphP4 from R. palustris CGA009 lacks the biliverdin binding cysteine and does not bind a chromophore (42).With the rapidly growing number of bacterial genome sequences, many new bacterial phytochromes are being discovered. Thus, a large and increasing number of newly identified phytochromes remain spectroscopically uncharacterized. We established an in vivo photometry approach which allowed the rapid acquisition of spectral information about phytochromes from intact bacterial cells. In the beginning period of plant phytochrome research, in vivo photometry was extensively applied (4, 6, 29, 34). This method, in fact, allowed the identification of phytochromes for the first time in plant tissues (6), which led to the purification of phytochromes from plant extracts (37). Here, we apply in vivo photometry for the first time to organisms outside the plant kingdom. This method is especially useful for studying species with single phytochrome genes. The approach is also helpful for comparing properties of native phytochromes in vivo and of their recombinant proteins in vitro.In the present study, we concentrate on nonphotosynthetic species of the order Rhizobiales which belongs to the Alphaproteobacteria. The family Rhizobiaceae comprises plant-interacting soil bacteria. A. tumefaciens and Agrobacterium vitis can transfer genes into plants to induce plant tumors, whereas many other Rhizobiaceae can live as plant symbionts in nodules of stems or roots in which they assimilate molecular nitrogen to produce NH₄⁺, which is used by the plant for synthesis of amino acids and other nitrogen-containing molecules. A. tumefaciens C58 contains two phytochromes, termed Agp1 (or AtBphP1) and Agp2 (or AtBphP2), that have been characterized as recombinant proteins (14, 18, 26, 35) and whose spectral activities have been measured in extracts of wild-type and knockout mutants (31). A large number of phytochromes from photosynthetic Bradyrhizobium and Rhodopseudomonas species, which also belong to the order Rhizobiales, have been characterized as recombinant proteins (11), some of which have already been noted above.It turned out that most of our analyzed phytochromes undergo dark conversion of Pr to Pfr and thus belong to the group of bathy phytochromes. Such phytochromes, which absorb at around 750 nm, clearly dominate among Rhizobiales. We propose that this specific property reflects an adaptation to the light regime in the soil. Our studies also suggest that bacterial phytochromes with a C-terminal response regulator have evolved from a bathy phytochrome progenitor.