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Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. In plants, phytochromes play a central role in growth and differentiation during the entire life cycle. Phytochromes of plants and other groups of archaeplastida have a common evolutionary origin in prokaryotes, but the exact prokaryotic origin is as yet uncertain. Two possibilities are presently discussed: either, archaeplastidal phytochromes arose from the last eukaryotic common ancestor (LECA) or they arose from the cyanobacterial endosymbiont that gave rise to plastids. We first constructed standard phylogenetic trees based on N-terminal protein sequences of the chromophore module. As usual, variation of algorithms and parameters led to different trees. A relationship between cyanobacteria and archaeplastida was observed in 7 out of 36 trees. The lack of consistency between results obtained from variation of parameters of tree constructions reflects the uncertainty of archaeplastidal origin. To gain more information about a possible cyanobacterial and archaeplastidal relationship, we performed phylogenetic studies based on the amino acids that line the chromophore pockets. These amino acids are highly conserved and could provide more accurate information about long evolutionary time scales, but the reduction of traits could also lead to insignificant results. From 30 selected chromophore-binding amino acids, 6 were invariant. The subsequent studies were thus based on the information dependent on 24 or fewer amino acid positions. Again, multiple trees were constructed to get information about the robustness of relationships. The very low number of information-containing traits resulted in low bootstrap values and many indistinguishable leaves. However, the major groups fungi, bacteria, cyanobacteria, and plants remained united. Without exception, cyanobacteria and archaeplastida were always closely linked. In this respect, the results were more robust than those of the classic approach, based on long contiguous sequences. We therefore consider cyanobacteria as the most likely origin of archaeplastidal phytochromes.

Deactivation processes of photoexcited (λex = 580 nm) phycocyanobilin (PCB) in methanol were investigated by means of UV/Vis and mid-IR femtosecond (fs) transient absorption (TA) as well as static fluorescence spectroscopy, supported by density-functional-theory calculations of three relevant ground state conformers, PCBA, PCBB and PCBC, their relative electronic state energies and normal mode vibrational analysis. UV/Vis fs-TA reveals time constants of 2.0, 18 and 67 ps, describing decay of PCBB*, of PCBA* and thermal re-equilibration of PCBA, PCBB and PCBC, respectively, in line with the model by Dietzek et al. (Chem Phys Lett 515:163, 2011) and predecessors. Significant substantiation and extension of this model is achieved first via mid-IR fs-TA, i.e. identification of molecular structures and their dynamics, with time constants of 2.6, 21 and 40 ps, respectively. Second, transient IR continuum absorption (CA) is observed in the region above 1755 cm−1 (CA1) and between 1550 and 1450 cm−1 (CA2), indicative for the IR absorption of highly polarizable protons in hydrogen bonding networks (X–H…Y). This allows to characterize chromophore protonation/deprotonation processes, associated with the electronic and structural dynamics, on a molecular level. The PCB photocycle is suggested to be closed via a long living (> 1 ns), PCBC-like (i.e. deprotonated), fluorescent species.

The soil bacterium Agrobacterium fabrum C58 infects plants by a unique DNA transfer mechanism. A. fabrum has two phytochrome photoreceptors, Agp1 and Agp2. We found that DNA transfer into plants by A. fabrum is down regulated by light and that phytochrome knockout mutants have diminished DNA transfer rates. The regulation pattern matches with that of bacterial conjugation reported earlier. Growth, swimming and interbacterial competition were also affected in phytochrome knockout mutants, although these effects were often not affected by light. We can thus distinguish between light-regulated and light-independent phytochrome responses. In microarray studies, transcription of only 4 genes was affected by light, indicating that most light responses are regulated post-transcriptionally. In a mass spectrometery-based proteomic study, 24 proteins were different between light and dark grown bacteria, whereas 382 proteins differed between wild type and phytochrome knockout mutants, pointing again to light-dependent and light-independent roles of Agp1 and Agp2.

Research for biotechnological applications of cyanobacteria focuses on synthetic pathways and bioreactor design, while little effort is devoted to introduce new, promising organisms in the field. Applications are most often based on recombinant work, and the establishment of transformation can be a risky, time-consuming procedure. In this work we demonstrate the natural transformation of the filamentous cyanobacterium Phormidium lacuna and insertion of a selection marker into the genome by homologous integration. This is the first example for natural transformation of a member of the order Oscillatoriales. We found that Phormidium lacuna is polyploid, each cell has about 20-100 chromosomes. Transformed filaments were resistant against up to 15 mg/ml of kanamycin, and the high resistance feature allowed for rapid segregation into all chromosomes. Formerly, natural transformation in cyanobacteria has been considered a rare and exclusive feature of a few unicellular species. Our finding suggests that natural competence is more distributed among cyanobacteria than previously thought. This is supported by bioinformatic analyses which show that all protein factors for natural transformation are present in the majority of the analyzed cyanobacteria.

International audience; The repair of sun-induced DNA lesions by photolyases is driven by a photoinduced electron transfer from a fully reduced FAD to the damaged DNA. A chain of several aromatic residues connecting FAD to solvent ensures the prior photoreduction of the FAD cofactor. In PhrA, a class III CPD photolyase, two branching tryptophan charge transfer pathways have been characterized. According to previous experiments, both pathways play a role in the FAD photoreduction. To provide a molecular insight to the charge transfer abilities of both pathways, we perform multiscales simulations where the protein motion and the positive charge are simultaneously propagated. Our computational approach reveals that one pathway drives a very fast charge transfer whereas the other pathway provides a very good thermodynamic stabilization of the positive charge. During the simulations, the positive charge firstly moves on the fast triad, while a reorganization of the close FAD À environment occurs. Then, backward transfers can lead to the propagation of the positive charge on the second pathway. After one nanosecond, we observe a nearly equal probability to find the charge at ending tryptophan of either pathway; eventually the charge distribution will likely evolve towards a charge stabilization on the last tryptophan of the slowest pathway. Our results highlight the role the protein environment, which manages the association of a kinetic and a thermodynamic pathways to trigger a fast and efficient FAD photoreduction.

Phytochromes are modular photoreceptors of plants, bacteria and fungi that use light as a source of information to regulate fundamental physiological processes. Interconversion between the active and inactive states is accomplished by a photoinduced reaction sequence which couples the sensor with the output module. However, the underlying molecular mechanism is yet not fully understood due to the lack of structural data of functionally relevant intermediate states. Here we report the crystal structure of a Meta-F intermediate state of an Agp2 variant from Agrobacterium fabrum. This intermediate, the identity of which was verified by resonance Raman spectroscopy, was formed by irradiation of the parent Pfr state and displays significant reorientations of almost all amino acids surrounding the chromophore. Structural comparisons allow identifying structural motifs that might serve as conformational switch for initiating the functional secondary structure change that is linked to the (de-)activation of these photoreceptors., Phytochromes are photoreceptors that are present in plants, bacteria and fungi. Here the authors present crystal structures of the phytochrome Agp2 from Agrobacterium fabrum in the parent Pfr state as well as a functional Meta-F intermediate and discuss mechanistic implications for photoconversion.

The phytochrome family of light-switchable proteins has long been studied by biochemical, spectroscopic and crystallographic means, while a direct probe for global conformational signal propagation has been lacking. Using solution X-ray scattering, we find that the photosensory cores of several bacterial phytochromes undergo similar large-scale structural changes upon red-light excitation. The data establish that phytochromes with ordinary and inverted photocycles share a structural signaling mechanism and that a particular conserved histidine, previously proposed to be involved in signal propagation, in fact tunes photoresponse.

FAD photoreduction mechanism by different aromatic residues in a phylogenetically ancient photolyase., Cryptochromes and photolyases form a flavoprotein family in which the FAD chromophore undergoes light induced changes of its redox state. During this process, termed photoreduction, electrons flow from the surface via conserved amino acid residues to FAD. The bacterial (6–4) photolyase PhrB belongs to a phylogenetically ancient group. Photoreduction of PhrB differs from the typical pattern because the amino acid of the electron cascade next to FAD is a tyrosine (Tyr391), whereas photolyases and cryptochromes of other groups have a tryptophan as direct electron donor of FAD. Mutagenesis studies have identified Trp342 and Trp390 as essential for charge transfer. Trp342 is located at the periphery of PhrB while Trp390 connects Trp342 and Tyr391. The role of Tyr391, which lies between Trp390 and FAD, is however unclear as its replacement by phenylalanine did not block photoreduction. Experiments reported here, which replace Tyr391 by Ala, show that photoreduction is blocked, underlining the relevance of Tyr/Phe at position 391 and indicating that charge transfer occurs via the triad 391-390-342. This raises the question, why PhrB positions a tyrosine at this location, having a less favourable ionisation potential than tryptophan, which occurs at this position in many proteins of the photolyase/cryptochrome family. Tunnelling matrix calculations show that tyrosine or phenylalanine can be involved in a productive bridged electron transfer between FAD and Trp390, in line with experimental findings. Since replacement of Tyr391 by Trp resulted in loss of FAD and DMRL chromophores, electron transfer cannot be studied experimentally in this mutant, but calculations on a mutant model suggest that Trp might participate in the electron transfer cascade. Charge transfer simulations reveal an unusual stabilization of the positive charge on site 391 compared to other photolyases or cryptochromes. Water molecules near Tyr391 offer a polar environment which stabilizes the positive charge on this site, thereby lowering the energetic barrier intrinsic to tyrosine. This opens a second charge transfer channel in addition to tunnelling through the tyrosine barrier, based on hopping and therefore transient oxidation of Tyr391, which enables a fast charge transfer similar to proteins utilizing a tryptophan-triad. Our results suggest that evolution of the first site of the redox chain has just been possible by tuning the protein structure and environment to manage a downhill hole transfer process from FAD to solvent.

Phytochromes act as photoswitches between the red- and far-red absorbing parent states of phytochromes (Pr and Pfr). Plant phytochromes display an additional thermal conversion route from the physiologically active Pfr to Pr. The same reaction pattern is found in prototypical biliverdin-binding bacteriophytochromes in contrast to the reverse thermal transformation in bathy bacteriophytochromes. However, the molecular origin of the different thermal stabilities of the Pfr states in prototypical and bathy bacteriophytochromes is not known. We analyzed the structures of the chromophore binding pockets in the Pfr states of various bathy and prototypical biliverdin-binding phytochromes using a combined spectroscopic-theoretical approach. For the Pfr state of the bathy phytochrome from Pseudomonas aeruginosa, the very good agreement between calculated and experimental Raman spectra of the biliverdin cofactor is in line with important conclusions of previous crystallographic analyses, particularly the ZZEssa configuration of the chromophore and its mode of covalent attachment to the protein. The highly homogeneous chromophore conformation seems to be a unique property of the Pfr states of bathy phytochromes. This is in sharp contrast to the Pfr states of prototypical phytochromes that display conformational equilibria between two sub-states exhibiting small structural differences at the terminal methine bridges A-B and C-D. These differences may mainly root in the interactions of the cofactor with the highly conserved Asp-194 that occur via its carboxylate function in bathy phytochromes. The weaker interactions via the carbonyl function in prototypical phytochromes may lead to a higher structural flexibility of the chromophore pocket opening a reaction channel for the thermal (ZZE → ZZZ) Pfr to Pr back-conversion.

Photoisomerization of biliverdin (BV) chromophore triggers the photoresponse in native Agp1 bacteriophytochrome. We discuss heterogeneity in phytochrome Pr form to account for the shape of the absorption profile. We investigated different regions of the absorption profile by angle balanced polarization resolved femtosecond VIS pump–IR probe spectroscopy. We studied the Pr form of Agp1 with its natural chromophore and with a sterically locked 18Et-BV (locked Agp1). We followed the dynamics and orientations of the carbonyl stretching vibrations of ring D and ring A in their ground and electronically excited states. Photoisomerization of ring D is reflected by strong signals of the ring D carbonyl vibration. In contrast, orientational data on ring A show no rotation of ring A upon photoexcitation. Orientational data allow excluding a ZZZasa geometry and corroborates a nontwisted ZZZssa geometry of the chromophore. We found no proof for heterogeneity but identified a new, to our knowledge, electronic transition in the absorption profile at 644 nm (S0→S2). Excitation of the S0→S2 transition will introduce a more complex photodynamics compared with S0→S1 transition. Our approach provides fundamental information on disentanglement of absorption profiles, identification of chromophore structures, and determination of molecular groups involved in the photoisomerization process of photoreceptors.

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red light–absorbing form, Pr, and the far-red-light–absorbing form, Pfr. Here we performed in vitro and in vivo studies using locked phycocyanobilin derivatives, termed 15 Z anti phycocyanobilin (15ZaPCB) and 15 E anti PCB (15EaPCB). Recombinant bacterial and plant phytochromes incorporated either chromophore in a noncovalent or covalent manner. All adducts were photoinactive. The absorption spectra of the 15ZaPCB and 15EaPCB adducts were comparable with those of the Pr and Pfr form, respectively. Feeding of 15EaPCB, but not 15ZaPCB, to protonemal filaments of the moss Ceratodon purpureus resulted in increased chlorophyll accumulation, modulation of gravitropism, and induction of side branches in darkness. The effect of locked chromophores on phytochrome responses, such as induction of seed germination, inhibition of hypocotyl elongation, induction of cotyledon opening, randomization of gravitropism, and gene regulation, were investigated in wild-type Arabidopsis thaliana and the phytochrome-chromophore–deficient long hypocotyl mutant hy1. All phytochrome responses were induced in darkness by 15EaPCB, not only in the mutant but also in the wild type. These studies show that the 15Ea stereochemistry of the chromophore results in the formation of active Pfr-like phytochrome in the cell. Locked chromophores might be used to investigate phytochrome responses in many other organisms without the need to isolate mutants. The induction of phytochrome responses in the hy1 mutant by 15EaPCB were however less efficient than by red light irradiation given to biliverdin-rescued seeds or seedlings.

Photolyases and cryptochromes are evolutionarily related flavoproteins with distinct functions. While photolyases can repair UV-induced DNA lesions in a light-dependent manner, cryptochromes regulate growth, development and the circadian clock in plants and animals. Here we report about two photolyase-related proteins, named PhrA and PhrB, found in the phytopathogen Agrobacterium tumefaciens. PhrA belongs to the class III cyclobutane pyrimidine dimer (CPD) photolyases, the sister class of plant cryptochromes, while PhrB belongs to a new class represented in at least 350 bacterial organisms. Both proteins contain flavin adenine dinucleotide (FAD) as a primary catalytic cofactor, which is photoreduceable by blue light. Spectral analysis of PhrA confirmed the presence of 5,10-methenyltetrahydrofolate (MTHF) as antenna cofactor. PhrB comprises also an additional chromophore, absorbing in the short wavelength region but its spectrum is distinct from known antenna cofactors in other photolyases. Homology modeling suggests that PhrB contains an Fe-S cluster as cofactor which was confirmed by elemental analysis and EPR spectroscopy. According to protein sequence alignments the classical tryptophan photoreduction pathway is present in PhrA but absent in PhrB. Although PhrB is clearly distinguished from other photolyases including PhrA it is, like PhrA, required for in vivo photoreactivation. Moreover, PhrA can repair UV-induced DNA lesions in vitro. Thus, A. tumefaciens contains two photolyase homologs of which PhrB represents the first member of the cryptochrome/photolyase family (CPF) that contains an iron-sulfur cluster.

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK(a) from 11.1 ( wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild- type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild- type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc -> Pfr transition and most likely coupled to the transient proton re- uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state. Fil: von Stetten, David. Technische Universität Berlin; Alemania Fil: Seibeck, Sven. Freie Universität Berlin.; Alemania Fil: Michael, Norbert. Freie Universität Berlin.; Alemania Fil: Scheerer, Patrick. Charité Universitätsmedizin Berlin; Alemania Fil: Mroginski, Maria Andrea. Technische Universität Berlin; Alemania Fil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Technische Universität Berlin; Alemania Fil: Krauss, Norbert. Freie Universität Berlin.; Alemania Fil: Heyn, Maarten P.. Charité Universitätsmedizin Berlin; Alemania Fil: Hildebrandt, Peter. Technische Universität Berlin; Alemania Fil: Borucki, Berthold. Freie Universität Berlin.; Alemania Fil: Lamparter, Tilman. Freie Universität Berlin.; Alemania

Phytochromes are photochromic photoreceptors with a bilin chromophore that are found in plants and bacteria. The soil bacterium Agrobacterium tumefaciens contains two genes that code for phytochrome-homologous proteins, termed Agrobacterium phytochrome 1 and 2 (Agp1 and Agp2). To analyze its biochemical and spectral properties, Agp1 was purified from the clone of an E. coli overexpressor. The protein was assembled with the chromophores phycocyanobilin and biliverdin, which is the putative natural chromophore, to photoactive holoprotein species. Like other bacterial phytochromes, Agp1 acts as light-regulated His kinase. The biliverdin adduct of Agp1 represents a previously uncharacterized type of phytochrome photoreceptor, because photoreversion from the far-red absorbing form to the red-absorbing form is very inefficient, a feature that is combined with a rapid dark reversion. Biliverdin bound covalently to the protein; blocking experiments and site-directed mutagenesis identified a Cys at position 20 as the binding site. This particular position is outside the region where plant and some cyanobacterial phytochromes attach their chromophore and thus represents a previously uncharacterized binding site. Sequence comparisons imply that the region around Cys-20 is a ring D binding motif in phytochromes.

Phytochromes are photoreceptors that occur in plants, fungi and bacteria, among others in the phytopathogen Agrobacterium tumefaciens. We constructed single and double knockout mutants of the two A. tumefaciens phytochromes Agp1 and Agp2. In liquid culture, the double mutant revealed a reduced growth rate, whereas the growth rates of the single mutants did not differ significantly from that of the wild type. Using these mutants, we analyzed the spectral properties of native A. tumefaciens phytochromes. A wild-type A. tumefaciens cell contains about 10 molecules of Agp1 and about 19 molecules of Agp2. Dark conversion of native Agp1 and Agp2 proceeds from Pfr to Pr and from Pr to Pfr, respectively, as has already been reported for the recombinant proteins. The spectral properties of recombinant and native Agp2 were significantly different. Mixing experiments with extracts from the double mutant and recombinant Agp2 imply that the spectral properties of Agp2 are modulated by components of the extract.