Time-resolved fluorescence anisotropy with Atto 488-labeled phytochrome Agp1 from Agrobacterium fabrum.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time-resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto-488 was attached to each mutant, and time-resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full-length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr-to-Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr.

Dynamic interplays between three redox cofactors in a DNA photolyase revealed by spectral decomposition.

DNA repair catalyzed by photolyases is accomplished by a light-dependent electron transfer event from a fully reduced flavin adenine dinucleotide to a DNA lesion site. Prokaryotic DNA photolyase, PhrB, possesses a ribolumazine cofactor and a four-iron-four-sulfur cluster in addition to the catalytic flavin, but their functional roles are poorly understood. Here, we employ time-resolved absorption spectroscopy to probe light-induced responses in both solution and single crystals of PhrB. We jointly analyze a large collection of light-induced difference spectra from the wild-type and mutant PhrB obtained under different light and redox conditions. By applying singular value decomposition to 159 time series, we dissect light-induced spectral changes and examine the dynamic interplay between three cofactors. Our findings suggest that these cofactors form an interdependent redox network to coordinate light-induced redox responses. We propose that the ribolumazine cofactor serves as a photoprotective pigment under intense light or prolonged illumination, while the iron-sulfur cluster acts as a transient electron cache to maintain balance between two otherwise independent photoreactions of the flavin and ribolumazine.

Phytochrome-Interacting Proteins.

Phytochromes are photoreceptors of plants, fungi, slime molds bacteria and heterokonts. These biliproteins sense red and far-red light and undergo light-induced changes between the two spectral forms, Pr and Pfr. Photoconversion triggered by light induces conformational changes in the bilin chromophore around the ring C-D-connecting methine bridge and is followed by conformational changes in the protein. For plant phytochromes, multiple phytochrome interacting proteins that mediate signal transduction, nuclear translocation or protein degradation have been identified. Few interacting proteins are known as bacterial or fungal phytochromes. Here, we describe how the interacting partners were identified, what is known about the different interactions and in which context of signal transduction these interactions are to be seen. The three-dimensional arrangement of these interacting partners is not known. Using an artificial intelligence system-based modeling software, a few predicted and modulated examples of interactions of bacterial phytochromes with their interaction partners are interpreted.

Evidence for evolutionary relationship between archaeplastidal and cyanobacterial phytochromes based on their chromophore pockets.

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.

The involvement of type IV pili and the phytochrome CphA in gliding motility, lateral motility and photophobotaxis of the cyanobacterium Phormidium lacuna.

Phormidium lacuna is a naturally competent, filamentous cyanobacterium that belongs to the order Oscillatoriales. The filaments are motile on agar and other surfaces and display rapid lateral movements in liquid culture. Furthermore, they exhibit a photophobotactic response, a phototactic response towards light that is projected vertically onto the area covered by the culture. However, the molecular mechanisms underlying these phenomena are unclear. We performed the first molecular studies on the motility of an Oscillatoriales member. We generated mutants in which a kanamycin resistance cassette (KanR) was integrated in the phytochrome gene cphA and in various genes of the type IV pilin apparatus. pilM, pilN, pilQ and pilT mutants were defective in gliding motility, lateral movements and photophobotaxis, indicating that type IV pili are involved in all three kinds of motility. pilB mutants were only partially blocked in terms of their responses. pilB is the proposed ATPase for expelling of the filament in type IV pili. The genome reveals proteins sharing weak pilB homology in the ATPase region, these might explain the incomplete phenotype. The cphA mutant revealed a significantly reduced photophobotactic response towards red light. Therefore, our results imply that CphA acts as one of several photophobotaxis photoreceptors or that it could modulate the photophobotaxis response.

Fungal phytochrome chromophore biosynthesis at mitochondria.

Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome-dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme-derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C-terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000-fold higher than the affinity of the holoprotein, suggesting a "kiss-and-go" mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.

Phytochrome Mediated Responses in Agrobacterium fabrum: Growth, Motility and Plant Infection.

The soil bacterium and plant pathogen Agrobacterium fabrum C58 has two phytochrome photoreceptors, Agp1 and Agp2. We found that plant infection and tumor induction by A. fabrum is down-regulated by light and that phytochrome knockout mutants of A. fabrum have diminished infection rates. The regulation pattern of infection matches with that of bacterial conjugation reported earlier, suggesting similar regulatory mechanisms. In the regulation of conjugation and plant infection, phytochromes are active in darkness. This is a major difference to plant phytochromes, which are typically active after irradiation. We also found that propagation and motility were affected in agp1- and agp2- knockout mutants, although propagation was not always affected by light. The regulatory patterns can partially but not completely be explained by modulated histidine kinase activities of Agp1 and Agp2. In a mass spectrometry-based proteomic study, 24 proteins were different between light and dark grown A. fabrum, whereas 382 proteins differed between wild type and phytochrome knockout mutants, pointing again to light independent roles of Agp1 and Agp2.

Phytochromes in Agrobacterium fabrum.

The focus of this review is on the phytochromes Agp1 and Agp2 of Agrobacterium fabrum. These are involved in regulation of conjugation, gene transfer into plants, and other effects. Since crystal structures of both phytochromes are known, the phytochrome system of A. fabrum provides a tool for following the entire signal transduction cascade starting from light induced conformational changes to protein interaction and the triggering of DNA transfer processes.

Two hybrid histidine kinases, TcsB and the phytochrome FphA, are involved in temperature sensing in Aspergillus nidulans.

The adaptation of microorganisms to different temperatures is an advantage in habitats with steadily changing conditions and raises the question about temperature sensing. Here we show that in the filamentous fungus Aspergillus nidulans, the hybrid histidine kinase TcsB and phytochrome are involved in temperature-induced gene transcription. Temperature-activated phytochrome fed the signal into the HOG MAP kinase pathway. There is evidence that the photoreceptor phytochrome fulfills a temperature sensory role in plants and bacteria. The effects in plants are based on dark reversion from the active form of phytochrome, Pfr, to the inactive form, Pr. Elevated temperature leads to higher dark reversion rates, and hence, temperature sensing depends on light. In A. nidulans and in Alternaria alternata, the temperature response was light-independent. In order to understand the primary temperature response of phytochrome, we performed spectral analyses of recombinant FphA from both fungi. Spectral properties after heat stress resembled the spectrum of free biliverdin, suggesting conformational changes and a softening of the binding pocket of phytochrome, possibly mimicking photoactivation. We propose a novel function for fungal phytochrome as temperature sensor.

Perceived distress, personality characteristics, coping strategies and psychosocial impairments in a national German multicenter cohort of patients with Crohn's disease and ulcerative colitis.

BACKGROUND AND AIMS: This study examined differences in personality, psychological distress, and stress coping in inflammatory bowel disease (IBD) depending on type of disease and disease activity. We compared patients suffering from Crohn's disease (CD) and ulcerative colitis (UC) with controls. While the literature is replete with distinctive features of the pathogenesis of IBD, the specific differences in psychological impairments are not well studied. METHODS: In this German national multicenter study, participants were recruited from 32 centers. Two hundred ninety-seven questionnaires were included, delivering vast information on disease status and psychological well-being based on validated instruments with a total of 285 variables. RESULTS: CD patients were more affected by psychological impairments than patients suffering from UC or controls. Importantly, patients with active CD scored higher in neuroticism (p < 0.01), psychological distress (p < 0.001) and maladaptive stress coping (escape, p = 0.03; rumination, p < 0.03), but less need for social support (p = 0.001) than controls. In contrast, patients suffering from active UC showed psychological distress (p < 0.04) and maladaptive coping (avoidance, p < 0.03; escape, p = 0.01). Patients in remission seemed to be less affected. In particular, patients with UC in remission were not inflicted by psychological impairments. The group of CD patients in remission however, showed insecurity (p < 0.01) and paranoid ideation (p = 0.04). CONCLUSIONS: We identified specific aspects of psychological impairment in IBD depending on disease and disease activity. Our results underscore the need for psychological support and treatment particularly in active CD.

Molecular Recognition: How Photosynthesis Anchors the Mobile Antenna.

True to its name, light-harvesting complex II (LHC II) harvests light energy for photosystem II (PS II). However, LHC II can stray, harvesting light energy for photosystem I (PS I) instead. Cryo-electron microscopy (cryo-EM) now shows how this mobile antenna becomes so attached to its new partner.

Evidence for weak interaction between phytochromes Agp1 and Agp2 from Agrobacterium fabrum.

During bacterial conjugation, plasmid DNA is transferred from cell to cell. In Agrobacterium fabrum, conjugation is regulated by the phytochrome photoreceptors Agp1 and Agp2. Both contribute equally to this regulation. Agp1 and Agp2 are histidine kinases, but, for Agp2, we found no autophosphorylation activity. A clear autophosphorylation signal, however, was obtained with mutants in which the phosphoaccepting Asp of the C-terminal response regulator domain is replaced. Thus, the Agp2 histidine kinase differs from the classical transphosphorylation pattern. We performed size exclusion, photoconversion, dark reversion, autophosphorylation, chromophore assembly kinetics and fluorescence resonance energy transfer measurements on mixed Agp1/Agp2 samples. These assays pointed to an interaction between both proteins. This could partially explain the coaction of both phytochromes in the cell.

Two aspartate residues close to the lesion binding site of Agrobacterium (6-4) photolyase are required for Mg2+ stimulation of DNA repair.

Prokaryotic (6-4) photolyases branch at the base of the evolution of cryptochromes and photolyases. Prototypical members contain an iron-sulphur cluster which was lost in the evolution of the other groups. In the Agrobacterium (6-4) photolyase PhrB, the repair of DNA lesions containing UV-induced (6-4) pyrimidine dimers is stimulated by Mg2+ . We propose that Mg2+ is required for efficient lesion binding and for charge stabilization after electron transfer from the FADH- chromophore to the DNA lesion. Furthermore, two highly conserved Asp residues close to the DNA-binding site are essential for the effect of Mg2+ . Simulations show that two Mg2+ bind to the region around these residues. On the other hand, DNA repair by eukaryotic (6-4) photolyases is not increased by Mg2+ . In these photolyases, structurally overlapping regions contain no Asp but positively charged Lys or Arg. During the evolution of photolyases, the role of Mg2+ in charge stabilization and enhancement of DNA binding was therefore taken over by a postiviely charged amino acid. Besides PhrB, another prokaryotic (6-4) photolyase from the marine cyanobacterium Prochlorococcus marinus, PromaPL, which contains no iron-sulphur cluster, was also investigated. This photolyase is stimulated by Mg2+ as well. The evolutionary loss of the iron-sulphur cluster due to limiting iron concentrations can occur in a marine environment as a result of iron deprivation. However, the evolutionary replacement of Mg2+ by a positively charged amino acid is unlikely to occur in a marine environment because the concentration of divalent cations in seawater is always sufficient. We therefore assume that this transition could have occurred in a freshwater environment.

Structural snapshot of a bacterial phytochrome in its functional intermediate state.

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.

Functional role of an unusual tyrosine residue in the electron transfer chain of a prokaryotic (6-4) 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.

Oligomeric states in sodium ion-dependent regulation of cyanobacterial histidine kinase-2.

Two-component signal transduction systems (TCSs) consist of sensor histidine kinases and response regulators. TCSs mediate adaptation to environmental changes in bacteria, plants, fungi and protists. Histidine kinase 2 (Hik2) is a sensor histidine kinase found in all known cyanobacteria and as chloroplast sensor kinase in eukaryotic algae and plants. Sodium ions have been shown to inhibit the autophosphorylation activity of Hik2 that precedes phosphoryl transfer to response regulators, but the mechanism of inhibition has not been determined. We report on the mechanism of Hik2 activation and inactivation probed by chemical cross-linking and size exclusion chromatography together with direct visualisation of the kinase using negative-stain transmission electron microscopy of single particles. We show that the functional form of Hik2 is a higher-order oligomer such as a hexamer or octamer. Increased NaCl concentration converts the active hexamer into an inactive tetramer. The action of NaCl appears to be confined to the Hik2 kinase domain.

Phytochromes from Agrobacterium fabrum.

Agrobacterium fabrum is a widely used model bacterium for gene transfer from pro- to eukaryote, for genetics and metabolism. The phytochrome system of Agrobacterium, encompassing the two phytochromes Agp1 and Agp2, has provided deep insight into phytochrome action in a bacterial organism. This review summarizes recent results on phytochrome evolution, phytochrome regulation of conjugation and plant infection and biochemical studies including the crystal structure of Agp1-PCM, the photosensory core module of Agp1.

Intersubunit distances in full-length, dimeric, bacterial phytochrome Agp1, as measured by pulsed electron-electron double resonance (PELDOR) between different spin label positions, remain unchanged upon photoconversion.

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.

Crystal Structures of Bacterial (6-4) Photolyase Mutants with Impaired DNA Repair Activity.

PhrB from Agrobacterium fabrum is the first prokaryotic photolyase which repairs (6-4) UV DNA photoproducts. The protein harbors three cofactors: the enzymatically active FAD chromophore, a second chromophore, 6,7-dimethyl-8-ribityllumazine (DMRL) and a cubane-type Fe-S cluster. Tyr424 of PhrB is part of the DNA-binding site and could provide an electron link to the Fe-S cluster. The PhrBY424F mutant showed reduced binding of lesion DNA and loss of DNA repair. The mutant PhrBI51W is characterized by the loss of the DMRL chromophore, reduced photoreduction and reduced DNA repair capacity. We have determined the crystal structures of both mutants and found that both mutations only affect local protein environments, whereas the overall fold remained unchanged. The crystal structure of PhrBY424F revealed a water network extending to His366, which are part of the lesion-binding site. The crystal structure of PhrBI51W shows how the bulky Trp leads to structural rearrangements in the DMRL chromophore pocket. Spectral characterizations of PhrBI51W suggest that DMRL serves as an antenna chromophore for photoreduction and DNA repair in the wild type. The energy transfer from DMRL to FAD could represent a phylogenetically ancient process.

Divalent Cations Increase DNA Repair Activities of Bacterial (6-4) Photolyases.

The (6-4) photolyases of the FeS-BCP group can be considered as the most ancient type among the large family of cryptochrome and photolyase flavoproteins. In contrast to other photolyases, they contain an Fe-S cluster of unknown function, a DMRL chromophore, an interdomain loop, which could interact with DNA, and a long C-terminal extension. We compared DNA repair and photoreduction of two members of the FeS-BCP family, Agrobacterium fabrum PhrB and Rhodobacter sphaeroides RsCryB, with a eukaryotic (6-4) photolyase from Ostreococcus, OsCPF, and a member of the class III CPD photolyases, PhrA from A. fabrum. We found that the low DNA repair effectivity of FeS-BCP proteins is largely stimulated by Mg2+ and other divalent cations, whereas no effect of divalent cations was observed in OsCPF and PhrA. The (6-4) repair activity in the presence of Mg2+ is comparable with the repair activities of the other two photolyases. The photoreduction, on the other hand, is negatively affected by Mg2+ in PhrB, but stimulated by Mg2+ in PhrA. A clear relationship of Mg2+ dependency on DNA repair with the evolutionary position conflicts with Mg2+ dependency of photoreduction. We discuss the Mg2+ effect in the context of structural data and DNA binding.