Tilman Lamparter

Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site

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.

Mosses as model systems

Mosses hold many attractions as model organisms for research in plant science. Their position as the simplest of land plants makes them central to the study of plant evolution, particularly in shedding light on how their aquatic predecessors evolved to survive on land. The use of mosses for developmental studies hinges on the ability to observe development in living material at the level of the individual cell. However, more recently techniques for the molecular analysis of mosses have provided tools for new approaches for determining the mechanisms controlling plant development, incorporating both cell and molecular biology.

Light-induced Proton Release of Phytochrome Is Coupled to the Transient Deprotonation of the Tetrapyrrole Chromophore

The Pr → Pfr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-RC were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in Pr and ZZEssa in Pfr, with all pyrrole nitrogens being protonated. Photoexcitation of Pr is followed by (at least) three thermal relaxation components in the formation of Pfr with time constants of 230 μs and 3.1 and 260 ms. H2O/D2O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-RC, respectively. Resonance Raman measurements of Meta-RC indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-RC are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the Pr → Pfr photocon-version mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the Pfr formation that switch between the active and the inactive state of the photoreceptor.

Evolution of cyanobacterial and plant phytochromes

Phytochromes are broadly distributed photochromic photoreceptors that are most sensitive in the red and far‐red region of the visible spectrum. Three different bilins can be used as chromophores: plant phytochromes incorporate phytochromobilin, while phycocyanobilin serves as a chromophore of some cyanobacterial phytochromes, whereas all other phytochromes, including cyanobacterial orthologs incorporate biliverdin. During the evolution of plant and cyanobacterial phytochromes, the chromophore binding site has changed from a cysteine close to the N‐terminus of the protein, the biliverdin attachment site, to a cysteine which lies within the so‐called GAF domain and serves as phytochromobilin or phycocyanobilin attachment site. Since phylogenetic analyses imply that plant phytochromes are not direct successors of cyanobacterial phytochromes, chromophore exchange and the switch of the chromophore binding site has probably occurred at least twice in evolution. This may be regarded as an example for convergent evolution at the molecular level.

Ultrafast dynamics of phytochrome from the cyanobacterium synechocystis, reconstituted with phycocyanobilin and phycoerythrobilin.

Femtosecond time-resolved transient absorption spectroscopy was employed to characterize for the first time the primary photoisomerization dynamics of a bacterial phytochrome system in the two thermally stable states of the photocycle. The 85-kDa phytochrome Cph1 from the cyanobacterium Synechocystis PCC 6803 expressed in Escherichia coli was reconstituted with phycocyanobilin (Cph1-PCB) and phycoerythrobilin (Cph1-PEB). The red-light-absorbing form Pr of Cph1-PCB shows an approximately 150 fs relaxation in the S(1) state after photoexcitation at 650 nm. The subsequent Z-E isomerization between rings C and D of the linear tetrapyrrole-chromophore is best described by a distribution of rate constants with the first moment at (16 ps)(-1). Excitation at 615 nm leads to a slightly broadened distribution. The reverse E-Z isomerization, starting from the far-red-absorbing form Pfr, is characterized by two shorter time constants of 0.54 and 3.2 ps. In the case of Cph1-PEB, double-bond isomerization does not take place, and the excited-state lifetime extends into the nanosecond regime. Besides a stimulated emission rise time between 40 and 150 fs, no fast relaxation processes are observed. This suggests that the chromophore-protein interaction along rings A, B, and C does not contribute much to the picosecond dynamics observed in Cph1-PCB but rather the region around ring D near the isomerizing C(15) [double bond] C(16) double bond. The primary reaction dynamics of Cph1-PCB at ambient temperature is found to exhibit very similar features as those described for plant type A phytochrome, i.e., a relatively slow Pr, and a fast Pfr, photoreaction. This suggests that the initial reactions were established already before evolution of plant phytochromes began.

Highly conserved residues Asp-197 and His-250 in agp1 phytochrome control the proton affinity of the chromophore and Pfr formation

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 pKa 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 photo-induced 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.

Biliverdin Binds Covalently to Agrobacterium Phytochrome Agp1 via Its Ring A Vinyl Side Chain

The widely distributed phytochrome photoreceptors carry a bilin chromophore, which is covalently attached to the protein during a lyase reaction. In plant phytochromes, the natural chromophore is coupled by a thioether bond between its ring A ethylidene side chain and a conserved cysteine residue within the so-called GAF domain of the protein. Many bacterial phytochromes carry biliverdin as natural chromophore, which is coupled in a different manner to the protein. In phytochrome Agp1 of Agrobacterium tumefaciens, biliverdin is covalently attached to a cysteine residue close to the N terminus (position 20). By testing different natural and synthetic biliverdin derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment. Only those bilins that have ring A vinyl side chain were covalently attached, whereas bilins with an ethylidene or ethyl side chain were bound in a noncovalent manner. Phycocyanobilin, which belongs to the latter group, was however covalently attached to a mutant in which a cysteine was introduced into the GAF domain of Agp1 (position 249). It is proposed that the regions around positions 20 and 249 are in close contact and contribute both to the chromophore pocket. In competition experiments it was found that phycocyanobilin and biliverdin bind with similar strength to the wild type protein. However, in the V249C mutant, phycocyanobilin bound much more strongly than biliverdin. This finding could explain why during phytochrome evolution in cyanobacteria, the chromophore-binding site swapped from the N terminus into the GAF domain.

Crystal structure of a prokaryotic (6-4) photolyase with an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore

The (6-4) photolyases use blue light to reverse UV-induced (6-4) photoproducts in DNA. This (6-4) photorepair was thought to be restricted to eukaryotes. Here we report a prokaryotic (6-4) photolyase, PhrB from Agrobacterium tumefaciens, and propose that (6-4) photolyases are broadly distributed in prokaryotes. The crystal structure of photolyase related protein B (PhrB) at 1.45 Å resolution suggests a DNA binding mode different from that of the eukaryotic counterparts. A His-His-X-X-Arg motif is located within the proposed DNA lesion contact site of PhrB. This motif is structurally conserved in eukaryotic (6-4) photolyases for which the second His is essential for the (6-4) photolyase function. The PhrB structure contains 6,7-dimethyl-8-ribityllumazine as an antenna chromophore and a [4Fe-4S] cluster bound to the catalytic domain. A significant part of the Fe-S fold strikingly resembles that of the large subunit of eukaryotic and archaeal primases, suggesting that the PhrB-like photolyases branched at the base of the evolution of the cryptochrome/photolyase family. Our study presents a unique prokaryotic (6-4) photolyase and proposes that the prokaryotic (6-4) photolyases are the ancestors of the cryptochrome/photolyase family.

Assembly of Synthetic Locked Chromophores with Agrobacterium Phytochromes Agp1 and Agp2

Phytochromes are photoreceptors with a bilin chromophore in which light triggers the conversion between the red-absorbing form Pr and the far-red-absorbing form Pfr. Agrobacterium tumefaciens has two phytochromes, Agp1 and Agp2, with antagonistic properties: in darkness, Agp1 converts slowly from Pfr to Pr, whereas Agp2 converts slowly from Pr to Pfr. In a previous study, we have assembled Agp1 with synthetic locked chromophores 15Za, 15Zs, 15Ea, and 15Es in which the C15=C16 double bond is fixed in either the E or Z configuration and the C14–C15 single bond is fixed in either the syn (s) or anti (a) conformation. In the present study, the locked chromophores 5Za and 5Zs were used for assembly with Agp1; in these chromophores, the C4=C5 double bond is fixed in the Z configuration, and the C5–C6 single bond is fixed in either the syn or anti conformation. All locked chromophores were also assembled with Agp2. The data showed that in both phytochromes the Pr chromophore adopts a C4=C5 Z C5–C6 syn C15=C16 Z C14–C15 anti stereochemistry and that in the Pfr chromophore the C15=C16 double bond has isomerized to the E configuration, whereas the C14–C15 single bond remains in the anti conformation. Photoconversion shifted the absorption maxima of the 5Zs adducts to shorter wavelengths, whereas the 5Za adducts were shifted to longer wavelengths. Thus, the C5–C6 single bond of the Pfr chromophore is rather in an anti conformation, supporting the previous suggestion that during photoconversion of phytochromes, a rotation around the ring A-B connecting single bond occurs.

Light affects motility and infectivity of Agrobacterium tumefaciens

Response to changes in light conditions involves a variety of receptors that can modulate gene expression, enzyme activity and/or motility. For the study of light-regulated effects of Agrobacterium tumefaciens, we used a global analysis approach - proteomics - and compared the protein patterns of dark- and light-grown bacteria. These analyses revealed a significant reduction of FlaA and FlaB - proteins of the flagellum - when the cells were grown in light. The light effect was confirmed by SDS-PAGE with isolated flagella. Quantitative PCR experiments showed a 10-fold increase of the transcription level of flaA, flaB and flaC within 20 min after the transfer from light to darkness. Electron microscopy revealed that these molecular events result in a light-induced reduction of the number of flagella per cell. These changes have major physiological consequences regarding motility, which is considerably reduced with exposure to light. The inhibitory effect of light on the motility is not unique to A. tumefaciens and was also seen in other species of the Rhizobiaceae. Previous studies suggested that the flagella function is significant for bacteria-plant interactions and bacterial virulence. In our studies, light reduced the attachment of A. tumefaciens to tomato roots and the virulence of the bacteria in a cucumber infection assay.