Characterization of the Blue-Light-Activated Adenylyl Cyclase mPAC by Flash Photolysis and FTIR Spectroscopy.

The recently discovered photo-activated adenylyl cyclase (mPAC from Microcoleus chthonoplastes) is the first PAC that owes a light-, oxygen- and voltage-sensitive (LOV) domain for blue-light sensing. The photoreaction of the mPAC receptor was studied by time-resolved UV/vis and light-induced Fourier transform infrared (FTIR) absorption difference spectroscopy. The photocycle comprises of the typical triplet state LOV715 and the thio-adduct state LOV390 . While the adduct state decays with a time constant of 8 s, the lifetime of the triplet state is with 656 ns significantly shorter than in all other reported LOV domains. The light-induced FTIR difference spectrum shows the typical bands of the LOV390 and LOV450 intermediates. The negative S-H stretching vibration at 2573 cm-1 is asymmetric suggesting two rotamer configurations of the protonated side chain of C194. A positive band at 3632 cm-1 is observed, which is assigned to an internal water molecule. In contrast to other LOV domains, mPAC exhibits a second positive feature at 3674 cm-1 which is due to the O-H stretch of a second intrinsic water molecule and the side chain of Y476. We conclude that the latter might be involved in the dimerization of the cyclase domain which is crucial for ATP binding.

Revisiting chromatin binding of the Arabidopsis UV-B photoreceptor UVR8.

BACKGROUND: Plants perceive UV-B through the UV RESISTANCE LOCUS 8 (UVR8) photoreceptor and UVR8 activation leads to changes in gene expression such as those associated with UV-B acclimation and stress tolerance. Albeit functionally unrelated, UVR8 shows some homology with RCC1 (Regulator of Chromatin Condensation 1) proteins from non-plant organisms at the sequence level. These proteins act as guanine nucleotide exchange factors for Ran GTPases and bind chromatin via histones. Subsequent to the revelation of this sequence homology, evidence was presented showing that UVR8 activity involves interaction with chromatin at the loci of some target genes through histone binding. This suggested a UVR8 mode-of-action intimately and directly linked with gene transcription. However, several aspects of UVR8 chromatin association remained undefined, namely the impact of UV-B on the process and how UVR8 chromatin association related to the transcription factor ELONGATED HYPOCOTYL 5 (HY5), which is important for UV-B signalling and has overlapping chromatin targets. Therefore, we have investigated UVR8 chromatin association in further detail. RESULTS: Unlike the claims of previous studies, our chromatin immunoprecipitation (ChIP) experiments do not confirm UVR8 chromatin association. In contrast to human RCC1, recombinant UVR8 also does not bind nucleosomes in vitro. Moreover, fusion of a VP16 activation domain to UVR8 did not alter expression of proposed UVR8 target genes in transient gene expression assays. Finally, comparison of the Drosophila DmRCC1 and the Arabidopsis UVR8 crystal structures revealed that critical histone- and DNA-interaction residues apparent in DmRCC1 are not conserved in UVR8. CONCLUSION: This has led us to conclude that the cellular activity of UVR8 likely does not involve its specific binding to chromatin at target genes.

A cyanobacterial light activated adenylyl cyclase partially restores development of a Dictyostelium discoideum, adenylyl cyclase a null mutant.

A light-regulated adenylyl cyclase, mPAC, was previously identified from the cyanobacterium Microcoleus chthonoplastes PCC7420. MPAC consists of a flavin-based blue light-sensing LOV domain and a catalytic domain. In this work, we expressed mPAC in an adenylate cyclase A null mutant (aca-) of the eukaryote Dictyostelium discoideum and tested to what extent light activation of mPAC could restore the cAMP-dependent developmental programme of this organism. Amoebas of Dictyostelium, a well-established model organism, generate and respond to cAMP pulses, which cause them to aggregate and construct fruiting bodies. mPAC was expressed under control of a constitutive actin-15 promoter in D. discoideum and displayed low basal adenylyl cyclase activity in darkness that was about five-fold stimulated by blue light. mPAC expression in aca- cells marginally restored aggregation and fruiting body formation in darkness. However, more and larger fruiting bodies were formed when mPAC expressing cells were incubated in light. Extending former applications of light-regulated AC, these results demonstrate that mPAC can be used to manipulate multicellular development in eukaryotes in a light dependent manner.

Combined mutagenesis and kinetics characterization of the bilin-binding GAF domain of the protein Slr1393 from the Cyanobacterium Synechocystis PCC6803.

The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red-absorbing parental and a green-absorbing photoproduct state (λmax =649 and 536 nm, respectively). Conversions in both directions were followed by time-resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 µs, 390 µs, and 1.5 ms) for the red-to-green conversion, and 1.2 µs, 340 µs, and 1 ms for the green-to-red conversion. In addition to the wild-type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site-directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red-absorbing, WT-like protein. Irradiation, however, not only converted it into the green-absorbing form, but also produced a 660 nm, further-red-shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.

A LOV-domain-mediated blue-light-activated adenylate (adenylyl) cyclase from the cyanobacterium Microcoleus chthonoplastes PCC 7420.

Genome screening of the cyanobacterium Microcoleus chthonoplastes PCC 7420 identified a gene encoding a protein (483 amino acids, 54.2 kDa in size) characteristic of a BL (blue light)-regulated adenylate (adenylyl) cyclase function. The photoreceptive part showed signatures of a LOV (light, oxygen, voltage) domain. The gene product, mPAC (Microcoleus photoactivated adenylate cyclase), exhibited the LOV-specific three-peaked absorption band (λmax=450 nm) and underwent conversion into the photoadduct form (λmax=390 nm) upon BL-irradiation. The lifetime for thermal recovery into the parent state was determined as 16 s at 20°C (25 s at 11°C). The adenylate cyclase function showed a constitutive activity (in the dark) that was in-vitro-amplified by a factor of 30 under BL-irradiation. Turnover of the purified protein at saturating light and pH 8 is estimated to 1 cAMP/mPAC per s at 25°C (2 cAMP/mPAC per s at 35°C). The lifetime of light-activated cAMP production after a BL flash was ~14 s at 20°C. The temperature optimum was determined to 35°C and the pH optimum to 8.0. The value for half-maximal activating light intensity is 6 W/m2 (at 35°C). A comparison of mPAC and the BLUF (BL using FAD) protein bPAC (Beggiatoa PAC), as purified proteins and expressed in Xenopus laevis oocytes, yielded higher constitutive activity for mPAC in the dark, but also when illuminated with BL.

A structural model for the full-length blue light-sensing protein YtvA from Bacillus subtilis, based on EPR spectroscopy.

A model for the full-length structure of the blue light-sensing protein YtvA from Bacillus subtilis has been determined by EPR spectroscopy, performed on spin labels selectively inserted at amino acid positions 54, 80, 117 and 179. Our data indicate that YtvA forms a dimer in solution and enable us, based on the known structures of the individual domains and modelling, to propose a three-dimensional model for the full length protein. Most importantly, this includes the YtvA N-terminus that has so far not been identified in any structural model. We show that our data are in agreement with the crystal structure of an engineered LOV-domain protein, YF1, that shows the N-terminus of the protein to be helical and to fold back in between the ß-sheets of the two LOV domains, and argue for an identical arrangement in YtvA. While we could not detect any structural changes upon blue-light activation of the protein, this structural model now forms an ideal basis for identifying residues as targets for further spin labelling studies to detect potential conformational changes upon irradiation of the protein.

The amino acids surrounding the flavin 7a-methyl group determine the UVA spectral features of a LOV protein.

Flavin-binding light, oxygen, and voltage (LOV) domains are UVA/blue-light-sensing protein units that form a reversible flavin mononucleotide-cysteine adduct upon light induction. In their dark-adapted state, LOV domains exhibit the typical spectral features of fully oxidized riboflavin derivatives. A survey on the absorption spectra of various LOV domains revealed that the UVA spectral range is the most variable region (whereas the absorption band at 450 nm is virtually unchanged), showing essentially two distinct patterns found in plant phototropin LOV1 and LOV2 domains, respectively. In this work, we have identified a residue directly interacting with the isoalloxazine methyl group at C(7a) as the major UVA spectral tuner. In YtvA from Bacillus subtilis, this amino acid is threonine 30, and its mutation into apolar residues converts the LOV2-like spectrum of native YtvA into a LOV1-like pattern. Mutation T30A also accelerates the photocycle ca. 4-fold. Together with control mutations at different positions, our results experimentally confirm the previously calculated direction of the transition dipole moment for the UVA ππ* state and identify the mechanisms underlying spectral tuning in the LOV domains.

A photochromic bacterial photoreceptor with potential for super-resolution microscopy.

We introduce a novel fluorescent reporter with potential for super-resolution microscopy, based on the bacterial photoreceptor YtvA. YtvA (from Bacillus subtilis) comprises a photosensitive flavin-based LOV domain, efficiently photo-switchable between fluorescent and non-fluorescent states. We demonstrate Fluorescence PhotoActivation Localization Microscopy (FPALM) studies of live Escherichia coli cells, expressing YtvA molecules.

Modulation of the photocycle of a LOV domain photoreceptor by the hydrogen-bonding network.

An extended hydrogen-bonding (HB) network stabilizes the isoalloxazine ring of the flavin mononucleotide (FMN) chromophore within the photosensing LOV domain of blue-light protein receptors, via interactions between the C(2)═O, N(3)H, C(4)═O, and N(5) groups and conserved glutamine and asparagine residues. In this work we studied the influence of the HB network on the efficiency, kinetics, and energetics of a LOV protein photocycle, involving the reversible formation of a FMN-cysteine covalent adduct. The following results were found for mutations of the conserved amino acids N94, N104, and Q123 in the Bacillus subtilis LOV protein YtvA: (i) Increased (N104D, N94D) or strongly reduced (N94A) rate of adduct formation; this latter mutation extends the lifetime of the flavin triplet state, i.e., adduct formation, more than 60-fold, from 2 µs for the wild-type (WT) protein to 129 µs. (ii) Acceleration of the overall photocycle for N94S, N94A, and Q123N, with recovery lifetimes 20, 45, and 85 times faster than for YtvA-WT, respectively. (iii) Slight modifications of FMN spectral features, correlated with the polarization of low-energy transitions. (iv) Strongly reduced (N94S) or suppressed (Q123N) structural volume changes accompanying adduct formation, as determined by optoacoustic spectroscopy. (v) Minor effects on the quantum yield, with the exception of a considerable reduction for Q123N, i.e., 0.22 vs 0.49 for YtvA-WT. The data stress the importance of the HB network in modulating the photocycle of LOV domains, while at the same time establishing a link with functional responses.

A polyphasic approach for the differentiation of environmental Vibrio isolates from temperate waters.

Climate change and marine traffic lead to changing species communities in the oceans. Due to increasing seawater temperatures, pathogenic Vibrio species could become significant even in temperate waters. We classified mesophilic Vibrio isolates from the German Bight (North Sea) using a polyphasic approach with special emphasis on Vibrio parahaemolyticus. Matrix-assisted laser desorption/ionization time-of-flight MS was used as a primary screen to classify isolates, 16S rRNA gene and rpoB gene sequencing to identify species. Potential V. parahaemolyticus isolates were screened for regulatory or virulence-related genes (toxR, tlh, tdh, trh). To investigate genomic diversity, we applied repetitive-sequence-based PCRs. Results were evaluated and methods compared using multivariate statistical analysis. Most isolates were classified as V. parahaemolyticus or Vibrio alginolyticus. Reliable differentiation between both species was achieved by rpoB sequencing and toxR detection. Among the fingerprinting methods, ERIC-PCR showed the highest discriminatory power, displaying three separated clusters. These clusters represent the species V. parahaemolyticus, V. alginolyticus and one group in between. The frequent detection of V. parahaemolyticus in the German Bight reveals the urgency for further monitoring. In this context, a polyphasic approach, such as defined in this study, is needed to differentiate populations of V. parahaemolyticus and V. alginolyticus.