Cyanochromes Are Blue/Green Light Photoreversible Photoreceptors Defined by a Stable Double Cysteine Linkage to a Phycoviolobilin-type Chromophore

Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C3¹ carbon of the ethylidene side chain and the C4 or C5 carbons in the A–B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb → Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals.Phytochromes (Phys)³ comprise a large and diverse superfamily of photoreceptors that regulate a wide range of physiological responses in plants, fungi, bacteria, and cyanobacteria (1–3). They are unique among photoreceptors by being able to photoconvert between two stable states, a red light-absorbing Pr form that is typically the dark-adapted and biologically inactive conformer and a far-red light-absorbing Pfr form that requires light for its production and is typically the biologically active conformer. By interconverting between Pr and Pfr, Phys act as light-regulated switches in controlling processes ranging from phototaxis and pigmentation in bacteria to seed germination, photomorphogenesis, and flowering time in higher plants.Light absorption by Phys is directed by a bilin (or linear tetrapyrrole) chromophore produced by the oxidative cleavage of heme. Although bacterial and fungal Phys use the immediate cleavage product biliverdin (BV), cyanobacterial and higher plant Phys use phycocyanobilin (PCB) and phytochromobilin, respectively, produced by enzymatic reduction of BV (1, 2). The bilin is then covalently bound autocatalytically to the photosensory unit of the apoprotein, which typically contains a sequence of Per/Arndt/Sim (PAS), cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF), and Phy-associated (PHY) domains. Intimate contact between the bilin and surrounding protein residues then generates the unique photochromic properties of Phys. Recent three-dimensional structures of the Pr form of several bacterial Phys (BphPs) and two cyanobacterial Phys (Cphs) have shown that the bilin is deeply buried within the GAF domain in a ZZZssa configuration and that the connection between the GAF and PAS domains is stabilized by a rare figure-of-eight knot involving the region upstream of the PAS domain being lassoed by a conserved loop within the GAF domain (4–9). Although the structure of Pfr remains unsolved, various physicochemical studies have proposed that photoconversion involves a rotation of one of the three methine bridges between the pyrrole rings (1, 10–14). This rotation then induces much slower thermally driven movements of the protein to initiate signal transduction.In microorganisms, Pfr can activate a variety of signaling systems using output motifs directly appended to the C-terminal end of the photosensory region. The most prevalent are histidine kinase domains that then begin specific two-component phosphorelays (3, 15, 16). Although the output of plant Phys remains unclear, the presence of a C-terminal HK-related domain suggests that they also work as light-regulated protein kinases (17).In addition to the canonical Phys, it has become apparent through phylogenetic and biochemical studies that a heterogeneous collection of Phy-like photoreceptors exists (e.g. Refs. 3 and 18). These include Phys that prefer Pfr as the dark-adapted state (7, 19, 20), Phys that photoconvert from Pr to shorter wavelength-absorbing “near red” or Pnr forms (6, 21), and Phy-like photoreceptors that bind bilins but instead photoconvert between forms with maximal absorption other than red and far-red light (22–25). Often these Phy-like sequences are missing key residues or domains common among canonical Phys, suggesting that they employ novel bilins as chromophores, bind the bilin in different architectures, and/or use distinct photochemistries.One subclass of novel Phy-like photoreceptors present in a number of cyanobacteria, which we have designated cyanochromes (or Cycs) to better distinguish them from Cphs, is exemplified by Synechocystis sp. PCC6803 (Syn) PixJ (or TaxD1, locus sll0041) and its relatives. Syn-PixJ was discovered based on its involvement in blue light-mediated phototaxis in this mesophilic cyanobacterium (26, 27) with its close homolog Te-PixJ (locus tll0569) then found in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 by sequence similarity (28). Like Cphs, the cyanochromes tested thus far covalently bind PCB but then generate photoreceptors that convert between blue and green light-absorbing forms designated Pb and Pg, respectively (22, 24, 29). Subsequent studies proposed that PCB is converted to phycoviolobilin (PVB) upon attachment to the apoprotein (30). PVB differs from PCB by having a methylene instead of a methine bridge between the A and B pyrrole rings, which blue-shifts the absorption of the chromophore by shortening the π-conjugation system. Phototransformation of Pb to Pg could then occur by a mechanism similar to Phys.How Te-PixJ and related cyanochromes bind PCB to generate more blue-shifted PVB-type chromophores remains unclear. Like Cphs, two cyanochromes examples link PCB via a thioether linkage between a cysteine in the Cyc-GAF domain and the C3¹ carbon of the ethylidene side chain of ring A (24, 28). Additionally, loss of the C4C5 double bond is necessary to generate PVB. One model by Ishizuka et al. (30) from studies with Te-PixJ proposed that the double bond moves from the C4-C5 position to the C2-C3 position by an autoisomerase activity intrinsic to the GAF domain. A more recent model by Rockwell et al. (24) using another Syn-PixJ relative in T. elongatus, Tlr0924, invoked the possibility of a second cysteine that also participates in PCB ligation. This cysteine was proposed to bind the bilin at the C10 position via a reversible thioether linkage. In the dark-adapted Pb state, the second linkage would then be formed to generate a rubin-like chromophore attached to the bridge between the B and C pyrrole rings. This bond would then break upon photoconversion to generate the more π-conjugated green light-absorbing photoproduct Pg.In this report, we employed a number of physicochemical approaches to help resolve the unique chromophore architecture and photochemical properties of cyanochromes, using Te-PixJ as the example. By independently mutagenizing the cysteine that binds the A ring ethylidene (Cys-522 (22)) and that proposed by Rockwell et al. (24) to reversibly bind the bilin at a second site (Cys-494), we demonstrate that both residues form light-stable covalent adducts with a PVB-type chromophore. In addition, we employed various spectroscopic methods to show that the bound PVB is fully protonated as both Pb and Pg, that only one pyrrole ring is active during photoconversion, and that the polypeptide may undergo extensive remodeling as Pb converts to Pg. We identified a set of conserved amino acids in Te-PixJ important for cyanochrome photochemistry, including several that when substituted generate yellow or red fluorescent chromoproteins potentially useful for cell biological applications. Phylogenetic analyses show that cyanochromes are widespread among cyanobacteria with their closest relatives being members of the red/far-red light-absorbing Phy subfamily defined by the absence of the N-terminal PAS domain (31).     

Respirometric response and microbial succession of nitrifying sludge to <Emphasis Type="Italic">m</Emphasis>-cresol pulses in a sequencing batch reactor

A nitrifying consortium was kinetically, stoichiometrically and molecularly characterized via the in situ pulse respirometric method and pyrosequencing analysis before and after the addition of m-cresol (25 mg C L^?1) in a sequencing batch reactor (SBR). Five important kinetic and stoichiometric parameters were determined: the maximum oxygen uptake rate, the maximum nitrification rate, the oxidation yield, the biomass growth yield, and the substrate affinity constant. An inhibitory effect was observed in the nitrification process with a recovery of this by up to eight SBR cycles after m-cresol was added to the system. However, full recovery of the nitrification process was not observed, as the maximum oxygen uptake rate was 25% lower than that of the previous operation without m-cresol addition. Furthermore, the pyrosequencing analyses of the nitrifying consortium after the addition of only two pulses of 25 mg C L^?1 m-cresol showed an important microbial community change represented by a decrease in the nitrifying populations and an increase in the populations degrading phenolic compounds.     

Heme oxidation in a chimeric protein of the alpha-selective Neisseriae meningitidis heme oxygenase with the distal helix of the delta-selective Pseudomonas aeruginosa

Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeruginosa (pa-HO) share significant sequence identity (37%). In nm-HO, biliverdin IXalpha is the sole product of the reaction, whereas pa-HO yields predominantly biliverdin IXdelta. We have previously shown by NMR that the in-plane conformation of the heme in pa-HO is significantly different from that of nm-HO as a result of distinct interactions of the heme propionates with the protein scaffold [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. In the report presented here, we have extended these studies to investigate the role of the distal helix by preparing a chimera of nm-HO (nm-HOch), in which distal helix residues 107-142 of nm-HO have been replaced with the corresponding residues of the delta-regioselective pa-HO (112-147). Electronic absorption spectra, resonance Raman and FTIR spectroscopic studies confirm that the orientation and hydrogen bonding properties of the proximal His ligand are not significantly altered in the chimera relative those of the wild-type proteins. The catalytic turnover of the nm-HOch-heme complex yields almost exclusively alpha-biliverdin and a small but reproducible amount of delta-biliverdin. NMR spectroscopic studies reveal that the altered regioselectivity in the chimeric protein likely stems from a dynamic equilibrium between two alternate in-plane conformations of the heme (in-plane heme disorder). Replacement of K16 with Ala and Met31 with Lys in the chimeric protein in an effort to tune key polypeptide-heme propionate contacts largely stabilizes the in-plane conformer conducive to delta-meso hydroxylation.     

Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses

Background

The current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein.

Methods

We analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation.

Results

The proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections.

Conclusion

These results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform.     

Bone modeling response to voluntary exercise in the hindlimb of mice

The functional adaptation of juvenile mammalian limb bone to mechanical loading is necessary to maintain bone strength. Diaphyseal size and shape are modified during growth through the process of bone modeling. Although bone modeling is a well-documented response to increased mechanical stress on growing diaphyseal bone, the effect of proximodistal location on bone modeling remains unclear. Distal limb elements in cursorial mammals are longer and thinner, most likely to conserve energy during locomotion because they require less energy to move. Therefore, distal elements are hypothesized to experience greater mechanical loading during locomotion and may be expected to exhibit a greater modeling response to exercise. In this study, histomorphometric comparisons are made between femora and tibiae of mice treated with voluntary exercise and a control group (N = 20). We find that femora of exercised mice exhibit both greater bone growth rates and growth areas than do controls (P < 0.05). The femora of exercised mice also have significantly greater cortical area, bending rigidity, and torsional rigidity (P < 0.05), although bending and torsional rigidity are comparable when standardized by bone length. Histomorphometric and cross-section geometric properties of the tibial midshaft of exercised and control mice did not differ significantly, although tibial length was significantly greater in exercised mice (P < 0.05). Femora of exercised mice were able to adapt to increased mechanical loading through increases in compressive, bending, and torsional rigidity. No such adaptations were found in the tibia. It is unclear if this is a biomechanical adaptation to greater stress in proximal elements or if distal elements are ontogenetically constrained in a tradeoff of bone strength of distal elements for bioenergetic efficiency during locomotion.     

The MMP/TIMP system in the nervous system

The matrix metalloproteinases (MMP) belong to a growing family of secreted or membrane-bound (MT-MMP) enzymes that cleave protein components of the extracellular matrix and bioactive factors involved in intercellular signaling. MMP activity is counterbalanced by their four physiological inhibitors, the tissue inhibitors of MMP (TIMPs). Together, MMP and TIMP control cell-cell and cell-matrix interactions associated with physiological processes. However, the breakdown of the protease-inhibitor balance may lead to the loss of tissue homeostasis and the development of degenerative and tumorigenic processes in various tissues. The emerging idea is that the MMP/TIMP system also plays a major role in the pathology and physiology of the nervous system and that mastering MMP activity will set the basis for new and more efficient therapeutic strategies against nervous system disorders.     

beta-Phosphorothioate analogs of nucleotide sugars are resistant to hydrolytic degradation and utilized efficiently by glycosyltransferases.

The alpha- and beta-phosphorothioate analogs of UDP-Gal and UDP-Glc, in which a sulfur is exchanged for a non-bridging oxygen at one of the phosphate groups, have been synthesized and tested for their resistance to enzymatic degradation and for their usefulness in glycosyltransferase reactions. The alpha analogs were found to be no more resistant to hydrolysis than the native nucleotide sugars, but as previously reported (R. B. Marchase et al. (1987) Biochim. Biophys. Acta 916: 157) the beta S analogs were approximately 10 times more resistant. The beta S analog and native UDP-Glc were found to have comparable Km's when used in assays for glucosylphosphoryl dolichol synthase with rat liver and hen oviduct microsomes, although the apparent Vmax of the reaction was about twofold higher for the analog, presumably due to its resistance to degradation. Partially purified 4 beta-galactosyltransferase exhibited a Vmax with (beta S)UDP-Gal that was only slightly lower than that with UDP-Gal and a Km that was slightly increased. The effectiveness of the analog was especially apparent in assays for 4 beta-galactosyltransferase on intact sperm and in rat liver homogenates, in which hydrolysis of the normal substrate was very rapid and net incorporation was at least 4 times greater with the beta S analog in each system.     

Repertoire Sharing and Song Similarity between Great Tit Males Decline with Distance between Forest Fragments

Birdsong can play a critical role in establishing a territory and finding a mate among individuals from local and foreign populations. Variation in birdsong among populations can be influenced by habitat fragmentation and might affect successful dispersal among habitat fragments. We studied variation in great tit song in a long‐term study population distributed over nine forest fragments. All individual males recorded had a known dispersal history within the fragmented forest habitat. We found spatial structure of declining song‐type sharing with distance, with a marked drop from an individual’s own forest fragment to another across a habitat gap. We also found decreasing song similarity among increasingly distant fragments in terms of temporal and spectral characteristics of shared song types. The change in acoustic structure was more gradual and seemed less affected by habitat discontinuity but also showed a tight correlation with dispersal index among forest fragments. Immigrant birds shared fewer song types with neighbouring birds that were born within the same forest fragment, but not less compared to birds born in another forest fragment within the study area. Our data provide detailed insight into the relationship between song differentiation and male dispersal and contribute to our understanding of the potential role of song in reproductive exchange and avian speciation. The fact that birds in small forest fragments shared more songs than birds in larger forest fragments confirms that song analysis has potential as a tool for conservation in rare species.