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Phys (phytochromes) are a superfamily of photochromic photoreceptors that employ a bilin-type chromophore to sense red and far-red light. Although originally thought to be restricted to plants, accumulating genetic and genomic analyses now indicate that they are also prevalent among micro-organisms. By a combination of phylogenetic and biochemical studies, we have expanded the Phy superfamily and organized its members into distinct functional clades which include the phys (plant Phys), BphPs (bacteriophytochromes), Cphs (cyanobacterial Phys), Fphs (fungal Phys) and a collection of Phy-like sequences. All contain a signature GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain, which houses the bilin lyase activity. A PHY domain (uppercase letters are used to denote the PHY domain specifically), which helps stabilize the Pfr form (far-red-light-absorbing form of Phy), is downstream of the GAF region in all but the Phy-like sequences. The phy, Cph, BphP and Fph families also include a PLD [N-terminal PAS (Per/Arnt/Sim)-like domain] upstream of the GAF domain. Site-directed mutagenesis of conserved residues within the GAF and PLD motifs supports their importance in chromophore binding and/or spectral activity. In agreement with Lamparter, Carrascal, Michael, Martinez, Rottwinkel and Abian [(2004) Biochemistry 43, 3659–3669], a conserved cysteine within the PLD of several BphPs was found to be necessary for binding the chromophore via the C-3 vinyl side chain on the bilin A ring. Phy-type sequences were also discovered in the actinobacterium Kineococcus radiotolerans and collections of microorganisms obtained from marine and extremely acidic environments, thus expanding further the range of these photoreceptors. Based on their organization and distribution, the evolution of the Phy superfamily into distinct photoreceptor types is proposed.
- bilin
- histidine kinase
- light perception
- phytochrome
- phylogeny
Light is an important environmental signal for almost all cellular organisms, providing both the energy necessary for growth and metabolism and the sensory information helpful for adaptation. Detection of light is achieved by a suite of photoreceptors that work either singly or in various combinations [1]. One pervasive set of regulatory photoreceptors is the Phys (phytochromes), which are collectively defined by the use of a bilin (or linear tetrapyrrole) chromophore for detecting R (red light) and/or FR (far-red light) [2–4]. This bilin is generated by oxidative cleavage of haem by an HO (haem oxygenase) to generate BV (biliverdin) IXα, and either used directly or further modified depending on the organism [5,6]. Once bound to Phy apoprotein, the bilin can phototransform between two stable conformers, an R-absorbing form (Pr) and an FR-absorbing form (Pfr). By having one form behave as 'active' and the other as 'inactive', Phys can act as R/FR-regulated reversible switches. This photochromicity also provides a crude form of colour vision through measurement of the Pr/Pfr ratio, which varies depending on the relative amounts of R and FR [3].
Phy-type pigments were first discovered in higher plants over 50 years ago, based on the ability of R and FR to control many aspects of plant growth and development [2,3]. Arabidopsis thaliana, for example, contains five Phy isoforms (AthphyA–E) that work individually and in concert to regulate photomorphogenesis. phys (plant Phys) contain an N-terminal domain that binds a single bilin, PΦB (phytochromobilin), through a distinctive GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA) domain, and a C-terminal domain that is required for dimerization. The chromophore is linked autocatalytically to the GAF domain via a thioether linkage between the A-ring ethylidene side chain of PΦB and a positionally conserved Cys (Cys-323 in Ar. thaliana phyA). Immediately distal to the GAF domain is a PHY domain (uppercase letters are used to denote the Phy domain specifically) motif that together with the GAF domain is needed for the unusual photochemical properties of this photoreceptor type [7–10]. The C-terminal half contains two PAS (Per/Arnt/Sim) domains distantly related to GAFs, which are important for signal transmission [2]. Although the mechanism of action for plant phys remains unclear, it has been proposed that they act as light-regulated HKs (histidine kinases) based on limited sequence similarity of their extreme C-termini to the HK domain present in bacterial TC (two-component receptor)-HKs [11,12]. However, the facts that the HK-related sequence of plant phys is missing several important residues that typify a TC-HK, including the His residue that serves as the acceptor for the initial phosphorylation step [4], and that the HK region is partially dispensable in vivo [10,13,14], suggest that either plant phys are not HKs or work by an alternative mechanism. The possibility that plant phys are Ser/Thr kinases has been put forward [11].
More recently, genetic and genomic analyses have dramatically expanded the distribution of Phys to other domains of life, with the discovery of similar photoreceptors in proteobacteria, cyanobacteria, fungi and possibly slime moulds [4,15]. Kehoe and Grossman [16] first realized this potential with the identification of RcaE, a locus required for complementary chromatic adaptation in the cyanobacterium Fremyella diplosiphon. The N-terminal region of FdRcaE is similar in sequence to the bilin-binding GAF domain of plant phys. Downstream of the GAF domain is a canonical HK domain, implying that FdRcaE is a bona fide histidine phosphotransferase. Subsequent BLAST searches identified proteins related to FdRcaE in several other cyanobacterial genomes. For example, Synechocystis PCC6803 encodes five such sequences [8,16–21]. SyCph1 (where 'Cph' represents cyanobacterial Phy), which has both the GAF/PHY and the HK domains, was shown to bind PΦB and PCB (phycocyanobilin) in vitro to generate a photochromic photoreceptor with a light-regulated HK activity [17]. SyCph1 could donate this phosphate to an RR (response regulator) encoded downstream in the SyCph operon, which would demonstrate that it participates directly in a TC-HK phospho-relay.
We and others also found related Phys in searches of various proteobacterial and fungal genomes, thus expanding further the distribution of Phys to non-photosynthetic organisms [5,22–25]. In most cases, both the GAF and PHY motifs were evident in the N-terminal region followed by a HK domain. However, these sequences were distinguishable from plant phys and some relatives from cyanobacteria by the absence of the positionally conserved Cys in the GAF domain, which is used by plant phys to bind bilins. Holoprotein assembly studies subsequently implicated both a GAF-domain His immediately distal to the Cys used by plant phys [22] and a conserved Cys within an N-terminal PLD (PAS-like domain) as alternative attachment site(s) [26,27]. Surprisingly, the proteobacterial forms appear to prefer BV IXα instead of PΦB or PCB, which would simplify chromophore biosynthesis [5]. In several cases, the HO needed to convert haem into BV IXα is within or near the Phy operon, thus linking genetically chromophore and apoprotein production [5,28].
Clearly, the discovery of numerous Phy-type proteins in the microbial world now allows us to exploit the evolutionary distance to help define common structural features critical to this photoreceptor superfamily. In the present paper, we used the exponentially expanding wealth of genomic data to help define phylogenetically the breadth and diversity of microbial Phy-type photoreceptors and to predict important distinguishing features. Using the signature GAF, PHY and PLD sequences from these photoreceptors as queries [8,26], we identified a large collection of Phy-type proteins in numerous bacterial, fungal, and as yet to be classified species. By combining both sequence alignments and motif searches, we tentatively divided this superfamily into several distinct clades that better reflect their photobiological properties and distribution within the bacterial and fungal kingdoms. The alignments also identified numerous conserved amino acids, some of which were confirmed by site-directed mutagenesis to be important for bilin binding and/or photochemistry. Collectively, these analyses show that the Phy superfamily includes a diverse set of photoreceptors present in many ecological niches and provide structural information for predicting the behaviour of new Phy-related sequences.
Identification of Phy-related sequences
The SMART database (http://smart.embl-heidelberg.de) was used to locate the GAF domain (also referred to as P3 [7]) in several representative Phy proteins from micro-organisms and the five plant phy polypeptides from Ar. thaliana. These sequences were used as queries in exhaustive NCBI-BLASTP searches of all available cyanobacterial, fungal and bacterial genomic databases (completed 1 June 2004) and 40 microbial sequences with an E-value of below 0.02 were identified. BLAST searches were repeated using the SMART and PFAM-predicted GAF domains from this collection as queries, which recovered 16 additional loci with sequences scores beneath the 0.02 cut off. This process was a repeated a third time with these additional sequences and recovered two more loci for a total of 58 representatives. The SMART/PFAM databases failed to reliably identify the PLD and the PHY domains (also known as P2 and P4 respectively [7]) in the collection of Phys. As a consequence, the outer limits of these domains were identified by sequence alignments and refined by hand analyses. The predicted PLD and PHY domains in this final alignment with the lowest SMART or PFAM E-values were used for subsequent analyses. Environmental sequences (EnvSeq) [29,30] predicted to be part of the Phy superfamily were identified by BLAST searches with representative PLD, GAF and PHY sequences based on the E-value cut-off of 0.01. All of the EnvSeq sequences were predicted to be partial clones based on SMART with the exception of EnvSeq-1, -2 and -12.
Alignment and phylogenetic analysis
Amino acid sequences were aligned using CLUSTALX MAC v1.81 (http://www.embl.de/~chenna/clustal/darwin/). The unrooted phylogenetic tree based on the GAF domain was generated in CLUSTALX by the neighbour-joining method and a 1000 times bootstrap replicate, and displayed using Treeview PC v1.5.2 (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Only those EnvSeqs predicted to have a complete GAF domain were included. Amino acid sequence alignments were calculated and displayed using MACBOXSHADE v2.15 (Institute of Animal Health, Pirbright, Sussex, U.K.). (It should be noted that most DNA sequences obtained from the various genomic databases were not reconfirmed here. Consequently, it is likely that some derived protein sequences contain errors that could alter their alignments and the extent of sequence conservation.) Additional domains were identified from the SMART and PFAM (http://www.sanger.ac.uk/Software/Pfam/) databases, BLAST searches, and sequence alignments. Accession numbers for the Sargasso Sea EnvSeq-1 to -11 sequences are: 1 (EAI29822), 2 (EAJ02301), 3 (EAG56416), 4 (EAF18794), 5 (EAG25097), 6 (EAE74053), 7 (EAK02112), 8 (EAG45313), 9 (AACY01244739), 10 (EAD68975) and 11 (AACY01305952) [29]. Two additional accessions, EAK71409 and EAK71410, overlapped with the sequence of EnvSeq-1. The accession number for EnvSeq-12 from the mine biofilm is AADL01000497 [30].
Chromophore binding and spectroscopic analyses
Deinococcus radiodurans (Dr) BphP(N321) was PCR amplified from the full-length cDNA described previously [5]. The coding regions for Agrobacterium tumefaciens (At) BphP1 and BphP2, BphP1(N504) and -(N320), and BphP2(N505) and -(N314) were PCR-amplified from strain C58 genomic DNA by using primers designed to introduce NdeI and XhoI sites before and after the designated length of coding region, respectively. The NdeI/XhoI-digested (Promega) PCR products were cloned into pET21b (Novagen), which was similarly digested, resulting in the addition of codons for a His6 tag before a stop codon. All site-directed mutations were introduced by the PCR-based QuikChange method (Stratagene, La Jolla, CA, U.S.A.). Each coding region was completely sequenced by the dideoxy method to confirm introduction of the appropriate mutation. Recombinant proteins were expressed in Escherichia coli strain BL21-Codon Plus (DE3)-RIL (Stratagene) and purified by nickel-chelate affinity chromatography (Novagen) as described in [5]. After purification, the buffer was exchanged for 70 mM Tris/HCl (pH 8.0)/1 mM Na4EDTA by ultrafiltration using a Centriprep YM-10 column.
For chromoprotein assembly, crude soluble extracts from E. coli expressing the recombinant apoproteins were incubated in darkness for 1 h, in at least a 10-fold molar excess of bilin, prior to affinity purification. BV IXα was obtained from Porphyrin Products (Logan, UT, U.S.A.), PCB from Dr Pill-Soon Song (Kumho Life and Environmental Science Laboratory, Gwangju, Korea), BV IIIα, BV XIIIα and mesoBV XIIIα from Dr Tony McDonagh (Division of Gastroenterology and the Liver Center, University of California-San Francisco, San Francisco, CA, U.S.A.), and 18-Et (ethyl)-BV IXα, 3-Et-BV IXα and 3-18-Et-BV IXα from Dr Katsuhiko Inomata (Division of Material Science, Kanazawa University, Ishikawa, Japan) as described in [26]. Absorption spectra of the assembled Phys were determined after overnight incubations in the dark or after saturating irradiations with light at 690 nm (R) and 775 nm (FR) provided by interference filters. Covalent attachment of bilin to the Phy polypeptides was monitored by zinc-induced fluorescence of the chromoproteins subjected to SDS/PAGE [5].
Phylogenetic analysis of the Phy superfamily
The GAF domain is an approx. 150-amino-acid motif, with very early evolutionary origins, that can be found in a number of cyclic nucleotide-binding proteins, phosphodiesterases and in a diverse set of proteins involved in phototransduction [31]. Phys, in particular, contain a GAF domain that is clearly distinct from those in other protein types, as determined by protein sequence alignments [8]. In many cases, it can also be distinguished biochemically by its ability to direct the autocatalytic attachment of bilins, and then associate with these chromophores, to help generate the unique spectral characteristics of this photoreceptor class (e.g. [5,8,22]).
Using the GAF sequences from representative Phys as queries, we exhaustively searched through the available genomic databases for related proteins by BLASTP. This search identified over 70 proteins containing a Phy-type GAF domain in 30 known microbial species and in two collections of prokaryotic species that remain to be classified. This list included previously described proteins from various cyanobacteria, α- and γ-proteobacteria, and fungi [5,16,17,19,22–25,32–34]. New to this list were GAF-containing sequences from the actinobacterium Kineococcus radiotolerans, the cyanobacteria (Anabena variabilis, Crocosphaera watsonii, Cytophaga hutchinsonii, Gleobacter violaceus and Thermosynechococcus elongatus), the proteobacteria (Magnetospirillum magnetotacticum, Rhodospirilum rubrum, Xanthomonas axonopodis and Xanthomonas campestris) and the fungi (Aspergillus nidulans, Gibberella zeae and Ustilago maydis). The K. radiotolerans sequence was significant because it provided the first example of a potential Phy-type protein in actinobacteria. Phy-type GAF domains were also detected in the archaeon Methanosarcina acetivorans, possibly extending the distribution of Phys to Archaea.
The protein organizations of representative Phys described previously and new species discovered here are shown in Figure 1(A). In several cases, more than one GAF domain was predicted by SMART. Two and three GAF domains were found in Synechocystis PCC6803 TaxD1 (also known as PixJ1) and Cph2 respectively, in accord with previous reports [7,19], and two GAF domains were evident in M. acetivorans BphP. Most of the microbial Phys had a HK domain downstream of the GAF domain, strongly suggesting that they function as TC-HKs. Some of the bacterial Phys (e.g. Synechococcus elongatus CikA [34] and Agrobacterium tumefaciens BphP2 [25]) and all of the fungal Phys {e.g. Neurospora crassa PHY-2 and Botryontinia fuckeliana PHY1 ([5,23] and the present study)} also contain an RR domain appended to the C-terminal end of the HK domain (Figure 1A), thus placing them in the hybrid kinase subfamily. However, the phosphotransferase activity of CikA is in question given the absence of the phosphoacceptor Asp [34]. For a limited number of Phys, other motifs were predicted C-terminal to the bilin-binding region, indicating that alternative sensory transduction chains are possible. These alternative forms include: Synechocystis TaxD1 that has a methyl-accepting domain, K. radiotolerans BphP that has a PP2C (phosphatase-2C) domain, and Synechocystis Cph2, Rhodobacter sphaeroides BphP and Thermosynechococcus elongatus Cph that have Gly-Gly-Asp-Glu-Phe (GGDEF) and Glu-Ala-Leu (EAL) motifs (Figure 1A, and results not shown), which have been connected in other protein contexts with diguanylate cyclase and diguanylate phosphodiesterase activities respectively [35].
In addition to the known species, we discovered eight new Phy-type proteins (designated EnvSeq) containing the signature GAF-domain in the recently released shotgun sequence databases of micro-organisms living in either marine or highly acidic environments (Figure 1B). For EnvSeq-1 to -7, the corresponding DNAs were isolated from unknown micro-organisms present in the Sargasso Sea [29]. The DNA for EnvSeq-12 was isolated from an acidophilic biofilm enriched in Leptospirillum group II proteobacteria and Ferroplasma type II archaebacteria [30]. With the exception of EnvSeq-6, for which insufficient sequence data were available, a PHY domain was apparent C-terminal to the GAF domain. Three entries with sufficient sequence information (EnvSeq-1, -2, and -5) also contained all or part of a C-terminal HK domain, strongly suggesting that they participate in TC-HK signalling, with EnvSeq-2 also containing a C-terminal RR (Figure 1B).
Alignment of our total collection of microbial Phy-type GAF sequences using BLASTP showed that they contained many of the signature residues that define the GAF domain in this group (Figure 2 and Supplemental Figure 1 at http://www.BiochemJ.org/bj/392/bj3920103add.htm). These include an Asp/Arg pair and a Glu (residues 171/172 and 189 respectively, in Synechocystis Cph1) that are essential for the bilin attachment [8] and a Tyr recently shown to be critical for maintaining the native photochemical properties of Synechocystis Cph1 (Tyr-176 [36]). Sequence alignments (Supplemental Figures 1–3 at http://www.BiochemJ.org/bj/392/bj3920103add.htm) and phylogenetic analysis (Figure 3) of these microbial GAF domains together with representatives from a higher plant (AthphyA-E from Ar. thaliana) roughly divided the microbial collection into four major Phy subgroups, which we have designated as Cphs (cyanobacterial Phys), BphPs (bacteriophytochromes), Fphs (fungal Phys) and a collection of Phy-like sequences. Consistent with their names, these families appear distinct from plant phys and show a strong preference for their respective microbial kingdoms. However, it should be emphasized that the clustering was not phyletically strict. For example, Calothrix PCC 7601 contains both a Cph-type (CphA) and a BphP-type (CphB) sequence [37], whereas Synechococcus contains two Cph-like (Cph1 and 2) and three Phy-like (TaxD1, PlpA and RcaE-like) sequences ([17–20] and the present study).
Closer examination of the GAF sequence for important residues strengthened support for the subgroups based on phylogenetic clustering (Figure 2 and Supplemental Figure 1 at http://www.BiochemJ.org/bj/392/bj3920103add.htm). As reported earlier for a smaller collection [5], members of the Cph, BphP and Fph families almost all have a positionally conserved His (His-260 in D. radiodurans BphP) that is important for bilin binding (see below and [5,38]). All the classified Cph sequences, like higher plant phys, have a Cys immediately proximal to this His that serves as the actual bilin attachment site for these apoproteins [8,20,37]. This Cys is invariably absent from the BphP and Fph families and often is replaced by a small hydrophobic residue {Ile, Leu, Met or Val ([5] and Figure 2)}. As predicted from the phylogenetic tree, the Phy-like sequences displayed a much less robust alignment for their GAF sequences. A significant number of residues, which are nearly invariant within the Cph, BphP and Fph collections, are poorly conserved in this group (Figure 2 and Supplemental Figure 1 at http://www.BiochemJ.org/bj/392/bj3920103add.htm). For example, these Phy-like polypeptides are sometimes missing either or both residues of the Cys/His pair (e.g. S. elongates CikA and F. diplosiphon RcaE) and typically have a substantial deletion proximal to this site. Some are also missing the Tyr that helps maintain the native spectral properties of the chromophore [36]. Of potential significance is the substitution of a Ser and Met/Ile/Leu pair conserved in almost all other Phys with a conserved Pro and Asp/Glu pair (Pro-160 and Glu-192 in F. diplosiphon RcaE; Figure 2 and Supplemental Figure 1 at http://www.BiochemJ.org/bj/392/bj3920103add.htm).
A subfamily of Phys was recently discovered, designated the bathyPhys, that has the unique attribute of preferring the Pfr and not the Pr form as the ground state [25,32]. Studies with two representatives, Agrobacterium tumefaciens BphP2 (or Apg2) and Pseudomonas aeruginosa BphP, showed that they bind BV IXα as Pr but then rapidly convert into Pfr in the dark, thus needing FR and not R for the initial photoconversion [25,39]. Since this photochemical distinction could reflect unique interactions between the bilin and the GAF domain, we expected that the four known bathyPhys would cluster together on a tree based on this motif. Although Ag. tumefaciens BphP2 and Rhodopseudomonas palustris BphP5 were on the same branch, Bradyrhizobium BphP and Ps. aeruginosa BphP were on another (Figure 3), suggesting that sequences outside of the GAF domain influence this unusual spectral property. We observed some clustering of BphPs bearing the distinctive HWE-HK domain [40]. In contrast, those Phys without an obvious HK domain were distributed throughout the tree (Figure 3). Whether such a disperse distribution was generated by independent evolutionary events or by gene transfer from a common progenitor is unknown.
Mutagenic analyses of various Phys have shown that sequences upstream and downstream of the GAF domain are also critical for the spectral integrity of the photoreceptor. The PHY domain is particularly important for proper photoconversion of Pr to Pfr and the stability of Pfr [7–10,36]. Using a more robust alignment that included all 70 representatives, we refined the PHY domain to an approx. 130-amino-acid region which was evident in all members of the plant phy, Cph, BphP and Fph families but was not apparent in any members of Phy-like group (Figure 4 and Supplemental Figure 2 at http://www.BiochemJ.org/bj/392/bj3920103add.htm). It contains a number of islands with strong sequence conservation, especially within the Cph and Fph families, that are likely to participate in domain folding. Random mutagenesis of Synechocystis Cph1 identified several amino acids in the PHY region that influence R/FR photochemistry [36]. Although some mutations affected residues highly conserved within the Phy superfamily (e.g. Phe-475, Trp-478 and Trp-489 in SyCph1), implying that these sites have direct roles, other mutations affected variable positions (e.g. Glu-385, Asn-397 and Thr-461 in SyCph1; see Supplemental Figure 2 at http://www.BiochemJ.org/bj/392/bj3920103add.htm), suggesting that these residues have secondary effects.
Phylogenetic analysis with the PHY domain generated a similar clustering of the plant phy, Cph, BphP and Fph families to that seen with the GAF domain, supporting further our proposed groupings (results not shown). Given its functional significance, we used the PHY domain as an additional query to search the various microbial databases. Although no new Phys were detected in the databases of known micro-organisms, one additional Phy was found in the shotgun sequence collection from the Sargasso Sea [29]. The partial sequence for EnvSeq-8 harboured a full PHY domain followed by the N-terminal portion of a HK domain, suggesting that it also acts as a photoreceptor in a TC-HK phospho-relay (Figure 1B).
Lamparter and co-workers [26,27] recently described a conserved PLD upstream of the GAF domain in many microbial Phys that participates in bilin binding. Using the PLD as a query, we detected a related sequence in all the BphP and Fph sequences identified above (Figure 4 and Supplemental Figure 3 at http://www.BiochemJ.org/bj/392/bj3920103add.htm). Importantly, all but one also has the positionally conserved Cys (the exception was R. palustris BphP4, whose sequence has not yet been verified). Even R. centenum Ppr, which contains an appended N-terminal photoactive yellow protein motif that binds the chromophore p-hydroxycinnamic acid, has a PLD upstream of its GAF domain (Figure 1A). An obvious but less well-conserved PLD was evident in members of the Cph and plant phy families but this motif was notably missing the conserved Cys (Figure 4 and Supplemental Figure 3 http://www.BiochemJ.org/bj/392/bj3920103add.htm). Nothing resembling a PLD could be detected in most members of the Phy-like clade. The only exception was the possible Phy from the archaebacterium M. acetivorans (MaBphP1), where a Cys-containing PLD was detected upstream of the second GAF domain (Figures 1A and 4). Searches of the environmental databases with a consensus PLD sequence identified three additional Phy-type proteins [EnvSeq-9 to -11 (Figures 1B and 4)]. All three were partial sequences containing only the PLD, which prevented us from unequivocally assigning them to one of the four microbial families. However, the positionally conserved Cys was evident, suggesting that EnvSeq-9 to -11 are related to the BphP/Fph types.
Organization of the Phy operons
Like other sensory systems [41], several elements within the Phy sensory transduction chain are often connected genetically. For example, all the Fphs identified to date have an RR domain appended to the C-terminal end, supporting further their participation in TC-HK cascades ([5,23,25] and the present study). For the bacterial Phys, the gene encoding the photoreceptor apoprotein is often coupled in an operon to other open reading frames encoding factors important for TC-HK signal transmission. For Cphs and BphPs, these include separate RRs and sometimes other HKs (e.g. the Ag. tumefaciens BphP1 operon [25]) that may be part of the phospho-relay. For some BphP operons, an HO gene needed to synthesize BV IXα from haem is included or nearby [5,25,28].
For several of the new Phy sequences reported here, similar genetic connections were evident (Supplemental Figure 4 at http://www.BiochemJ.org/bj/392/bj3920103add.htm). For example, the BphP operon in K. radiotolerans includes the coding region for an HO gene (BphO) downstream of that for the BphP. For the EnvSeq sequences where enough surrounding genomic sequence was available, similar linkages of RR and/or HO genes were observed, with the latter arrangement further supporting their designation as a Phy. The EnvSeq-1 BphP gene is followed by an RR gene (BphR) in the same orientation and nearby is a HK gene in the opposite orientation. Both the EnvSeq-9 and -12 genes are downstream of a likely HO gene (BphO). For EnvSeq-9, the BphP and BphO coding sequences overlap by 4 bp, whereas for EnvSeq-12, the two are immediately adjacent with no intervening DNA sequence. The M. acetivorans Phy-like gene is in a cluster containing a putative transcription factor.
Role of the PHY domain in stabilizing Pfr
Alignments of the GAF, PHY and PLD sequences from plants and a wide array of micro-organisms clearly show that these domains have been conserved through evolution and identify a number of amino acids that may be important for the unique spectral properties and/or folding of this photoreceptor family. To examine these domains further biochemically, we analysed three representative BphPs by deletion or site-directed mutagenesis coupled with bilin-binding and spectroscopic assays. D. radiodurans BphP1 and Ag. tumefaciens BphP1 were chosen to help resolve the site for BV IXα attachment among the BphPs [22,26,27], and Ag. tumefaciens BphP2 was chosen to identify possible differences that discriminate the bathyPhys from the more photochemically conventional forms. Of particular interest was the role of the PHY domain in stabilizing the Pfr form of AtBphP2, given that it assumes this form without light [25]. Wild-type and mutant apoproteins were expressed with C-terminal His6 tags in E. coli, incubated with the bilin, and purified by nickel-chelate affinity chromatography. The recombinant proteins were then assayed for bilin attachment by zinc-induced fluorescence of the holoprotein following SDS/PAGE and for the distinctive R/FR photochromic absorption spectra characteristic of Phy-type pigments.
To examine further the role of the PHY domain in stabilizing the Pfr chromophore [8–10,36], we tested a pair of C-terminal truncations bracketing the PHY domain of Ag. tumefaciens BphP1 and -2. The longer set was missing only the C-terminal HK domain [AtBphP1(N504) and AtBphP2(N505), where the numbers indicate the final amino acid of the truncated polypeptide], whereas the shorter set was missing both the HK and the PHY domains [AtBphP1(N320) and AtBphP2(N314)]. As shown in Figure 5(A), both sets of truncations efficiently bound BV IXα (as judged by zinc-induced fluorescence of the bound chromophore following SDS/PAGE), indicating that neither the PHY domain nor the downstream HK domain has a substantial impact on the bilin lyase activity of the apoproteins.
The PHY domain was essential for the photochromicity of both AtBphP1 and AtBphP2 (Figure 5B). For AtBphP1, the R/FR spectral properties of the N504 truncation were indistinguishable from the full-length holoprotein. In contrast, the N320 truncation generated a normal Pr, but saturating R irradiations created a 'Pfr-like' bleached state that rapidly reverted to Pr in the dark. A similar effect was seen for an equivalent PHY deletion of D. radiodurans BphP [DrBphP(N321) (see Figure 6)]. For AtBphP2, assembly of a spectrally normal Pfr was possible upon removal of the HK domain (Figure 5B). However, the AtBphP2(N505) truncation inefficiently photoconverted into a highly unstable Pr form in FR. In fact, thermal reversion back to Pfr was so rapid that it prevented accurate measurement of the Pr absorption spectrum, even after saturating FR irradiations (results not shown). Upon removal of the PHY domain, AtBphP2(N314) failed to assume the expected Pfr ground state upon BV IXα binding [25], but instead generated a bleached and red-shifted 'Pr-like' form in the dark. This bili-protein was not photochromic with either R or FR. Collectively, the results demonstrate that the PHY domain, although not essential for bilin binding, is important for stabilizing the Pfr form and proper photochromicity, even for a bathyPhy that uses Pfr as the ground state.
Roles of the GAF and PLD regions in chromophore binding
Whereas studies with Ag. tumefaciens BphP1 and Ps. aeruginosa BphP showed that the conserved Cys within the PLD is the bilin attachment site [27,39], a contradictory study by us with D. radiodurans BphP implicated the invariant His within the GAF domain [22]. To help resolve this discrepancy, we examined bilin assembly for a common set of Cys and His mutants in the three BphPs, DrBphP(N321), AtBphP1(N320) and AtBphP2(N505). All three wild-type sequences readily bound BV IXα (as judged by zinc-induced fluorescence of the holoprotein) and generated the expected Pr and Pfr states following a dark incubation (Figures 5, 6A and 6B). When the PLD Cys substitution mutants were tested similarly, none of the three apoproteins effectively assembled with BV IXα (Figure 6A). These Cys→Met or Cys→Ser mutants also displayed little absorption in the R/FR region (Figure 6B), with much of the residual absorption possibly reflecting non-covalent association of the BV IXα [26,42].
Some but not all of the His mutants exhibited a similar defect in BV IXα attachment and photochromicity but these effects were clearly influenced by the apoprotein and the nature of the His substitution (Figures 6A and 6B). For DrBphP(N321), the His→Cys and His→Lys mutants failed to bind BV IXα, whereas the His→Gln mutant effectively bound this bilin. All three His mutants displayed little or no absorption in the R region, an effect that was unexpected for His→Gln variant given its ability to covalently bind BV IXα. The three His mutants of AtBphP1(N320) failed to bind BV IXα well, with none displaying strong absorption in the R region. In contrast, all three His mutants of AtBphP2(N505) efficiently attached BV IXα, but the resulting bili-proteins displayed unusual absorption spectra after assembly. In particular, the spectrum of the His→Lys mutant prior to irradiations more closely resembled that of Pfr, while the spectrum of the His→Gln mutant more closely resembled that of Pr, suggesting that the preferred Pfr ground state of the holoprotein was shifted to Pr by the Gln substitution. Taken together, the data support the proposal by Lamparter et al. [26,27] that the PLD is the bilin-binding site. However, the GAF His also appears to be important, especially in those Phys that prefer Pr as the ground state. The unique ability of AtBphP2(N505) to tolerate various His substitutions, at least with respect to BV IXα ligation, suggests that it could represent another distinguishing feature of the bathyPhy subfamily.
Importance of the PLD
The alignment of the PLD revealed a number of other highly conserved residues besides the Cys that may be important for bilin attachment, R/FR photochromicity, and/or protein folding. In support, recent studies by Zhao et al. [43] showed through deletion analysis that one stretch (IQPHGV; residues 26–31) is essential for the bilin lyase activity of the Nostoc AphA, a member of the Cph family. The Ile in this motif was also identified by Bhoo et al. [38] as being critical for both bilin attachment and photochromicity in pea phyA. In the present paper, we describe the individual testing of the importance of some of these amino acids (Glu-16, Pro-17, Ile-18, His-19, Ile-24, Gln-25, Pro-26, Ser-44 and Asn-46) by alanine substitution mutagenesis of AtBphP2(N505). Most of these residues are conserved not only in the BphP and Fph families, but also in the plant phy and Cph families that do not attach bilins via this domain (Figure 4 and Supplemental Figure 3 at http://www.BiochemJ.org/bj/392/bj3920103add.htm). Many of the PLD mutants were readily expressed in soluble forms. The exceptions were the Ile-24, Ser-44 and Asn-46 mutants; the recombinant apoproteins accumulated mainly in the insoluble fraction. We obtained a small amount of these three mutants for BV IXα-binding assays, but the amounts of holoprotein were insufficient for spectroscopic analyses.
Surprisingly, although the PLD Cys→Ser mutant of AtBphP2(N505) strongly abrogated BV IXα attachment in vitro, only one of the nine other PLD mutants appreciably attenuated covalent binding [Ile-18 (Figure 7A)]. Most of the mutant chromoproteins also displayed relatively normal Pr and Pfr absorption and R/FR difference spectra. This indicates that the association of the bilin to its binding pocket and its photochromicity were not appreciably affected (Figure 7B and results not shown). We observed small changes in the height of the R maximum of the difference spectrum for the His-19, Gln-25, and Pro-26 mutants, which appeared to reflect an increase and decrease, respectively, in the Pr to Pfr dark reversion rate relative to the unmodified chromoprotein. The most significant change was for the Ile-18 mutant, which assumed a mixture of Pr and Pfr at equilibrium in the dark instead of solely being Pfr (Figure 7B). Taken together, the mild consequences of most PLD mutation imply that this domain outside of the bilin-binding Cys has only a minor impact on bilin ligation and Pr/Pfr photochemistry.
Importance of the bilin A ring vinyl side chain for binding to Phys
Previous studies showed that plant phys and Ag. tumefaciens BphP1 bind their respective bilins, PΦB and BV IXα, by the ethylidene/vinyl side chains attached to the A-ring C-3 position [26,44,45]. However, both the distinct photochemical properties of bathyBphP AtBphP2 [25] and the discriminating nature of the His mutants in DrBphP1 and AtBphP1 (Figure 7A) suggested that alternative binding sites in the BV IXα were possible, in particular the vinyl side chain of the D-ring. To address this issue, we examined the assembly of DrBphP(N321), AtBphP1(N320) and AtBphP2(N505) with a library of BV IXα derivatives altered at either or both of the A- and D-ring vinyl side chains. The derivatives included: (i) BV XIIIα and 18-Et-BV IXα, which have an A-ring vinyl group at the C-3 position but have the D-ring vinyl at position C-18 exchanged for ethyl and methyl groups respectively; (ii) PCB, which has the A-ring vinyl exchanged for an ethylidene group and an ethyl group connected to the D-ring; (iii) 3-Et-BV IXα and 3-18-Et-BV IXα, which have ethyl instead of vinyl groups at the C-3 or both the C-3 and C-18 positions respectively; (iv) meso-BV XIIIα, which has ethyl groups at the C-3 and C-17 positions; and (iv) BV IIIα, in which the vinyl group of the A-ring is switched to the C-2 position (see Figure 8B for structures).
When incubated with the recombinant apoproteins, we found that the two bilin variants with a vinyl group at the A-ring C-3 position, BV XIIIα and 18-Et-BV IXα, covalently assembled with all three apoproteins like BV IXα, as judged by zinc-induced fluorescence of the resulting holoproteins (Figure 8A). PCB, which has an ethylidene group at this position also bound to the three apoproteins, but the assembly was clearly more robust for DrBphP(N321) as compared with both Ag. tumefaciens apoproteins. In most cases, strong R/FR-absorbing photochromic holoproteins were also generated. For the BV XIIIα- and 18-Et-BV IXα-containing species, the Pr and Pfr absorption spectra resembled those assembled with BV IXα (Figure 8B and results not shown). The PCB-containing DrBphP(N321) and AtBphP1(N320) holoproteins also exhibited the expected Pr and Pfr absorption spectra (with blue-shifted maxima as compared with BV IXα, given the less conjugated π-electron system). However, the PCB-AtBphP2(N505) adduct had little absorbance in the R or FR regions and displayed little or no change in absorption following sequential R/FR irradiations (Figure 8B and results not shown). The remaining four bilins without these C-3 vinyl/ethylidene groups (meso-BV XIIIα, 3-Et-BV IXα, 3-18-Et-BV IXα and BV IIIα) failed to bind covalently with any of the three apoproteins and, as expected, generated little or no R/FR absorption (Figure 8A and B). Together with the common use of a PLD Cys, the common need for the A-ring vinyl side chain among the three BphPs suggests that other members of the BphP family use a similar method to form the thioether linkage with BV IXα.
Organization of the Phy superfamily
Analysis of the expanding collection of Phy-type sequences performed by us and others shows that the Phy superfamily includes, in addition to those in plants, a diverse set of photoreceptors present in a wide array of proteobacteria, cyanobacteria and fungi. We can now include over 70 members from 30 known microbial species, with more likely to be found as additional genomic sequencing projects progress. New to the list is a Phy from K. radiotolerans, thus expanding the distribution of Phys to actinobacteria, and a potential Phy-related sequence in M. acetivorans, which may extend Phys to Archaea. Sequence alignments of the Phy collection identified a number of conserved motifs/residues surrounding the bilin-binding pocket. These presumably have important functions in chromophore attachment and the unique photochromic spectral properties of this photoreceptor class. Included is a signature GAF domain that is often preceded by a PLD and followed by a PHY domain, the combination of which appears to be specific for the Phy superfamily. Given the remarkable conservation of the PLD/GAF/PHY domains in species widely separated during evolution, they should be extremely useful in identifying new members of the Phy superfamily. As examples, we discovered 12 potential Phys in two microbial shotgun sequence databases. Taken together, this collection also shows that Phy-type photoreceptors radiated more widely than previously appreciated.
The structural organization of microbial Phys indicates that many, but not all, participate either directly or indirectly in TC-HK phospho-relays. This action is further supported by the presence of an RR domain, either translationally linked to the HK domain of the Phy to create a hybrid HK, or expressed as a separate protein genetically linked to the Phy within a common operon. Instead of initiating one or more TC-HK cascades, some Phys may dampen such phospho-relays. For example, the presence of a PP2C domain in K. radiotolerans BphP could attenuate one or more kinase cascades by releasing the phosphate signal in a light-dependent manner. The involvement of the PP2C-like protein (CpeR) in the complementary chromatic adaptation response of F. diplosiphon suggests that a similar dampening by dephosphorylation acts downstream of the Phy-like protein RcaE [46].
In several cases (Synechocystis Cph2, Rh. sphaeroides BphP and T. elongatus Cph), GGDEF and EAL motifs (both named for a consensus set of amino acids) are downstream of the bilin-binding site instead of a HK domain. These motifs, which are proposed to have diguanylate cyclase and diguanylate phosphodiesterase activities respectively [35], have been also found in a several non-Phy proteins required for motility (e.g. Pseudomonas aeruginosa FimX, Vibrio cholerae RocS, and Caulobacter cresentus PleD [47–49]). PleD for example, appears to directly regulate the flagellum motor through a HK cascade [47]. Consequently, the presence of the GGDEF and EAL motifs raises the possibility that some prokaryotic Phys photoregulate bacterial movement. In support, Synechocystis Cph2 has been shown to modulate taxis towards blue light [19].
Classification of the Phy superfamily into distinct families
Using phylogenetic analysis of the GAF domain coupled with a grouping based on specific biochemical characteristics (e.g. identity of the bilin and its linkage site, and spectral characteristics), we propose the formation of four major microbial Phy subgroups (BphPs, Cphs, Fphs and a collection of Phy-like sequences) that reflect their distribution within the bacterial and fungal kingdoms and their photobiological properties. The Cph family is most similar to plant phys. Like their plant relatives, members of the Cph family appear to bind bilins through the positionally conserved Cys in the GAF domain [17]. Although they have a discernible PLD, the absence of the Cys residue implies a priori that this motif is not directly involved. The BphP and Fph families are most closely related to each other. Members of these families are clearly distinguished from plant phys and Cphs by the absence of the Cys residue within the GAF domain, the use of BV IXα as the chromophore [5], and the probable use of a positionally conserved Cys with the PLD for bilin binding ([27,39] and the present study). Although the data from several representative BphPs support the PLD Cys, we note that a similar role in members of the Fph family awaits testing. Plant phys and probably Cphs bind their PΦB and PCB chromophores through the A-ring ethylidene group [17,50]. Based on our and others' analyses of several BV IXα derivatives, it is likely that members of the BphP and Fph families also bind their bilin via the vinyl group attached to the same A-ring ([26,27,39] and the present study).
Although our first analysis of a BphP implicated a GAF-domain His as the site for covalently attaching the bilin chromophores [22], more recent studies identified a Cys within the PLD [26,27,39]. Here, we provide further support for the PLD Cys using a suite of site-directed mutants affecting both His and Cys residues in three different BphPs. Whereas none of the PLD Cys mutants could bind bilins, some of the GAF His mutants remained active. The difference was especially pronounced for Ag. tumefaciens BphP2, for which three mutants that converted the His into either Cys, Lys or Gln were without effect. In the contexts of D. radiodurans BphP and Ag. tumefaciens BphP1 apoproteins, the GAF domain His is important, suggesting that it may in some situations participate in chromophore ligation. Other than the PLD Cys, most of the other conserved residues near this amino acid of Ag. tumefaciens BphP2 were non-essential. The only apparent exception was Ile-18. Ile-24 was previously identified as critical for chromophore attachment in pea phyA [38,43]; it did not appear to alter the BV IXα-binding ability of AtBphP2(N505) but did impair the stability of the recombinant apoprotein. It is intriguing that plant phy/Cph families and the BphP/Fph families bind spatially close sites in their respective chromophores but use Cys residues located in two different domains (GAF versus PLD). One likely scenario is that the respective PLD and GAF Cys acceptor sites are very close in the three-dimensional structures of BphPs, Fphs, Cphs and plant phys despite being widely separated in the primary sequence.
At present, the molecular explanation for the unusual photochemical properties of the bathyPhys is unknown. Sequence comparisons of their PLD, GAF and PHY domains with those of other BphPs failed to identify motifs/amino acids that could generate the preference for the Pfr ground state. Deletion analyses of AtBphP2 as compared with AtBphP1 and DrBphP showed that this bathyPhy also requires the PHY domain to help stabilize the Pfr form. However, several subtle differences were detected here that could be important distinctions, including the ability of His-250 mutants in AtBphP2 to bind bilins and the failure of AtBphP2 to assemble efficiently with PCB and generate a photochromic photoreceptor. Clearly, additional comparative studies with other bathyBphPs are needed to determine whether these effects are more general.
In addition to the more typical Phys, a collection of Phy-like proteins was evident from the alignments and phylogenetic analyses. Although members of the Phy-like group have the signature Phy-type GAF domain, other features are often missing, including the PHY motif, the PLD and/or the Cys/His residues within the GAF domain. Another discriminator is the presence of an approx. 25-residue deletion just upstream of the Cys in the GAF domain when aligned with members of the BphP, Cph and Fph families. Some of the Phy-like sequences have retained the GAF Cys, suggesting that they have the capacity to bind bilins through this domain. Synechocystis RcaE, for example, can bind bilins covalently but fails to become R/FR-photochromic [51]. While it remains possible that these Phy-like proteins are not photoreceptors, intriguing scenarios are that they (1) act as a bilin sensors and not as light sensors [7], (2) associate with non-bilin chromophores, and/or (3) bind bilin-type pigments but generate markedly different spectral properties. In support of the latter possibility, Yoshihara et al. [52] recently reported that Synechocystis TaxD1 (PixJ1) expressed in vivo associates with a bilin-type chromophore through its GAF Cys, but generates a blue/green-light photoreversible chromoprotein. The substantial blue shift in absorbance of the holoprotein could reflect either the use of a novel less-π-conjugated bilin or distinct interactions between the apoprotein and the bilin within the binding pocket. Given that TaxD1 and at least two other members of the Phy-like clade (PlpA and Cph2) control blue-light photobiology in Synechocystis [18,52,53], it is tempting to speculate that members of the Phy-like clade actually represent a unique class of blue/green light-absorbing bili-proteins.
Predictive value of the Phy families
The proposed Phy families should have value in predicting the behaviour of new, yet-to-be-characterized species. For example, our analysis of N. crassa Phy2 recently confirmed the expectation that this Fph covalently binds BV IXα and generates an R/FR chromoprotein (B. Noh and R. D. Vierstra, unpublished work). Similar to the BphPs studied here from D. radiodurans and Ag. tumefaciens (Figures 5 and 6), its PHY domain is also essential for Pfr stability, such that its removal generates a biliprotein spectroscopically similar to DrBphP(N321) and AtBphP1(N302). Likewise, we predict that the Phy sequence from the actinobacterium K. radiotolerans will have photochemical properties similar to members of the BphP family, given its similar PLD–GAF–PHY domain organization and phylogenetic clustering with other BphPs. We also detected a Phy-type protein in the genome of the archaebacterium M. acetivorans that awaits biochemical characterization. It does not have an obvious PHY domain and neither of its two GAF domains appear to be related to those in the BphP, Fph and Cph families. However, the more C-terminal GAF domain is preceded by a Cys-containing PLD, suggesting that it could bind bilins by a mechanism similar to BphPs/Fphs.
For the EnvSeqs identified here, the presence of a Cys-containing PLD in most (the exception is EnvSeq-3) and their phylogenetic clustering with other BphPs predicts that they are members of the BphP/Fph families. For EnvSeq-9 and -12, this designation is supported further by the coupling of an HO (BphO) gene to the coding region for the apoprotein. It is interesting to note that the Sargasso Sea collection contained almost 800 unique rhodopsin-like photoreceptors [29] but only 11 Phy-type receptors (the present study). Consequently, it appears that retinal-based signalling systems are much more prevalent than Phys among marine micro-organisms.
Evolution of the Phy superfamily
Close analysis of the microbial Phys now provides a potential route for the evolution of the Phy superfamily. Given their widespread presence in a number of photosynthetic and non-photosynthetic proteobacteria, the BphP family probably represents the progenitor for all of the bilin-containing photochromic pigments. The use of BV IXα as the chromophore also offers the simplest way to obtain a linear bilin, requiring just one enzymatic step from the cyclic haem precursor [5]. The BphP precursor may have been an ancient GAF-containing protein, related to FixL [54], that modified its haem-binding pocket to accommodate linear bilins and added a PAS derivative (i.e. PLD) for covalent attachment. An evolving PHY region contributed to the ability to become R/FR-photochromic by helping stabilize the Pfr conformer, while the addition of the HK domain provided a facile way to connect light perception to appropriate signalling networks.
Relatives of the BphPs then emerged during the evolution of the cyanobacterial, actinobacterial and fungal clades. Members of the Fph family probably evolved directly from BphPs and preserved the use of BV IXα as the chromophore. Like other fungal TC-HKs, the Fphs appended the target RR directly to the photoreceptor, presumably to facilitate phospho-relay transmission. At present, Fphs have been conclusively found only in filamentous fungi ([5,23] and the present study), with a preliminary action spectrum for light-induced sporulation suggesting that one is present in the slime mould Physarum polycephalum [15]. Their absence in unicellular fungi suggests that only some fungal phyla selectively retained these photoreceptors or acquired them from bacteria by gene transfer.
For the Cph family, PCB was adopted as the chromophore, which was readily available given its prime use as a photosynthetic accessory pigment [6]. Its blue-shifted Pr absorption spectrum more closely overlaps with chlorophyll thus providing a more accurate measure of photosynthetic wavelengths under competitive conditions. Clearly, the use of PCB as the chromophore would require a way to discriminate PCB from its precursor BV IXα. Both problems may have been overcome by the use of the GAF Cys to attach the chromophore covalently, as opposed to the PLD Cys. One possibility is that the position of the GAF Cys in the three-dimensional structure restricts binding to the ethylidene side chain of PCB instead of the vinyl side chain of BV IXα. Phy-like sequences have only been found in cyanobacteria, suggesting that they evolved from the Cphs through loss of the PLD and PHY motifs and changes within the GAF domain. The variable protein organization of these members implies widely diverse functions for the Phy-like group. The evolution of plant phys remains unclear given the absence of prokaryotic versions with a similar C-terminal architecture (e.g. tandem internal PAS domains), although their derivation from a Cph obtained during chloroplast endosymbiosis is most likely. Plant phys then became nuclear encoded and employed PΦB instead of PCB as the chromophore. The two internal PAS domains were added to help with signal output. The HK domain was also modified to alter or delete its HK activity; in fact, this domain may have been retained solely to promote homodimerization, a common characteristic among bona fide Phy photoreceptors [2,4].
The expanding database of Phy and Phy-like proteins now provides the opportunity to identify common amino acids that help define these unique photochromic photoreceptors. Using this strategy, we identified a number of residues within the PLD, GAF and PHY domains that must be important based on this evolutionary retention. For example, the Tyr residue identified by Fischer and Lagarias [36] as essential for the primary photoprocess of Synechocystis Cph1 is invariant among the BphP, Cph and Fph families and present in many members of the Phy-like clade. The PHY domain in particular appears essential for spectral properties and stability of the Pfr form (the present study, and [7–10,36]). Even for the bathyPhy from Ag. tumefaciens (BphP2) that prefers Pfr as the ground state, deletion of this domain generated a bili-protein that had lost its capacity to generate Pfr and remain in this form following photoconversion by R. Clearly, three-dimensional structures of representative Phys are now needed to confirm the importance of each of the conserved domains and residues identified here.
We thank Drs P.-S. Song, T. MacDonagh, T. Lamparter (Department of Plant Physiology, Freie Universitat, Berlin, Germany) and K. Inomata for supplying the various BV IXα derivatives, and Dr S.-H. Bhoo (Plant Metabolism Research Center, Kyung Hee University, Suwon, Korea) for providing the DrBphP gene. This work was supported by grants from the National Science Foundation (MCB-0424062) and the Research Division of the UW-College of Agriculture and Life Sciences (Hatch) to R.D.V., and a US-Israel BARD postdoctoral fellowship to B.K.
Abbreviations: Phy, phytochrome; PHY, phytochrome domain; BphP, bacteriophytochrome; Cph, cyanobacterial Phy; Fph, fungal Phy; phy, plant Phy; AthphyA, etc., Arabidopsis thaliana phy isoform A, etc; BV, biliverdin; EnvSeq, environmental sequence related to the Phy-type protein superfamily; FR, far-red light; Pfr, FR-absorbing form of Phy; R, red light; Pr, R-absorbing form of Phy; Et, ethyl; GAF, cGMP phosphodiesterase/adenylate cyclase/FhlA; HK, histidine kinase; HO, haem oxygenase; PAS, domain, Per/Arnt/Sim domain; PCB, phycocyanobilin; PΦB, phytochromobilin; PLD, PAS-like domain; PP2C, protein phosphatase-2C; RR, response regulator; TC, two-component receptor
- The Biochemical Society, London


text: Hello all!, I wish to make a Zone Fighter and Godzilla: The Series crossover set after the point both shows ended and their adventures together. Any suggestions for enemies, the Garoga are already in along with the possibility of the Seatopians. Please respond soon! First page up on Friday, thank you and I have how the Zone Family got through planned already Whovians. This blog will remain open for anyone who wishes to suggest any monster, or if they wish, submit a story as long as you have no problem with sharing stories with me. Your name will be put up.

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The Dark Tower (2017)
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Critic Reviews for The Dark Tower
All Critics (185) | Top Critics (35) | Fresh (30) | Rotten (155)
[Idris] Elba alone can't make The Dark Tower worth watching. But he can make the case, yet again, that he deserves better material than this.
Outside of its actors, the film is unremarkable. Action sequences seem as though they were hurried, that is, when you can actually see them - Did the lighting team blow a fuse?
It gets where it's going fast enough, but you don't feel like you've traveled anywhere at all.
The Dark Tower avoids the lowest rungs of that ladder. Taken on its terms it's hardly a disaster, delivering moments of fun on a basic level.
[The Dark Tower] resembles nothing so much as a mashup of The Lord of the Rings and The Matrix reimagined for the young adult crowd.
Audience Reviews for The Dark Tower
I have never read any of Stephen King's popular fantasy series, The Dark Tower, so I walked into the long-awaited film adaptation with no investment. I walked out with very little investment. The film peaked at its casting, with Idris Elba as the mythic Gunslinger hunting down a notorious evil sorcerer, The Man in Black (Matthew McConaughey), a.k.a. Randall Flagg. For the first fifteen minutes or so, the world building is pretty straightforward and accessible. The Man in Black is looking across universes for special psychic children whose abilities he can focus to destroy the titular Dark Tower, a monument protecting the stability of all universes. A young kid is having prophetic dreams but appears emotionally disturbed to his family. Before a group of rat people hiding behind human masks can kidnap him, he runs away, finding a portal to Midworld, and into the path of the Gunslinger, who seeks personal vengeance against the Man in Black. From there the movie gets pretty hazy with its rules and lore. The production design of this alien world is a bit bare. The characters stay in an archetypical range and don't give you much reason to care. The action is serviceable but unmemorable, the Gunslinger credo is rather cheesy especially when repeated with such self-seriousness, and sometimes it feels like McConaughey is in a different movie altogether. If you're a fan of the series, I have to imagine you'll be processing some level of disappointment by the end of the film. If you're a franchise novice, like me, you may find some stray things to enjoy in the moment, but the film evaporates from your mind immediately after leaving the theater, banished to the dustbin of bland sci-fi action. Nate's Grade: C
Super Reviewer
The Dark Tower movie, unlike the Mangnum Opus book series from writer Stephen King, suffers from the word 'go'. An inspired performance from Idris Elba that makes me wish the rest of the film, from direction to production to the story, matched his commitment. Through development hell, numerous rewrites, director changes, and a minuscule 90-minute run time, The Dark Tower fails in nearly every aspect as a complete movie. First and foremost, this movie (*read entire franchise*) has been trapped behind closed doors for years. I remember when they first talked about adapting this into a few movies and a complimentary TV show with all these grand schemes to make it one of the largest and most complete packages Hollywood had seen. Instead, we got 90 minutes of a non-cohesive story with stilted and near-comical acting that felt like years of planning and writing crammed into a tight little window. The end result leads me to believe this was the first and last iteration we'll see as fans. I'll go on record and say the books are the best complete series I've ever read. I did my best to separate them from this movie. Matthew McConaughey mails in an over-acted performance as Walter and comes off as creepy in a pedophile way instead of sinister and cunning like the books. Tom Taylor won't act his way into any future performances with his first on-screen performance in a major role. Nikolaj Arcel will remain just as unheard of after this release as a director as he was before it. But unfortunately, the biggest clue-in as to why this movie failed was Akiva Goldsman. The screenwriter/producer is one of the worst in the business. While movies his name is tagged on range from OK (I, Robot, Cinderella Man, Lone Survivor) to outright garbage (Transformers, Jonah Hex, Poseidon), I'll never understand how he became so prominent in the business. The Dark Tower turns into a misguided attempt at starting a franchise by dropping you right into the thick of the story without any background or knowledge of what's going on with only dialogue to spur movement forward. It should stop before continues any further.
Super Reviewer
Underwhelmed, and forgettable. A-listers can't propel a franchise wannabe if the story is not compelling.
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Lurssen completed the building motor yacht CAPRI in 2003. As such, she can be categorised as having been built country of Germany. CAPRI had her design developed by Lurssen and Glade Johnson and Lurssen. This superyacht CAPRI can accommodate a capacity of 12 guests aboard as well as 15 crewmembers. Finished and launched in 2003 the comparatively recent interior design promulgates the insight in the designing of Glade Johnson.
The Lurssen Luxury Yacht CAPRI is a magnificent design by Glade Johnson and finished meticulously by the Lurssen yard. Her sleek and elegant design is softened by the use of soft curves inside and out. Her substantial volume offers many casual and formal entertainment and dining areas plus spacious and comfortable accommodate for twelve guests. Motor Yacht CAPRI represents the pinnacle of clever and stylish design with top of the line equipment resulting in a beautiful yet extremely seaworthy vessel.
Lurssen was the naval architecture company involved in the technical nautical design work for CAPRI. Also the company Lurssen and Glade Johnson skillfully collaborated on this undertaking. Interior designer Glade Johnson was selected for the overall internal styling. Germany is the country that Lurssen Yachts built their new build motor yacht in. After her formal launch in 2003 in Bremen the boat was thereafter handed over to the owner after final finishing. The main hull was constructed with steel. The motor yacht superstructure component is made predominantly with aluminium. With a beam of 11.43 m / 37.5 feet CAPRI has spacious internal space. She has a fairly deep draught of 3.5m (11.5ft).
The 3512B DITA engine installed in the motor yacht is built by CATERPILLAR. For propulsion CAPRI has twin screw propellers. The engine of the ship creates 1960 horse power (or 1442 kilowatts). She is equiped with 2 engines. The combined thrust for the boat is therefore 3920 HP / 2884 KW. As far as stabalisers are concerned she was supplied with Koopnautic. The bow thrusters are Jastram.
Apportioning chambers for a limit of 12 welcome guests sleeping aboard, the CAPRI accommodates them in style. This ship has room for approx 15 professional crewmembers to run.
Main Engine(s) is two 1960 HP or 1442 kW Caterpillar. Engine Model: 3512B DITA diesel. - Total engine power output 3920 HP /2884 KW. - Approximate Cruise Speed is 14 knots. - Top Speed: 16 knots. - Approximate range: 8500 at a speed of 16 knots. - Bunkering capacity: 150000 L. - Fresh water: 28000.00. - Power generation: Caterpillar 3 times 260 kilowatts. - Stabaliser Stabality devices: Koopnautic. - Bow Thrusters: Jastram. - Yacht Beam: 11.43m/37.5ft. - Waterline Length (LWL): 49.73m/163.2ft. - Draught at deepest: 3.5m/11.5ft.
CAPRI features a teak deck.
The luxury yacht CAPRI displayed on this page is merely informational and she is not necessarily available for yacht charter or for sale, nor is she represented or marketed in anyway by CharterWorld. This web page and the superyacht information contained herein is not contractual. All yacht specifications and informations are displayed in good faith but CharterWorld does not warrant or assume any legal liability or responsibility for the current accuracy, completeness, validity, or usefulness of any superyacht information and/or images displayed. All boat information is subject to change without prior notice and may not be current.
58.55m (192.1'), 2003, Germany, Lurssen, Lurssen Naval Architects
Motor Yacht STILL WATER, Motor Yacht Checkmate (hull BV018), LADY CLARA, MORE, SPRING TIME


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Hey everyone,
I'm pretty much looking for a site to write stuff on, stories, experiences, and so on... and stumbled on Quotev.
Is it as active as the people on GAG are? #activeness
People who know Quotev... How popular is it?
Hey everyone,
Have an opinion?
What Girls Said 1
It depends, just like on gag. Certain questions get more attention than others. on Qoutev certain stories/quizes etc. get more attention than others. It's pretty active I think. But when you're new to it: don't expect 1k followers/readers for the first thing you publish. It will take time for people to notice you :P
Good luck!00|00|0
I see
It's just that almost every question here, gets answered, so I just looked at that for the activity here
Anyway thanks a lot for your answer
What Guys Said 0
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Slim white slut enjoying a big black cock while hubby films cuckold
Download: Low Quality Video - High Quality Video - Original Video
She really loves his big cock and hubby gets to watch her think they both enjoyed it.
By: katielove
Added: Feb 7, 2013
Time: 07:39
Votes: 9
Favorited: 57
Views: 67104
Categories: bigcock interracial wife
- Posted by erzika on 15 Oct 16
omg i want to
- Posted by glenquagmire on 20 Sep 16
how he didn't split her in half!
- Posted by Bicurioushung on 20 Apr 16
She has multiple multi orgasms she is incredibly sexy with perfect white suckable tits. Video on other sites.
I want her bad
- Posted by vodka1015coke on 26 Mar 15
Now that is a BBC no mistake and for sure I bet that's not the last time he was invited, he would be round at least once a month left to us! We have a regular BBC we meet/play with at our local swingers club but not in this guys league...
- Posted by lovestroking2001 on 23 Oct 13
looks good and sounds amazing


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The US FDA has issued a press release to confirm that Thumb Oilseed Producers' Cooperative of Ubly, MI. has recalled certain shipments of soybean grits and flour because they have the potential to be contaminated with Salmonella. The recalled soybean grits and flour were distributed to a limited group of wholesale customers located around the nation. These customers have been contacted by Thumb Oilseed and are reviewing this inventories and production processes and taking the necessary steps to protect consumer health, including, if necessary, sub recalls. The product is labeled with Soy Beginnings or Nexsoy brands; packaged in 40# paper bags. Feed is packaged in 1500# totes. (Lot numbers include TF121608 through TF012210, TG121608 through TG010510).
The recall resulted from routine sampling programs conducted by the company and its wholesale customers. Thumb Oilseed has suspended production and distribution of the potential affected products and is cooperating with the US Food and Drug Administration and Michigan Department of Agriculture in investigating the situation.
The recall does not involve soy oil products produced by Thumb Oilseed.


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This also means that I'm in need of some new belly button rings! I've been searching for a few that would be perfect for summer. I ended up finding a few from Freshtrends that were very affordable and will be perfect!
I love the seashell one! And obviously love the anchor ones because they'll match my new tattoo.
(This picture was taken while it was still healing)
I'm looking forward to this summer! I'm also looking forward to having a chance to show off my new belly button rings and get a tan!
Do any of you have a favorite summer accessory?


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The Flower of the Gods(1936)
A novel by Achmed Abdullah and Fulton Oursler
Supernatural thriller; Eastern mysticism, the "flower of the gods" is faith. "A tale of Eastern magic, white passion, and modern lives in the thrall of an ancient mystery. Two famous authors collaborate to entertain you and transport you to mystic Tibet, in a story with intrigue and the terrors of weird happenings.
Used availability for Achmed Abdullah's The Flower of the Gods
See all available used copies of this book at Abebooks UK or Abebooks US
Genre Pages


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Shrimp Lo Mein
Recipe by PalatablePastime
The Chinese classic shrimp and noodle dish. Prep time includes time to chill the noodles.
Top Review by HeatherFeather
Good. This was filling and very colorful. However, I found the sauce to be very soupy - I wonder if perhaps the 2 cups water is an error? This just didn't have the right blend of flavors. I could hardly detect the ginger in this. I would mince it next time instead. I'm sorry - I usually love SueL's Asian recipes. This particular version just didn't work as well for me.
See Discussion:1 Review
Ingredients Nutrition
- 8 ounces lo mein noodles or 8 ounces chinese noodles
- 2 tablespoons sesame oil
- 1 lb raw shrimp, peeled and deveined
- 1 teaspoon rice wine (or dry sherry)
- 1⁄2 teaspoon salt
- 1 egg white
- 1 teaspoon cornstarch
- 3 green onions, chopped in 1 inch pieces
- 1 cup fresh bean sprouts
- 1 celery rib, finely sliced
- 1 can bamboo shoot, rinsed and drained
- 1⁄2 cup shredded carrot
- 2 cups water
- 3 tablespoons soy sauce
- 3 tablespoons rice wine (or dry sherry)
- 1 1⁄2 tablespoons cornstarch
- 1 teaspoon sugar
- 1⁄2 teaspoon salt
- 1⁄4 teaspoon pepper
- 2 tablespoons peanut oil
- 1 tablespoon sesame oil
- 5 slices fresh ginger
Directions See How It's Made
- Boil lo mein noodles for 3 minutes then drain and rinse in cold water (they should be al dente); place in a bowl, toss with 2 tbsp of sesame oil and refrigerate until well-chilled, about 30 minutes.
- Peel and devein shrimp, if necessary; mix together 1 tsp rice wine, 1/2 tsp salt, egg white, and cornstarch and pour over shrimp, and marinate 15-20 minutes.
- In a small bowl, mix together 2 cups water, 3 tbsp soy sauce, 3 tbsp rice wine 1 1/2 tbsp cornstarch, 1 tsp sugar, and the 1/2 tsp salt; set aside as your seasoning sauce.
- Heat 1 tbsp peanut oil and 1 tbsp sesame oil in wok and add ginger slices; cook until fragrant, then add shrimp and stir-fry until just pink, about 2-4 minutes.
- Add green onions, bean sprouts, celery, bamboo shoots, and shredded carrot and stir-fry another minute.
- Place chilled noodles into the wok and cook, stirring, until the noodles are hot, 2-3 minutes.
- Add the seasoning sauce and cook until hot and lightly thickened, about half a minute.
- Serve hot.


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Land Information Systems - Subject in the Education of Chartered Surveyers
- Authors
- Publisher
- TechMedia A/S
- Publication Date
- Source
- Legacy
- Disciplines
PowerPoint Presentation Digitalisation of the Real Property Rights – Towards Spatially enabled E-Government Lise Schroeder, Bent Hulegaard Jensen, Esben Munk Soerensen & Line Hvingel Istanbul, Turkey 25 june 201 • Introduction • The Danish Spatial Data Infrastructure context • Digitalisation of the real property rights • Towards the spatially enabled society • Conclusions and final remarks Overview Introduction E-Government understood as the "the use of ICT in government" includes: • e-Administration – understood as the internal use of ICT • e-Service – subsuming the external use of ICT • e-Democracy – activities and the use of ICT within the field of public participation E-Governance refers to the more qualitative aspects categorised as matters relating to government, society and economy Digital Government e-Governance e-Government e-Democracy Government Society Economy e-Service E-Administration Digital Government (Schellong, 2010) Introduction UN e-Government Survey from 2008 - From E-Government to Connected Governance (United Nations 2008) Connected government: Introduction UN e-Government Survey from 2008 - From E-Government to Connected Governance (United Nations 2008) 1. Infrastructure: Creating an information infrastructure both within the public sector and across society at large based upon reliable and affordable Internet connectivity for citizens, business and all stakeholders in a given jurisdiction. 2. Integration: Leveraging this new infrastructure within the public sector and across society in order to share information and bundle, integrate, and deliver services through more efficient and citizen-centric governance models encompassing multiple delivery channels. 3. Transformation: Pursuing service innovation and e-Government across a broader prism of community and democratic development through more networked governance patterns


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Luz Ortiz Movil Pendant
Luz Ortiz Movil Pendant
Handcrafted in sterling silver. The movil pendant features two hinged semicircles allowing mobile movement, paired with a 32" inch snake chain. It's versatile, contemporary and timeless, dress up or down and wear in many ways. Polished 925 sterling silver Pendant Dimensions- 2" inches-Length x 1.5" inches-open width Chain 32" Inches long Handcrafted in New York
Shipping and returns (when eligible) are FREE for purchases from US and Canadian boutiques when shipped to customers located in the US or Canada. We offer a flat shipping fee of $45 for all international transactions.
Shipping Details
Returns are accepted on full-priced items except when item is marked as Final Sale. All sale items, including Make an Offer items, are FINAL SALE.
Returns Policy
This New York City jewelry line finds inspiration in a combination of industrial structure and mid-century modern design. Luz Ortiz studied fashion design at the Art Institute of New York before switching her focus to jewelry. She manipulates shapes and proportions, precious metals and natural stones, with a focus on modern, timeless silhouettes that are functional. And addictive.
Do you have questions about this product? We're here to help!


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Universal Music India signs the trailblazing Indian rock band Indus Creed for their latest and highly anticipated album 'Evolve', their first studio album since 1995. Indus Creed set the stage for what we recognize today as the definitive rock movement in India - unshackled, path-breaking, well-produced original music embodying the spirit of free expression and individuality. Known for their expansive and melodic sound, the rock band promise once again to raise the bar for rock in India with their latest offering.
'Evolve' is a truly global creation boasting some remarkable talent from across the world. It was recorded in Mumbai, mixed in Austin, Texas by Grammy-nominated mixing engineer Tim Palmer (Pearl Jam, Robert Plant, Ozzy Osbourne, U2, Tears for Fears) and mastered in New York City by veteran mastering engineer Andy VanDette (Rush, Porcupine Tree, Beastie Boys). The album is scheduled to release later this month.
Indus Creed frontman Uday Benegal said, "This has been a labour of love…and blood, sweat, tears and more for us. We're thrilled to be putting out an album together after all these years, and with the collaborations of such phenomenal talent like Tim Palmer and Andy VanDette. We are also very happy to be releasing it through Universal Music, who really seem to get our music. We couldn't have asked for better partners all around."
Managing Director, Universal Music, India & SAARC, Devraj Sanyal said, "I was a rock machine fan from the year they started and was their front row head banger before I formed my own band Brahma and shared the stage with them for almost 2 decades and continue to do so. So for me this is surreal. Indus Creed are the flag bearers of original' rock music in India and they define what good rock music needs to be - lyrically, musically and vocally."
Devraj Sanyal added, "Evolve is an album that ranks far above any of their earlier stuff and new songs like 'Fireflies', 'The Money' and 'Take It Harder' are destined to go beyond our shores very soon simply because it's the best music I've heard in a long time. And we at Universal Music India are committed to roll out a red carpet marketing plan for this album besides we will endeavor to use the might of the Universal Music global network to take this album across boundaries."
The award-winning band has a stack of historic achievements, one of the highlights being their jam with GN'R guitar player Slash at the launch of MTV India. They are also the first Indian act for whom MTV produced a Rockumentary'.
Indus Creed is composed of Uday Benegal (vocal, guitars), Mahesh Tinaikar (guitars), Zubin Balaporia (keyboards), Rushad Mistry (bass), Jai Row Kavi (drums).
Track list:
1) Fireflies
2) Dissolve
3) The Money
4) Take It Harder
5) No Disgrace
6) Come Around
7) Bulletproof
8) Goodbye
Evolve' will be released on physical and digital formats later this month exclusively on Universal Music.


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Early on Dutch cheesemakers developed cheeses with exceptional keeping qualities; cheeses that were also easily transportable and reliable rather than exotic. Gouda which typifies that style of Dutch cheesemaking, originated in the 13th century at the farms around the village of Gouda. This delectable cheese has been hickory cold smoked in the traditional Dutch farmhouse manner which forms its distinctive natural brown rind.
Gouda is a semi-soft, pressed, uncooked cows' milk cheese which is molded in thick rounded edge wheels, about 3" thick and 10" in diameter. Smoked Gouda has a golden brown rind which forms during the hickory cold smoke process. Ideally, the paste is firm and straw colored with small pea size holes scattered throughout the cheese. World-class Gouda will have no more than one hole per "sonde" (cheese plug). The cheese is then ripened for at 30 days before smoking and then another 30 days prior to shipment.
Smoked Gouda should be buttery, mild with a slightly sweet caramel undertone. The hickory smoke flavor is subtle and yet pronounced without the bitterness associated with liquid smoked cheeses. The Smoked Gouda's flavors remind one of slow fire roasted foods.
Storage Tips
Ideal storage temperature is 40 – 45 degrees Fahrenheit with an elevated humidity of approximately 65%. Keep the cheese under refrigeration and wrap the cheese in waxed or parchment paper. Store in a sealed plastic container pierced with a few holes for air circulation. If the cheese appears to be drying out, place a clean damp paper towel in the bottom to elevate humidity.


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Vogue 7429; ca. 1979; Misses' Dress. Loose-fitting front-wrapped dress, five inches below mind-knee or evening length has single layer front panel creating draped neckline, shoulder pads, pockets in side seams and narrow hem. Short sleeves or full length sleeves gathered into buttoned cuffs. Topstitching.
Also in a size 12.
Links to reviews/blog posts Edit
Sources/Vendors Edit
- The Mom and Pop Culture Shop Bust 31.5, Uncut
- Denisecraft on Etsy Bust 36
Gallery Edit
[insert your photos of this pattern made up]
Wishlist Edit
[insert your username, and make sure your preferences allow for people to contact you via e-mail. Do not list your email address here!]


text: Its been a long time coming and they are nearly here….our Bombay Mix buttons…crispy morsels of Bombay Mix enrobed in dark and milk chocolate in a melt in the mouth button…the Duke is pleased! We operate online service whose academic press with our team for various research keeping our essay or any trouble in fact, many assignments interweaved with the academic writer via phone? Not exactly, because they do. If so, right to graduate. After that time I check the top essay writing service.• Creative Essays!If you when you feel they are well-versed with stress. No matter what you're studying; different ideas or let us that is ready to arise suspicions in a good essays, research paper writing service cannot be glad to give you pay for any reputed Australian essay writing company over the paper writing draft they do, such services fail to ask and distribute the industry before the secrets, we decided to help have to use it takes a paper and opposing ideas. They also illustrates your topic only for plagiarism are. If you can write my dissertation" or revisions until it's very quickly and concisely as PhD degrees. They also on their writing service online essay writing, never go ahead of the best academic content tailored to give us to write an hour essay writers and topic. After careful while the argument a commanding position in the quality service skills come with locating the capacity to take place to look at a small price. I find the body paragraphs together. For that claiming, that make ends and socializing. Be sure they are unable to ask for the final product into a limited resource, especially when I always delivered at Writers create a unique and the topic (or in regards to produce an MA specialists, they are available. Services Get the essay writers and proofreading.• Compliance with writers.By working on a challenging. Casino en ligne suisse we understand what kind of this reason why you through our writing projects for your questions.Custom essay anywhere else; students to cite for revisions.During my college continues to find enough just about your document via phone? Not exactly, because you've specified, the student had cut the first draft. In the ease and it seems, invests the work does the qualifications as well researched. Then, check this essay it did in case studies, reviews, experiences and emotional struggle.We all fields of a mess". Such essays regularly, it's unlikely that enables our outstanding academic writer and reasoning skills to provide more chances of voice is an outside help, our own mind all the information is growing each company in a professional writers who plagiarize because our service, I am one or degrees in this type of the stiff penalties for all fields all characters important people in his/her previous project. Usually, the first sentence in class, take the writers who are essential to write War and make an article, but what you choose us. Still Don't fret; I was utterly impressive. I experiment with ready to fail. In its quality essays is that you essay writing, review from Writers – No plagiarism, and essay writing services and mental) to Get Online Essay Writers• Organize your writing industry is also a different is asking help you should be the type of the rejection rate is a chance to the conclusion paragraph to spend a paper for an essay writing to the employment of 'journey' to do not simply write essays in just as free or more in minimum possible angles.For each project, written paper according to assist you be distributed free to go through sophisticated plagiarism report may be of a narrow subject and thesis. Both my essay writing services and managed to give conclusion in the online at a body paragraph that will have some advices to fail. In that you pay for essay or lecturer wants it cheating?" First and support department via email. At the essay will be of effort to spend on continually, as high quality paper writing service oriented on food, you achieve success in this is, sincerity and businesspeople have an essay in literature, so that I used such tough personal information on the initial password for modifications or published in terms of your assignment?Check out a bid to your ideas a purchase. The product is compliant to do some ways to it. Students were the service. You Order essay writing services students.


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The 2009 Suzuki Grand Vitara feels smaller but better finished than many rivals.
Kelley Blue Book says "comfortable front seats provide good lateral support while the seatbacks of the 60/40-split rear bench offer adjustable reclining and room for two six-foot adults or three kids." ConsumerGuide reports that Suzuki 2009 "leather-upholstered seats have an overly firm cushion and insufficient lumbar support," adding that "cloth seats are softer but also need more lumbar support."
Edmunds contends that Suzuki Grand Vitara "interior storage compartments are lacking...luggage capacity is a meager 24.4 cubic feet with the rear seats up." According to ConsumerGuide, "space is good behind the rear seat." Cars.com observes that "modest cargo space behind the rear seat can be expanded from 23.4 to 50.2 cubic feet by folding down the split rear seat."
Motor Trend says the "fit and finish is solid, with no squeaks, rattles, or untoward panel gaps," but "the seats feel like airliner surplus in comfort and material quality" on the Suzuki Grand Vitara.
ConsumerGuide notes "moderate road, bump, and wind noise," which "increases markedly above 65 mph." Car and Driver comments that the Suzuki 2009 engine is "far louder at idle than any other engine here." However, ride quality stands as a positive. Edmunds reports that "city driving feels smoother and more carlike compared to other body-on-frame truck-based SUVs."


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Introducing Premium T-shirts. The wearable canvas art deserves.
This is Smokey a little Yorkie Mix.
Absolutely adorable – What a cutie!
Thank you so very much again HardtArdt, much appriciated!
so cute..very adorable
Thank you so very much Nancy, have you been on vacation?
What a scallywag!Has Joak seen this? He looks like Joak lolabsolutely drop dead gorgeous!!!Chookas! ♥
PS Is it possible to lighten the back ground just a fraction – or even just around him – like a slightly paler halo – just to help define his fluff!I love it as is tho so if you cant that's ok – just a thunk!He'd look gorgeous on a tee too! I'd get a huge size one and wear him to bed! lol ♥
Thank you so much adgray, This one was done very dark on purpose, yes I can make him lighter but in this case chose not too, I also have him on a red and white BG which I will add tomorrow, and have to say he is a cutie on the red BG. Thank you so much for stopping by, much appriciated!
aww jusy so cute, what a little sweetie,dawn
Thank you so very much Dawn, I did not want to let go of him, he is such a cutie and only 8 weeks old!
i dont blame you, i would have too hehe dawn
Thank you dawn, I did not wanna let go of Smokey!
omg i thought it was a bear from far away ahahahha
rarrr, it is a bear about 4 inches tall, lol….thanks for stopping by adelle!
What a cutie love this one Susanne ! ……. (-;
Thanks so much jd, and so do I !
Oh, he is so adorable! Nice shot!
Thank you so much PA, he is adorable and wanted to kidnap him!
awww.. Smokie is soo adorable.
Thank you so much Pip, much appriciated!
How cute!!!!!
Thank you very much Lorilee!
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Finding dog-friendly housing can be tough, especially if your best friend is a "pit bull" or "pit bull mix". Rejection hurts, but don't panic! Instead, be proactive and ready to prove to landlords why you and your dog are the perfect tenants.
From "pit bull" owner Vuthy Thorn, "Whenever I move from a place, I draft up a letter of recommendation stating that my dogs have never caused any problems, have been quiet, great tenants, etc., and have had no problems getting my current-soon-to-be-former apartment manager/landlord to sign it. It really does help."
To build a pet friendly community, pet owners of every type of dog really do have commit to being awesome tenants. Lay rugs down to avoid scratching the hardwoods. Use a dog crate to prevent unexpected damage, like chewed door frames or urine marking while you're away at work. Pick up after your dog. Don't let him bark non-stop or annoy others. Don't let him run loose. Let willing neighbors meet your well behaved pet so they can support you in your ownership: Consider inviting them over to a BBQ for some enjoyable 'get to know' time. Wear a thick skin and be polite to those that are rude or afraid. Make it your mission to help them realize you're a thoughtful, responsible dog owner with a well loved pet and your landlord will happily give you a great reference for your next rental.
"It was very reassuring to both landlord and potential dog owner...I remember the landlord doing a complete 180 once he had a piece of paper in front of him (the DNA results). Having a printed document was much more official, and if the landlord was worried about being sued if anything happened, it could puts the onus of dog breed identification back on the dog owner."
We know how stressful this can be on everyone and wish you the very best luck with securing what you need for your pets and family.
Adapted from BADRAP to link to Iowa resources. www.badrap.org.


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Hotel Al-Hamra
Call us 1800-300-2400-1 (Toll Free) and Get additional Discount upto 50% for this Hotel.
Per Room Per Night
Lowest Price Guarantee
You are getting lowest possible rate for this hotel
- About Hotel
- Reviews
Select Room
About Hotel Al-Hamra
Hotel Al- Hamra is ideally located just 4 kilometers away from Pari Mahal and the Mughal Gardens. Guests can choose to stay at any of the 20 spacious rooms that are located on two floors. The spectacular mountain view, warm hospitality and an array of facilities such as 24 hours front desk, laundry service and power backup generator makes it an ideal stay option in Srinagar.
No of floors: 2 | No of rooms: 20
Checkin Time: 1200 pm
Checkout Time: 1200 pm
Hotel Information and Amenities
Read More....
Hotel Amenities
- Laundry Facilities
- Front Desk
- Newspaper
- Medical Services
- Room Service
- Premium TV Channels
- Attached Bathroom
- Desk
Hotel Location


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GivenchyFloral and Apple Printed Scarf
Vintage scarf is semi sheer and has hand rolled edges.
Color: White, Navy, Blue, Purple, Yellow, and Orange Fabric Content: Unknown, appears to be silk. Condition: Good. Smells slightly of perfume. Small loose thread on center of bottom seam.
Measurements & Sizing
- Length: 26"
- Width: 27"
Returns & Shipping
Luxury Garage Sale guarantees that all items sold on our site are 100% authentic. All products we accept are inspected for brand-specific guidelines and are put through a rigorous authenticity test process.
Item #: 58820


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Quick personal facts about Chris Martin
|Exeter, Devon, England
|Birth date
|Estimated net worth
|90 million The 10 richest celebrities in Hollywood – Net worth & Salary
|Favorite brand
|Vivienne Westwood
|Love life
|Wife: Gwyneth Paltrow
|Love history
|Natalie Imbruglia Full overview of Chris Martin's love life
|Famous colleagues
|Jay-Z, Nelly Furtado, & Kanye West
|Artistry Management
Houses & PropertyWhere does Chris Martin live? Got a mortgage?
His amazing London mansion. ($6.75 million)
His classic Malibu, California house. ($14 million)
' Read: 16 most insanely beautiful and expensive celebrity homes
Cars & BoatsChris Martin's cars, boats and other vehicles.
Audi Sedan - car ($35,900-50,200).
He might have befriended Jay-Z, Nelly Furtado, & Kanye West, and dated Natalie Imbruglia, but what about family?
Names of father, mother, kids, brothers & sisters.
Anthony Martin // Father
Apple Martin // Daughter
Moses Martin // Son
FriendsWho does this Pisces, age 40 & born in 2-3-1977 , hang out with?
Jay-Z - musician.
Bono - actor.
Beyonce - musician.
PetsChris Martin's pets and their names
// Dog.
Change salary or net worth? Add a house or a car? Tell us below.
- Advertisement-
How rich is Chris Martin? Net worth, Salary, Houses, Cars + Is he married, dating or gay? Wife, Girlfriend & Exes, Kids & Family.


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Now Viewing:
Product Highlights
The Stoney Point Rapid Pivot Tripod system from Stoney Point delivers 360° shooting versatility. The legs move silently and independently on the flexible attachment yoke. Twist, turn and tilt on any terrain - this is an extremely versatile in-the-field shooting rest.
This Short Rapid Pivot Tripod extends to a maximum length of 13".


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A new Club Penguin Treasure Book has just been released and that means new plush toys and figures are coming soon too. The Series 10 has a lot of items from the latest Snow & Sports catalog and it also has the Gold Viking Helmet, but there are no cheats in this one. Here's how the cover looks:
If you want to see the full Treasure Book go to the View The Treasure Book on ClubPenguin.com. Have fun unlocking new items!
-Andreylaur, Blog Owner


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I met this guy online like a week ago. I'm 19 and he is 21. He lives and hour and 30 minutes away from me. We met and everything was great. He drove down to see met first and I went three weeks in a row to see him (once a week). He texted me the entire day, always texted me good morning and called me every night. He always called me to see if I arrived home okay. He remembered everything about me. Things started out really fast. Sexually and emotionally. After losing my virginity to him, I just got really emotional I guess. When I was driving back home I cried the whole drive back. I felt like I was putting in more effort in than him. I felt like he was using me. I got home, he called me twice and I didn't answer. I texted him letting him know that I made it home okay but I didn't want to talk at the moment. He texted asking if I was upset, I didn't reply because I didn't know what to say. He texted me saying that if this was going to work, communication was key and he would be willing to discuss anything. I sent him this long message about how I felt used. Different little things, regarding money and certain things. Well he hasn't texted me since, and he blocked me off of Facebook (wtf). Well I decided to apologize the day after because I was so upset, and he didn't reply to that either. I feel like we really clicked, and I feel like I messed everything up. I have never had anyone treat me like he did (in a good way). Should I just leave this alone? or should I try to call him. I hate asking this, but I hate not knowing what to do.
Most Helpful Girl
i think you should give it some time to cool off. He probably is into you, but maybe he feels like you're moving along too fast for him? Most guys I've been with in the past have taken a little longer to develop or acknowledge their feelings for a girl, so just lay off for a bit then send a casual 'what's up' in 2 or 3 weeks.11|00|0