The origin and evolution of photosynthesis have long remained enigmatic due to a lack of sequence information of photosynthesis genes across the entire photosynthetic domain. To probe early evolutionary history of photosynthesis, we obtained new sequence information of a number of photosynthesis genes from the green sulfur bacterium Chlorobium tepidum and the green nonsulfur bacterium Chloroflexus aurantiacus. A total of 31 open reading frames that encode enzymes involved in bacteriochlorophyll/porphyrin biosynthesis, carotenoid biosynthesis, and photosynthetic electron transfer were identified in about 100 kilobase pairs of genomic sequence. Phylogenetic analyses of multiple magnesium-tetrapyrrole biosynthesis genes using a combination of distance, maximum parsimony, and maximum likelihood methods indicate that heliobacteria are closest to the last common ancestor of all oxygenic photosynthetic lineages and that green sulfur bacteria and green nonsulfur bacteria are each other's closest relatives. Parsimony and distance analyses further identify purple bacteria as the earliest emerging photosynthetic lineage. These results challenge previous conclusions based on 16S ribosomal RNA and Hsp60/Hsp70 analyses that green nonsulfur bacteria or heliobacteria are the earliest phototrophs. The overall consensus of our phylogenetic analysis, that bacteriochlorophyll biosynthesis evolved before chlorophyll biosynthesis, also argues against the long-held Granick hypothesis.

Recognition of double-stranded RNA by Toll-like receptor 3 (TLR3) will increase the production of cytokines and chemokines through transcriptional activation by the NF-B protein.Over 136 single-nucleotide polymorphisms (SNPs) in TLR3 have been identified in the human population. Of these, four alter the sequence of the TLR3 protein. Molecular modeling suggests that two of the SNPs, N284I and L412F, could affect the packing of the leucine-rich repeating units in TLR3. Notably, L412F is reported to be present in 20% of the population and is higher in the asthmatic population. To examine whether the four SNPs affect TLR3 function, each were cloned and tested for their ability to activate the expression of TLR3-dependent reporter constructs. SNP N284I was nearly completely defective for activating reporter activity, and L412F was reduced in activity. These two SNPs did not obviously affect the level of TLR3 expression or their intracellular location in vesicles. However, N284I and L412F were underrepresented on the cell surface, as determined by flow cytometry analysis, and were not efficiently secreted into the culture medium when expressed as the soluble ectodomain. They were also reduced in their ability to act in a dominant negative fashion on the wild type TLR3 allele. These observations suggest that N284I and L412F affect the activities of TLR3 needed for proper signaling.

The origin of photosynthesis is a fundamental biological question that has eluded researchers for decades. The complexity of the origin and evolution of photosynthesis is a result of multiple photosynthetic components having independent evolutionary pathways. Indeed, evolutionary scenarios have been established for only a few photosynthetic components. Phylogenetic analysis of Mg-tetrapyrrole biosynthesis genes indicates that most anoxygenic photosynthetic organisms are ancestral to oxygen-evolving cyanobacteria and that the purple bacterial lineage may contain the most ancestral form of this pigment biosynthesis pathway. The evolutionary path of type I and type II reaction center apoproteins is still unresolved owing to the fact that a unified evolutionary tree cannot be generated for these divergent reaction center subunits. However, evidence for a cytochrome b origin for the type II reaction center apoproteins is emerging. Based on the combined information for both photopigments and reaction centers, a unified theory for the evolution of reaction center holoproteins is provided. Further insight into the evolution of photosynthesis will have to rely on additional broader sampling of photosynthesis genes from divergent photosynthetic bacteria.

Proteins that recognize pathogen-associated molecular patterns are key factors in the cascade of events from the detection to the elimination of an invading organism. This form of innate immunity is conserved in eukaryotes. For example, the Drosophila melanogaster Toll protein is responsible for resistance to fungal and bacterial infections (3, 4), and plants can encode disease-resistance proteins that are important in determining the outcome of infection (5). The vertebrate pathogendetecting proteins called Toll-like receptors (TLRs) 5 are key players in the activation of both the innate and adaptive arms of the immune system (6 -9).The TLRs and related pathogen sensors contain leucine-rich repeat motifs that form docking sites for pathogen ligands or adaptors that bind pathogen ligands, the binding of which will activate signal transduction pathway(s) (10 -12). TLR3 recognizes double-stranded RNA and may be a part of a redundant sensor system to detect viral infections (13-15). Although specific features in the ligands required to interact with TLR3 remain to be identified, TLR3 is activated by polyinosinepolycytidylic acid (poly(I:C)) and has been reported to be activated by RNAs extracted from necrotic cells (16).A number of issues concerning TLR3 structure and function remain to be elucidated. For example, TLR3 can apparently act both on the surface of the plasma membrane, as it does in fibroblasts, and by attaching to the membranes of intracellular vacuoles, where it is proposed to act in immature dendritic cells (17,18). The trafficking of TLRs should be influenced by glycosylation in general, and N-linked glycosylation of TLR2 and TLR4 has been shown to play essential roles in its localization (19,20). A significant recent advance in TLR3 was the elucidation of a 2.1 Å structure of the soluble ectodomain by Choe et al. (1). Bell et al. (2) independently elucidated a highly similar structure. In both studies, the crystallized ectodomains were produced in a baculovirus expression system and formed a horseshoe-shaped solenoid structure that was extensively decorated with glycosyl modifications, some of which were partially resolved in the structure. Whether the glycosylations are important in TLR3 localization and/or function were not directly addressed in these works (1, 2). However, de Bouteiller et al. (21) showed that a change of Asn-247 to an arginine in TLR3 negatively affected TLR3 activity.We expressed the extracellular domain (ECD) of the human TLR3 in human embryonic kidney cells (HEK 293T) and demonstrated that it was modified with N-linked glycosylations. Using the GlcNAc-transferase inhibitor tunicamycin, a concentration-dependent inhibition of TLR3 activity was observed. Systematic mutational analysis of the predicted N-linked glycosylation sites identified two asparagine residues in leucine-rich repeats 8 and 15 that are important for TLR3 activity. The mutant proteins remain expressed at levels similar to wild type. In addition, because our ectodomain was produced in human cells as opp...

A DNA sequence has been obtained for a 35.6-kb genomic segment from Heliobacillus mobilis that contains a major cluster of photosynthesis genes. A total of 30 ORFs were identified, 20 of which encode enzymes for bacteriochlorophyll and carotenoid biosynthesis, reaction-center (RC) apoprotein, and cytochromes for cyclic electron transport. Donor side electron-transfer components to the RC include a putative RC-associated cytochrome c 553 and a unique four-large-subunit cytochrome bc complex consisting of Rieske Fe-S protein (encoded by petC), cytochrome b 6 (petB), subunit IV (petD), and a diheme cytochrome c (petX). Phylogenetic analysis of various photosynthesis gene products indicates a consistent grouping of oxygenic lineages that are distinct and descendent from anoxygenic lineages. In addition, H. mobilis was placed as the closest relative to cyanobacteria, which form a monophyletic origin to chloroplast-based photosynthetic lineages. The consensus of the photosynthesis gene trees also indicates that purple bacteria are the earliest emerging photosynthetic lineage. Our analysis also indicates that an ancient gene-duplication event giving rise to the paralogous bchI and bchD genes predates the divergence of all photosynthetic groups. In addition, our analysis of gene duplication of the photosystem I and photosystem II core polypeptides supports a ''heterologous fusion model'' for the origin and evolution of oxygenic photosynthesis.Bacterial photosynthesis is found in five eubacterial phyla: cyanobacteria, purple bacteria, heliobacteria, green sulfur bacteria, and green nonsulfur bacteria. Analysis of photosystems from purple and green nonsulfur bacteria shows that these organisms synthesize primitive photosystems that are ancestral to the more complex oxygen-evolving photosystem II (PSII) from cyanobacteria and chloroplasts (1). Additional studies of heliobacteria and green sulfur bacteria indicate that they synthesize photosystems that are ancestral to the photosystem I (PSI) complex from cyanobacteria and chloroplasts (2).With the exception of the sequence analysis of PSI and PSII structural polypeptides, there is little information on the origin and evolution of photosynthesis. Complete sequence information on photosynthesis genes is only available for the purple nonsulfur bacterium, Rhodobacter capsulatus, which has a major clustering of photosynthesis genes (3), and for the cyanobacterium Synechocystis sp. PCC 6803, for which sequence analysis has been completed for the entire chromosome. Information on photosynthesis genes from the three other eubacterial photosynthetic phyla is limited mainly to the reaction-center (RC) core polypeptides and a few cytochromes.With the aim of providing more substantive information on the evolution of photosynthesis, we have undertaken an extensive characterization of photosynthesis genes from Heliobacillus mobilis. A gene cluster was found to encode enzymes for bacteriochlorophyll and carotenoid biosynthesis, as well as the RC core polypeptide and cytochromes i...

We generated random transposon insertion mutants to identify genes involved in light input pathways to the circadian clock of the cyanobacterium Synechococcus elongatus PCC 7942. Two mutants, AMC408-M1 and AMC408-M2, were isolated that responded to a 5-h dark pulse differently from the wild-type strain. The two mutants carried independent transposon insertions in an open reading frame here named ldpA (for lightdependent period). Although the mutants were isolated by a phase shift screening protocol, the actual defect is a conditional alteration in the circadian period. The mutants retain the wild-type ability to phase shift the circadian gene expression (bioluminescent reporter) rhythm if the timing of administration of the dark pulse is corrected for a 1-h shortening of the circadian period in the mutant. Further analysis indicated that the conditional short-period mutant phenotype results from insensitivity to light gradients that normally modulate the circadian period in S. elongatus, lengthening the period at low light intensities. The ldpA gene encodes a polypeptide that predicts a 7Fe-8S cluster-binding motif expected to be involved in redox reactions. We suggest that the LdpA protein modulates the circadian clock as an indirect function of light intensity by sensing changes in cellular physiology.

A three-dimensional model of the photosystem I1 (PSII) reaction center from the cyanobacterium Synechocystis sp. PCC 6803 was generated based on homology with the anoxygenic purple bacterial photosynthetic reaction centers of Rhodobacter sphaeroides and Rhodopseudomonas viridis, for which the X-ray crystallographic structures are available. The model was constructed with an alignment of Dl and D2 sequences with the L and M subunits of the bacterial reaction center, respectively, and by using as a scaffold the structurally conserved regions (SCRs) from bacterial templates. The structurally variant regions were built using a novel sequence-specific approach of searching for the best-matched protein segments in the Protein Data Bank with the "basic local alignment search tool" (Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ, 1990, J Mol Biol 215:403-410). and imposing the matching conformational preference on the corresponding D l and D2 regions. The structure thus obtained was refined by energy minimization. The modeled D l and D2 proteins contain five transmembrane a-helices each, with cofactors (4 chlorophylls, 2 pheophytins, 2 plastoquinones, and a non-heme iron) essential for PSII primary photochemistry embedded in them. A p-carotene, considered important for PSII photoprotection, was also included in the model. Four different possible conformations of the primary electron donor P680 chlorophylls were proposed, one based on the homology with the bacterial template and the other three on existing experimental suggestions in literature. The P680 conformation based on homology was preferred because it has the lowest energy.Redox active tyrosine residues important for P680' reduction as well as residues important for PSI1 cofactor binding were analyzed. Residues involved in interprotein interactions in the model were also identified. Herbicide 3-(3,4-dichlorophenyI)-I , 1 -dimethylurea (DCMU) was also modeled in the plastoquinone QB binding niche using the structural information available from a DCMU-binding bacterial reaction center.A bicarbonate anion, known to play a role in PSII, but not in anoxygenic photosynthetic bacteria, was modeled in the non-heme iron site, providing a bidentate ligand to the iron. By modifying the previous hypothesis of Blubaugh and Govindjee (1988, Photosyn Res 1 9 3 -1 2 8 ) , we modeled a second bicarbonate and a water molecule in the Qe site and we proposed a hypothesis to explain the mechanism of QB protonation mediated by bicarbonate and water. The bicarbonate, stabilized by DLR257, donates a proton to Qi-through the intermediate of DLH252; and a water molecule donates another proton to Qi-. Based on the discovery of a "water transport channel" in the bacterial reaction center, an analogous channel for transporting water and bicarbonate is proposed in our PSII model. The putative channel appears to be primarily positively charged near QB and the non-heme iron, in contrast to the polarity distribution in the bacterial water transport channel. The constructed model has ...

Essential Bioinformatics is a concise yet comprehensive textbook of bioinformatics, which provides a broad introduction to the entire field. Written specifically for a life science audience, the basics of bioinformatics are explained, followed by discussions of the state-of-the-art computational tools available to solve biological research problems. All key areas of bioinformatics are covered including biological databases, sequence alignment, genes and promoter prediction, molecular phylogenetics, structural bioinformatics, genomics and proteomics. The book emphasizes how computational methods work and compares the strengths and weaknesses of different methods. This balanced yet easily accessible text will be invaluable to students who do not have sophisticated computational backgrounds. Technical details of computational algorithms are explained with a minimum use of mathematical formulae; graphical illustrations are used in their place to aid understanding. The effective synthesis of existing literature as well as in-depth and up-to-date coverage of all key topics in bioinformatics make this an ideal textbook for all bioinformatics courses taken by life science students and for researchers wishing to develop their knowledge of bioinformatics to facilitate their own research.