Protochlorophyllide synthesis by recombinant cyclases from eukaryotic oxygenic phototrophs and the dependence on Ycf54

The unique isocyclic E ring of chlorophylls contributes to their role as light-absorbing pigments in photosynthesis. The formation of the E ring is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase, and the O2-dependent cyclase in prokaryotes consists of a diiron protein AcsF, augmented in cyanobacteria by an auxiliary subunit Ycf54. Here, we establish the composition of plant and algal cyclases, by demonstrating the in vivo heterologous activity of O2-dependent cyclases from the green alga Chlamydomonas reinhardtii and the model plant Arabidopsis thaliana in the anoxygenic photosynthetic bacterium Rubrivivax gelatinosus and in the non-photosynthetic bacterium Escherichia coli. In each case, an AcsF homolog is the core catalytic subunit, but there is an absolute requirement for an algal/plant counterpart of Ycf54, so the necessity for an auxiliary subunit is ubiquitous among oxygenic phototrophs. A C-terminal ∼40 aa extension, which is present specifically in green algal and plant Ycf54 proteins, may play an important role in the normal function of the protein as a cyclase subunit

Absence of the cbb3 terminal oxidase reveals an active oxygen-dependent cyclase involved in bacteriochlorophyll biosynthesis in Rhodobacter sphaeroides.

The characteristic green color associated with chlorophyll pigments results from the formation of an isocyclic fifth ring on the tetrapyrrole macrocyle during the biosynthesis of these important molecules. This reaction is catalyzed by two unrelated cyclase enzymes employing different chemistries. Oxygenic phototrophs such as plants and cyanobacteria utilize an oxygen-dependent enzyme, the major component of which is a diiron protein named AcsF, while BchE, an oxygen-sensitive [4Fe-4S] cluster protein, dominates in phototrophs inhabiting anoxic environments, such as the purple phototrophic bacterium Rhodobacter sphaeroides We identify a potential acsF in this organism and assay for activity of the encoded protein in a strain lacking bchE under various aeration regimes. Initially, cells lacking bchE did not demonstrate AcsF activity under any condition tested. However, on removal of a gene encoding a subunit of the cbb3-type respiratory terminal oxidase, cells cultured under regimes ranging from oxic to microoxic exhibited cyclase activity, confirming the activity of the oxygen-dependent enzyme in this model organism. Potential reasons for the utilization of an oxygen-dependent enzyme in anoxygenic phototrophs are discussed. IMPORTANCE: The formation of the E ring of (bacterio)chlorophyll pigments is the least well-characterized step in their biosynthesis, remaining enigmatic for over 60 years. Two unrelated enzymes catalyze this cyclization step; O2-dependent and O2-independent forms dominate in oxygenic and anoxygenic phototrophs, respectively. We uncover the activity of an O2-dependent enzyme in the anoxygenic purple phototrophic bacterium Rhodobacter sphaeroides, initially by inactivation of the high affinity terminal respiratory oxidase, cytochrome cbb3 We propose that the O2-dependent form allows for the biosynthesis of a low level of bacteriochlorophyll under oxic conditions, so that a rapid initiation of photosynthetic processes is possible for this bacterium upon a reduction of oxygen tension

Adapting Real Quantifier Elimination Methods for Conflict Set Computation

The satisfiability problem in real closed fields is decidable. In the context of satisfiability modulo theories, the problem restricted to conjunctive sets of literals, that is, sets of polynomial constraints, is of particular importance. One of the central problems is the computation of good explanations of the unsatisfiability of such sets, i.e.\ obtaining a small subset of the input constraints whose conjunction is already unsatisfiable. We adapt two commonly used real quantifier elimination methods, cylindrical algebraic decomposition and virtual substitution, to provide such conflict sets and demonstrate the performance of our method in practice

Truth Table Invariant Cylindrical Algebraic Decomposition by Regular Chains

A new algorithm to compute cylindrical algebraic decompositions (CADs) is presented, building on two recent advances. Firstly, the output is truth table invariant (a TTICAD) meaning given formulae have constant truth value on each cell of the decomposition. Secondly, the computation uses regular chains theory to first build a cylindrical decomposition of complex space (CCD) incrementally by polynomial. Significant modification of the regular chains technology was used to achieve the more sophisticated invariance criteria. Experimental results on an implementation in the RegularChains Library for Maple verify that combining these advances gives an algorithm superior to its individual components and competitive with the state of the art

Engineering chlorophyll, bacteriochlorophyll, and carotenoid biosynthetic pathways in Escherichia coli

The biosynthesis of chlorophylls (Chls) and bacteriochlorophylls (BChls) represents a key aspect of photosynthesis research. Our previous work assembled the complete pathway for the synthesis of Chl a in Escherichia coli; here we engineer the more complex BChl a pathway in the same heterotrophic host. Coexpression of 18 genes enabled E. coli to produce BChl a, verifying that we have identified the minimum set of genes for the BChl a biosynthesis pathway. The protochlorophyllide reduction step was mediated by the bchNBL genes, and this same module was used to modify the Chl a pathway previously constructed in E. coli, eliminating the need for the light-dependent protochlorophyllide reductase. Furthermore, we demonstrate the feasibility of synthesizing more than one family of photosynthetic pigments in one host by engineering E. coli strains that accumulate the carotenoids neurosporene and β-carotene in addition to BChl a

Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina

The major photopigment of the cyanobacterium Acaryochloris marina is chlorophyll d , while its direct biosynthetic precursor, chlorophyll a , is also present in the cell. These pigments, along with the majority of chlorophylls utilized by oxygenic pho- totrophs, carry an ethyl group at the C-8 position of the molecule, having undergone reduction of a vinyl group during biosyn- thesis. Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB. The genome of Acaryochloris marina contains open reading frames (ORFs) encoding proteins displaying high sequence similarity to BciA or BciB, although they are annotated as genes involved in transcriptional control ( nmrA ) and methanogenesis ( frhB ), respectively. These genes were introduced into an 8-vinyl chlorophyll a -producing delta bciB strain of Synechocystis sp. strain PCC 6803, and both were shown to restore synthesis of the pigment with an ethyl group at C-8, demonstrating their activities as 8-vinyl reductases. We propose that nmrA and frhB be reassigned as bciA and bciB , respectively; transcript and proteomic analysis of Acaryochloris marina reveal that both bciA and bciB are expressed and their encoded proteins are present in the cell, possibly in order to ensure that all synthesized chlorophyll pigment carries an ethyl group at C-8. Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed

Chern-Simons like term generation in an extended model of QED under external conditions

The possibility of a Chern-Simons like term generation in an extended model of QED, in which a Lorentz and CPT non-covariant interaction term for fermions is present, has been investigated at finite temperature and in the presence of a background color magnetic field. To this end, the photon polarization operator in an external constant axial-vector field has been considered. One-loop contributions to its antisymmetric component due to fermions in the linear order of the axial-vector field have been obtained. Moreover, the first nontrivial correction to the induced CS term due to the presence of a weak constant homogeneous color magnetic field has been derived.Comment: RevTex, 10 pages with 3 figure

Equivariant cohomology and analytic descriptions of ring isomorphisms

In this paper we consider a class of connected closed $G$-manifolds with a non-empty finite fixed point set, each $M$ of which is totally non-homologous to zero in $M_G$ (or $G$-equivariantly formal), where $G={\Bbb Z}_2$. With the help of the equivariant index, we give an explicit description of the equivariant cohomology of such a $G$-manifold in terms of algebra, so that we can obtain analytic descriptions of ring isomorphisms among equivariant cohomology rings of such $G$-manifolds, and a necessary and sufficient condition that the equivariant cohomology rings of such two $G$-manifolds are isomorphic. This also leads us to analyze how many there are equivariant cohomology rings up to isomorphism for such $G$-manifolds in 2- and 3-dimensional cases.Comment: 20 pages, updated version with two references adde

Supermodels for early LHC

We investigate what new physics signatures the LHC can discover in the 2009-2010 run, beyond the expected sensitivity of the Tevatron data by 2010. We construct "supermodels", for which the LHC sensitivity even with only 10 inverse picobarn is greater than that of the Tevatron with 10 inverse femtobarn. The simplest supermodels involve s-channel resonances in the quark-antiquark and especially in the quark-quark channels. We concentrate on easily visible final states with small standard model backgrounds, and find that there are simple searches, besides those for Z' states, which could discover new physics in early LHC data. Many of these are well-suited to test searches for "more conventional" models, often discussed for multi-inverse femtobarn data sets.Comment: 11 pages, 6 figures; v2, references adde