Daniel P. Canniffe

A Cyanobacterial Chlorophyll Synthase-HliD Complex Associates with the Ycf39 Protein and the YidC/Alb3 Insertase

Chlorophyll synthase attaches geranylgeraniol to the chlorophyll macrocycle and was used as bait to retrieve an enzymatically active complex comprising the synthase, the high-light-inducible protein HliD, Ycf39, and the YidC/Alb3 insertase. These data reveal a link between chlorophyll biosynthesis and Sec/YidC-dependent cotranslational insertion of nascent photosystem polypeptides into membranes. Macromolecular membrane assemblies of chlorophyll-protein complexes efficiently harvest and trap light energy for photosynthesis. To investigate the delivery of chlorophylls to the newly synthesized photosystem apoproteins, a terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), was tagged in the cyanobacterium Synechocystis PCC 6803 (Synechocystis) and used as bait in pull-down experiments. We retrieved an enzymatically active complex comprising ChlG and the high-light-inducible protein HliD, which associates with the Ycf39 protein, a putative assembly factor for photosystem II, and with the YidC/Alb3 insertase. 2D electrophoresis and immunoblotting also provided evidence for the presence of SecY and ribosome subunits. The isolated complex contained chlorophyll, chlorophyllide, and carotenoid pigments. Deletion of hliD elevated the level of the ChlG substrate, chlorophyllide, more than 6-fold; HliD is apparently required for assembly of FLAG-ChlG into larger complexes with other proteins such as Ycf39. These data reveal a link between chlorophyll biosynthesis and the Sec/YidC-dependent cotranslational insertion of nascent photosystem polypeptides into membranes. We expect that this close physical linkage coordinates the arrival of pigments and nascent apoproteins to produce photosynthetic pigment-protein complexes with minimal risk of accumulating phototoxic unbound chlorophylls.

Rapid resonance Raman microspectroscopy to probe carbon dioxide fixation by single cells in microbial communities

Photosynthetic microorganisms play crucial roles in aquatic ecosystems and are the major primary producers in global marine ecosystems. The discovery of new bacteria and microalgae that play key roles in CO2 fixation is hampered by the lack of methods to identify hitherto-unculturable microorganisms. To overcome this problem we studied single microbial cells using stable-isotope probing (SIP) together with resonance Raman (RR) microspectroscopy of carotenoids, the light-absorbing pigments present in most photosynthetic microorganisms. We show that fixation of 13CO2 into carotenoids produces a red shift in single-cell RR (SCRR) spectra and that this SCRR–SIP technique is sufficiently sensitive to detect as little as 10% of 13C incorporation. Mass spectrometry (MS) analysis of labelled cellular proteins verifies that the red shift in carotenoid SCRR spectra acts as a reporter of the 13C content of single cells. Millisecond Raman imaging of cells in mixed cultures and natural seawater samples was used to identify cells actively fixing CO2, demonstrating that the SCRR–SIP is a noninvasive method for the rapid and quantitative detection of CO2 fixation at the single cell level in a microbial community. The SCRR–SIP technique may provide a direct method for screening environmental samples, and could help to reveal the ecophysiology of hitherto-unculturable microorganisms, linking microbial species to their ecological function in the natural environment.

Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium-protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803

Background: The cyclase step in chlorophyll biosynthesis remains uncharacterized. Results: Ycf54 forms a complex with other oxidative cyclase components, and a ycf54 mutant accumulates the cyclase substrate. Conclusion: Ycf54 is essential for cyclase function. Significance: Identification of all of the components of chlorophyll biosynthesis is a step closer. The cyclase step in chlorophyll (Chl) biosynthesis has not been characterized biochemically, although there are some plausible candidates for cyclase subunits. Two of these, Sll1214 and Sll1874 from the cyanobacterium Synechocystis 6803, were FLAG-tagged in vivo and used as bait in separate pulldown experiments. Mass spectrometry identified Ycf54 as an interaction partner in each case, and this interaction was confirmed by a reciprocal pulldown using FLAG-tagged Ycf54 as bait. Inactivation of the ycf54 gene (slr1780) in Synechocystis 6803 resulted in a strain that exhibited significantly reduced Chl levels. A detailed analysis of Chl precursors in the ycf54 mutant revealed accumulation of very high levels of Mg-protoporphyrin IX methyl ester and only traces of protochlorophyllide, the product of the cyclase, were detected. Western blotting demonstrated that levels of the cyclase component Sll1214 and the Chl biosynthesis enzymes Mg-protoporphyrin IX methyltransferase and protochlorophyllide reductase are significantly impaired in the ycf54 mutant. Ycf54 is, therefore, essential for the activity and stability of the oxidative cyclase. We discuss a possible role of Ycf54 as an auxiliary factor essential for the assembly of a cyclase complex or even a large multienzyme catalytic center.

Identification of an 8-vinyl reductase involved in bacteriochlorophyll biosynthesis in Rhodobacter sphaeroides and evidence for the existence of a third distinct class of the enzyme

The purple phototrophic bacterium Rhodobacter sphaeroides utilises bacteriochlorophyll a for light harvesting and photochemistry. The synthesis of this photopigment includes the reduction of a vinyl group at the C8 position to an ethyl group, catalysed by a C8-vinyl reductase. An active form of this enzyme has not been identified in R. sphaeroides, but its genome contains two candidate ORFs (open reading frames) similar to those reported to encode C8-vinyl reductases in the closely related Rhodobacter capsulatus (bchJ), and in plants and green sulfur bacteria (rsp_3070). To determine which gene encodes the active enzyme, knock-out mutants in both genes were constructed. Surprisingly, mutants in which one or both genes were deleted still retained the ability to synthesize C8-ethyl bacteriochlorophyll. These genes were subsequently expressed in a cyanobacterial mutant that cannot synthesize C8-ethyl chlorophyll a. R. sphaeroides rsp_3070 was able to restore synthesis of the WT (wild-type) C8-ethyl chlorophyll a in the mutant, whereas bchJ did not. The results of the present study demonstrate that Rsp_3070 is a functional C8-vinyl reductase and that R. sphaeroides utilises at least two enzymes to catalyse this reaction, indicating the existence of a third class, while there remains no direct evidence for the activity of BchJ as a C8-vinyl reductase.

Elucidation of the preferred routes of C8-vinyl reduction in chlorophyll and bacteriochlorophyll biosynthesis

Most of the chlorophylls and bacteriochlorophylls utilized for light harvesting by phototrophic organisms carry an ethyl group at the C8 position of the molecule, the product of a C8-vinyl reductase acting on a chlorophyll/bacteriochlorophyll biosynthetic precursor. Two unrelated classes of C8-vinyl reductase are known to exist, BciA and BciB, found in the purple phototroph Rhodobacter sphaeroides and the cyanobacterium Synechocystis sp. PCC6803 respectively. We constructed strains of each bacterium with the native C8-vinyl reductase swapped for the other class of the enzyme, and combined these replacements with a series of deletions of the native bch and chl genes. In vivo data indicate that the preferred substrates for both classes of the enzyme is C8-vinyl chlorophyllide, with C8-vinyl protochlorophyllide reduced only under conditions in which this pigment accumulates as a result of perturbed formation of chlorophyllide.

Absence of the cbb3 Terminal Oxidase Reveals an Active Oxygen-Dependent Cyclase Involved in Bacteriochlorophyll Biosynthesis in Rhodobacter sphaeroides

ABSTRACT The characteristic green color associated with chlorophyll pigments results from the formation of an isocyclic fifth ring on the tetrapyrrole macrocycle 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 micro-oxic 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 bacteriochlorophyll 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.

Three classes of oxygen-dependent cyclase involved in chlorophyll and bacteriochlorophyll biosynthesis

Significance (Bacterio)chlorophylls harvest and convert the solar energy that powers the biosphere. The absorption properties of these pigments are determined in part by formation of the isocyclic ring, which confers their characteristic green color. This last remaining uncharacterized biosynthetic step has remained enigmatic for more than 60 y, and only a single subunit of the O2-dependent cyclase, AcsF, has been identified. Here we demonstrate that Ycf54 is a bona fide subunit of this enzyme in oxygenic phototrophs; identify a new cyclase subunit, BciE, in Alphaproteobacteria; and confirm that the AcsF found in other classes of bacterial phototrophs is the principal form of the cyclase, requiring neither Ycf54 nor BciE for activity. The biosynthesis of (bacterio)chlorophyll pigments is among the most productive biological pathways on Earth. Photosynthesis relies on these modified tetrapyrroles for the capture of solar radiation and its conversion to chemical energy. (Bacterio)chlorophylls have an isocyclic fifth ring, the formation of which has remained enigmatic for more than 60 y. This reaction is catalyzed by two unrelated cyclase enzymes using different chemistries. The majority of anoxygenic phototrophic bacteria use BchE, an O2-sensitive [4Fe-4S] cluster protein, whereas plants, cyanobacteria, and some phototrophic bacteria possess an O2-dependent enzyme, the major catalytic component of which is a diiron protein, AcsF. Plant and cyanobacterial mutants in ycf54 display impaired function of the O2-dependent enzyme, accumulating the reaction substrate. Swapping cyclases between cyanobacteria and purple phototrophic bacteria reveals three classes of the O2-dependent enzyme. AcsF from the purple betaproteobacterium Rubrivivax (Rvi.) gelatinosus rescues the loss not only of its cyanobacterial ortholog, cycI, in Synechocystis sp. PCC 6803, but also of ycf54; conversely, coexpression of cyanobacterial cycI and ycf54 is required to complement the loss of acsF in Rvi. gelatinosus. These results indicate that Ycf54 is a cyclase subunit in oxygenic phototrophs, and that different classes of the enzyme exist based on their requirement for an additional subunit. AcsF is the cyclase in Rvi. gelatinosus, whereas alphaproteobacterial cyclases require a newly discovered protein that we term BciE, encoded by a gene conserved in these organisms. These data delineate three classes of O2-dependent cyclase in chlorophototrophic organisms from higher plants to bacteria, and their evolution is discussed herein.

Engineered biosynthesis of bacteriochlorophyll b in Rhodobacter sphaeroides

Bacteriochlorophyll b has the most red-shifted absorbance maximum of all naturally occurring photopigments. It has a characteristic ethylidene group at the C8 position in place of the more common ethyl group, the product of a C8-vinyl reductase, which is carried by the majority of chlorophylls and bacteriochlorophylls used in photosynthesis. The subsequent and first step exclusive to bacteriochlorophyll biosynthesis, the reduction of the C7 = C8 bond, is catalyzed by chlorophyllide oxidoreductase. It has been demonstrated that the enzyme from bacteriochlorophyll a-utilizing bacteria can catalyze the formation of compounds carrying an ethyl group at C8 from both ethyl- and vinyl-carrying substrates, indicating a surprising additional C8-vinyl reductase function, while the enzyme from organisms producing BChl b could only catalyze C7 = C8 reduction with a vinyl substrate, but this product carried an ethylidene group at the C8 position. We have replaced the native chlorophyllide oxidoreductase-encoding genes of Rhodobacter sphaeroides with those from Blastochloris viridis, but the switch from bacteriochlorophyll a to b biosynthesis is only detected when the native conventional C8-vinyl reductase is absent. We propose a non-enzymatic mechanism for ethylidene group formation based on the absence of cellular C8-vinyl reductase activity.

Biosynthesis of Chlorophyll a in a Purple Bacterial Phototroph and Assembly into a Plant Chlorophyll–Protein Complex

Improvements to photosynthetic efficiency could be achieved by manipulating pigment biosynthetic pathways of photosynthetic organisms in order to increase the spectral coverage for light absorption. The development of organisms that can produce both bacteriochlorophylls and chlorophylls is one way to achieve this aim, and accordingly we have engineered the bacteriochlorophyll-utilizing anoxygenic phototroph Rhodobacter sphaeroides to make chlorophyll a. Bacteriochlorophyll and chlorophyll share a common biosynthetic pathway up to the precursor chlorophyllide. Deletion of genes responsible for the bacteriochlorophyll-specific modifications of chlorophyllide and replacement of the native bacteriochlorophyll synthase with a cyanobacterial chlorophyll synthase resulted in the production of chlorophyll a. This pigment could be assembled in vivo into the plant water-soluble chlorophyll protein, heterologously produced in Rhodobacter sphaeroides, which represents a proof-of-principle for the engineering of novel antenna complexes that enhance the spectral range of photosynthesis.

Highly confined surface imaging by solid immersion total internal reflection fluorescence microscopy

We report the use of a high-refractive-index aplanatic solid immersion lens (ASIL) in total internal reflection fluorescence (TIRF) microscopy. This new solid immersion total internal reflection fluorescence (SITIRF) microscopy allows highly confined surface imaging with a significantly reduced imaging depth compared with conventional TIRF microscopy. We explore the application of a high refractive index, low optical dispersion material zirconium dioxide in the SITIRF microscope and also introduce a novel system design which enables the SITIRF microscope to work either in the epi-fluorescence or TIRF modes with variable illumination angles. We use both synthetic and biological samples to demonstrate that the imaging depth in the SITIRF microscope can be confined to a few tens of nanometers. SITIRF microscopy has the advantages of performing highly selective imaging and high-resolution high-contrast imaging. Potential applications in biological imaging and future developments of SITIRF microscopy are proposed.