Richard M. Alvey

Expression of genes in cyanobacteria: adaptation of endogenous plasmids as platforms for high-level gene expression in Synechococcus sp. PCC 7002.

Synechococcus sp. PCC 7002 is an ideal model cyanobacterium for functional genomics and biotechnological applications through metabolic engineering. A gene expression system that takes advantage of its multiple, endogenous plasmids has been constructed in this cyanobacterium. The method involves the integration of foreign DNA cassettes with selectable markers into neutral sites that can be located on any of the several endogenous plasmids of this organism. We have exploited the natural transformability and powerful homologous recombination capacity of this organism by using linear DNA fragments for transformation. This approach overcomes barriers that have made the introduction and expression of foreign genes problematic in the past. Foremost among these is the natural restriction endonuclease barrier that can cleave transforming circular plasmid DNAs before they can be replicated in the cell. We describe herein the general methodology for expressing foreign and homologous genes in Synechococcus sp. PCC 7002, a comparison of several commonly used promoters, and provide examples of how this approach has successfully been used in complementation analyses and overproduction of proteins with affinity tags.

Electronic coherence lineshapes reveal hidden excitonic correlations in photosynthetic light harvesting

The effective absorption cross-section of a molecule (acceptor) can be greatly increased by associating it with a cluster of molecules that absorb light and transfer the excitation energy to the acceptor molecule. The basic mechanism of such light harvesting by Förster resonance energy transfer (FRET) is well established, but recent experiments have revealed a new feature whereby excitation is coherently shared among donor and acceptor molecules during FRET. In the present study, two-dimensional electronic spectroscopy was used to examine energy transfer at ambient temperature in a naturally occurring light-harvesting protein (PE545 of the marine cryptophyte alga Rhodomonas sp. strain CS24). Quantum beating was observed across a range of excitation frequencies. The shapes of those features in the two-dimensional spectra were examined. Through simulations, we show that two-dimensional electronic spectroscopy provides a probe of the adiabaticity of the free energy landscape underlying light harvesting.

Biosynthesis of Cyanobacterial Phycobiliproteins in Escherichia coli: Chromophorylation Efficiency and Specificity of All Bilin Lyases from Synechococcus sp. Strain PCC 7002

ABSTRACT Phycobiliproteins are water-soluble, light-harvesting proteins that are highly fluorescent due to linear tetrapyrrole chromophores, which makes them valuable as probes. Enzymes called bilin lyases usually attach these bilin chromophores to specific cysteine residues within the alpha and beta subunits via thioether linkages. A multiplasmid coexpression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. strain PCC 7002 in Escherichia coli. This system efficiently produced chromophorylated allophycocyanin (ApcA/ApcB) and α-phycocyanin with holoprotein yields ranging from 3 to 12 mg liter−1 of culture. This heterologous expression system was used to demonstrate that the CpcS-I and CpcU proteins are both required to attach phycocyanobilin (PCB) to allophycocyanin subunits ApcD (αAP-B) and ApcF (β18). The N-terminal, allophycocyanin-like domain of ApcE (LCM99) was produced in soluble form and was shown to have intrinsic bilin lyase activity. Lastly, this in vivo system was used to evaluate the efficiency of the bilin lyases for production of β-phycocyanin.

Attachment of noncognate chromophores to CpcA of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 by heterologous expression in Escherichia coli.

Many cyanobacteria use brilliantly pigmented, multisubunit macromolecular structures known as phycobilisomes as antenna to enhance light harvesting for photosynthesis. Recent studies have defined the enzymes that synthesize phycobilin chromophores as well as many of the phycobilin lyase enzymes that attach these chromophores to their cognate apoproteins. The ability of the phycocyanin α-subunit (CpcA) to bind alternative linear tetrapyrrole chromophores was examined through the use of a heterologous expression system in Escherichia coli. E. coli strains produced phycocyanobilin, phytochromobilin, or phycoerythrobilin when they expressed 3Z-phycocyanobilin:ferredoxin oxidoreductase (PcyA), 3Z-phytochromobilin:ferredoxin oxidoreductase (HY2) from Arabidopsis thaliana, or phycoerythrobilin synthase (PebS) from the myovirus P-SSM4, respectively. CpcA from Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 was coexpressed in these strains with the phycocyanin α-subunit phycocyanobilin lyase, CpcE/CpcF, or the phycoerythrocyanin α-subunit phycocyanobilin isomerizing lyase, PecE/PecF, from Noctoc sp. PCC 7120. Both lyases were capable of attaching three different linear tetrapyrrole chromophores to CpcA; thus, up to six different CpcA variants, each with a unique chromophore, could be produced with this system. One of these chromophores, denoted phytoviolobilin, has not yet been observed naturally. The recombinant proteins had unexpected and potentially useful properties, which included very high fluorescence quantum yields and photochemical activity. Chimeric lyases PecE/CpcF and CpcE/PecF were used to show that the isomerizing activity that converts phycocyanobilin to phycoviolobilin resides with PecF and not PecE. Finally, spectroscopic properties of recombinant phycocyanin R-PCIII, in which the CpcA subunits carry a phycoerythrobilin chromophore, are described.

Inverse transcriptional activities during complementary chromatic adaptation are controlled by the response regulator RcaC binding to red and green light-responsive promoters

Complementary chromatic adaptation (CCA) provides cyanobacteria with the ability to shift between red and blue‐green phenotypes that are optimized for absorption of different wavelengths of light. Controlled by the ratio of green to red light, this process results from differential expression of two groups of operons, many of which encode proteins involved in photosynthetic light harvesting antennae biogenesis. In the freshwater species Fremyella diplosiphon, the inverse regulation of these two classes is complex and occurs through different mechanisms. It also involves a two‐component pathway that includes a phytochrome‐class photoreceptor and the response regulator RcaC. Here we uncover the mechanism through which this system controls CCA by demonstrating that RcaC binds to the L Box within promoters of both classes of light‐regulated operons. We provide functional evidence that complementary regulation of these operons occurs by RcaCs simultaneous activation and repression of transcription in red light. We identify rcaC and L Boxes in the genome of a marine cyanobacterium capable of CCA, suggesting widespread use of this control system. These results provide important insights into the long‐standing enigma of CCA regulation and complete the first description of an entire two‐component system controlled by a phytochrome‐class photoreceptor.

Phycobiliprotein Biosynthesis in Cyanobacteria: Structure and Function of Enzymes Involved in Post-translational Modification

Cyanobacterial phycobiliproteins are brilliantly colored due to the presence of covalently attached chromophores called bilins, linear tetrapyrroles derived from heme. For most phycobiliproteins, these post-translational modifications are catalyzed by enzymes called bilin lyases; these enzymes ensure that the appropriate bilins are attached to the correct cysteine residues with the proper stereochemistry on each phycobiliprotein subunit. Phycobiliproteins also contain a unique, post-translational modification, the methylation of a conserved asparagine (Asn) present at beta-72, which occurs on the beta-subunits of all phycobiliproteins. We have identified and characterized several new families of bilin lyases, which are responsible for attaching PCB to phycobiliproteins as well as the Asn methyl transferase for beta-subunits in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. All of the enzymes responsible for synthesis of holo-phycobiliproteins are now known for this cyanobacterium, and a brief discussion of each enzyme family and its role in the biosynthesis of phycobiliproteins is presented here. In addition, the first structure of a bilin lyase has recently been solved (PDB ID: 3BDR). This structure shows that the bilin lyases are most similar to the lipocalin protein structural family, which also includes the bilin-binding protein found in some butterflies.

Effects of Modified Phycobilin Biosynthesis in the Cyanobacterium Synechococcus sp. Strain PCC 7002

The pathway for phycocyanobilin biosynthesis in Synechococcus sp. strain PCC 7002 comprises two enzymes: heme oxygenase and phycocyanobilin synthase (PcyA). The phycobilin content of cells can be modified by overexpressing genes encoding alternative enzymes for biliverdin reduction. Overexpression of the pebAB and HY2 genes, encoding alternative ferredoxin-dependent biliverdin reductases, caused unique effects due to the overproduction of phycoerythrobilin and phytochromobilin, respectively. Colonies overexpressing pebAB became reddish brown and visually resembled strains that naturally produce phycoerythrin. This was almost exclusively due to the replacement of phycocyanobilin by phycoerythrobilin on the phycocyanin α-subunit. This phenotype was unstable, and such strains rapidly reverted to the wild-type appearance, presumably due to strong selective pressure to inactivate pebAB expression. Overproduction of phytochromobilin, synthesized by the Arabidopsis thaliana HY2 product, was tolerated much better. Cells overexpressing HY2 were only slightly less pigmented and blue-green than the wild type. Although the pcyA gene could not be inactivated in the wild type, pcyA was easily inactivated when cells expressed HY2. These results indicate that phytochromobilin can functionally substitute for phycocyanobilin in Synechococcus sp. strain PCC 7002. Although functional phycobilisomes were assembled in this strain, the overall phycobiliprotein content of cells was lower, the efficiency of energy transfer by these phycobilisomes was lower than for wild-type phycobilisomes, and the absorption cross-section of the cells was reduced relative to that of the wild type because of an increased spectral overlap of the modified phycobiliproteins with chlorophyll a. As a result, the strain producing phycobiliproteins carrying phytochromobilin grew much more slowly at low light intensity.

Genomic analysis reveals key aspects of prokaryotic symbiosis in the phototrophic consortium “Chlorochromatium aggregatum”

Background‘Chlorochromatium aggregatum’ is a phototrophic consortium, a symbiosis that may represent the highest degree of mutual interdependence between two unrelated bacteria not associated with a eukaryotic host. ‘Chlorochromatium aggregatum’ is a motile, barrel-shaped aggregate formed from a single cell of ‘Candidatus Symbiobacter mobilis”, a polarly flagellated, non-pigmented, heterotrophic bacterium, which is surrounded by approximately 15 epibiont cells of Chlorobium chlorochromatii, a non-motile photolithoautotrophic green sulfur bacterium.ResultsWe analyzed the complete genome sequences of both organisms to understand the basis for this symbiosis. Chl. chlorochromatii has acquired relatively few symbiosis-specific genes; most acquired genes are predicted to modify the cell wall or function in cell-cell adhesion. In striking contrast, ‘Ca. S. mobilis’ appears to have undergone massive gene loss, is probably no longer capable of independent growth, and thus may only reproduce when consortia divide. A detailed model for the energetic and metabolic bases of the dependency of ‘Ca. S. mobilis’ on Chl. chlorochromatii is described.ConclusionsGenomic analyses suggest that three types of interactions lead to a highly sophisticated relationship between these two organisms. Firstly, extensive metabolic exchange, involving carbon, nitrogen, and sulfur sources as well as vitamins, occurs from the epibiont to the central bacterium. Secondly, ‘Ca. S. mobilis’ can sense and move towards light and sulfide, resources that only directly benefit the epibiont. Thirdly, electron cycling mechanisms, particularly those mediated by quinones and potentially involving shared protonmotive force, could provide an important basis for energy exchange in this and other symbiotic relationships.

Characterization of the Activities of the CpeY, CpeZ, and CpeS Bilin Lyases in Phycoerythrin Biosynthesis in Fremyella diplosiphon Strain UTEX 481

When grown in green light, Fremyella diplosiphon strain UTEX 481 produces the red-colored protein phycoerythrin (PE) to maximize photosynthetic light harvesting. PE is composed of two subunits, CpeA and CpeB, which carry two and three phycoerythrobilin (PEB) chromophores, respectively, that are attached to specific Cys residues via thioether linkages. Specific bilin lyases are hypothesized to catalyze each PEB ligation. Using a heterologous, coexpression system in Escherichia coli, the PEB ligation activities of putative lyase subunits CpeY, CpeZ, and CpeS were tested on the CpeA and CpeB subunits from F. diplosiphon. Purified His6-tagged CpeA, obtained by coexpressing cpeA, cpeYZ, and the genes for PEB synthesis, had absorbance and fluorescence emission maxima at 566 and 574 nm, respectively. CpeY alone, but not CpeZ, could ligate PEB to CpeA, but the yield of CpeA-PEB was lower than achieved with CpeY and CpeZ together. Studies with site-specific variants of CpeA(C82S and C139S), together with mass spectrometric analysis of trypsin-digested CpeA-PEB, revealed that CpeY/CpeZ attached PEB at Cys82 of CpeA. The CpeS bilin lyase ligated PEB at both Cys82 and Cys139 of CpeA but very inefficiently; the yield of PEB ligated at Cys82 was much lower than observed with CpeY or CpeY/CpeZ. However, CpeS efficiently attached PEB to Cys80 of CpeB but neither CpeY, CpeZ, nor CpeY/CpeZ could ligate PEB to CpeB.

Structural and Biochemical Characterization of the Bilin Lyase CpcS from Thermosynechococcus Elongatus

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded β barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced.