Excitation energy transfer to Photosystem I in filaments and heterocysts of Nostoc punctiforme

AbstractCyanobacteria adapt to varying light conditions by controlling the amount of excitation energy to the photosystems. On the minute time scale this leads to redirection of the excitation energy, usually referred to as state transitions, which involves movement of the phycobilisomes. We have studied short-term light adaptation in isolated heterocysts and intact filaments from the cyanobacterium Nostoc punctiforme ATCC 29133. In N.punctiforme vegetative cells differentiate into heterocysts where nitrogen fixation takes place. Photosystem II is inactivated in the heterocysts, and the abundancy of Photosystem I is increased relative to the vegetative cells. To study light-induced changes in energy transfer to Photosystem I, pre-illumination was made to dark adapted isolated heterocysts. Illumination wavelengths were chosen to excite Photosystem I (708nm) or phycobilisomes (560nm) specifically. In heterocysts that were pre-illuminated at 708nm, fluorescence from the phycobilisome terminal emitter was observed in the 77K emission spectrum. However, illumination with 560nm light caused quenching of the emission from the terminal emitter, with a simultaneous increase in the emission at 750nm, indicating that the 560nm pre-illumination caused trimerization of Photosystem I. Excitation spectra showed that 560nm pre-illumination led to an increase in excitation transfer from the phycobilisomes to trimeric Photosystem I. Illumination at 708nm did not lead to increased energy transfer from the phycobilisome to Photosystem I compared to dark adapted samples. The measurements were repeated using intact filaments containing vegetative cells, and found to give very similar results as the heterocysts. This demonstrates that molecular events leading to increased excitation energy transfer to Photosystem I, including trimerization, are independent of Photosystem II activity

Global distribution of a chlorophyll f cyanobacterial marker

Some cyanobacteria use light outside the visible spectrum for oxygenic photosynthesis. The far-red light (FRL) region is made accessible through a complex acclimation process that involves the formation of new phycobilisomes and photosystems containing chlorophyll f. Diverse cyanobacteria ranging from unicellular to branched-filamentous forms show this response. These organisms have been isolated from shaded environments such as microbial mats, soil, rock, and stromatolites. However, the full spread of chlorophyll f-containing species in nature is still unknown. Currently, discovering new chlorophyll f cyanobacteria involves lengthy incubation times under selective far-red light. We have used a marker gene to detect chlorophyll f organisms in environmental samples and metagenomic data. This marker, apcE2, encodes a phycobilisome linker associated with FRL-photosynthesis. By focusing on a far-red motif within the sequence, degenerate PCR and BLAST searches can effectively discriminate against the normal chlorophyll a-associated apcE. Even short recovered sequences carry enough information for phylogenetic placement. Markers of chlorophyll f photosynthesis were found in metagenomic datasets from diverse environments around the globe, including cyanobacterial symbionts, hypersaline lakes, corals, and the Arctic/Antarctic regions. This additional information enabled higher phylogenetic resolution supporting the hypothesis that vertical descent, as opposed to horizontal gene transfer, is largely responsible for this phenotype’s distribution

Origin and evolution of water oxidation before the last common ancestor of the Cyanobacteria

Photosystem II, the water oxidizing enzyme, altered the course of evolution by filling the atmosphere with oxygen. Here, we reconstruct the origin and evolution of water oxidation at an unprecedented level of detail by studying the phylogeny of all D1 subunits, the main protein coordinating the water oxidizing cluster (Mn4CaO5) of Photosystem II. We show that D1 exists in several forms making well-defined clades, some of which could have evolved before the origin of water oxidation and presenting many atypical characteristics. The most ancient form is found in the genome of Gloeobacter kilaueensis JS-1 and this has a C-terminus with a higher sequence identity to D2 than to any other D1. Two other groups of early evolving D1 correspond to those expressed under prolonged far-red illumination and in darkness. These atypical D1 forms are characterized by a dramatically different Mn4CaO5 binding site and a Photosystem II containing such a site may assemble an unconventional metal cluster. The first D1 forms with a full set of ligands to the Mn4CaO5 cluster are grouped with D1 proteins expressed only under low oxygen concentrations and the latest evolving form is the dominant type of D1 found in all cyanobacteria and plastids. In addition, we show that the plastid ancestor had a D1 more similar to those in early branching Synechococcus. We suggest each one of these forms of D1 originated from transitional forms at different stages towards the innovation and optimization of water oxidation before the last common ancestor of all known cyanobacteria

A fresh look at the evolution and diversification of photochemical reaction centers

In this review, I reexamine the origin and diversification of photochemical reaction centers based on the known phylogenetic relations of the core subunits, and with the aid of sequence and structural alignments. I show, for example, that the protein folds at the C-terminus of the D1 and D2 subunits of Photosystem II, which are essential for the coordination of the water-oxidizing complex, were already in place in the most ancestral Type II reaction center subunit. I then evaluate the evolution of reaction centers in the context of the rise and expansion of the different groups of bacteria based on recent large-scale phylogenetic analyses. I find that the Heliobacteriaceae family of Firmicutes appears to be the earliest branching of the known groups of phototrophic bacteria; however, the origin of photochemical reaction centers and chlorophyll synthesis cannot be placed in this group. Moreover, it becomes evident that the Acidobacteria and the Proteobacteria shared a more recent common phototrophic ancestor, and this is also likely for the Chloroflexi and the Cyanobacteria. Finally, I argue that the discrepancies among the phylogenies of the reaction center proteins, chlorophyll synthesis enzymes, and the species tree of bacteria are best explained if both types of photochemical reaction centers evolved before the diversification of the known phyla of phototrophic bacteria. The primordial phototrophic ancestor must have had both Type I and Type II reaction centers

The Heterocysts of Nostoc punctiforme : From Proteomics to Energy Transfer

The aim of this thesis is to provide a thorough characterization of the photosynthetic machinery from the heterocysts of Nostoc punctiforme strain ATCC 29133. In this thesis I describe the protocols I have optimized for the isolation of thylakoids from vegetative cells, the purification of heterocysts and the isolation of thylakoids from the purified heterocysts. The composition of the thylakoid membranes was studied by two dimensional electrophoresis and mass-spectrometry. Further insight into the functionality of the photosynthetic complexes was obtained by EPR, electron transport measurements through Photosystem II (PSII), and fluorescence spectroscopy. The proteome of the heterocysts thylakoids compared to that of the vegetative cell was found to be dominated by Photosystem I (PSI) and ATP-synthase complexes, both essential for keeping high nitrogenase activities. Surprisingly, we found a significant amount of assembled monomeric PSII complexes in the heterocysts thylakoid membranes. We measured in vitro light-driven electron transfer from PSII in heterocysts using an artificial electron donor, suggesting that under certain circumstances heterocysts might activate PSII. Parallel to my main research I also worked in a collaboration to elucidate the total proteome of Nostoc sp. strain 7120 and Nostoc punctiforme using quantitative shotgun proteomics. Several hundred proteins were quantified for both species. It was possible to trace the detailed changes that occurred in the energy and nitrogen metabolism of a heterocyst after differentiation. Moreover, the presence of PSII proteins identified in our membrane proteome was also confirmed and extended. Lastly, I studied how the heterocysts are capable of responding to variations in light quality as compared to vegetative cells. Using 77 K fluorescence spectroscopy on heterocysts and vegetative cells previously illuminated with light at specific wavelengths, I was able to demonstrate that heterocysts still possess a possibly modified but functional antenna system, capable of harvesting light and transferring energy preferentially to PSI. The characterization of the membrane and total proteome permitted to draw a more comprehensive and integrated picture of the interplay between the distinct metabolic processes that are carried out in each cell type at the same time; from oxygenic photosynthesis and carbon fixation in the vegetative cells to the anoxygenic cyclic photophosphorylation essential to power nitrogen assimilation in the heterocysts

Evolution of bacteria in relation to BchF and reaction center proteins.

<p><b>A</b> shows a phylogenetic tree of BchF overlaid over a phylogenomic tree of prokaryotes. B and C show those of Type I and Type II reaction center proteins over the phylogenomic tree, respectively. The phylogenomic tree that depicts the evolutionary relationship of different phyla of bacteria was obtained from Segata et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151250#pone.0151250.ref017" target="_blank">17</a>], which was published open access. It was constructed using hundreds of proteins and thousands of genomes and the phylogenetic method, sequence alignments, and the phylogenetic tree are freely available from the author’s website (<a href="http://huttenhower.sph.harvard.edu/phylophlan" target="_blank">http://huttenhower.sph.harvard.edu/phylophlan</a>). It is overall very similar to other phylogenomic trees, and the relationship of phyla containing phototrophic strains is virtually identical to those presented before, see for example Jun et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151250#pone.0151250.ref020" target="_blank">20</a>], who implemented an alignment free approach. The phyla of bacteria highlighted in color represent those with photochemical reaction centers, with the exception of Actinobacteria that only recently was suggested to have been ancestrally phototrophic [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151250#pone.0151250.ref002" target="_blank">2</a>]. The colored transparent lines that are overlaid on top of the phylogenomic tree represent the evolution of the selected proteins. The phylogenetic relations of Type I (<b>B</b>) and Type II (<b>C</b>) reaction centers are well-established and were recently reviewed in detail [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151250#pone.0151250.ref001" target="_blank">1</a>].</p

On the origin of oxygenic photosynthesis and Cyanobacteria

Oxygenic phototrophs have played a fundamental role in Earth's history by enabling the rise of atmospheric oxygen (O2) and paving the way for animal evolution. Understanding the origin of oxygenic photosynthesis and Cyanobacteria are key when piecing together the events around Earth's oxygenation. It is likely that photosynthesis evolved within bacterial lineages that are not extant, so it can be challenging when studying the early history of photosynthesis. Recent genomic and molecular evolution studies have transformed our understanding about the evolution of photosynthetic reaction centres and the evolution of Cyanobacteria. The evidence reviewed here highlights some of the most recent advances on the origin of photosynthesis both at the genomic and gene family level