Photosynthesis Research (v.126, #1)

Photosynthetic organisms tightly regulate the energy arriving to the reaction centers in order to avoid photodamage or imbalance between the photosystems. To this purpose, cyanobacteria have developed mechanisms involving relatively rapid (seconds to minutes) changes in the photosynthetic apparatus. In this review, two of these processes will be described: orange carotenoid protein(OCP)-related photoprotection and state transitions which optimize energy distribution between the two photosystems. The photoactive OCP is a light intensity sensor and an energy dissipater. Photoactivation depends on light intensity and only the red-active OCP form, by interacting with phycobilisome cores, increases thermal energy dissipation at the level of the antenna. A second protein, the “fluorescence recovery protein”, is needed to recover full antenna capacity under low light conditions. This protein accelerates OCP conversion to the inactive orange form and plays a role in dislodging the red OCP protein from the phycobilisome. The mechanism of state transitions is still controversial. Changes in the redox state of the plastoquinone pool induce movement of phycobilisomes and/or photosystems leading to redistribution of energy absorbed by phycobilisomes between PSII and PSI and/or to changes in excitation energy spillover between photosystems. The different steps going from the induction of redox changes to movement of phycobilisomes or photosystems remain to be elucidated.
Keywords: Cyanobacteria; Carotenoid; Photoprotection; State transitions; Fluorescence

13C flux analysis of cyanobacterial metabolism by Adeola O. Adebiyi; Lara J. Jazmin; Jamey D. Young (19-32).
13C metabolic flux analysis (MFA) has made important contributions to our understanding of the physiology of model strains of E. coli and yeast, and it has been widely used to guide metabolic engineering efforts in these microorganisms. Recent advancements in 13C MFA methodology combined with publicly available software tools are creating new opportunities to extend this approach to examine less characterized microbes. In particular, growing interest in the use of cyanobacteria as industrial hosts for photosynthetic production of biofuels and biochemicals has led to a critical need to better understand how cyanobacterial metabolic fluxes are regulated in response to changes in growth conditions or introduction of heterologous pathways. In this contribution, we review several prior studies that have applied isotopic steady-state 13C MFA to examine heterotrophic or mixotrophic growth of cyanobacteria, as well as recent studies that have pioneered the use of isotopically nonstationary MFA (INST-MFA) to study autotrophic cultures. We also provide recommendations for the design and analysis of INST-MFA experiments in cyanobacteria, based on our previous experience and a series of simulation studies used to assess the selection of measurements and sample time points. We anticipate that this emerging knowledgebase of prior 13C MFA studies, optimized experimental protocols, and public software tools will catalyze increasing use of 13C MFA techniques by the cyanobacteria research community.
Keywords: Metabolic flux analysis; Cyanobacteria; Isotope labeling experiment; Optimal experiment design; Isotopically nonstationary MFA

Live-cell imaging of cyanobacteria by Rayka Yokoo; Rachel D. Hood; David F. Savage (33-46).
Cyanobacteria are a diverse bacterial phylum whose members possess a high degree of ultrastructural organization and unique gene regulatory mechanisms. Unraveling this complexity will require the use of live-cell fluorescence microscopy, but is impeded by the inherent fluorescent background associated with light-harvesting pigments and the need to feed photosynthetic cells light. Here, we outline a roadmap for overcoming these challenges. Specifically, we show that although basic cyanobacterial biology creates challenging experimental constraints, these restrictions can be mitigated by the careful choice of fluorophores and microscope instrumentation. Many of these choices are motivated by recent successful live-cell studies. We therefore also highlight how live-cell imaging has advanced our understanding of bacterial microcompartments, circadian rhythm, and the organization and segregation of the bacterial nucleoid.
Keywords: Cyanobacteria; Synechococcus ; Live-cell microscopy; Circadian rhythm; Carboxysome

Proteomic approaches in research of cyanobacterial photosynthesis by Natalia Battchikova; Martina Angeleri; Eva-Mari Aro (47-70).
Oxygenic photosynthesis in cyanobacteria, algae, and plants is carried out by a fabulous pigment-protein machinery that is amazingly complicated in structure and function. Many different approaches have been undertaken to characterize the most important aspects of photosynthesis, and proteomics has become the essential component in this research. Here we describe various methods which have been used in proteomic research of cyanobacteria, and demonstrate how proteomics is implemented into on-going studies of photosynthesis in cyanobacterial cells.
Keywords: Cyanobacteria; Photosynthesis; Proteomics; Mass spectrometry; Protein identification; Protein quantitation; Post-translational modification

Shedding new light on viral photosynthesis by Richard J. Puxty; Andrew D. Millard; David J. Evans; David J. Scanlan (71-97).
Viruses infecting the environmentally important marine cyanobacteria Prochlorococcus and Synechococcus encode ‘auxiliary metabolic genes’ (AMGs) involved in the light and dark reactions of photosynthesis. Here, we discuss progress on the inventory of such AMGs in the ever-increasing number of viral genome sequences as well as in metagenomic datasets. We contextualise these gene acquisitions with reference to a hypothesised fitness gain to the phage. We also report new evidence with regard to the sequence and predicted structural properties of viral petE genes encoding the soluble electron carrier plastocyanin. Viral copies of PetE exhibit extensive modifications to the N-terminal signal peptide and possess several novel residues in a region responsible for interaction with redox partners. We also highlight potential knowledge gaps in this field and discuss future opportunities to discover novel phage–host interactions involved in the photosynthetic process.
Keywords: Cyanophage; Photosynthesis; Auxiliary metabolic genes; Plastocyanin

Cyanobacteria have evolved a carbon-concentrating mechanism (CCM) which has enabled them to inhabit diverse environments encompassing a range of inorganic carbon (Ci: $${ ext{HCO}}_{3}^{ - }$$ HCO 3 - and CO2) concentrations. Several uptake systems facilitate inorganic carbon accumulation in the cell, which can in turn be fixed by ribulose 1,5-bisphosphate carboxylase/oxygenase. Here we survey the distribution of genes encoding known Ci uptake systems in cyanobacterial genomes and, using a pfam- and gene context-based approach, identify in the marine (alpha) cyanobacteria a heretofore unrecognized number of putative counterparts to the well-known Ci transporters of beta cyanobacteria. In addition, our analysis shows that there is a huge repertoire of transport systems in cyanobacteria of unknown function, many with homology to characterized Ci transporters. These can be viewed as prospective targets for conversion into ancillary Ci transporters through bioengineering. Increasing intracellular Ci concentration coupled with efforts to increase carbon fixation will be beneficial for the downstream conversion of fixed carbon into value-added products including biofuels. In addition to CCM transporter homologs, we also survey the occurrence of rhodopsin homologs in cyanobacteria, including bacteriorhodopsin, a class of retinal-binding, light-activated proton pumps. Because they are light driven and because of the apparent ease of altering their ion selectivity, we use this as an example of re-purposing an endogenous transporter for the augmentation of Ci uptake by cyanobacteria and potentially chloroplasts.
Keywords: pfam; Rhodopsin; Inorganic carbon transport; Cyanobacteria; Carbon fixation; Carbon-concentrating mechanism; Genomic context; Synthetic biology; Bioinformatics

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.
Keywords: Cyanobacteria; Acidobacteria; Chloroflexi; Heliobacteria; Chlorobi; Photosystem

Challenges of metagenomics and single-cell genomics approaches for exploring cyanobacterial diversity by Michelle Davison; Eric Hall; Richard Zare; Devaki Bhaya (135-146).
Cyanobacteria have played a crucial role in the history of early earth and continue to be instrumental in shaping our planet, yet applications of cutting edge technology have not yet been widely used to explore cyanobacterial diversity. To provide adequate background, we briefly review current sequencing technologies and their innovative uses in genomics and metagenomics. Next, we focus on current cell capture technologies and the challenges of using them with cyanobacteria. We illustrate the utility in coupling breakthroughs in DNA amplification with cell capture platforms, with an example of microfluidic isolation and subsequent targeted amplicon sequencing from individual terrestrial thermophilic cyanobacteria. Single cells of thermophilic, unicellular Synechococcus sp. JA-2-3-B′a(2-13) (Syn OS-B′) were sorted in a microfluidic device, lysed, and subjected to whole genome amplification by multiple displacement amplification. We amplified regions from specific CRISPR spacer arrays, which are known to be highly diverse, contain semi-palindromic repeats which form secondary structure, and can be difficult to amplify. Cell capture, lysis, and genome amplification on a microfluidic device have been optimized, setting a stage for further investigations of individual cyanobacterial cells isolated directly from natural populations.
Keywords: Multiple displacement amplification (MDA); Whole genome amplification (WGA); Single cell; Microfluidics; Cyanobacteria; CRISPR

Initiation is a key control point for the regulation of translation in prokaryotes and prokaryotic-like translation systems such as those in plant chloroplasts. Genome sequencing and biochemical studies are increasingly demonstrating differences in many aspects of translation between well-studied microbes such as Escherichia coli and lesser studied groups such as cyanobacteria. Analyses of chloroplast translation have revealed its prokaryotic origin but also uncovered many unique aspects that do not exist in E. coli. Recently, a novel form of posttranscriptional regulation by light color was discovered in the filamentous cyanobacterium Fremyella diplosiphon that requires a putative stem-loop and involves the use of two different prokaryotic translation initiation factor 3s (IF3s). Multiple (up to five) putative IF3s have now been found to be encoded in 22 % of sequenced cyanobacterial genomes and 26 % of plant nuclear genomes. The lack of similar light-color regulation of gene expression in most of these species suggests that IF3s play roles in regulating gene expression in response to other environmental and developmental cues. In the plant Arabidopsis, two nuclear-encoded IF3s have been shown to localize to the chloroplasts, and the mRNA levels encoding these vary significantly in certain organ and tissue types and during several phases of development. Collectively, the accumulated data suggest that in about one quarter of photosynthetic prokaryotes and eukaryotes, IF3 gene families are used to regulate gene expression in addition to their traditional roles in translation initiation. Models for how this might be accomplished in prokaryotes versus eukaryotic plastids are presented.
Keywords: Light regulation; Transcription attenuation; RNA-binding protein; Stem-loop; 5′ Leader

The Photosystem II D1-K238E mutation enhances electrical current production using cyanobacterial thylakoid membranes in a bio-photoelectrochemical cell by Shirley Larom; Dan Kallmann; Gadiel Saper; Roy Pinhassi; Avner Rothschild; Hen Dotan; Guy Ankonina; Gadi Schuster; Noam Adir (161-169).
The conversion of solar energy (SEC) to storable chemical energy by photosynthesis has been performed by photosynthetic organisms, including oxygenic cyanobacteria for over 3 billion years. We have previously shown that crude thylakoid membranes from the cyanobacterium Synechocytis sp. PCC 6803 can reduce the electron transfer (ET) protein cytochrome c even in the presence of the PSII inhibitor DCMU. Mutation of lysine 238 of the Photosystem II D1 protein to glutamic acid increased the cytochrome reduction rates, indicating the possible position of this unknown ET pathway. In this contribution, we show that D1-K238E is rather unique, as other mutations to K238, or to other residues in the same vicinity, are not as successful in cytochrome c reduction. This observation indicates the sensitivity of ET reactions to minor changes. As the next step in obtaining useful SEC from biological material, we describe the use of crude Synechocystis membranes in a bio-photovoltaic cell containing an N-acetyl cysteine-modified gold electrode. We show the production of significant current for prolonged time durations, in the presence of DCMU. Surprisingly, the presence of cytochrome c was not found to be necessary for ET to the bio-voltaic cell.
Keywords: Photosynthesis; Cyanobacteria; Solar energy conversion; Cytochrome c; Electrochemistry

Genetic and genomic analysis of RNases in model cyanobacteria by Jeffrey C. Cameron; Gina C. Gordon; Brian F. Pfleger (171-183).
Cyanobacteria are diverse photosynthetic microbes with the ability to convert CO2 into useful products. However, metabolic engineering of cyanobacteria remains challenging because of the limited resources for modifying the expression of endogenous and exogenous biochemical pathways. Fine-tuned control of protein production will be critical to optimize the biological conversion of CO2 into desirable molecules. Messenger RNAs (mRNAs) are labile intermediates that play critical roles in determining the translation rate and steady-state protein concentrations in the cell. The majority of studies on mRNA turnover have focused on the model heterotrophic bacteria Escherichia coli and Bacillus subtilis. These studies have elucidated many RNA modifying and processing enzymes and have highlighted the differences between these Gram-negative and Gram-positive bacteria, respectively. In contrast, much less is known about mRNA turnover in cyanobacteria. We generated a compendium of the major ribonucleases (RNases) and provide an in-depth analysis of RNase III-like enzymes in commonly studied and diverse cyanobacteria. Furthermore, using targeted gene deletion, we genetically dissected the RNases in Synechococcus sp. PCC 7002, one of the fastest growing and industrially attractive cyanobacterial strains. We found that all three cyanobacterial homologs of RNase III and a member of the RNase II/R family are not essential under standard laboratory conditions, while homologs of RNase E/G, RNase J1/J2, PNPase, and a different member of the RNase II/R family appear to be essential for growth. This work will enhance our understanding of native control of gene expression and will facilitate the development of an RNA-based toolkit for metabolic engineering in cyanobacteria.
Keywords: mRNA; RNA; Ribonuclease; Photosynthesis; Cyanobacteria; Synthetic biology; Biofuels; Comparative genomics