Ferredoxin5 Deletion Affects Metabolism of Algae during the Different Phases of Sulfur Deprivation.

Ferredoxin5 (FDX5), a minor ferredoxin protein in the alga Chlamydomonas (Chlamydomonas reinhardtii), helps maintain thylakoid membrane integrity in the dark. Sulfur (S) deprivation has been used to achieve prolonged hydrogen production in green algae. Here, we propose that FDX5 is involved in algal responses to S-deprivation as well as to the dark. Specifically, we tested the role of FDX5 in both the initial aerobic and subsequent anaerobic phases of S-deprivation. Under S-deprived conditions, absence of FDX5 causes a distinct delay in achieving anoxia by affecting photosynthetic O2 evolution, accompanied by reduced acetate uptake, lower starch accumulation, and delayed/lower fermentative metabolite production, including photohydrogen. We attribute these differences to transcriptional and/or posttranslational regulation of acetyl-CoA synthetase and ADP-Glc pyrophosphorylase, and increased stability of the PSII D1 protein. Interestingly, increased levels of FDX2 and FDX1 were observed in the mutant under oxic, S-replete conditions, strengthening our previously proposed hypothesis that other ferredoxins compensate in response to a lack of FDX5. Taken together, the results of our omics and pull-down experiments confirmed biochemical and physiological results, suggesting that FDX5 may have other effects on Chlamydomonas metabolism through its interaction with multiple redox partners.

Glycogen Synthesis and Metabolite Overflow Contribute to Energy Balancing in Cyanobacteria.

Understanding how living cells manage high-energy metabolites such as ATP and NADPH is essential for understanding energy transformations in the biosphere. Using light as the energy input, we find that energy charge (ratio of ATP over ADP+ATP) in the cyanobacterium Synechocystis sp. PCC 6803 varies in different growth stages, with a peak upon entry into the rapid growth phase, as well as a positive correlation with light intensity. In contrast, a mutant that can no longer synthesize the main carbon storage compound glycogen showed higher energy charge. The overflow of organic acids in this mutant under nitrogen depletion could also be triggered under high light in nitrogen-replete conditions, with an energy input level dependency. These findings suggest that energy charge in cyanobacteria is tightly linked to growth and carbon partition and that energy management is of key significance for their application as photosynthetic carbon dioxide-assimilating cell factories.

Development of a Rhodobacter capsulatus self-reporting model system for optimizing light-dependent, [FeFe]-hydrogenase-driven H2 production.

The photosynthetic bacterium Rhodobacter capsulatus normally photoproduces H2 as a by-product of its nitrogenase-catalyzed nitrogen-fixing activity. Such H2 production, however, is expensive from a metabolic perspective, requiring nearly four times as many photons as the equivalent algal hydrogenase-based system (Ghirardi et al., 2009 Photobiological hydrogen-producing systems. Chem Soc Rev 38(1):52-61). Here, we report the insertion of a Clostridium acetobutylicum [FeFe]-hydrogenase and its three attendant hydrogenase assembly proteins into an R. capsulatus strain lacking its native uptake hydrogenase. Further, this strain is modified to fluoresce upon sensing H2 . The resulting strain photoproduces H2 and self-reports its own H2 production through fluorescence. This model system represents a unique method of developing hydrogenase-based H2 production in R. capsulatus, may serve as a powerful system for in vivo directed evolution of hydrogenases and hydrogenase-associated genes, and provides a means of screening for increased metabolic production of H2 . Biotechnol. Bioeng. 2017;114: 291-297. © 2016 Wiley Periodicals, Inc.

Crystal structure and biochemical characterization of Chlamydomonas FDX2 reveal two residues that, when mutated, partially confer FDX2 the redox potential and catalytic properties of FDX1.

The green alga Chlamydomonas reinhardtii contains six plastidic [2Fe2S]-cluster ferredoxins (FDXs), with FDX1 as the predominant isoform under photoautotrophic growth. FDX2 is highly similar to FDX1 and has been shown to interact with specific enzymes (such as nitrite reductase), as well as to share interactors with FDX1, such as the hydrogenases (HYDA), ferredoxin:NAD(P) reductase I (FNR1), and pyruvate:ferredoxin oxidoreductase (PFR1), albeit performing at low catalytic rates. Here we report the FDX2 crystal structure solved at 1.18 Å resolution. Based on differences between the Chlorella fusca FDX1 and C. reinhardtii FDX2 structures, we generated and purified point-mutated versions of the FDX2 protein and assayed them in vitro for their ability to catalyze hydrogen and NADPH photo-production. The data show that structural differences at two amino acid positions contribute to functional differences between FDX1 and FDX2, suggesting that FDX2 might have evolved from FDX1 toward a different physiological role in the cell. Moreover, we demonstrate that the mutations affect both the midpoint potentials of the FDX and kinetics of the FNR reaction, possibly due to altered binding between FDX and FNR. An effect on H2 photo-production rates was also observed, although the kinetics of the reaction were not further characterized.

Implementation of photobiological H2 production: the O 2 sensitivity of hydrogenases.

The search for the ultimate carbon-free fuel has intensified in recent years, with a major focus on photoproduction of H2. Biological sources of H2 include oxygenic photosynthetic green algae and cyanobacteria, both of which contain hydrogenase enzymes. Although algal and cyanobacterial hydrogenases perform the same enzymatic reaction through metallo-clusters, their hydrogenases have evolved separately, are expressed differently (transcription of algal hydrogenases is anaerobically induced, while bacterial hydrogenases are constitutively expressed), and display different sensitivity to O2 inactivation. Among various physiological factors, the sensitivity of hydrogenases to O2 has been one of the major factors preventing implementation of biological systems for commercial production of renewable H2. This review addresses recent strategies aimed at engineering increased O2 tolerance into hydrogenases (as of now mainly unsuccessful), as well as towards the development of methods to bypass the O2 sensitivity of hydrogenases (successful but still yielding low solar conversion efficiencies). The author concludes with a description of current approaches from various laboratories to incorporate multiple genetic traits into either algae or cyanobacteria to jointly address limiting factors other than the hydrogenase O2 sensitivity and achieve more sustained H2 photoproduction activity.

Enhancing photo-catalytic production of organic acids in the cyanobacterium Synechocystis sp.†PCC 6803 ΔglgC, a strain incapable of glycogen storage.

A key objective in microbial biofuels strain development is to maximize carbon flux to target products while minimizing cell biomass accumulation, such that ideally the algae and bacteria would operate in a photo-catalytic state. A brief period of such a physiological state has recently been demonstrated in the cyanobacterium Synechocystis sp. PCC 6803 ΔglgC strain incapable of glycogen storage. When deprived of nitrogen, the ΔglgC excretes the organic acids alpha-ketoglutarate and pyruvate for a number of days without increasing cell biomass. This study examines the relationship between the growth state and the photo-catalytic state, and characterizes the metabolic adaptability of the photo-catalytic state to increasing light intensity. It is found that the culture can transition naturally from the growth state into the photo-catalytic state when provided with limited nitrogen supply during the growth phase. Photosynthetic capacity and pigments are lost over time in the photo-catalytic state. Reversal to growth state is observed with re-addition of nitrogen nutrient, accompanied by restoration of photosynthetic capacity and pigment levels in the cells. While the overall productivity increased under high light conditions, the ratio of alpha-ketoglutarate/pyruvate is altered, suggesting that carbon partition between the two products is adaptable to environmental conditions.

Phosphoketolase pathway contributes to carbon metabolism in cyanobacteria.

Central carbon metabolism in cyanobacteria comprises the Calvin-Benson-Bassham (CBB) cycle, glycolysis, the pentose phosphate (PP) pathway and the tricarboxylic acid (TCA) cycle. Redundancy in this complex metabolic network renders the rational engineering of cyanobacterial metabolism for the generation of biomass, biofuels and chemicals a challenge. Here we report the presence of a functional phosphoketolase pathway, which splits xylulose-5-phosphate (or fructose-6-phosphate) to acetate precursor acetyl phosphate, in an engineered strain of the model cyanobacterium Synechocystis (ΔglgC/xylAB), in which glycogen synthesis is blocked, and xylose catabolism enabled through the introduction of xylose isomerase and xylulokinase. We show that this mutant strain is able to metabolise xylose to acetate on nitrogen starvation. To see whether acetate production in the mutant is linked to the activity of phosphoketolase, we disrupted a putative phosphoketolase gene (slr0453) in the ΔglgC/xylAB strain, and monitored metabolic flux using (13)C labelling; acetate and 2-oxoglutarate production was reduced in the light. A metabolic flux analysis, based on isotopic data, suggests that the phosphoketolase pathway metabolises over 30% of the carbon consumed by ΔglgC/xylAB during photomixotrophic growth on xylose and CO2. Disruption of the putative phosphoketolase gene in wild-type Synechocystis also led to a deficiency in acetate production in the dark, indicative of a contribution of the phosphoketolase pathway to heterotrophic metabolism. We suggest that the phosphoketolase pathway, previously uncharacterized in photosynthetic organisms, confers flexibility in energy and carbon metabolism in cyanobacteria, and could be exploited to increase the efficiency of cyanobacterial carbon metabolism and photosynthetic productivity.

Engineering photosynthetic organisms for the production of biohydrogen.

Oxygenic photosynthetic organisms such as green algae are capable of absorbing sunlight and converting the chemical energy into hydrogen gas. This process takes advantage of the photosynthetic apparatus of these organisms which links water oxidation to H2 production. Biological H2 has therefore the potential to be an alternative fuel of the future and shows great promise for generating large scale sustainable energy. Microalgae are able to produce H2 under light anoxic or dark anoxic condition by activating 3 different pathways that utilize the hydrogenases as catalysts. In this review, we highlight the principal barriers that prevent hydrogen production in green algae and how those limitations are being addressed, through metabolic and genetic engineering.  We also discuss the major challenges and bottlenecks facing the development of future commercial algal photobiological systems for H2 production. Finally we provide suggestions for future strategies and potential new techniques to be developed towards an integrated system with optimized hydrogen production.

High-throughput biosensor discriminates between different algal H2 -photoproducing strains.

A number of species of microalgae and cyanobacteria photosynthetically produce H2 gas by coupling water oxidation with the reduction of protons to molecular hydrogen, generating renewable energy from sunlight and water. Photosynthetic H2 production, however, is transitory, and there is considerable interest in increasing and extending it for commercial applications. Here we report a Petri-plate version of our previous, microplate-based assay that detects photosynthetic H2 production by algae. The assay consists of an agar overlay of H2 -sensing Rhodobacter capsulatus bacteria carrying a green fluorescent protein that responds to H2 produced by single algal colonies in the bottom agar layer. The assay distinguishes between algal strains that photoproduce H2 at different levels under high light intensities, and it does so in a simple, inexpensive, and high-throughput manner. The assay will be useful for screening both natural populations and mutant libraries for strains having increased H2 production, and useful for identifying various genetic factors that physiologically or genetically alter algal hydrogen production.

Identification of global ferredoxin interaction networks in Chlamydomonas reinhardtii.

Ferredoxins (FDXs) can distribute electrons originating from photosynthetic water oxidation, fermentation, and other reductant-generating pathways to specific redox enzymes in different organisms. The six FDXs identified in Chlamydomonas reinhardtii are not fully characterized in terms of their biological function. In this report, we present data from the following: (a) yeast two-hybrid screens, identifying interaction partners for each Chlamydomonas FDX; (b) pairwise yeast two-hybrid assays measuring FDX interactions with proteins from selected biochemical pathways; (c) affinity pulldown assays that, in some cases, confirm and even expand the interaction network for FDX1 and FDX2; and (d) in vitro NADP(+) reduction and H2 photo-production assays mediated by each FDX that verify their role in these two pathways. Our results demonstrate new potential roles for FDX1 in redox metabolism and carbohydrate and fatty acid biosynthesis, for FDX2 in anaerobic metabolism, and possibly in state transition. Our data also suggest that FDX3 is involved in nitrogen assimilation, FDX4 in glycolysis and response to reactive oxygen species, and FDX5 in hydrogenase maturation. Finally, we provide experimental evidence that FDX1 serves as the primary electron donor to two important biological pathways, NADPH and H2 photo-production, whereas FDX2 is capable of driving these reactions at less than half the rate observed for FDX1.

De novo transcriptomic analysis of hydrogen production in the green alga Chlamydomonas moewusii through RNA-Seq.

BACKGROUND: Microalgae can make a significant contribution towards meeting global renewable energy needs in both carbon-based and hydrogen (H2) biofuel. The development of energy-related products from algae could be accelerated with improvements in systems biology tools, and recent advances in sequencing technology provide a platform for enhanced transcriptomic analyses. However, these techniques are still heavily reliant upon available genomic sequence data. Chlamydomonas moewusii is a unicellular green alga capable of evolving molecular H2 under both dark and light anaerobic conditions, and has high hydrogenase activity that can be rapidly induced. However, to date, there is no systematic investigation of transcriptomic profiling during induction of H2 photoproduction in this organism. RESULTS: In this work, RNA-Seq was applied to investigate transcriptomic profiles during the dark anaerobic induction of H2 photoproduction. 156 million reads generated from 7 samples were then used for de novo assembly after data trimming. BlastX results against NCBI database and Blast2GO results were used to interpret the functions of the assembled 34,136 contigs, which were then used as the reference contigs for RNA-Seq analysis. Our results indicated that more contigs were differentially expressed during the period of early and higher H2 photoproduction, and fewer contigs were differentially expressed when H2-photoproduction rates decreased. In addition, C. moewusii and C. reinhardtii share core functional pathways, and transcripts for H2 photoproduction and anaerobic metabolite production were identified in both organisms. C. moewusii also possesses similar metabolic flexibility as C. reinhardtii, and the difference between C. moewusii and C. reinhardtii on hydrogenase expression and anaerobic fermentative pathways involved in redox balancing may explain their different profiles of hydrogenase activity and secreted anaerobic metabolites. CONCLUSIONS: Herein, we have described a workflow using commercial software to analyze RNA-Seq data without reference genome sequence information, which can be applied to other unsequenced microorganisms. This study provided biological insights into the anaerobic fermentation and H2 photoproduction of C. moewusii, and the first transcriptomic RNA-Seq dataset of C. moewusii generated in this study also offer baseline data for further investigation (e.g. regulatory proteins related to fermentative pathway discussed in this study) of this organism as a H2-photoproduction strain.

Agenciamentos coletivos na experimentação do PACTO Trabalho / Collective assemblage in the experimentation of the 'PACTO Trabalho' group

Este artigo apresenta um relato de experiência em Terapia Ocupacional, o PACTO Trabalho, que durante 6 anos de existência objetivou propor um modo possível de geração de renda, discutindo coletivamente questões relativas ao trabalho junto aos participantes em situação de vulnerabilidade social associada a questões de saúde mental. O método do work in progress ? emprestado do campo das artes ? foi utilizado para desenvolver, operar e construir coletivamente esta intervenção, pautado pelas demandas individuais, mas voltado para a construção de um dispositivo coletivo. Considerando a centralidade do trabalho na constituição subjetiva e da vida social, foram desenvolvidas ações e parcerias, sendo centrais as articulações com diversas redes ligadas diretamente a projetos de geração de renda, redes de suporte social, de saúde, de transporte, de ação jurídica. A complexidade das histórias de vida dos participantes, marcadas por fragilidades, necessitou sustentações múltiplas.

This article presents the experience of an Occupational Therapy group, the ?Pacto Trabalho?. For six years, the objective of this group has been to propose possible ways of generating income, by collectively discussing issues related to work together with the participants in a socially vulnerable situation associated to mental health problems. The work-in-progress method, borrowed from the of the arts, was used to collectively develop, operate and construct these interventions based on individual demands, but oriented towards the construction of a collective device. Considering that work is a core issue in the subjective constitution and social life, actions were taken and partnerships were made, mainly through the articulation with several networks directly linked to projects of income generation, and social, health, transportation and legal action support networks. The complexity of the participants? lives, marked by fragilities, required multiple supports.

Agenciamentos coletivos na experimentação do PACTO Trabalho / Collective assemblage in the experimentation of the ?PACTO Trabalho? group

Este artigo apresenta um relato de experiência em Terapia Ocupacional, o PACTO Trabalho, que durante 6 anos de existência objetivou propor um modo possível de geração de renda, discutindo coletivamente questões relativas ao trabalho junto aos participantes em situação de vulnerabilidade social associada a questões de saúde mental. O método do work in progress ? emprestado do campo das artes ? foi utilizado para desenvolver, operar e construir coletivamente esta intervenção, pautado pelas demandas individuais, mas voltado para a construção de um dispositivo coletivo. Considerando a centralidade do trabalho na constituição subjetiva e da vida social, foram desenvolvidas ações e parcerias, sendo centrais as articulações com diversas redes ligadas diretamente a projetos de geração de renda, redes de suporte social, de saúde, de transporte, de ação jurídica. A complexidade das histórias de vida dos participantes, marcadas por fragilidades, necessitou sustentações múltiplas.(AU)

This article presents the experience of an Occupational Therapy group, the ?Pacto Trabalho?. For six years, the objective of this group has been to propose possible ways of generating income, by collectively discussing issues related to work together with the participants in a socially vulnerable situation associated to mental health problems. The work-in-progress method, borrowed from the of the arts, was used to collectively develop, operate and construct these interventions based on individual demands, but oriented towards the construction of a collective device. Considering that work is a core issue in the subjective constitution and social life, actions were taken and partnerships were made, mainly through the articulation with several networks directly linked to projects of income generation, and social, health, transportation and legal action support networks. The complexity of the participants? lives, marked by fragilities, required multiple supports.(AU)

Angle-resolved XPS analysis and characterization of monolayer and multilayer silane films for DNA coupling to silica.

We measure silane density and Sulfo-EMCS cross-linker coupling efficiency on aminosilane films by high-resolution X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) measurements. We then characterize DNA immobilization and hybridization on these films by (32)P-radiometry. We find that the silane film structure controls the efficiency of the subsequent steps toward DNA hybridization. A self-limited silane monolayer produced from 3-aminopropyldimethylethoxysilane (APDMES) provides a silane surface density of ~3 nm(-2). Thin (1 h deposition) and thick (19 h deposition) multilayer films are generated from 3-aminopropyltriethoxysilane (APTES), resulting in surfaces with increased roughness compared to the APDMES monolayer. Increased silane surface density is estimated for the 19 h APTES film, due to a ∼32% increase in surface area compared to the APDMES monolayer. High cross-linker coupling efficiencies are measured for all three silane films. DNA immobilization densities are similar for the APDMES monolayer and 1 h APTES. However, the DNA immobilization density is double for the 19 h APTES, suggesting that increased surface area allows for a higher probe attachment. The APDMES monolayer has the lowest DNA target density and hybridization efficiency. This is attributed to the steric hindrance as the random packing limit is approached for DNA double helices (dsDNA, diameter ≥ 2 nm) on a plane. The heterogeneity and roughness of the APTES films reduce this steric hindrance and allow for tighter packing of DNA double helices, resulting in higher hybridization densities and efficiencies. The low steric hindrance of the thin, one to two layer APTES film provides the highest hybridization efficiency of nearly 88%, with 0.21 dsDNA/nm(2). The XPS data also reveal water on the cross-linker-treated surface that is implicated in device aging.

Novel FixL homologues in Chlamydomonas reinhardtii bind heme and O(2).

Genome inspection revealed nine putative heme-binding, FixL-homologous proteins in Chlamydomonas reinhardtii. The heme-binding domains from two of these proteins, FXL1 and FXL5 were cloned, expressed in Escherichia coli, purified and characterized. The recombinant FXL1 and FXL5 domains stained positively for heme, while mutations in the putative ligand-binding histidine FXL1-H200S and FXL5-H200S resulted in loss of heme binding. The FXL1 and FXL5 [Fe(II), bound O(2)] had Soret absorption maxima around 415 nm, and weaker absorptions at longer wavelengths, in concurrence with the literature. Ligand-binding measurements showed that FXL1 and FXL5 bind O(2) with moderate affinity, 135 and 222 µM, respectively. This suggests that Chlamydomonas may use the FXL proteins in O(2)-sensing mechanisms analogous to that reported in nitrogen-fixing bacteria to regulate gene expression.

Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: Insight into the role of HYDA2 in H2 production.

Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H(2) chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H(2) production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H(2) photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H(2) production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H(2)-photoproduction rates did not exceed those of wild-type cells.

Percursos de pesquisa e estratégias de ensino no campo da assistência em terapia ocupacional / Pathways of researching and strategies to teaching occupational therapy on social assistance field

Este relato de experiência apresenta aspectos de uma abordagem de ensino-aprendizagem na graduação em terapia ocupacional. Buscou-se trabalhar simultaneamente elementos de ensino e de assistência, num percurso de formação centrado nas atividades de campo de uma pesquisa em terapia ocupacional social. Pretendeu-se motivar o interesse pela pesquisa acadêmica enquanto uma prática profissional, tendo como estratégia a participação de estudantes no campo de pesquisa e nas discussões com a equipe. Estabeleceu-se uma experiência de ensino que, ao longo de um semestre letivo, permitiu um exercício reflexivo acerca da formulação de tecnologia social em percursos de assistência voltados à integração sócio-econômica de populações economicamente periféricas. Partiu-se de uma pesquisa acerca de uma ocupação potencialmente geradora de renda, proposta a destinatários de uma instituição de assistência social, para refletir a propósito das práticas previstas no campo da pesquisa como parte integrante das ações de assistência em terapia ocupacional. Observou-se que a estratégia didática de encontros entre discentes, docentes e terapeutas ocupacionais integrantes da pesquisa, permitiu ativar, simultaneamente, capacidades críticas individuais e coletivas além de intensificar a reflexividade no processo de ensino-aprendizagem. Algumas temáticas emergentes nesses encontros são apresentadas como forma de ilustrar essa experiência.

This paper discusses one strategy to teaching Occupational Therapy to undergraduates and offers examples of its implementation in practice. It examines a course which brought together classroom teaching and field practice activities so as to lead students to have a first-hand experience of research in Social Occupational Therapy. The course intended to foster student interest in academic research by getting students involved in field work and in debates with peers, professors and practicing therapists. Its format led students to think critically about the building up of social technologies to promote the social-economic integration of economically peripheral populations. The course took as its starting point the study of a potentially income-generating squatted area to help students think about academic research as an intrinsic and important part of assistance policy-designing in Occupational Therapy. At the end of the course, it became clear that the teaching strategy of bringing together students, teachers and occupational therapists in a discussion group, and the opportunity for actual field work, greatly enhanced students' individual and collective critical skills.

High-resolution X-ray photoelectron spectroscopy of mixed silane monolayers for DNA attachment.

The amine density of 3-aminopropyldimethylethoxysilane (APDMES) films on silica is controlled to determine its effect on DNA probe density and subsequent DNA hybridization. The amine density is tailored by controlling the surface reaction time of (1) APDMES, or (2) n-propyldimethylchlorosilane (PDMCS, which is not amine terminated) and then reacting it with APDMES to form a mixed monolayer. High-resolution X-ray photoelectron spectroscopy (XPS) is used to quantify silane surface coverage of both pure and mixed monolayers on silica; the XPS data demonstrate control of amine density in both pure APDMES and PDMCS/APDMES mixed monolayers. A linear correlation between the atomic concentration of N atoms from the amine and Si atoms from the APDMES in pure APDMES films allows us to calculate the PDMCS/APDMES ratio in the mixed monolayers. Fluorescence from attached DNA probes and from hybridized DNA decreases as the percentage of APDMES in the mixed monolayer is decreased by dilution with PDMCS.

Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A.

[FeFe]-hydrogenases (HYDA) link the production of molecular H(2) to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H(2)-photoproduction activity observed is as O(2) sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases.