Carbon Fixation in the Chemolithoautotrophic Bacterium Aquifex aeolicus Involves Two Low-Potential Ferredoxins as Partners of the PFOR and OGOR Enzymes.

Aquifex aeolicus is a microaerophilic hydrogen- and sulfur -oxidizing bacterium that assimilates CO2 via the reverse tricarboxylic acid cycle (rTCA). Key enzymes of this pathway are pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), which are responsible, respectively, for the reductive carboxylation of acetyl-CoA to pyruvate and of succinyl-CoA to 2-oxoglutarate, two energetically unfavorable reactions that require a strong reduction potential. We have confirmed, by biochemistry and proteomics, that A. aeolicus possesses a pentameric version of these enzyme complexes ((αßγδε)2) and that they are highly abundant in the cell. In addition, we have purified and characterized, from the soluble fraction of A. aeolicus, two low redox potential and oxygen-stable [4Fe-4S] ferredoxins (Fd6 and Fd7, E0 = -440 and -460 mV, respectively) and shown that they can physically interact and exchange electrons with both PFOR and OGOR, suggesting that they could be the physiological electron donors of the system in vivo. Shotgun proteomics indicated that all the enzymes assumed to be involved in the rTCA cycle are produced in the A. aeolicus cells. A number of additional enzymes, previously suggested to be part of a putative partial Wood-Ljungdahl pathway used for the synthesis of serine and glycine from CO2 were identified by mass spectrometry, but their abundance in the cell seems to be much lower than that of the rTCA cycle. Their possible involvement in carbon assimilation is discussed.

Targeting TOR signaling for enhanced lipid productivity in algae.

Microalgae can produce large quantities of triacylglycerols (TAGs) and other neutral lipids that are suitable for making biofuels and as feedstocks for green chemistry. However, TAGs accumulate under stress conditions that also stop growth, leading to a trade-off between biomass production and TAG yield. Recently, in the model marine diatom Phaeodactylum tricornutum it was shown that inhibition of the target of rapamycin (TOR) kinase boosts lipid productivity by promoting TAG production without stopping growth. We believe that basic knowledge in this emerging field is required to develop innovative strategies to improve neutral lipid accumulation in oleaginous microalgae. In this minireview, we discuss current research on the TOR signaling pathway with a focus on its control on lipid homeostasis. We first provide an overview of the well characterized roles of TOR in mammalian lipogenesis, adipogenesis and lipolysis. We then present evidence of a role for TOR in controlling TAG accumulation in microalgae, and draw parallels between the situation in animals, plants and microalgae to propose a model of TOR signaling for TAG accumulation in microalgae.

Exploring the microbiome of the "star" freshwater diatom Asterionella formosa in a laboratory context.

Most of our knowledge on the mechanisms underlying diatom-bacterial interactions has been acquired through studies involving isolation of culturable partners. Here, we established a laboratory model of intermediate complexity between complex natural communities and laboratory pure culture models. We investigated the whole community formed by the freshwater diatom Asterionella formosa and its associated bacteria in a laboratory context, including both culturable and unculturable bacteria. Combining cellular and molecular approaches, we showed that in laboratory cultures, A. formosa microbiome was dynamic and comprised of numerous bacterial species (mainly Proteobacteria and Bacteroidetes). Using metagenomics, we explored several metabolic potentials present within the bacterial community. Our analyses suggested that bacteria were heterotrophic although a third of them (Alpha- and Beta-proteobacteria) could also be phototrophic. About 60% of the bacteria, phylogenetically diverse, could metabolize glycolate. The capacity to synthesize molecules such as B vitamins appeared unevenly distributed among bacteria. Altogether, our results brought insights into the bacterial diversity found in diatom-bacterial communities and hinted at metabolic interdependencies within the community that could result in diatom-bacterial and bacterial-bacterial interactions. The present work allowed us to explore the functional architecture of the bacterial community associated with A. formosa in culture and is complementary to field studies.

Diversity of CO2-concentrating mechanisms and responses to CO2 concentration in marine and freshwater diatoms.

The presence of CO2-concentrating mechanisms (CCMs) is believed to be one of the characteristics that allows diatoms to thrive in many environments and to be major contributors to global productivity. Here, the type of CCM and the responses to variable CO2 concentration were studied in marine and freshwater diatoms. At 400 ppm, there was a large diversity in physiological and biochemical mechanisms among the species. While Phaeodactylum tricornutum mainly used HCO3-, Thalassiosira pseudonana mainly used CO2. Carbonic anhydrase was an important component of the CCM in all species and C4 metabolism was absent, even with T. weissflogii. For all species, at 20 000 ppm, the affinity for dissolved inorganic carbon was lower than at 400 ppm CO2 and the reliance on CO2 was higher. Despite the difference in availability of inorganic carbon in marine and fresh waters, there were only small differences in CCMs between species from the two environments, and Navicula pelliculosa behaved similarly when grown in the two environments. The results suggest that species-specific differences are great, and more important than environmental differences in determining the nature and effectiveness of the CCM in diatoms.

Complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa.

We report the complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa. The large 61.9 kb circular sequence encodes 34 proteins and 25 tRNAs that are universally conserved in other sequenced diatoms. We fully resolved a unique 24 kb region containing highly conserved repeated sequence units, possibly collocating with an origin of replication.

Direct and indirect influence of sulfur availability on phytoplankton evolutionary trajectories.

The sulfate facilitation hypothesis suggests that changes in ocean sulfate concentration influenced the rise to dominance of phytoplankton species of the red lineage. The mechanistic reasons for this phenomenon are not yet understood. We started to address this question by investigating the differences in S utilization by algae of the green and red lineages and in cyanobacteria cultured in the presence of either 5 mmol · L-1 (approximately equivalent to Paleozoic ocean concentrations) or 30 mmol · L-1 (corresponding to post-Mesozoic/extant concentrations) sulfate. The activities of the main enzymes involved in SO42- assimilation changed in response to changes in growth sulfate concentration. ATP sulfurylase showed different kinetics in the various taxa, with an especially odd behavior for the dinoflagellate. Sulfate availability had a modest effect on cell organic composition. Species-specific differences in the use of some elements were instead obvious in algae grown in the presence of different sulfate concentrations, overall confirming that algae of the red lineage do better at high sulfate than algae of the green lineage. The increase in sulfate concentration may thus have had an impact on phytoplankton radiation both through changes in their enzymatic machinery and through indirect repercussion on elemental usage.

Redox regulation of ATP sulfurylase in microalgae.

ATP sulfurylase (ATPS) catalyzes the first step of sulfur assimilation in photosynthetic organisms. An ATPS type A is mostly present in freshwater cyanobacteria, with four conserved cysteine residues. Oceanic cyanobacteria and most eukaryotic algae instead, possess an ATPS-B containing seven to ten cysteines; five of them are conserved, but only one in the same position as ATPS-A. We investigated the role of cysteines on the regulation of the different algal enzymes. We found that the activity of ATPS-B from four different microorganisms was enhanced when reduced and decreased when oxidized. The LC-MS/MS analysis of the ATPS-B from the marine diatom Thalassiosira pseudonana showed that the residue Cys-247 was presumably involved in the redox regulation. The absence of this residue in the ATPS-A of the freshwater cyanobacterium Synechocystis sp. instead, was consistent with its lack of regulation. Some other conserved cysteine residues in the ATPS from T. pseduonana and not in Synechocystis sp.were accessible to redox agents and possibly play a role in the enzyme regulation. Furthermore, the fact that oceanic cyanobacteria have ATPS-B structurally and functionally closer to that from most of eukaryotic algae than to the ATPS-A from other cyanobacteria suggests that life in the sea or freshwater may have driven the evolution of ATPS.

Diversity and regulation of ATP sulfurylase in photosynthetic organisms.

ATP sulfurylase (ATPS) catalyzes the first committed step in the sulfate assimilation pathway, the activation of sulfate prior to its reduction. ATPS has been studied in only a few model organisms and even in these cases to a much smaller extent than the sulfate reduction and cysteine synthesis enzymes. This is possibly because the latter were considered of greater regulatory importance for sulfate assimilation. Recent evidences (reported in this paper) challenge this view and suggest that ATPS may have a crucial regulatory role in sulfate assimilation, at least in algae. In the ensuing text, we summarize the current knowledge on ATPS, with special attention to the processes that control its activity and gene(s) expression in algae. Special attention is given to algae ATPS proteins. The focus on algae is the consequence of the fact that a comprehensive investigation of ATPS revealed that the algal enzymes, especially those that are most likely involved in the pathway of sulfate reduction to cysteine, possess features that are not present in other organisms. Remarkably, algal ATPS proteins show a great diversity of isoforms and a high content of cysteine residues, whose positions are often conserved. According to the occurrence of cysteine residues, the ATPS of eukaryotic algae is closer to that of marine cyanobacteria of the genera Synechococcus and Prochlorococcus and is more distant from that of freshwater cyanobacteria. These characteristics might have evolved in parallel with the radiation of algae in the oceans and the increase of sulfate concentration in seawater.