Marine coccolithophorid algae are thought to play a significant role in carbon cycling due to their ability to incorporate dissolved inorganic carbon (DIC) into both calcite and photosynthetic products. Among coccolithophorids, Emiliania huxleyi is the most prolific, forming massive blooms that affect the global environment. In addition to its ecological importance, the elaborate calcite structures (coccoliths) are being investigated for the design of potential materials for science and biotechnological devices. To date, most of the research focus in this organism has involved the partitioning of DIC between calcification and photosynthesis, primarily using measurements of an external versus internal carbonic anhydrase (CA) activity under defined conditions. The actual genes, proteins, and pathways employed in these processes have not been identified and characterized Single-celled marine algae fix inorganic carbon via the Calvin-Benson-Bassham cycle, resulting in the formation of 2 mol of phosphoglyceric acid from CO 2 and its five-carbon acceptor. The enzyme responsible for this reaction, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), requires CO 2 for carbon fixation, and kinetic analyses of these enzymes from several photosynthetic microorganisms have shown that RubisCO enzymes have poor binding affinity for this substrate. The predominant form of dissolved inorganic carbon (DIC) in the oceans is bicarbonate (ϳ2 mM), and concentrations of aqueous CO 2 are very low (10 M), well below the half-saturation constants of most marine algal RubisCO enzymes (6). This requires that unicellular algae increase intracellular DIC levels either via the direct transport of HCO 3 Ϫ or by the activity of an external carbonic anhydrase (CA) (CA ext ). All carbon-concentrating mechanisms (CCMs) described to date in marine phytoplankton involve the zinc metalloenzyme carbonic anhydrase; therefore, CO 2 acquisition via these enzymes is dependent upon the availability of trace metals (Zn, Co, and Cd), the concentrations of which are extremely low (nanomolar to picomolar levels) in ocean surface waters. These conditions place severe constraints on CO 2 availability for marine phytoplankton via CA, yet growth kinetics and global distribution data on coccolithophorids and diatoms suggest that they are not generally CO 2 limited for photosynthesis under most environmental conditions. Considering the low concentrations and dramatic fluctuations in these trace metals, it is logical to envision scenarios under which CA activity is repressed and an alternative CCM mechanism is induced, such as the CCM pathways observed in C 4 and Crassulacean acid metabolism (CAM) plants. An example of such a mechanism was recently described for the marine diatom Thalassiosira weissflogii. The results of that elegant study provided solid evidence for C 4 photosynthesis in a unicellular alga (26). Another study showed a similar C 4 mechanism within a single photosynthetic cell in a terrestrial plant (44), and thus, the argument requiring a Kran...

We report the complete genome of Thermofilum pendens, a deeply branching, hyperthermophilic member of the order Thermoproteales in the archaeal kingdom Crenarchaeota. T. pendens is a sulfur-dependent, anaerobic heterotroph isolated from a solfatara in Iceland. It is an extracellular commensal, requiring an extract of Thermoproteus tenax for growth, and the genome sequence reveals that biosynthetic pathways for purines, most amino acids, and most cofactors are absent. In fact, T. pendens has fewer biosynthetic enzymes than obligate intracellular parasites, although it does not display other features that are common among obligate parasites and thus does not appear to be in the process of becoming a parasite. It appears that T. pendens has adapted to life in an environment rich in nutrients. T. pendens was known previously to utilize peptides as an energy source, but the genome revealed a substantial ability to grow on carbohydrates. T. pendens is the first crenarchaeote and only the second archaeon found to have a transporter of the phosphotransferase system. In addition to fermentation, T. pendens may obtain energy from sulfur reduction with hydrogen and formate as electron donors. It may also be capable of sulfur-independent growth on formate with formate hydrogen lyase. Additional novel features are the presence of a monomethylamine:corrinoid methyltransferase, the first time that this enzyme has been found outside the Methanosarcinales, and the presence of a presenilin-related protein.The predicted highly expressed proteins do not include proteins encoded by housekeeping genes and instead include ABC transporters for carbohydrates and peptides and clustered regularly interspaced short palindromic repeat-associated proteins.Crenarchaeota is one of the two major divisions of the Archaea, and it is the least well represented taxon in terms of genome sequences. Only seven crenarchaeal genomes have been sequenced and published so far, and three of these are genomes of members of the genus Sulfolobus. For the order Thermoproteales, the complete sequence of only one organism, Pyrobaculum aerophilum, has been determined and published so far, although several more species of Pyrobaculum, Caldivirga maquilingensis, and Thermoproteus tenax have been or are currently being sequenced (29). Thermofilum pendens represents a deep branch in the order Thermoproteales, and this organism grows only in rich medium with a fraction of the polar lipids of T. tenax (64), a property that has not been seen before in archaea. Therefore, it was an attractive sequencing target. We report here the genome sequence of T. pendens and analysis of the type strain, T. pendens Hrk5.T. pendens is an anaerobic, sulfur-dependent hyperthermophile isolated from a solfatara in Iceland. It forms long thin filaments and may have an unusual mode of reproduction in which spherical bulges form at one end of the cell. It requires complex media and a lipid extract from the related organism T. tenax for growth (64). The unknown lipid may be a cellular componen...

Background: Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes.

Methanocaldococcus jannaschii, a deeply rooted hyperthermophilic anaerobic methanarchaeon from a deep-sea hydrothermal vent, carries an NADH oxidase (Nox) homologue (MJ0649). According to the characteristics described here, MJ0649 represents an unusual member within group 3 of the flavin-dependent disulfide reductase (FDR) family. This FDR group comprises Nox, NADH peroxidases (Npx) and coenzyme A disulfide reductases (CoADRs); each carries a Cys residue that forms Cys-sulfenic acid during catalysis. A sequence analysis identified MJ0649 as a CoADR homologue. However, recombinant MJ0649 (rMJNox), expressed in Escherichia coli and purified to homogeneity an 86 kDa homodimer with 0.27 mol FAD (mol subunit) "1 , showed Nox but not CoADR activity. Incubation with FAD increased FAD content to 1 mol (mol subunit) "1 and improved NADH oxidase activity 3.4-fold. The FAD-incubated enzyme was characterized further. The optimum pH and temperature were ¢10 and ¢95 6C, respectively. At pH 7 and 83 6C, apparent K m values for NADH and O 2 were 3 mM and 1.9 mM, respectively, and the specific activity at 1.4 mM O 2 was 60 mmol min "1 mg "1 ; 62 % of NADH-derived reducing equivalents were recovered as H 2 O 2 and the rest probably generated H 2 O. rMjNox had poor NADPH oxidase, NADH peroxidase and superoxide formation activities. It reduced ferricyanide, plumbagin and 5,59-dithiobis(2-nitrobenzoic acid), but not disulfide coenzyme A and disulfide coenzyme M. Due to a high K m , O 2 is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown.

Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.