N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas.

N(6)-methyldeoxyadenosine (6mA or m(6)A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here, we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms.

A single-culture bioprocess of Methanothermobacter thermautotrophicus to upgrade digester biogas by CO2 -to-CH4 conversion with H2.

We optimized and tested a postbioprocessing step with a single-culture archaeon to upgrade biogas (i.e., increase methane content) from anaerobic digesters via conversion of CO(2) into CH(4) by feeding H(2) gas. We optimized a culture of the thermophilic methanogen Methanothermobacter thermautotrophicus using: (1) a synthetic H(2)/CO(2) mixture; (2) the same mixture with pressurization; (3) a synthetic biogas with different CH(4) contents and H(2); and (4) an industrial, untreated biogas and H(2). A laboratory culture with a robust growth (dry weight of 6.4-7.4 g/L; OD600 of 13.6-15.4), a volumetric methane production rate of 21 L/L culture-day, and a H(2) conversion efficiency of 89% was moved to an industrial anaerobic digester facility, where it was restarted and fed untreated biogas with a methane content of ~70% at a rate such that CO(2) was in excess of the stoichiometric requirements in relation to H(2). Over an 8-day operating period, the dry weight of the culture initially decreased slightly before stabilizing at an elevated level of ~8 g/L to achieve a volumetric methane production rate of 21 L/L culture-day and a H(2) conversion efficiency of 62%. While some microbial contamination of the culture was observed via microscopy, it did not affect the methane production rate of the culture.

Chromophore formation in DsRed occurs by a branched pathway.

Like GFP, the fluorescent protein DsRed has a chromophore that forms autocatalytically within the folded protein, but the mechanism of DsRed chromophore formation has been unclear. It was proposed that an initial oxidation generates a green chromophore, and that a final oxidation yields the red chromophore. However, this model does not adequately explain why a mature DsRed sample contains a mixture of green and red chromophores. We present evidence that the maturation pathway for DsRed branches upstream of chromophore formation. After an initial oxidation step, a final oxidation to form the acylimine of the red chromophore is in kinetic competition with a dehydration to form the green chromophore. This scheme explains why green and red chromophores are alternative end points of the maturation pathway.

Chlamydomonas reinhardtii formin FOR1 and profilin PRF1 are optimized for acute rapid actin filament assembly.

The regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in Chlamydomonas is the fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed-end elongation at equimolar concentrations to actin. However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-bound actin. Furthermore, for1 and prf1-1 mutants, as well as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 cooperate to selectively and rapidly assemble F-actin at the right time and place.

Likelihood-based inference for multi-color optical mapping.

Multi-color optical mapping is a new technique being developed to obtain detailed physical maps (indicating relative positions of various recognition sites) of DNA molecules. We consider a study design in which the data consist of noisy observations of multiple copies of a DNA molecule marked with colors at recognition sites. The primary goal is to estimate a physical map. A secondary goal is to estimate error rates associated with the experiment, which are potentially useful for analysis and refinement of the biochemical steps in the mapping procedure. We propose statistical models for various sources of error and use maximum likelihood estimation (MLE) to construct a physical map and estimate error rates. To overcome difficulties arising in the maximization process, a latent-variable Markov chain version of the model is proposed, and the EM algorithm is used for maximization. In addition, a simulated annealing procedure is applied to maximize the profile likelihood over the discrete space of sequences of colors. We apply the methods to simulated data on the bacteriophage lambda genome.

Improvement of photosynthetic CO2 fixation at high light intensity through reduction of chlorophyll antenna size.

At elevated light intensities (greater than approximately 200 microE/[m2 x s]), the kinetic imbalance between the rate of photon excitation and thermally activated electron transport results in saturation of the rate of photosynthesis. Since maximum terrestrial solar radiation can reach 200 microE/(m2 x s), a significant opportunity exists to improve photosynthetic efficiency at elevated light intensities by achieving a kinetic balance between photon excitation and electron transport, especially in designed large-scale photosynthetic reactors in which a low-cost and efficient biomass production system is desired. One such strategy is a reduction in chlorophyll (chl) antenna size in relation to the reaction center that it serves. In this article, we report recent progress in this area of research. Light-saturation studies for CO2 fixation were performed on an antenna-deficient mutant of Chlamydomonas (DS521) and the wild type (DES15) with 700 ppm of CO2 in air. The light-saturated rate for CO2 assimilation in the mutant DS521 was about two times higher (187 micromol/[h x mg of chl]) than that of the wild type, DES15 (95 micromol/[h x mg of chl]). Significantly, a partial linearization of the light-saturation curve was also observed. These results confirmed that DS521 has a smaller relative chl antenna size and demonstrated that reduction of relative antenna size can improve the overall efficiency of photon utilization at higher light intensities. The antenna-deficient mutant DS521 can provide significant resistance to photoinhibition, in addition to improvement in the overall efficiency of CO2 fixation at high light. The experimental data reported herein support the idea that reduction in chl antenna size could have significant implications for both fundamental understanding of photosynthesis and potential application to improve photosynthetic CO2 fixation efficiency.

The redox state of the plastoquinone pool directly modulates minimum chlorophyll fluorescence yield in Chlamydomonas reinhardtii.

The effect of the plastoquionone (PQ) pool oxidation state on minimum chlorophyll fluorescence was studied in the green alga Chlamydomonas reinhardtii. In wild type and a mutant strain that lacks both photosystems but retains light harvesting complexes, oxygen depletion induced a rise in minimum chlorophyll fluorescence. An increase in minimum fluorescence yield is also observed when the PQ pool becomes reduced in the presence of oxygen and after application of an ionophore that collapses the transmembrane proton gradient. Together these results indicate that minimum chlorophyll fluorescence is modulated by the PQ oxidation state.

The Chlamydomonas genome reveals the evolution of key animal and plant functions.

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

Nanometer-localized multiple single-molecule fluorescence microscopy.

Fitting the image of a single molecule to the point spread function of an optical system greatly improves the precision with which single molecules can be located. Centroid localization with nanometer precision has been achieved when a sufficient number of photons are collected. However, if multiple single molecules reside within a diffraction-limited spot, this localization approach does not work. This paper demonstrates nanometer-localized multiple single-molecule (NALMS) fluorescence microscopy by using both centroid localization and photobleaching of the single fluorophores. Short duplex DNA strands are used as nanoscale "rulers" to validate the NALMS microscopy approach. Nanometer accuracy is demonstrated for two to five single molecules within a diffraction-limited area. NALMS microscopy will greatly facilitate single-molecule study of biological systems because it covers the gap between fluorescence resonance energy transfer-based (<10 nm) and diffraction-limited microscopy (>100 nm) measurements of the distance between two fluorophores. Application of NALMS microscopy to DNA mapping with <10-nm (i.e., 30-base) resolution is demonstrated.