Laurens Mets

Two-dimensional polyacrylamide gel electrophoresis: an improved method for ribosomal proteins.

Abstract A two-dimensional electrophoresis system for analysis of ribosomal proteins with several advantages over previous systems is described. The general features of this system are: (1) first-dimension separation on the basis of mobility at pH 5.0 in 8 m urea and 4% polyacrylamide; (2) second-dimension separation on the basis of molecular weight using dodecyl sulfate detergent; (3) rapid electrophoretic shift between first- and second-dimension separation conditions; (4) high resolution separation can be obtained on 10-cm 2 slabs with proteins from approximately 100 μg of ribosomal subunits; (5) capacity for handling up to 10 samples at a time, with electrophoresis complete within about 10 hr; and (6) the apparatus is relatively simple and inexpensive to construct and use.

Herbicide resistance and cross-resistance: changes at three distinct sites in the herbicide-binding protein.

Plants and algae resistant to the commonly used s-triazine herbicides display a wide spectrum of cross-resistance to other herbicides that act in a similar manner. Analysis of uniparental mutants of the green alga Chlamydomonas reinhardi showed that three different amino acid residues in the 32-kilodalton thylakoid membrane protein can be independently altered to produce three different patterns of resistance to s-triazine and urea-type herbicides. These results clarify the molecular basis for herbicide resistance and cross-resistance. Two of the mutations do not alter normal electron transport and thus may have applications of agronomic interest.

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.

Direct observation of ultrafast energy transfer in PSI core antenna

A femtosecond fluorescence upconversion apparatus was used to measure the fluorescence decay in the PSI core antenna of a PSI-only mutant of Chlamydomonas reinhardtii. The fluorescence depolarization measurements reveal a fast depolarization with a time constant in the range of 150–300 fs irrespective of the excitation and/or detection wavelengths. We associate this time with the energy transfer timescale. The isotropic fluorescence decay shows a decay component of ≈ 5 ps and is suggested to result from spectral equilibration within the antenna.

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.

Localization of two novel chloroplast genome functions: trans-splicing of RNA and protochlorophyllide reduction

SummaryChloroplast DNA deletions in the unicellular green alga Chlamydomonas reinhardtii localize two novel chloroplast gene functions. One of these, tscA, is required in trans for splicing, in trans, of the first and second, but not the second and third, exons of the RNA of the chloroplast gene psaA. Previously, no chloroplast genes were known to be required in trans for the splicing of chloroplast RNA. The other chloroplast gene function is required for light-independent reduction of protochlorophyllide, a key step in the algal pathway of chlorophyll biosynthesis. Both functions reside in the same 4 kbp region of chloroplast DNA.

First DNA sequence of a chloroplast mutation: A missense alteration in the ribulosebisphosphate car☐ylase large subunit gene

Sequence comparison of the chloroplast genes of the large subunit of ribulosebisphosphate carboxylase from wild-type and from a uniparental mutant of the green unicellular alga Chlamydomonas reinhardii has revealed a single nucleotide change. The corresponding Gly to Asp amino acid substitution would introduce a negative charge into the presumptive substrate binding region of the enzyme and would explain the inactivity of the mutant protein. This is the first chloroplast mutation whose DNA sequence is known. Our results establish the first exact point of correlation between the physical map of the chloroplast genome of C. reinhardii and a specific genetic locus.

Mendelian and Uniparental Alterations in Erythromycin Binding by Plastid Ribosomes

Erythromycin binds specifically to the 52S subunit of the chloroplast ribosome of Chlamydomonas reinhardi. A number of erythromycin-resistant mutants whose ribosomes have lost their affinity for the antibiotic have been isolated, but the sedimentation properties of their ribosomes are indistinguishable from those of the wild-type strain. These mutants represent at least three genetic loci. Two of them show Mendelian inheritance, and one of them is inherited in a uniparental manner.

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 CO2 into CH4 by feeding H2 gas. We optimized a culture of the thermophilic methanogen Methanothermobacter thermautotrophicus using: (1) a synthetic H2/CO2 mixture; (2) the same mixture with pressurization; (3) a synthetic biogas with different CH4 contents and H2; and (4) an industrial, untreated biogas and H2. 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 H2 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 CO2 was in excess of the stoichiometric requirements in relation to H2. 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 H2 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.

PICOSECOND FLUORESCENCE STUDY OF PHOTOSYNTHETIC MUTANTS OF Chlamydomonas reinhardii: ORIGIN OF THE FLUORESCENCE DECAY KINETICS OF CHLOROPLASTS

Abstract— The fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardii which lack photosystem II (PS II), photosystem I (PS I), and both PS II and PS I have been measured. The PS II mutant strain8–36C exhibits fluorescence decay lifetime components of 53, 424 and 2197 ps. The fluorescence decay of a PS I mutant strain12–7 contains two major fluorescence decay components with lifetimes of 152 and 424 ps. The fluorescence decay of mutant strain C2, which lacks both PS II and PS I, is nearly single exponential with a lifetime of 2561 ± 222 ps. In simulations in which it is assumed that wild‐type decays are a simple sum of the major decay components of the isolated parts of the photosynthetic unit as measured in the mutants, curves are obtained that fit the wild‐type C. reinhardii fluorescence decay data when the absorption cross‐sections of PS II and PS I are weighted approximately equally. The 89 ps lifetime component in the wild‐type is an average of 53 and 152 ps components arising from excitation transfer to and trapping in PS I and PS II. The single step transfer time in PS I is estimated to be between 100 and 700 fs depending on assumptions about array size. We find that between two and four visits to the PS I reaction center are required before final trapping.