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Must and wine may be contaminated with elevated copper concentrations by the use of fungicides or in course of the vinification process. Hitherto only a few practicable and harmless procedures exist to reduce an excess of copper from must and wine. For this reason we investigated the biosorption of copper by eight wine-relevant Lactobacillus species. Both, living and heat-inactivated cells revealed a significant degree of Cu adsorption. It was shown that Cu binding correlated positively with an increasing pH value of the environment. The highest binding capacity of the tested lactic acid bacteria was found for L. buchneri DSM 20057 with a maximum of 46.17 μg Cu bound per mg cell in deionized water. In must, wine and grape juice Cu was removed less effective which is not solely attributed to low pH-values, but also to specific medium parameters such as intrinsic metal cations, organic acids or phenolic compounds. Nevertheless, about 0.5-1.0 μg Cu per ml could be removed from wine samples, which is sufficient enough to lower critical copper concentrations., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Algae with secondary plastids such as diatoms maintain two different eukaryotic cytoplasms. One of them, the so-called periplastidal compartment (PPC), is the naturally minimized cytoplasm of a eukaryotic endosymbiont. In order to investigate the protein composition of the PPC of diatoms, we applied knowledge of the targeting signals of PPC-directed proteins in searches of the genome data for proteins acting in the PPC and proved their in vivo localization via expressing green fluorescent protein (GFP) fusions. Our investigation increased the knowledge of the protein content of the PPC approximately 3-fold and thereby indicated that this narrow compartment was functionally reduced to some important cellular functions with nearly no housekeeping biochemical pathways.

The plastids of cryptophytes, haptophytes, and heterokontophytes (stramenopiles) (together once known as chromists) are surrounded by four membranes, reflecting the origin of these plastids through secondary endosymbiosis. They share this trait with apicomplexans, which are alveolates, the plastids of which have been suggested to stem from the same secondary symbiotic event and therefore form a phylogenetic clade, the chromalveolates. The chromists are quantitatively the most important eukaryotic contributors to primary production in marine ecosystems. The mechanisms of protein import across their four plastid membranes are still poorly understood. Components of an endoplasmic reticulum-associated degradation (ERAD) machinery in cryptophytes, partially encoded by the reduced genome of the secondary symbiont (the nucleomorph), are implicated in protein transport across the second outermost plastid membrane. Here, we show that the haptophyte Emiliania huxleyi, like cryptophytes, stramenopiles, and apicomplexans, possesses a nuclear-encoded symbiont-specific ERAD machinery (SELMA, symbiont-specific ERAD-like machinery) in addition to the host ERAD system, with targeting signals that are able to direct green fluorescent protein or yellow fluorescent protein to the predicted cellular localization in transformed cells of the stramenopile Phaeodactylum tricornutum. Phylogenies of the duplicated ERAD factors reveal that all SELMA components trace back to a red algal origin. In contrast, the host copies of cryptophytes and haptophytes associate with the green lineage to the exclusion of stramenopiles and alveolates. Although all chromalveolates with four membrane-bound plastids possess the SELMA system, this has apparently not arisen in a single endosymbiotic event. Thus, our data do not support the chromalveolate hypothesis.

Brick stones collected from different production facilities were studied for their elemental compositions under forensic aspects using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), instrumental neutron activation analysis (INAA) and X-ray fluorescence analysis (XRF). The aim of these examinations was to assess the potential of these methods in forensic comparison analyses of brick stones. The accuracy of the analysis methods was evaluated using NIST standard reference materials (679, 98b and 97b). In order to compare the stones to each other, multivariate data analysis was used. The evaluation of the INAA results (based on the concentrations of V, Na, K, Sm, U, Sc, Fe, Co, Rb and Cs) using principal component analysis (PCA) and cluster analysis is presented as an example. The results derived from the different analytical methods are consistent. It was shown that elemental analysis using the described methods is a valuable tool for forensic examinations of brick stones.

Samples from 20 reverse paintings on glass from different regions have been analyzed by NAA with the aim to deduce the place and date of their origin. A separation of earlier and later paintings was due to different concentrations of K and Na, because a sodium-containing flux came into use after 1870. Since in southern Germany quartz sand, and in the eastern area quartz rock had been used for glass manufacture, specific impurities could be used to distinguish southern from eastern glasses.

Diatoms have played a decisive role in the ecosystem for millions of years as one of the foremost set of oxygen synthesizers on earth and as one of the most important sources of biomass in oceans. Previously, diatoms have been almost exclusively limited to academic research with little consideration of their practical uses beyond the most rudimentary of applications. Efforts have been made to establish them as decisively useful in such commercial and industrial applications as the carbon neutral synthesis of fuels, pharmaceuticals, health foods, biomolecules, materials relevant to nanotechnology, and bioremediators of contaminated water. Progress in the technologies of diatom molecular biology such as genome projects from model organisms, as well as culturing conditions and photobioreactor efficiency, may be able to be combined in the near future to make diatoms a lucrative source of novel substances with widespread relevance.

Most of the coding capacity of primary plastids is reserved for expressing some central components of the photosynthesis machinery and the translation apparatus. Thus, for the bulk of biochemical and cell biological reactions performed within the primary plastids, many nucleus-encoded components have to be transported posttranslationally into the organelle. The same is true for plastids surrounded by more than two membranes, where additional cellular compartments have to be supplied with nucleus-encoded proteins, leading to a corresponding increase in complexity of topogenic signals, transport and sorting machineries. In this review, we summarize recent progress in elucidating protein transport across up to five plastid membranes in plastids evolved in secondary endosymbiosis. Current data indicate that the mechanisms for protein transport across multiple membranes have evolved by altering pre-existing ones to new requirements in secondary plastids.

Background: The gene expression system of chloroplasts is far more complex than that of their cyanobacterial progenitor. This gain in complexity affects in particular RNA metabolism, specifically the transcription and maturation of RNA. Mature chloroplast RNA is generated by a plethora of nuclear-encoded proteins acquired or recruited during plant evolution, comprising additional RNA polymerases and sigma factors, and sequence-specific RNA maturation factors promoting RNA splicing, editing, end formation and translatability. Despite years of intensive research, we still lack a comprehensive explanation for this complexity., Results: We inspected the available literature and genome databases for information on components of RNA metabolism in land plant chloroplasts. In particular, new inventions of chloroplast-specific mechanisms and the expansion of some gene/protein families detected in land plants lead us to suggest that the primary function of the additional nuclear-encoded components found in chloroplasts is the transgenomic suppression of point mutations, fixation of which occurred due to an enhanced genetic drift exhibited by chloroplast genomes. We further speculate that a fast evolution of transgenomic suppressors occurred after the water-to-land transition of plants., Conclusion: Our inspections indicate that several chloroplast-specific mechanisms evolved in land plants to remedy point mutations that occurred after the water-to-land transition. Thus, the complexity of chloroplast gene expression evolved to guarantee the functionality of chloroplast genetic information and may not, with some exceptions, be involved in regulatory functions.

Many algal groups evolved by engulfment and intracellular reduction of a eukaryotic phototroph within a heterotrophic cell. Via this process, so-called secondary plastids evolved, surrounded by three or four membranes. In these organisms most of the genetic material encoding plastid functions is localized in the cell nucleus, with the result that many proteins have to pass three, four, or even five membranes to reach their final destination within the plastid. In this article, we review recent models and findings that help to explain important cellular mechanisms involved in the complex process of protein transport into secondary plastids.

Sedoheptulose-1,7-bisphosphatase (SBPase) and fructose-1,6-bisphosphatase (FBPase) are essential nuclear-encoded enzymes involved in land plant Calvin cycle and gluconeogenesis. In this study, we cloned seven SBP and seven FBP cDNAs/genes and established sequences from all lineages of photosynthetic eukaryotes, in order to investigate their origin and evolution. Our data are best explained by a single recruitment of plastid-targeted SBP in Plantae after primary endosymbiosis and a further distribution to algae with complex plastids. While SBP is universally found in photosynthetic lineages, its presence in apicomplexa, ciliates, trypanosomes, and ascomycetes is surprising given that no metabolic function beyond the one in the plastid Calvin cycle is described so far. Sequences of haptophytes, cryptophytes, diatoms, and peridinin-containing dinoflagellates (complex red lineage) strongly group together in the SBP tree and the same assemblage is recovered for plastid-targeted FBP sequences, although this is less supported. Both SBP and plastid-targeted FBP are most likely of red algal origin. Including phosphoribulokinase, fructose bisphosphate aldolase, and glyceraldehyde-3-phosphate dehydrogenase, a total of five independent plastid-related nuclear-encoded markers support a common origin of all complex rhodoplasts via a single secondary endosymbiosis event. However, plastid phylogenies are incongruent with those of the host cell, as illustrated by the cytosolic FBP isoenzyme. These results are discussed in the context of Cavalier-Smith's far-reaching chromalveolate hypothesis. In our opinion, a more plausible evolutionary scenario would be the establishment of a unique secondary rhodoplast and its subsequent spread via tertiary endosymbioses.

The cryptophyte Guillardia theta harbors a plastid surrounded by four membranes. This turns protein targeting of nucleus-encoded endosymbiont localized proteins into quite a challenge, as the respective precursors have to pass either all four membranes to reach the plastid stroma or only the outermost two membranes to enter the periplastidal compartment. Therefore two sets of nuclear-encoded proteins imported into the endosymbiont can be distinguished and their topogenic signals may serve as good indicators for studying protein targeting and subsequent transport across the outermost membranes of the cryptophyte plastid. We isolated genes encoding enzymes involved in two different biochemical pathways, both of which are predicted to be localized inside the periplastidal compartment, and compared their topogenic signals to those of precursor proteins for the plastid stroma, which are encoded on either the nucleus or the nucleomorph. By this and exemplary in vitro and in vivo analyses of the topogenic signal of one protein localized in the periplastidal compartment, we present new data implicating the mechanism of targeting and transport of proteins to and across the outermost plastid membranes. Furthermore, we demonstrate that one single, but conserved amino acid is the triggering key for the discrimination between nucleus-encoded plastid and periplastidal proteins.

Background: Most genes introduced into phototrophic eukaryotes during the process of endosymbiosis are either lost or relocated into the host nuclear genome. In contrast, groEL homologues are found in different genome compartments among phototrophic eukaryotes. Comparative sequence analyses of recently available genome data, have allowed us to reconstruct the evolutionary history of these genes and propose a hypothesis that explains the unusual genome distribution of groEL homologues., Results: Our analyses indicate that while two distinct groEL genes were introduced into eukaryotes by a progenitor of plastids, these particular homologues have not been maintained in all evolutionary lineages. This is of significant interest, because two chaperone proteins always co-occur in oxygenic photosynthetic organisms. We infer strikingly different lineage specific processes of evolution involving deletion, duplication and targeting of groEL proteins., Conclusion: The requirement of two groEL homologues for chaperon function in phototrophs has provided a constraint that has shaped convergent evolutionary scenarios in divergent evolutionary lineages. GroEL provides a general evolutionary model for studying gene transfers and convergent evolutionary processes among eukaryotic lineages.

We show that phototaxis in cryptophytes is likely mediated by a two-rhodopsin-based photosensory mechanism similar to that recently demonstrated in the green alga Chlamydomonas reinhardtii, and for the first time, to our knowledge, report spectroscopic and charge movement properties of cryptophyte algal rhodopsins. The marine cryptophyte Guillardia theta exhibits positive phototaxis with maximum sensitivity at 450 nm and a secondary band above 500 nm. Variability of the relative sensitivities at these wavelengths and light-dependent inhibition of phototaxis in both bands by hydroxylamine suggest the involvement of two rhodopsin photoreceptors. In the related freshwater cryptophyte Cryptomonas sp. two photoreceptor currents similar to those mediated by the two sensory rhodopsins in green algae were recorded. Two cDNA sequences from G. theta and one from Cryptomonas encoding proteins homologous to type 1 opsins were identified. The photochemical reaction cycle of one Escherichia-coli-expressed rhodopsin from G. theta (GtR1) involves K-, M-, and O-like intermediates with relatively slow (approximately 80 ms) turnover time. GtR1 shows lack of light-driven proton pumping activity in E. coli cells, although carboxylated residues are at the positions of the Schiff base proton acceptor and donor as in proton pumping rhodopsins. The absorption spectrum, corresponding to the long-wavelength band of phototaxis sensitivity, makes this pigment a candidate for one of the G. theta sensory rhodopsins. A second rhodopsin from G. theta (GtR2) and the one from Cryptomonas have noncarboxylated residues at the donor position as in known sensory rhodopsins.

Nucleomorphs are remnant nuclei of eukaryotic, secondary endosymbionts exclusively found in cryptophytes and chlorarachniophytes. The nucleomorph of the cryptophyte Guillardia theta codes for 36 transfer RNA (tRNA) genes, 15 of them predicted to contain introns and 1 pseudo-tRNA. Some of the predicted intervening sequences are manifested at positions not known in Eukarya, even tRNAs with more than one intron were suggested. By isolating reverse transcriptase-polymerase chain reaction products of the spliced tRNAs we verify the processing of all predicted intron-harboring tRNAs and demonstrate the splicing of the smallest introns (3 nt) investigated so far. However, the spliced intervening sequences are in some cases shifted in respect to the predicted ones. Moreover, we show that introns, if inserted into the B-box of tRNA genes in the nucleomorph of cryptophytes, mimic promoter regions and do not abolish transcription by RNA polymerase III. Consequently, internal nucleomorph-encoded tRNA promoter regions are in some cases dissected from the sequence of the mature tRNAs. By reanalyzing tRNA introns of a recently sequenced red algae we furthermore show that splicing of introns at unusual positions may be introduced in cryptophytes by its secondary endosymbiont. However, in contrast to the rest of the symbiont genome, introns are not minimized in quantity but are instead scattered along the tRNA genes.

The mobilization of sulfur (SUF) system is one of three systems involved in iron-sulfur cluster biosynthesis and maintenance. In eukaryotes the SUF system is specific for the plastid and therefore of symbiotic origin. Analyses in cryptophytes showed a unique genetic compartmentalization of the SUF system, which evolved by at least two different gene transfer events. We analyzed one of the components, SufD, in the cryptophyte Guillardia theta and in Arabidopsis thaliana. We demonstrated that SufD fulfils house keeping functions during embryogenesis and in adult plants in A. thaliana.

Peridinin-containing dinoflagellates, a group of alveolate organisms, harbour small plasmids called minicircles. As most of these minicircles encode genes of cyanobacterial origin, which are also found in plastid genomes of stramenopiles, they were thought to represent the plastid genome of peridinin-containing dinoflagellates. The analyses of minicircle derived mRNAs and the 16S rRNA showed that extensive editing of minicircle gene transcripts is common for Ceratium horridum. Posttranscriptional changes occur predominantly by editing A into G, but other types of editing including a previously unreported A to C transversion were also detected. This leads to amino acid changes in most cases or, in one case, to the elimination of a stop-codon. Interestingly, the edited mRNAs show higher identities to homologous sequences of other peridinin-containing dinoflagellates than their genomic copy. Thus, our results imply that transcript editing of genes of cyanobacterial origin is species specific in peridinin-containing dinoflagellates and demonstrate that editing of genes of cyanobacterial origin is not restricted to land plants.

p-Hydroxyphenylacetate decarboxylase from Clostridium difficile catalyses the decarboxylation of p-hydroxyphenylacetate to yield the cytotoxic compound p-cresol. The three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme have been cloned and expressed in Escherichia coli. The recombinant enzymes were used to reconstitute a catalytically functional system in vitro. In contrast with the decarboxylase purified from C. difficile, which was an almost inactive homo-dimeric protein (beta(2)), the recombinant enzyme was a hetero-octameric (beta(4)gamma(4)), catalytically competent complex, which was activated using endogenous activating enzyme from C. difficile or recombinant activating enzyme to a specific activity of 7 U.mg(-1). Preliminary results suggest that phosphorylation of the small subunit is responsible for the change of the oligomeric state. These data point to an essential function of the small subunit of the decarboxylase and may indicate unique regulatory properties of the system.

The malaria parasite Plasmodium falciparum has an unusual organization of its secretory compartments. As an approach to a functional identification of auxiliary proteins involved in secretion, a parasite line was generated by drug selection that is resistant to brefeldin A, an inhibitor of the secretory pathway. In the resistant line, neither protein secretion nor parasite viability were affected by the drug. The analysis of a sec7 domain, a conserved structure of guanine nucleotide exchange factors (ARF-GEF) required for the activation of ADP-ribosylation factors, revealed a single methionine-isoleucine substitution in the resistant parasite line. ARF-GEFs are key molecules in the formation of transport vesicles and the main targets of brefeldin A. The methionine residue in this position of sec7 domains is highly conserved and confers brefeldin A sensitivity. Unlike other eukaryotes that have multiple ARF-GEFs, the plasmodial genome encodes a single sec7 domain. This domain shows a distinct structural difference to all sec7 domains analysed so far; two conserved subdomains that are essential for protein function are separated in the plasmodial protein by an insertion of 146 amino acids.

Chromophyte algae differ fundamentally from plants in possessing chloroplasts that contain chlorophyll c and that have a more complex bounding-membrane topology. Although chromophytes are known to be evolutionary chimaeras of a red alga and a non-photosynthetic host, which gave rise to their exceptional membrane complexity, their cell biology is poorly understood. Cryptomonads are the only chromophytes that still retain the enslaved red algal nucleus as a minute nucleomorph. Here we report complete sequences for all three nucleomorph chromosomes from the cryptomonad Guillardia theta. This tiny 551-kilobase eukaryotic genome is the most gene-dense known, with only 17 diminutive spliceosomal introns and 44 overlapping genes. Marked evolutionary compaction hundreds of millions of years ago eliminated nearly all the nucleomorph genes for metabolic functions, but left 30 for chloroplast-located proteins. To allow expression of these proteins, nucleomorphs retain hundreds of genetic-housekeeping genes. Nucleomorph DNA replication and periplastid protein synthesis require the import of many nuclear gene products across endoplasmic reticulum and periplastid membranes. The chromosomes have centromeres, but possibly only one loop domain, offering a means for studying eukaryotic chromosome replication, segregation and evolution.

Chloroplasts contain proteins that are encoded by different genetic systems, the plastid genome and the nuclear chromosomes. By comparing the gene content of plastid genomes of different taxa, some predictions about nuclear-encoded genes for plastid proteins are possible. However, early in evolution, many genes were transferred from the plastid to the cell nucleus and are therefore missing from all known plastid genomes and escape such predictions. By sequencing the miniaturized chromosomes of the nucleomorph of the cryptophyte Guillardia theta, as well as the plastid genome, we uncovered two genes encoding CbbX which are predicted to be involved in plastid function. Our findings suggest that (1) red-type plastid rbcLS genes evolved together with cbbX, which is related to cbbX genes of purple bacteria; (2) early in rhodoplast evolution, the cbbX gene was duplicated and transferred into the nucleus; (3) the plastid-encoded LysR transcriptional activator gene, rbcR, is homologous to rbcR and cbbR transcriptional activator genes of purple bacteria and cyanobacteria; and (4) the ancestral plastid probably harbored both types of form I RuBisCo.

Cells of several major algal groups are evolutionary chimeras of two radically different eukaryotic cells. Most of these "cells within cells" lost the nucleus of the former algal endosymbiont. But after hundreds of millions of years cryptomonads still retain the nucleus of their former red algal endosymbiont as a tiny relict organelle, the nucleomorph, which has three minute linear chromosomes, but their function and the nature of their ends have been unclear. We report extensive cryptomonad nucleomorph sequences (68.5 kb), from one end of each of the three chromosomes of Guillardia theta. Telomeres of the nucleomorph chromosomes differ dramatically from those of other eukaryotes, being repeats of the 23-mer sequence (AG)(7)AAG(6)A, not a typical hexamer (commonly TTAGGG). The subterminal regions comprising the rRNA cistrons and one protein-coding gene are exactly repeated at all three chromosome ends. Gene density (one per 0.8 kb) is the highest for any cellular genome. None of the 38 protein-coding genes has spliceosomal introns, in marked contrast to the chlorarachniophyte nucleomorph. Most identified nucleomorph genes are for gene expression or protein degradation; histone, tubulin, and putatively centrosomal ranbpm genes are probably important for chromosome segregation. No genes for primary or secondary metabolism have been found. Two of the three tRNA genes have introns, one in a hitherto undescribed location. Intergenic regions are exceptionally short; three genes transcribed by two different RNA polymerases overlap their neighbors. The reported sequences encode two essential chloroplast proteins, FtsZ and rubredoxin, thus explaining why cryptomonad nucleomorphs persist.

Cryptomonads, small biflagellate algae, contain four different genomes. In addition to the nucleus, mitochondrion, and chloroplast is a fourth DNA-containing organelle the nucleomorph. Nucleomorphs result from the successive reduction of the nucleus of an engulfed phototrophic eukaryotic endosymbiont by a secondary eukaryotic host cell. By sequencing the chloroplast genome and the nucleomorph chromosomes, we identified a groEL homologue in the genome of the chloroplast and a related cpn60 in one of the nucleomorph chromosomes. The nucleomorph-encoded Cpn60 and the chloroplast-encoded GroEL correspond in each case to one of the two divergent GroEL homologues in the cyanobacterium Synechocystis sp. PCC6803. The coexistence of divergent groEL/cpn60 genes in different genomes in one cell offers insights into gene transfer from evolving chloroplasts to cell nuclei and convergent gene evolution in chlorophyll a/b versus chlorophyll a/c/phycobilin eukaryotic lineages.