Phospho-mimicry mutant of atToc33 affects early development of Arabidopsis thaliana

The precursor protein receptor at the chloroplast outer membrane atToc33 is a GTPase, which can be inactivated by phosphorylation in vitro, being arrested in the GDP loaded state. To assess the physiological function of phosphorylation, attoc33 knock out mutants were complemented with a mutated construct mimicking the constitutively phosphorylated state. Our data suggest that the reduced functionality of the mutant protein can be compensated by its upregulation. Chloroplast biogenesis and photosynthetic activity are impaired in the mutants during the early developmental stage, which is consistent with the requirement of atToc33 in young photosynthetic tissues.     

Erysiphe abbreviata on cherry bark oak--morphology, phylogeny and taxonomy

Powdery mildew on cherry bark oak (Quercus falcate var. pagodifolia) collected in Tennessee, USA, was determined to be Erysiphe abbreviata, a species confined to North America. The diagnostically important anamorph of this species is described for the first time. Sequence analyses of the rDNA ITS region and D1/D2 domains of the 28S rDNA were used to obtain phylogenetic data for and taxonomic conclusions about this species. The structure of the anamorph (Oidium subgen. Pseudoidium) and the molecular data support the placement of this species in Erysiphe emend. (including Microsphaera) as a species separate from the Eurasian Erysiphe alphitoides.     

The Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2-alkyl-4(1H)quinolones

The apicomplexan parasite Toxoplasma gondii does not possess complex I of the mitochondrial respiratory chain, but has two genes encoding rotenone-insensitive, non-proton pumping type-II NADH dehydrogenases (NDH2s). The absence of such “alternative” NADH dehydrogenases in the human host defines these enzymes as potential drug targets. TgNDH2-I and TgNDH2-II are constitutively expressed in tachyzoites and bradyzoites and are localized to the mitochondrion as shown by epitope tagging. Functional expression of TgNDH2-I in the yeast Yarrowia lipolytica as an internal enzyme, with the active site facing the mitochondrial matrix, permitted growth in the presence of the complex I inhibitor DQA. Bisubstrate kinetics of TgNDH2-I measured within Y. lipolytica mitochondrial membrane preparations were in accordance with a ping-pong mechanism. Using inhibition kinetics we demonstrate here that 1-hydroxy-2-alkyl-4(1)quinolones with long alkyl chains of C₁₂ (HDQ) and C₁₄ are high affinity inhibitors for TgNDH2-I, while compounds with shorter side chains (C₅ and C₆) displayed significantly higher IC₅₀ values. The efficiency of the various quinolone derivatives to inhibit TgNDH2-I enzyme activity mirrors their inhibitory potency in vivo, suggesting that a long acyl site chain is critical for the inhibitory potential of these compounds.     

Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus

The interaction between the c(11)ring and the gammaepsilon complex, forming the rotor of the Ilyobacter tartaricus ATP synthase, was probed by surface plasmon resonance spectroscopy and in vitro reconstitution analysis. The results provide, for the first time, a direct and quantitative assessment of the stability of the rotor. The data indicated very tight binding between the c(11)ring and the gammaepsilon complex, with an apparent K(d) value of approximately 7.4nm. The rotor assembly was primarily dependent on the interaction of the cring with the gammasubunit, and binding of the cring to the free epsilon subunit was not observed. Mutagenesis of selected conserved amino acid residues of all three rotor components (cR45, cQ46, gammaE204, gammaF203 and epsilonH38) severely affected rotor assembly. The interaction kinetics between the gammaepsilon complex and c(11)ring mutants suggested that the assembly of the c(11)gammaepsiloncomplex was governed by interactions of low and high affinity. Low-affinity binding was observed between the polar loops of the cring subunits and the bottom part of the gamma subunit. High-affinity interactions, involving the two residues gammaE204 and epsilonH38, stabilized the holo-c(11)gammaepsilon complex. NMR experiments indicated the acquisition of conformational order in otherwise flexible C- and N-terminal regions of the gamma subunit on rotor assembly. The results of this study suggest that docking of the central stalk of the F(1)complex to the rotor ring of F(o) to form tight, but reversible, contacts provides an explanation for the relative ease of dissociation and reconstitution of F(1)F(o)complexes.     

Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain

The production of fuel ethanol from low‐cost lignocellulosic biomass currently suffers from several limitations. One of them is the presence of inhibitors in lignocellulosic hydrolysates that are released during pre‐treatment. These compounds inhibit growth and hamper the production of ethanol, thereby affecting process economics. To delineate the effects of such complex mixtures, we conducted a chemical analysis of four different real‐world lignocellulosic hydrolysates and determined their toxicological effect on yeast. By correlating the potential inhibitor abundance to the growth‐inhibiting properties of the corresponding hydrolysates, we identified furfural as an important contributor to hydrolysate toxicity for yeast. Subsequently, we conducted a targeted evolution experiment to improve growth behaviour of the half industrial Saccharomyces cerevisiae strain TMB3400 in the hydrolysates. After about 300 generations, representative clones from these evolved populations exhibited significantly reduced lag phases in medium containing the single inhibitor furfural, but also in hydrolysate‐supplemented medium. Furthermore, these strains were able to grow at concentrations of hydrolysates that effectively killed the parental strain and exhibited significantly improved bioconversion characteristics under industrially relevant conditions. The improved resistance of our evolved strains was based on their capacity to remain viable in a toxic environment during the prolonged, furfural induced lag phase.     

Dynamics of organic acid occurrence under flooding stress in the rhizosphere of three plant species from the water fluctuation zone of the Three Gorges Reservoir, P.R. China

The effects of flooding on rhizospheric organic acid concentrations of three abundant flooding tolerant plant species (Alternanthera philoxeroides Mart., Arundinella anomala Steud., Salix variegata Franch.) from the water fluctuation zone of the Three Gorges Reservoir (TGR, Yangtze River) were investigated. Soil solution samples of eight low molecular weight organic acids were obtained from rhizotrons using micro suction cups during 3 weeks of waterlogging, after 6 weeks flooding and after a 1 week recovery. To estimate the contribution of water temperature and microbial community, plants in sterile glass bead substrate and original Yangtze sediment were submerged in laboratory at +10°, +20° and +30°C. Waterlogged plants did seldom express a significantly different pattern of rhizospheric organic acid (OA) composition compared to control plants. Flooding caused no burst of organic acid concentration in soil solution: All species express a silencing strategy. Average OA levels were higher in A. anomala rhizosphere than in the other two species, but increased again after resurfacing in all species. Temperature had a stronger influence in sediment than in sterile setup. In contrast to field measurements, succinate, malate and citrate were detected in the sterile setup. Microbial contribution appeared to have great influence on increasing OA occurrence.     

An Unusual ERAD-Like Complex Is Targeted to the Apicoplast of Plasmodium falciparum

Many apicomplexan parasites, including Plasmodium falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event. The exact molecular mechanisms directing the transport of nuclear-encoded proteins to the apicoplast of P. falciparum are not well understood. Recently, in silico analyses revealed a second copy of proteins homologous to components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) system in organisms with secondary plastids, including the malaria parasite P. falciparum. These proteins are predicted to be endowed with an apicoplast targeting signal and are suggested to play a role in the transport of nuclear-encoded proteins to the apicoplast. Here, we have studied components of this ERAD-derived putative preprotein translocon complex in malaria parasites. Using transfection technology coupled with fluorescence imaging techniques we can demonstrate that the N terminus of several ERAD-derived components targets green fluorescent protein to the apicoplast. Furthermore, we confirm that full-length PfsDer1-1 and PfsUba1 (homologues of yeast ERAD components) localize to the apicoplast, where PfsDer1-1 tightly associates with membranes. Conversely, PfhDer1-1 (a host-specific copy of the Der1-1 protein) localizes to the ER. Our data suggest that ERAD components have been “rewired” to provide a conduit for protein transport to the apicoplast. Our results are discussed in relation to the nature of the apicoplast protein transport machinery.The apicomplexan parasite Plasmodium falciparum is the etiological agent of malaria tropica, the most severe form of human malaria, responsible for over 250 million infections and 1 million deaths annually (61). Many apicomplexan parasites, including P. falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event (27, 58). Although during the course of evolution this plastid organelle has lost the ability to carry out photosynthesis, it is still the site of several important biochemical pathways, including isoprenoid and heme biosynthesis, and as such is essential for parasite survival (60). As in other plastids, the vast majority of genes originally encoded on the plastid genome have been transferred to the nucleus of the host. As a result, their gene products (predicted to constitute up to 10% of all nucleus-encoded proteins) must be imported back into the apicoplast (12). The apicoplast is surrounded by four membranes (55), and this protein import process thus represents a major cell biological challenge and has attracted much research interest, not least due to the importance of P. falciparum as a human pathogen (16, 50).The signals directing transport of nucleus-encoded proteins to complex plastids, including the apicomplexan apicoplast, have been studied in great detail in recent years, and reveal that such proteins are endowed with specific N-terminal targeting sequences, referred to as a bipartite topogenic signals (BTS), that direct their transport to this compartment (50). BTS are composed of an N-terminal endoplasmic reticulum (ER)-type signal sequence, which initially allows proteins to enter the secretory system via the Sec61 complex (59). Following this, proteins are carried via a Golgi complex-independent transport step to the second outermost membrane, from where they are then translocated across the remaining three apicoplast membranes, directed by the second part of the BTS, the transit peptide (51). Based on evolutionary considerations, it has long been suggested that transport across the inner two apicoplast membranes occurs via a Toc/Tic-like (where Toc and Tic are translocons of the outer and inner chloroplast envelopes, respectively) protein translocase machinery, and this is supported by a recent publication that provides evidence for an essential role of a Toxoplasma gondii Tic20 homologue in this transport process (50, 57). Despite this progress, it is still unclear how proteins travel across the second and third outer apicoplast membranes. Several models have been discussed to account for this transport step, including vesicular shuttle and translocon-based mechanisms (recently reviewed in reference 19), but until recently no actual molecular equipment had been found which could account for these membrane translocation events. To address this question, Sommer et al. screened the nucleomorph genome of the chromalveolate cryptophyte Guillardia theta (which, similar to P. falciparum, contains a four-membrane-bound plastid organelle) for genes encoding potential translocon-related proteins (49). Surprisingly, the authors identified genes encoding proteins usually involved in the ER-associated protein degradation pathway (ERAD), which recognizes incorrectly folded protein substrates and retrotranslocates them to the cell cytosol for degradation by the ubiquitin (Ub)-proteasome system (35, 44). As such, the ERAD system functions as a translocation complex, capable of transporting proteins across a biological membrane. Further characterization of one of these proteins (G. theta Der1-1, a homologue of yeast Der1p, a component of the ERAD system) provided strong evidence for a plastid localization. These data suggested an attractive solution to the mechanistic problem of transport across the second and third outermost membrane of complex plastids by hypothesizing a role for an ERAD-derived protein translocon complex. Intriguingly, this study also identified several members of this ERAD-derived translocon complex (apicoplast ERAD [apERAD]) in the nuclear genome of P. falciparum endowed with an N-terminal BTS (49). The BTS derived from one of these proteins, P. falciparum sDer1-1 [PfsDer1-1], was sufficient to direct transport of green fluorescent protein (GFP) to the apicoplast of P. falciparum, suggesting that this ERAD-like machinery is ubiquitous among chromalveolates with four membrane-bound plastids (49). In this current report we extend our study of the P. falciparum apERAD complex.     

A comparative approach to study inhibition of grazing and lipid composition of a toxic and non-toxic clone of Chrysochromulina polylepis (Prymnesiophyceae)

Since the massive bloom in 1988 in the North Sea, the prymnesiophyte flagellate Chrysochromulina polylepis Manton et Parke has been known for its ichtyotoxicity. Laboratory experiments using two different clones of C. polylepis were conducted in a comparative approach. Both clones were nearly similar in size and shape, but differed in their toxicity, as demonstrated by the Artemia bioassay. In order to study the effects of toxic C. polylepis on protozooplankton grazers, grazing experiments were performed with the heterotrophic dinoflagellate Oxyrrhis marina Dujardin as grazer. A first experiment was carried out in order to follow batch culture growth and initial grazing of O. marina when fed toxic or non-toxic clones of C. polylepis. Ingestion of the toxic clone was 27% of ingestion when fed with the non-toxic clone. When O. marina was fed with the toxic clone, vacuoles within O. marina contained fewer food particles per cell and the cells attained slower division rate (58% of the division rate estimated for the non-toxic clone). A second experiment was conducted to determine the grazing and growth response of O. marina as a function of algal food concentration. Profound differences in ingestion, clearance, division and gross growth efficiency of O. marina when fed the two clones of C. polylepis again were apparent. However, even at algal concentrations of 400×10³ ml⁻¹, O. marina is not killed by the presence or by the ingestion of toxic C. polylepis, indicating that the toxin deters grazers. In addition to grazing experiments, lipid classes and fatty acids of both algal clones were analysed and compared in order to follow the hypothesis that toxicity of C. polylepis is caused by liposaccharides, lipids, or fatty acids. However, the chemical composition with respect to lipid classes and fatty acids of both clones were quite similar, making an involvement of these substances in the toxicity towards Artemia and O. marina unlikely.     

Lead concentrations and lead and strontium stable-isotope ratios in teeth of European roe deer (Capreolus capreolus)

The study reports lead concentrations and lead and strontium stable-isotope ratios in mandibular molars of roe deer from three different areas in western Germany. Lead concentrations in third molars ranged between 0.23 and 36.61 μg/g (dry weight). Comparing lead concentrations in first molars and third molars in a group of ca. 1.5- or ca. 2.5-year-old individuals from the same area revealed an effect of tooth age on tooth lead content. The higher lead concentrations in the first molars were attributed to the longer period of lead accumulation by the dentin of these teeth compared with the later-forming third molars. Differences in lead isotopic signatures of the teeth were observed between the three areas, presumably reflecting variation in exposure to different sources of environmental lead. We also found marked variation in the ⁸⁷Sr/⁸⁶Sr isotope ratios of the teeth, with only a small overlap in values between two of the areas. Strontium isotope analysis alone or in combination with lead isotope analyses can be a useful means of assessing the provenance of deer teeth of unknown geographical origin.