Crucial role of conserved lysine 277 in the fidelity of tRNA aminoacylation by Escherichia coli valyl-tRNA synthetase

Valyl-tRNA synthetase (ValRS) from Escherichia coli undergoes covalent valylation by a donor valyl adenylate synthesized by the enzyme itself. ValRS could also be modified, although to a lesser extent, by the noncognate isosteric substrate L-threonine from a donor threonyl adenylate synthesized by the synthetase itself, or by the nonsubstrate methionine from methionyl adenylate produced by catalytic amounts of methionyl-tRNA synthetase. MALDI mass spectrometry analysis designated lysines 154, 162, 170, 533, 554, 593, 894, 930, and 940 of ValRS as the target residues for the attachment of valine. Following autothreonylation, lysines 162, 170, 178, 277, 291, 554, 580, 593, 861, 894, and 930 were found to be modified. Finally, L-Met-labeled residues were lysines 118, 162, 170, 178, 277, and 938. Alignment of the available ValRS amino acid sequences showed that lysines 277 and 554 are strictly conserved (with the exception concerning replacement of Lys-277 with a methionine or a tyrosine in archaebacteria), suggesting that these residues might be functionally significant. Indeed, lysine 554 of ValRS is the first lysine of the Lys-Met-Ser-Lys-Ser signature of the catalytic site of class I aminoacyl-tRNA synthetases. Lys-277 which is labeled by L-threonine or L-methionine, and not by L-valine, is located at or near the editing site, in the three-dimensional structure of ValRS. The role of lysine 277 was evaluated by site-directed mutagenesis. The Lys277Ala mutant (K277A) exhibited a posttransfer Thr-tRNA(Val) editing rate that was significantly lower than that observed for the wild-type enzyme. In addition, the K277A substitution altered amino acid discrimination in the editing site, resulting in hydrolysis of the correctly charged cognate Val-tRNA(Val). Finally, significant amounts of mischarged Thr-tRNA(Val) were produced by the K277A mutant, and not by wild-type ValRS. Altogether, our results designate Lys-277 as a likely candidate for nucleophilic attack of misacylated tRNA in the editing site of ValRS.     

Marked seasonality and high spatial variability of protist communities in shallow freshwater systems

Small eukaryotes have key roles in aquatic ecosystems, influencing their local environment, global biogeochemical cycles and climate. Their impact depends on community structure, which varies along time. However, very few studies take into account temporal variation. This is especially true for small, shallow freshwater systems, which remain largely understudied despite their wide variety, global surface and intense microbial activity. We have monthly followed changes in the community structure of small microbial eukaryotes (0.2–5 μm cell diameter) for 2 years in four ponds and one brook located in North-Western France based on massive 18S rDNA amplicon 454 pyrosequencing. We detected a total of 3742 stringently defined operational taxonomic units (OTUs) encompassing all recognized eukaryotic supergroups and lineages of uncertain affiliation. Although geographically close, protist communities in the five ecosystems were contrasting, with very few shared OTUs, suggesting that environmental selection mainly drives community structure. The temporal dynamics of different high-rank taxa appeared complex and rapid at monthly scales. Despite this, a clear and reproducible seasonality was observed. As expected, low-abundance OTUs dominated the community. Although some of them appeared sporadically or remained at low frequencies during the survey, others occasionally reached relatively high abundances, sometimes recurrently. This shows that at least a fraction of low-abundance eukaryotes constitutes a seed bank. The annual proportion of primary producers, free-living heterotrophs and parasites appeared remarkably constant among the different ecosystems, suggesting underlying trends of ecosystem carrying capacity for these functional groups.     

Efficiently Tracking Selection in a Multiparental Population: The Case of Earliness in Wheat

Multiparental populations are innovative tools for fine mapping large numbers of loci. Here we explored the application of a wheat Multiparent Advanced Generation Inter-Cross (MAGIC) population for QTL mapping. This population was created by 12 generations of free recombination among 60 founder lines, following modification of the mating system from strict selfing to strict outcrossing using the ms1b nuclear male sterility gene. Available parents and a subset of 380 SSD lines of the resulting MAGIC population were phenotyped for earliness and genotyped with the 9K i-Select SNP array and additional markers in candidate genes controlling heading date. We demonstrated that 12 generations of strict outcrossing rapidly and drastically reduced linkage disequilibrium to very low levels even at short map distances and also greatly reduced the population structure exhibited among the parents. We developed a Bayesian method, based on allelic frequency, to estimate the contribution of each parent in the evolved population. To detect loci under selection and estimate selective pressure, we also developed a new method comparing shifts in allelic frequency between the initial and the evolved populations due to both selection and genetic drift with expectations under drift only. This evolutionary approach allowed us to identify 26 genomic areas under selection. Using association tests between flowering time and polymorphisms, 6 of these genomic areas appeared to carry flowering time QTL, 1 of which corresponds to Ppd-D1, a major gene involved in the photoperiod sensitivity. Frequency shifts at 4 of 6 areas were consistent with earlier flowering of the evolved population relative to the initial population. The use of this new outcrossing wheat population, mixing numerous initial parental lines through multiple generations of panmixia, is discussed in terms of power to detect genes under selection and association mapping. Furthermore we provide new statistical methods for use in future analyses of multiparental populations.     

Sunlight-Exposed Biofilm Microbial Communities Are Naturally Resistant to Chernobyl Ionizing-Radiation Levels

Background

The Chernobyl accident represents a long-term experiment on the effects of exposure to ionizing radiation at the ecosystem level. Though studies of these effects on plants and animals are abundant, the study of how Chernobyl radiation levels affect prokaryotic and eukaryotic microbial communities is practically non-existent, except for a few reports on human pathogens or soil microorganisms. Environments enduring extreme desiccation and UV radiation, such as sunlight exposed biofilms could in principle select for organisms highly resistant to ionizing radiation as well.

Methodology/Principal Findings

To test this hypothesis, we explored the diversity of microorganisms belonging to the three domains of life by cultivation-independent approaches in biofilms developing on concrete walls or pillars in the Chernobyl area exposed to different levels of radiation, and we compared them with a similar biofilm from a non-irradiated site in Northern Ireland. Actinobacteria, Alphaproteobacteria, Bacteroidetes, Acidobacteria and Deinococcales were the most consistently detected bacterial groups, whereas green algae (Chlorophyta) and ascomycete fungi (Ascomycota) dominated within the eukaryotes. Close relatives to the most radio-resistant organisms known, including Rubrobacter species, Deinococcales and melanized ascomycete fungi were always detected. The diversity of bacteria and eukaryotes found in the most highly irradiated samples was comparable to that of less irradiated Chernobyl sites and Northern Ireland. However, the study of mutation frequencies in non-coding ITS regions versus SSU rRNA genes in members of a same actinobacterial operational taxonomic unit (OTU) present in Chernobyl samples and Northern Ireland showed a positive correlation between increased radiation and mutation rates.

Conclusions/Significance

Our results show that biofilm microbial communities in the most irradiated samples are comparable to non-irradiated samples in terms of general diversity patterns, despite increased mutation levels at the single-OTU level. Therefore, biofilm communities growing in sunlight exposed substrates are capable of coping with increased mutation rates and appear pre-adapted to levels of ionizing radiation in Chernobyl due to their natural adaptation to periodical desiccation and ambient UV radiation.     

Expression of estrogen receptor beta in prostate carcinoma cells inhibits invasion and proliferation and triggers apoptosis

The involvement of estrogen receptor beta (ERbeta) in prostate carcinogenesis has been hypothesized. Several reports have shown that ERbeta expression was decreased when prostate cells undergo neoplastic transformation, suggesting that it could play a tumor-suppressor role. By restoring ERbeta expression in prostatic carcinoma cells by adenoviral delivery, we aimed to test this hypothesis. We observed that ERbeta strongly inhibited the invasiveness and the growth of these cells. In addition, ERbeta cells were undergoing apoptosis, as shown by quantification of Bax, poly(ADP-ribose) polymerase and caspase-3 expression. Our data suggest that ERbeta acts as a tumor-suppressor by its anti-proliferative, anti-invasive and pro-apoptotic properties.     

In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves

Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5-6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues.     

Linking chloroplast relocation to different responses of photosynthesis to blue and red radiation in low and high light-acclimated leaves of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.)

Low light (LL) and high light (HL)-acclimated plants of A. thaliana were exposed to blue (BB) or red (RR) light or to a mixture of blue and red light (BR) of incrementally increasing intensities. The light response of photosystem II was measured by pulse amplitude-modulated chlorophyll fluorescence and that of photosystem I by near infrared difference spectroscopy. The LL but not HL leaves exhibited blue light-specific responses which were assigned to relocation of chloroplasts from the dark to the light-avoidance arrangement. Blue light (BB and BR) decreased the minimum fluorescence (\(F_{0}^{\prime }\)) more than RR light. This extra reduction of the \(F_{0}^{\prime }\) was stronger than theoretically predicted for \(F_{0}^{\prime }\) quenching by energy dissipation but actual measurement and theory agreed in RR treatments. The extra \(F_{0}^{\prime }\) reduction was assigned to decreased light absorption of chloroplasts in the avoidance position. A maximum reduction of 30% was calculated. Increasing intensities of blue light affected the fluorescence parameters NPQ and q_P to a lesser degree than red light. After correcting for the optical effects of chloroplast relocation, the NPQ responded similarly to blue and red light. The same correction method diminished the color-specific variations in q_P but did not abolish it; thus strongly indicating the presence of another blue light effect which also moderates excitation pressure in PSII but cannot be ascribed to absorption variations. Only after RR exposure, a post-illumination overshoot of \(F_{0}^{\prime }\) and fast oxidation of PSI electron acceptors occurred, thus, suggesting an electron flow from stromal reductants to the plastoquinone pool.     

Mutation of the Protein Kinase C Site in Borna Disease Virus Phosphoprotein Abrogates Viral Interference with Neuronal Signaling and Restores Normal Synaptic Activity

Understanding the pathogenesis of infection by neurotropic viruses represents a major challenge and may improve our knowledge of many human neurological diseases for which viruses are thought to play a role. Borna disease virus (BDV) represents an attractive model system to analyze the molecular mechanisms whereby a virus can persist in the central nervous system (CNS) and lead to altered brain function, in the absence of overt cytolysis or inflammation. Recently, we showed that BDV selectively impairs neuronal plasticity through interfering with protein kinase C (PKC)–dependent signaling in neurons. Here, we tested the hypothesis that BDV phosphoprotein (P) may serve as a PKC decoy substrate when expressed in neurons, resulting in an interference with PKC-dependent signaling and impaired neuronal activity. By using a recombinant BDV with mutated PKC phosphorylation site on P, we demonstrate the central role of this protein in BDV pathogenesis. We first showed that the kinetics of dissemination of this recombinant virus was strongly delayed, suggesting that phosphorylation of P by PKC is required for optimal viral spread in neurons. Moreover, neurons infected with this mutant virus exhibited a normal pattern of phosphorylation of the PKC endogenous substrates MARCKS and SNAP-25. Finally, activity-dependent modulation of synaptic activity was restored, as assessed by measuring calcium dynamics in response to depolarization and the electrical properties of neuronal networks grown on microelectrode arrays. Therefore, preventing P phosphorylation by PKC abolishes viral interference with neuronal activity in response to stimulation. Our findings illustrate a novel example of viral interference with a differentiated neuronal function, mainly through competition with the PKC signaling pathway. In addition, we provide the first evidence that a viral protein can specifically interfere with stimulus-induced synaptic plasticity in neurons.