CGFS-type monothiol glutaredoxins from the cyanobacterium Synechocystis PCC6803 and other evolutionary distant model organisms possess a glutathione-ligated [2Fe-2S] cluster

When produced in Escherichia coli, the CGFS-type monothiol Grxs from this organism (EcGrx4p) and the model cyanobacterium Synechocystis (SyGrx3p) exist as a dimeric iron-sulfur containing holoprotein or as a monomeric apoprotein in solution. Spectroscopic and site-directed mutagenesis analyses show that the SyGrx3 holoprotein contains a subunit-bridging [2Fe-2S] cluster that is ligated by the catalytic cysteine located in the CGFS motif of each monomer and the cysteines of two molecules of glutathione. The biochemical characterization of several monothiol Grxs from the cyanobacteria Gloeobacter violaceus (GvGrx3p) and Thermosynechococcus elongatus (TeGrx3p), the yeast Saccharomyces cerevisiae (ScGrx3p, ScGrx4p, and ScGrx5p), the plant Arabidopsis thaliana (AtGrx5p), and human (HsGrx5p) indicate that the incorporation of a GSH-ligated [2Fe-2S] center is a common feature of prokaryotic and eukaryotic CGFS-active site monothiol Grxs. In light of these results, the involvement of these enzymes in the sensing of iron and/or the biogenesis and transfer of Fe-S cluster is discussed.     

Endoplasmic reticulum-quality control chaperones facilitate the biogenesis of Cf receptor-like proteins involved in pathogen resistance of tomato

Cf proteins are receptor-like proteins (RLPs) that mediate resistance of tomato (Solanum lycopersicum) to the foliar pathogen Cladosporium fulvum. These transmembrane immune receptors, which carry extracellular leucine-rich repeats that are subjected to posttranslational glycosylation, perceive effectors of the pathogen and trigger a defense response that results in plant resistance. To identify proteins required for the functionality of these RLPs, we performed immunopurification of a functional Cf-4-enhanced green fluorescent protein fusion protein transiently expressed in Nicotiana benthamiana, followed by mass spectrometry. The endoplasmic reticulum (ER) heat shock protein70 binding proteins (BiPs) and lectin-type calreticulins (CRTs), which are chaperones involved in ER-quality control, were copurifying with Cf-4-enhanced green fluorescent protein. The tomato and N. benthamiana genomes encode four BiP homologs and silencing experiments revealed that these BiPs are important for overall plant viability. For the three tomato CRTs, virus-induced gene silencing targeting the plant-specific CRT3a gene resulted in a significantly compromised Cf-4-mediated defense response and loss of full resistance to C. fulvum. We show that upon knockdown of CRT3a the Cf-4 protein accumulated, but the pool of Cf-4 protein carrying complex-type N-linked glycans was largely reduced. Together, our study on proteins required for Cf function reveals an important role for the CRT ER chaperone CRT3a in the biogenesis and functionality of this type of RLP involved in plant defense.     

Systematic review of Atractus schach (Serpentes: Dipsadidae) species complex from the Guiana Shield with description of three new species

The Guiana Shield harbours one of the best preserved and largest extents of tropical forest on Earth and an immense biodiversity. The herpetofauna of this region remains poorly known. The species-rich snake genus Atractus contains ～140 species, many with complicated taxonomic histories, including A. schach. Examination of specimens in museums and newly collected material from French Guiana has allowed the illustration of hemipenial morphology for the first time and an expanded diagnosis. Concatenated molecular phylogenetic (mitochondrial and nuclear genes) and phenotypic (morphometrics, external and hemipenial morphology) analyses confirm non-monophyly of the A. flammigerus group and indicate that A. schach is a species complex with three new species described here. The geographic distribution of A. schach sensu stricto is restricted to Guiana, Surinam, and French Guiana north of Tumucumaque massif. Populations tentatively assigned to A. schach from the east from French Guiana in the Roura lowlands to Almeirim, and from central Amazonia between the Negro and Trombetas rivers in Brazil are also recognized as new species. Our results suggest that populations from south of the Amazon River are not conspecific with those from the Guiana Shield.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:A7AE40BC-4716-4302-B3BE-1F43600B0A72     

Herbicide multiple-resistance in a Lolium rigidum biotype is endowed by multiple mechanisms: isolation of a subset with resistant acetyl-CoA carboxylase

The development of herbicide multiple-resistance in weed species represents a major threat to current agricultural practices. The mechanistic basis for herbicide multiple-resistance has been investigated in a population of the annual grass weed Lolium rigidum Gaud. (annual ryegrass) resistant to herbicides affecting 6 target sites. A subset of the resistant population (R₂ subset) has been isolated by germination on a medium containing the acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) inhibiting herbicide, sethoxydim ((2-[1-(ethoxyimino)butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one)). This 12% R₂ subset of the population is 600 times more resistant to sethoxydim and between 30 to 200 times more resistant to other ACCase inhibitors than the bulk of the R population. The subset has a form of ACCase which is 6 to 55 times less sensitive to inhibition by these herbicides than the enzyme present in the bulk of the resistant or in the susceptible population. There was no difference in the uptake and metabolic degradation of [4-¹⁴C]sethoxydim between the R₂ subset and the unselected R population. These results show the accumulation of different resistance mechanisms in that single population. Furthermore we propose that this accumulation of multiple resistance mechanisms is the basis for herbicide multiple-resistance in this biotype.     

Phytochemical analysis and in vitro evaluation of the biological activity against herpes simplex virus type 1 (HSV-1) of Cedrus libani A. Rich.

Cedrus libani are widely used as traditional medicine in Lebanon for treatment of different infection diseases. In the present study we reported the phytochemical composition analyzed by GC-MS of wood essential oil and cones and leaves ethanol extracts. The main components of wood essential oil were himachalol (22.50%), beta-himachalene (21.90%), and alpha-himachalene (10.50%). Leaves ethanol extract was characterized by a high content of germacrene d (29.40%). The same extract obtained from cones essentially contained alpha-pinene (51.0%) and beta-myrcene (13.0%). Moreover, we investigated extracts, essential oil, and identified compounds for their in vitro antiviral activities against herpes simplex virus type 1 (HSV-1). Cytotoxicity was evaluated by MTT assay in Vero cells. Cones and leaves ethanol extracts exhibited an interesting activity with IC50 of 0.50 and 0.66 mg/ml, respectively, at non-cytotoxic concentration. A comparable activity was found when essential oil was tested (IC50 of 0.44 mg/ml).     

mKikGR,a Monomeric Photoswitchable Fluorescent Protein

The recent demonstration and utilization of fluorescent proteins whose fluorescence can be switched on and off has greatly expanded the toolkit of molecular and cell biology. These photoswitchable proteins have facilitated the characterization of specifically tagged molecular species in the cell and have enabled fluorescence imaging of intracellular structures with a resolution far below the classical diffraction limit of light. Applications are limited, however, by the fast photobleaching, slow photoswitching, and oligomerization typical for photoswitchable proteins currently available. Here, we report the molecular cloning and spectroscopic characterization of mKikGR, a monomeric version of the previously reported KikGR that displays high photostability and switching rates. Furthermore, we present single-molecule imaging experiments that demonstrate that individual mKikGR proteins can be localized with a precision of better than 10 nanometers, suggesting their suitability for super-resolution imaging.     

Heterologous production, assembly, and secretion of a minicellulosome by Clostridium acetobutylicum ATCC 824

The gene man5K encoding the mannanase Man5K from Clostridium cellulolyticum was cloned alone or as an operon with the gene cipC1 encoding a truncated scaffoldin (miniCipC1) of the same origin in the solventogenic Clostridium acetobutylicum. The expression of the heterologous gene(s) was under the control of a weakened thiolase promoter Pthl. The recombinant strains of the solventogenic bacterium were both found to secrete active Man5K in the range of milligrams per liter. In the case of the strain expressing only man5K, a large fraction of the recombinant enzyme was truncated and lost the N-terminal dockerin domain, but it remained active towards galactomannan. When man5K was coexpressed with cipC1 in C. acetobutylicum, the recombinant strain secreted almost exclusively full-length mannanase, which bound to the scaffoldin miniCipC1, thus showing that complexation to the scaffoldin stabilized the enzyme. The secreted heterologous complex was found to be functional: it binds to crystalline cellulose via the carbohydrate binding module of the miniscaffoldin, and the complexed mannanase is active towards galactomannan. Taken together, these data show that C. acetobutylicum is a suitable host for the production, assembly, and secretion of heterologous minicellulosomes.