The Green Microalga Chlamydomonas reinhardtii Has a Single ω-3 Fatty Acid Desaturase That Localizes to the Chloroplast and Impacts Both Plastidic and Extraplastidic Membrane Lipids

The ω-3 polyunsaturated fatty acids account for more than 50% of total fatty acids in the green microalga Chlamydomonas reinhardtii, where they are present in both plastidic and extraplastidic membranes. In an effort to elucidate the lipid desaturation pathways in this model alga, a mutant with more than 65% reduction in total ω-3 fatty acids was isolated by screening an insertional mutant library using gas chromatography-based analysis of total fatty acids of cell pellets. Molecular genetics analyses revealed the insertion of a TOC1 transposon 113 bp upstream of the ATG start codon of a putative ω-3 desaturase (CrFAD7; locus Cre01.g038600). Nuclear genetic complementation of crfad7 using genomic DNA containing CrFAD7 restored the wild-type fatty acid profile. Under standard growth conditions, the mutant is indistinguishable from the wild type except for the fatty acid difference, but when exposed to short-term heat stress, its photosynthesis activity is more thermotolerant than the wild type. A comparative lipidomic analysis of the crfad7 mutant and the wild type revealed reductions in all ω-3 fatty acid-containing plastidic and extraplastidic glycerolipid molecular species. CrFAD7 was localized to the plastid by immunofluorescence in situ hybridization. Transformation of the crfad7 plastidial genome with a codon-optimized CrFAD7 restored the ω-3 fatty acid content of both plastidic and extraplastidic lipids. These results show that CrFAD7 is the only ω-3 fatty acid desaturase expressed in C. reinhardtii, and we discuss possible mechanisms of how a plastid-located desaturase may impact the ω-3 fatty acid content of extraplastidic lipids.Research on lipid metabolism in microalgae has flourished in recent years due to their potential as a rich source of ω-3 fatty acids (Guschina and Harwood, 2006; Khozin-Goldberg et al., 2011) and as a feedstock for biodiesel (Hu et al., 2008b; Rosenberg et al., 2008; Beer et al., 2009; Radakovits et al., 2010; Wijffels and Barbosa, 2010; Merchant et al., 2012; Work et al., 2012). Oils produced by microalgae resemble that of plants (Hu et al., 2008b), with the exception that they contain higher proportions of polyunsaturated fatty acid (PUFA) species (Harwood and Guschina, 2009). Desaturation of acyl groups in glycerolipids is catalyzed by fatty acid desaturases (FADs), which insert a C=C bond at a specifically defined position of an acyl chain (Shanklin and Cahoon, 1998). The degree of unsaturation of fatty acid components largely determines the chemical property and thus the utility of the oils produced. FADs have been one of the major tools for the genetic engineering of oil composition in land crops (Shanklin and Cahoon, 1998; Napier et al., 1999). In view of biodiesel applications, low PUFA content is advantageous in algal oil because of oxidation issues (Frankel, 1991).With the suites of sophisticated molecular genetic and genomic tools developed in the green microalga Chlamydomonas reinhardtii and the existence of substantial literature related to its cell biology, physiology, and biochemistry, this organism has emerged as a major model for research on algal oil (Radakovits et al., 2010; Merchant et al., 2012; Liu and Benning, 2013). Although the understanding of lipid metabolism in C. reinhardtii largely relies on sequence homologies to other models (Riekhof et al., 2005) and is still rather limited compared with the model plant Arabidopsis (Arabidopsis thaliana; Li-Beisson et al., 2010), functional studies based on mutants have started to provide important insights into the biosynthesis and turnover of membrane and storage lipids in this model alga (Riekhof et al., 2005; Work et al., 2010; Fan et al., 2011; Goodson et al., 2011; Boyle et al., 2012; Li et al., 2012a, 2012b; Yoon et al., 2012).In C. reinhardtii, C16 and C18 PUFAs (ω-3 + ω-6) make up to 60 mol% of total membrane fatty acids, of which more than 80% are ω-3 species (Giroud and Eichenberger, 1988; Siaut et al., 2011). Biochemical evidence for lipid-linked desaturation of fatty acyl chains has been established in C. reinhardtii over 20 years (Giroud and Eichenberger, 1989), but only two C. reinhardtii mutants affected in fatty acid desaturation have been described to date. These are crfad6 (hf-9), an insertional mutant for the plastidial ω-6 desaturase FAD6 (Sato et al., 1995), and microRNA-based silenced lines for the Δ4 desaturase CrΔ4FAD (Zäuner et al., 2012). The putative microsomal Δ12 desaturase FAD2 (Chi et al., 2008) and front-end ω-13 desaturase (Kajikawa et al., 2006) have been characterized by heterologous expression in the methylotrophic yeast Pichia pastoris, but no mutant is available. Moreover, although ω-3 PUFA is the most abundant fatty acid class in C. reinhardtii, the ω-3 desaturase remains uncharacterized, and no mutant with specific reduction in ω-3 content has been isolated so far.In Arabidopsis and C. reinhardtii, ω-3 PUFAs are present in both plastidic and extraplastidic lipids such as monogalactosyldiacylglycerol (MGDG) and phosphatidylethanolamine (PtdEtn), respectively (Mendiola-Morgenthaler et al., 1985; Giroud et al., 1988). While in plants there are distinct genes for plastidial and extraplastidial ω-3 FADs (Wallis and Browse, 2002), only one putative ω-3 desaturase seems encoded in the C. reinhardtii genome (version 5.0; Merchant et al., 2007). This raises several intriguing possibilities, including the existence of a mechanism to export ω-3 acyls from their site of biogenesis to other membranes or a dual localization of the ω-3 desaturase homolog (plastid and endoplasmic reticulum [ER]). In this study, we report the identification and characterization of a C. reinhardtii mutant defective in the promoter region of the putative ω-3 FAD encoded by the Cre01.g038600 locus. We show that while this enzyme is localized to plastids, impairment in its expression leads to a reduction of ω-3 fatty acids acylated to both plastidial and ER lipids. Additionally, using plastidial transformation of the mutant, it is demonstrated that the location of this desaturase in the plastid alone is sufficient to ensure normal ω-3 fatty acid content in extraplastidic lipids. Possible acyl desaturation and trafficking mechanisms implied by these findings are discussed.     

Diversity of Culturable Bacteria,from the Anaerobic Zone of the Meromictic Lake Pavin,Able to Perform Dissimilatory-Iron Reduction in Different in Vitro Conditions

A culture-dependent study was performed with the aim of assessing the carbon, electron and Fe(III) sources used for the dissimilatory Fe(III) reduction pathway and the diversity of culturable Fe(III)-reducers in the anoxic zone of the meromictic Lake Pavin. This metabolic pathway was investigated in enrichment cultures inoculated with water samples collected at 70 m depth in the anoxic zone of Lake Pavin. Combinations of different media, organic acids, and incubation gas phases were performed. The potential for Fe(III) reduction in the different growth conditions was assessed by measuring the accumulation of Fe(II) overtime. Bacterial community structure was determined in each growth conditions by Temporal Temperature gradient Gel Electrophoresis (TTGE) profiles of 16S rDNA genes and bands of interest in positive enrichments were sequenced. Comparisons of bacterial community structure between growth conditions revealed that the electron donor, the basal media as well as the Fe(III) source yielded to the selection of different bacterial populations, suggesting that Fe(III) reducers occupy different ecological niches in the anoxic zone of Lake Pavin. Facultative Fe(III) reducers, such as fermentative (e.g., Pseudomonas, Clostridium) and sulphate-reducing (e.g., Desulfovibrio sp.) bacteria, were retrieved in enrichments but well-known obligatory Fe(III) reducers (e.g., Geobacter) were not detected. A greater Fe(III) reduction was noted under H₂:CO₂ gas phase, suggesting that H₂ is used as an electron donor for Fe(III) reduction. Acetate was not used as a precursor for this terminal electron-accepting process, and a high Fe(III) reduction was observed with fumarate provided as the electron donor and carbon sources suggesting that this metabolite may be energetically more beneficial for Fe(III)-reducers.     

Structure and Conformation of the Branch Core Triribonucleotide Containing 2′-5′ and 3′-5′ Phosphodiester Linkages (A2′p5′G 3′p5′C) in Solution,Essential for Yeast mRNA Splicing,Deduced from 1H-NMR

Abstract

The non-exchangeable ¹H-NMR signals of the branch core trinucleotide of the lariat branch site (A^2′p5′G _3′p5′C), 1) and its derivatives 2 and 3 are completely assigned using one- and two- dimensional NMR techniques including NOE, COSY, NOESY, ¹H-¹HINADEQUATE and 2D-J-resolved spectroscopy. From the vicinal coupling constants in the individual ribose rings, NOE data and T₁ measurements, the following properties of the trimers are deduced.(i)The unique stacking behavior of the trimers is S^1′N _3′N, and the sugar rings exist predominantly in the N-conformation (3′-endo-2′-exo).(ii)The sugar-base orientations appear to be anti.(iii) The branched trimers exist in solution as single-stranded right-handed conformations resembling A-RNA with stacking between the adenine and guanine residues in aqueous solution at 21°C and pH 7.2.(iv) The calculated values for the torsion angles ε^t andγ⁺ for the trimers are 201–203° and 71–86%, respectively, while the percent β¹ values are higher for the guanine (87–92%) than the cytosine residues (73–77%). The computer generated depiction of the triribonucleotide 1 is also shown. These subtle structural features may act as recognition signals for this critical lariat branch site which is essential for the second step in yeast mRNA splicing.     

Advanced procedures for labeling of antibodies with quantum dots

Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs’ advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15 nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs’ amines with the sulfhydryl groups issued from the reduced Abs’ disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD–Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy–light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD–Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals.     

Spatial structure increases the benefits of antibiotic production in Streptomyces*

Bacteria in the soil compete for limited resources. One of the ways they might do this is by producing antibiotics, but the metabolic costs of antibiotics and their low concentrations have caused uncertainty about the ecological role of these products for the bacteria that produce them. Here, we examine the benefits of streptomycin production by the filamentous bacterium Streptomyces griseus. We first provide evidence that streptomycin production enables S. griseus to kill and invade the susceptible species, S. coelicolor, but not a streptomycin-resistant mutant of this species. Next, we show that the benefits of streptomycin production are density dependent, because production scales positively with cell number, and frequency dependent, with a threshold of invasion of S. griseus at around 1%. Finally, using serial transfer experiments where spatial structure is either maintained or destroyed, we show that spatial structure reduces the threshold frequency of invasion by more than 100-fold, indicating that antibiotic production can permit invasion from extreme rarity. Our results show that streptomycin is both an offensive and defensive weapon that facilitates invasion into occupied habitats and also protects against invasion by competitors. They also indicate that the benefits of antibiotic production rely on ecological interactions occurring at small local scales.     

Phenotypic and Genotypic Characterisation of Burkholderia cenocepacia J2315 Mutants Affected in Homoserine Lactone and Diffusible Signal Factor-Based Quorum Sensing Systems Suggests Interplay between Both Types of Systems

Many putative virulence factors of Burkholderia cenocepacia are controlled by various quorum sensing (QS) circuits. These QS systems either use N-acyl homoserine lactones (AHL) or cis-2-dodecenoic acid (“Burkholderia diffusible signal factor”, BDSF) as signalling molecules. Previous work suggested that there is little cross-talk between both types of systems. We constructed mutants in B. cenocepacia strain J2315, in which genes encoding CepI (BCAM1870), CciI (BCAM0239a) and the BDSF synthase (BCAM0581) were inactivated, and also constructed double (ΔcepIΔBCAM0581, ΔcciIΔBCAM0581 and ΔcepIΔcciI) mutants and a triple (ΔcepIΔcciIΔBCAM0581) mutant. Subsequently we investigated phenotypic properties (antibiotic susceptibility, biofilm formation, production of AHL and BDSF, protease activity and virulence in Caenorhabditis elegans) and measured gene expression in these mutants, and this in the presence and absence of added BDSF, AHL or both. The triple mutant was significantly more affected in biofilm formation, antimicrobial susceptibility, virulence in C. elegans, and protease production than either the single or double mutants. The ΔBCAM0581 mutant and the ΔcepIΔBCAM0581 and ΔcciIΔBCAM0581 double mutants produced significantly less AHL compared to the WT strain and the ΔcepI and ΔcciI single mutant, respectively. The expression of cepI and cciI in ΔBCAM0581, was approximately 3-fold and 7-fold (p<0.05) lower than in the WT, respectively. The observed differences in AHL production, expression of cepI and cciI and QS-controlled phenotypes in the ΔBCAM0581 mutant could (at least partially) be restored by addition of BDSF. Our data suggest that, in B. cenocepacia J2315, AHL and BDSF-based QS systems co-regulate the same set of genes, regulate different sets of genes that are involved in the same phenotypes and/or that the BDSF system controls the AHL-based QS system. As the expression of the gene encoding the C6-HSL synthase CciI (and to a lesser extent the C8-HSL synthase CepI) is partially controlled by BDSF, it seems likely that the BDSF QS systems controls AHL production through this system.     

Cateslytin,a Chromogranin A Derived Peptide Is Active against Staphylococcus aureus and Resistant to Degradation by Its Proteases

Innate immunity involving antimicrobial peptides represents an integrated and highly effective system of molecular and cellular mechanisms that protects host against infections. One of the most frequent hospital-acquired pathogens, Staphylococcus aureus, capable of producing proteolytic enzymes, which can degrade the host defence agents and tissue components. Numerous antimicrobial peptides derived from chromogranins, are secreted by nervous, endocrine and immune cells during stress conditions. These kill microorganisms by their lytic effect at micromolar range, using a pore-forming mechanism against Gram-positive bacteria, filamentous fungi and yeasts. In this study, we tested antimicrobial activity of chromogranin A-derived peptides (catestatin and cateslytin) against S. aureus and analysed S. aureus-mediated proteolysis of these peptides using HPLC, sequencing and MALDI-TOF mass spectrometry. Interestingly, this study is the first to demonstrate that cateslytin, the active domain of catestatin, is active against S. aureus and is interestingly resistant to degradation by S. aureus proteases.     

Structural Determinants for Protein adsorption/non-adsorption to Silica Surface

The understanding of the mechanisms involved in the interaction of proteins with inorganic surfaces is of major interest in both fundamental research and applications such as nanotechnology. However, despite intense research, the mechanisms and the structural determinants of protein/surface interactions are still unclear. We developed a strategy consisting in identifying, in a mixture of hundreds of soluble proteins, those proteins that are adsorbed on the surface and those that are not. If the two protein subsets are large enough, their statistical comparative analysis must reveal the physicochemical determinants relevant for adsorption versus non-adsorption. This methodology was tested with silica nanoparticles. We found that the adsorbed proteins contain a higher number of charged amino acids, particularly arginine, which is consistent with involvement of this basic amino acid in electrostatic interactions with silica. The analysis also identified a marked bias toward low aromatic amino acid content (phenylalanine, tryptophan, tyrosine and histidine) in adsorbed proteins. Structural analyses and molecular dynamics simulations of proteins from the two groups indicate that non-adsorbed proteins have twice as many π-π interactions and higher structural rigidity. The data are consistent with the notion that adsorption is correlated with the flexibility of the protein and with its ability to spread on the surface. Our findings led us to propose a refined model of protein adsorption.