Calcium carbonate formation by Synechococcus sp. strain PCC 8806 and Synechococcus sp. strain PCC 8807

Precipitation of CaCO3 catalyzed by the growth and physiology of cyanobacteria in the genus Synechococcus represents a potential mechanism for sequestration of atmospheric CO2 produced during the burning of coal for power generation. Synechococcus sp. strain PCC 8806 and Synechococcus sp. strain PCC 8807 were tested in microcosm experiments for their ability to calcify when exposed to a fixed calcium concentration of 3.4 mM and dissolved inorganic carbon concentrations of 0.5, 1.25 and 2.5 mM. Synechococcus sp. strain PCC 8806 removed calcium continuously over the duration of the experiment producing approximately 18.6 mg of solid phase calcium. Calcium removal occurred over a two-day time period when Synechococcus sp. strain PCC 8807 was tested and only 8.9 mg of solid phase calcium was produced. Creation of an alkaline growth environment catalyzed by the physiology of the cyanobacteria appeared to be the primary factor responsible for CaCO3 precipitation in these experiments.     

Enhancement of Carotenoid Biosynthesis in Transplastomic Tomatoes by Induced Lycopene-to-Provitamin A Conversion

Carotenoids are essential pigments of the photosynthetic apparatus and an indispensable component of the human diet. In addition to being potent antioxidants, they also provide the vitamin A precursor β-carotene. In tomato (Solanum lycopersicum) fruits, carotenoids accumulate in specialized plastids, the chromoplasts. How the carotenoid biosynthetic pathway is regulated and what limits total carotenoid accumulation in fruit chromoplasts is not well understood. Here, we have introduced the lycopene β-cyclase genes from the eubacterium Erwinia herbicola and the higher plant daffodil (Narcissus pseudonarcissus) into the tomato plastid genome. While expression of the bacterial enzyme did not strongly alter carotenoid composition, expression of the plant enzyme efficiently converted lycopene, the major storage carotenoid of the tomato fruit, into provitamin A (β-carotene). In green leaves of the transplastomic tomato plants, more lycopene was channeled into the β-branch of carotenoid biosynthesis, resulting in increased accumulation of xanthophyll cycle pigments and correspondingly reduced accumulation of the α-branch xanthophyll lutein. In fruits, most of the lycopene was converted into β-carotene with provitamin A levels reaching 1 mg per g dry weight. Unexpectedly, transplastomic tomatoes also showed a >50% increase in total carotenoid accumulation, indicating that lycopene β-cyclase expression enhanced the flux through the pathway in chromoplasts. Our results provide new insights into the regulation of carotenoid biosynthesis and demonstrate the potential of plastids genome engineering for the nutritional enhancement of food crops.Carotenoids are isoprenoid molecules that are synthesized by all photosynthetic organisms and also by some fungi and nonphotosynthetic bacteria. In plants, they participate in photosynthetic light harvesting and protection against light stress. In addition, carotenoids accumulate to large levels as storage metabolites in chromoplasts of flowers, fruits, and taproots. Carotenoids are also essential to animals, which, however, are unable to synthesize them de novo, and therefore must rely on dietary sources of carotenoids. β-Carotene is the main dietary precursor of vitamin A and therefore also referred to as provitamin A. Vitamin A deficiency in humans represents a global health problem affecting approximately one-third of the countries of the world (Mayer et al., 2008). Presumably due to their antioxidant activity, β-carotene and other carotenoid species also exert protective effects against cardiovascular diseases, certain cancers, and aging-related diseases (Collins, 1999).While the enzymology of the carotenoid biosynthetic pathways in plants and eubacteria is now reasonably well understood (Armstrong, 1997; Cunningham and Gantt, 1998; Hirschberg, 2001), understanding of the regulation of carotenoid biosynthesis is still rather poor (Bramley, 2002). Mainly using the tomato (Solanum lycopersicum) fruit as model system, the study of pigmentation mutants (Ronen et al., 2000; Isaacson et al., 2002; Galpaz et al., 2006) and transgenic approaches (Giuliano et al., 2000, 2008; Römer and Fraser, 2005; Fraser et al., 2007) have provided first insights into regulatory mechanisms operating in carotenogenesis. For example, constitutive expression of the phytoene desaturase (crtI) gene from the bacterium Erwinia uredovora resulted in elevated β-carotene accumulation in tomatoes, but also led to an unexpected reduction in total carotenoid levels (Römer et al., 2000). The reduction in total carotenoids is believed to be an effect of feedback regulation from β-carotene or one of its downstream metabolites (Bramley, 2002). However, fruit-specific overexpression of the native lycopene β-cyclase resulted in increased β-carotene accumulation, without a concomitant decrease in total carotenoids (Rosati et al., 2000). Why some genetic disturbances of carotenoid biosynthesis negatively affect total carotenoid accumulation and others do not (or even result in an increase; Dharmapuri et al., 2002; Fraser et al., 2002), remains to be established.Here we have used tomato plastid transformation to address the regulation of carotenoid biosynthesis exerted at the level of lycopene to β-carotene conversion by the enzyme lycopene β-cyclase (Fig. 1A). We show that plastid expression of a plant lycopene β-cyclase does not only trigger efficient conversion of lycopene to β-carotene, but unexpectedly also results in a >50% increase in total carotenoid accumulation. This contrasts moderately increased β-carotene levels and reduced total carotenoid accumulation upon expression of a bacterial lycopene β-cyclase (Wurbs et al., 2007) and suggests lycopene β-cyclase activity as an important regulatory point in plant and microbial carotenoid biosynthesis.Open in a separate windowFigure 1.Engineering of the carotenoid biosynthetic pathway by plastid transformation. A, Carotenoid biosynthetic pathway in higher plants. The pathway splits into an α-branch and a β-branch immediately downstream of lycopene, the major storage carotenoid of tomato fruits. The enzyme expressed from the tomato plastid genome in this study, lycopene β-cyclase, leads into the β-branch. B, Physical maps of the targeting region in the plastid genome (ptDNA) and the plastid transformation vectors pEcrtY and pNLyc constructed in this study. Genes above the line are transcribed from the left to the right, genes below the line are transcribed in the opposite direction. The transgenes are targeted to the intergenic region between the trnfM and trnfG genes (Ruf et al., 2001). The selectable marker gene aadA is driven by a chimeric rRNA operon promoter (Prrn; Svab and Maliga, 1993), fused to the 3′-UTR from the psbA gene (TpsbA), and flanked by two loxP sites to allow marker removal by Cre-mediated site-specific recombination (Zhou et al., 2008). The transgene expression cassette consists of the ribosomal RNA operon promoter fused to the 5′ leader from the gene 10 of phage T7 (Prrn-G10L; Kuroda and Maliga, 2001) and the 3′-UTR of the rps16 gene (Trps16). Restriction sites used for cloning or RFLP analysis are indicated, and the psaB-derived hybridization probe is denoted by a horizontal bar. Sites lost due to ligation to heterologous ends are in parentheses. C, Southern-blot analysis of tomato transplastomic lines carrying the lycopene β-cyclase gene from daffodil (S.l.-pNLyc) or from E. herbicola (S.l.-pEcrtY). Total cellular DNA was digested with BglII and hybridized to a radioactively labeled probe detecting the psaB region of the plastid genome, which flanks the transgene insertion site (section B). Fragment sizes are given in kb. wt, Wild type. D, Alignment (produced with ClustalW2) of the amino acid sequences of the lycopin β-cyclases from daffodil (Np) and E. herbicola (Eh). Asterisk (*) denotes residues identical in both sequences (marked in bold), colon (:) indicates conserved substitutions, and a dot indicates semiconserved substitutions. The N-terminal extension of the Np sequence is likely to harbor the transit peptide for protein import into plastids. The amino acids that changed due to correction of the Lyc sequence from daffodil (published sequence: GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"X98796.1","term_id":"1419692"}}X98796.1; corrected sequence: accession no. {"type":"entrez-nucleotide","attrs":{"text":"GQ327929","term_id":"254681605"}}GQ327929) are underlined. The corrections improve the sequence similarity in the N-terminal domains of the Np and Eh sequences.     

Suitability of human mesenchymal stem cells for gene therapy depends on the expansion medium

Great hope is set in the use of mesenchymal stem cells for gene therapy and regenerative medicine. Since the frequency of this subpopulation of stem cells in bone marrow is low, mesenchymal stem cells are expanded ex vivo and manipulated prior to experimental or clinical use. Different methods for isolation and expansion are available, but the particular effect on the stem cell character is unclear. While the isolation of mesenchymal stem cells by density centrifugation followed by selection of the plastic adherent fraction is frequently used, the composition of expansion media differs. Thus, in the present study we cultured mesenchymal stem cells isolated from five healthy young volunteers in three widely used expansion media and performed a detailed analysis of the effect on morphology, proliferation, clonogenicity, passaging, differentiation and senescence. By this way we clearly show that the type of expansion medium used determines the stem cell character and time of senescence which is critical for future gene therapeutic and regenerative approaches using mesenchymal stem cells.     

Arabidopsis mutants reveal multiple singlet oxygen signaling pathways involved in stress response and development

Shortly after the release of singlet oxygen (¹O₂) in chloroplasts drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. Factors involved in this retrograde signaling were identified by mutagenizing a transgenic flu line expressing a ¹O₂-responsive reporter gene. The reporter gene consisted of the luciferase open reading frame and the promoter of an AAA-ATPase gene (At3g28580) that was selectively activated by ¹O₂ but not by superoxide or hydrogen peroxide. A total of eight second-site mutants were identified that either constitutively activate the reporter gene and the endogenous AAA-ATPase irrespectively of whether ¹O₂ was generated or not (constitutive activators of AAA-ATPase, caa) or abrogated the ¹O₂-dependent up-regulation of these genes as seen in the transgenic parental flu line (non-activators of AAA-ATPase, naa). The characterization of the mutants strongly suggests that ¹O₂-signaling does not operate as an isolated linear pathway but rather forms an integral part of a signaling network that is modified by other signaling routes and impacts not only stress responses of plants but also their development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Aiswarya Baruah and Klára Šimková contributed equally to the article.     

ADAMTS7B, the full-length product of the ADAMTS7 gene, is a chondroitin sulfate proteoglycan containing a mucin domain

We have characterized ADAMTS7B, the authentic full-length protein product of the ADAMTS7 gene. ADAMTS7B has a domain organization similar to that of ADAMTS12, with a total of eight thrombospondin type 1 repeats in its ancillary domain. Of these, seven are arranged in two distinct clusters that are separated by a mucin domain. Unique to the ADAMTS family, ADAMTS7B is modified by attachment of the glycosaminoglycan chondroitin sulfate within the mucin domain, thus rendering it a proteoglycan. Glycosaminoglycan addition has potentially important implications for ADAMTS7B cellular localization and for substrate recognition. Although not an integral membrane protein, ADAMTS7B is retained near the cell surface of HEK293F cells via interactions involving both the ancillary domain and the prodomain. ADAMTS7B undergoes removal of the prodomain by a multistep furin-dependent mechanism. At least part of the final processing event, i.e. cleavage following Arg(220) (mouse sequence annotation), occurs at the cell surface. ADAMTS7B is an active metalloproteinase as shown by its ability to cleave alpha(2)-macroglobulin, but it does not cleave specific peptide bonds in versican and aggrecan attacked by ADAMTS proteases. Together with ADAMTS12, whose primary structure also predicts a mucin domain, ADAMTS7B constitutes a unique subgroup of the ADAMTS family.     

TIGRINA d, required for regulating the biosynthesis of tetrapyrroles in barley, is an ortholog of the FLU gene of Arabidopsis thaliana

Regulation of tetrapyrrole biosynthesis in higher plants has been attributed to negative feedback control of steps prior to delta-aminolevulinic acid (ALA) formation. One of the first mutants with a defect in this control had been identified in barley. The tigrina (tig) d mutant accumulates 10-15-fold higher amounts of protochlorophyllide than wild type, when grown in the dark. The identity of the TIGRINA d protein and its mode of action are not known yet. Initially this protein had been proposed to act as a repressor of genes that encode enzymes involved in early steps of ALA formation, but subsequent attempts to confirm this experimentally failed. Here we demonstrate that the TIGRINA d gene of barley is an ortholog of the FLU gene of Arabidopsis thaliana. The FLU protein is a nuclear-encoded plastid protein that plays a key role in negative feedback control of chlorophyll biosynthesis in higher plants. Sequencing of the FLU gene of barley revealed a frame shift mutation in the FLU gene of the tig d mutant that results in the loss of two tetratricopeptide repeats that in the FLU protein of Arabidopsis are essential for its biological activity. This mutation cosegregates strictly with the tigrina phenotype within the F1 population of a heterozygous tig d mutant, thus providing additional support for the flu gene being responsible for the tigrina phenotype of barley.     

Rapid induction of distinct stress responses after the release of singlet oxygen in Arabidopsis

The conditional fluorescent (flu) mutant of Arabidopsis accumulates the photosensitizer protochlorophyllide in the dark. After a dark-to-light shift, the generation of singlet oxygen, a nonradical reactive oxygen species, starts within the first minute of illumination and was shown to be confined to plastids. Immediately after the shift, plants stopped growing and developed necrotic lesions. These early stress responses of the flu mutant do not seem to result merely from physicochemical damage. Peroxidation of chloroplast membrane lipids in these plants started rapidly and led to the transient and selective accumulation of a stereospecific and regiospecific isomer of hydroxyoctadecatrieonic acid, free (13S)-HOTE, that could be attributed almost exclusively to the enzymatic oxidation of linolenic acid. Within the first 15 min of reillumination, distinct sets of genes were activated that were different from those induced by superoxide/hydrogen peroxide. Collectively, these results demonstrate that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways. Its biological activity in Arabidopsis exhibits a high degree of specificity that seems to be derived from the chemical identity of this reactive oxygen species and/or the intracellular location at which it is generated.     

Effect of Carbon and Energy Source on Bacterial Chromate Reduction

Studies were conducted to evaluate carbon and energy sources suitable to support hexavalent chromium (Cr(VI)) reduction by a bacterial consortium enriched from dichromate-contaminated aquifer sediments. The consortium was cultured under denitrifying conditions in a minimal, synthetic groundwater medium that was amended with various individual potential carbon and energy sources. The effects of these individual carbon and energy sources on Cr(VI) reduction and growth were measured. The consortium was found to readily reduce Cr(VI) with sucrose, acetate, L-asparagine, hydrogen plus carbon dioxide, ethanol, glycerol, glycolate, propylene glycol, or D-xylose as a carbon and energy source. Minimal Cr(VI) reduction was observed when the consortium was cultured with citrate, 2-ketoglutarate, L-lactate, pyruvate, succinate, or thiosulfate plus carbon dioxide as a carbon and energy source when compared with abiotic controls. The consortium grew on all of the above carbon and energy sources, with the highest cell densities reached using D-xylose and sucrose, demonstrating that the consortium is metabolically diverse and can reduce Cr(VI) using a variety of different carbon and energy sources. The results suggest that the potential exists for the enrichment of Cr(VI)-reducing microbial populations in situ by the addition of a sucrose-containing feedstock such as molasses, which is an economical and readily available carbon and energy source.