Project description:Phosphorus (P) is an essential nutrient for plant growth and development. Phosphate transporters (PHTs) are trans-membrane proteins that mediate the uptake and translocation of phosphate (Pi) in green plants. The PHT family including PHT1, PHT2, PHT3 and PHT4 subfamilies are well-studied in land plants; however, PHT genes in green algae are poorly documented and not comprehensively identified. Here, we analyzed the PHTs in a model green alga Chlamydomonas reinhardtii and found 25 putative PHT genes, which can be divided into four subfamilies. The subfamilies of CrPTA, CrPTB, CrPHT3, and CrPHT4 contain four, eleven, one, and nine genes, respectively. The structure, chromosomal distribution, subcellular localization, duplication, phylogenies, and motifs of these genes were systematically analyzed in silico. Expression profile analysis showed that CrPHT genes displayed differential expression patterns under P starvation condition. The expression levels of CrPTA1 and CrPTA3 were down-regulated, while the expression of most CrPTB genes was up-regulated under P starvation, which may be controlled by CrPSR1. The transcript abundance of most CrPHT3 and CrPHT4 genes was not significantly affected by P starvation except CrPHT4-3, CrPHT4-4, and CrPHT4-6. Our results provided basic information for understanding the evolution and features of the PHT family in green algae.
Project description:Eukaryotic green algae have become an increasingly popular platform for recombinant proteins production. In particular, Chlamydomonas reinhardtii, has garnered increased attention for having the necessary biochemical machinery to produce vaccines, human antibodies and next generation cancer targeting immunotoxins. While it has been shown that chloroplasts contain chaperones, peptidyl prolylisomerases and protein disulfide isomerases that facilitate these complex proteins folding and assembly, little has been done to determine which processes serve as rate-limiting steps for protein accumulation. In other expression systems, as Escherichia coli, Chinese hamster ovary cells, and insect cells, recombinant protein accumulation can be hampered by cell's inability to fold the target polypeptide into the native state, resulting in aggregation and degradation. To determine if chloroplasts' ability to oxidize proteins that require disulfide bonds into a stable conformation is a rate-limiting step of protein accumulation, three recombinant strains, each expressing a different recombinant protein, were analyzed. These recombinant proteins included fluorescent GFP, a reporter containing no disulfide bonds; Gaussia princeps luciferase, a luminescent reporter containing disulfide bonds; and an immunotoxin, an antibody-fusion protein containing disulfide bonds. Each strain was analyzed for its ability to accumulate proteins when supplemented with selenocystamine, a small molecule capable of catalyzing the formation of disulfide bonds. Selenocystamine supplementation led to an increase in luciferase and immunotoxin but not GFP accumulation. These results demonstrated that selenocystamine can increase the accumulation of proteins containing disulfide bonds and suggests that a rate-limiting step in chloroplast protein accumulation is the disulfide bonds formation in recombinant proteins native structure.
Project description:The green alga Hematococcus pluvialis accumulates large amounts of the antioxidant astaxanthin under inductive stress conditions, such as nitrogen starvation. The response to nitrogen starvation and high light leads to the accumulation of carbohydrates and fatty acids as well as increased activity of the tricarboxylic acid cycle. Although the behavior of individual pathways has been well investigated, little is known about the systemic effects of the stress response mechanism. Here we present time-resolved metabolite, enzyme activity, and physiological data that capture the metabolic response of H. pluvialis under nitrogen starvation and high light. The data were integrated into a putative genome-scale model of the green alga to in silico test hypotheses of underlying carbon partitioning. The model-based hypothesis testing reinforces the involvement of starch degradation to support fatty acid synthesis in the later stages of the stress response. In addition, our findings support a possible mechanism for the involvement of the increased activity of the tricarboxylic acid cycle in carbon repartitioning. Finally, the in vitro experiments and the in silico modeling presented here emphasize the predictive power of large scale integrative approaches to pinpoint metabolic adjustment to changing environments.
Project description:BackgroundNon-specific lipid transfer proteins (nsLTPs) are a group of small and basic proteins that can bind and transfer various lipid molecules to the apoplastic space. A typical nsLTP carries a conserved architecture termed eight-cysteine motif (8CM), a scaffold of loop-linked helices folding into a hydrophobic cavity for lipids binding. Encoded by a multigene family, nsLTPs are widely distributed in terrestrial plants from bryophytes to angiosperms with dozens of gene members in a single species. Although the nsLTPs in the most primitive plants such as Marchantia already reach 14 members and are divergent enough to form separate groups, so far none have been identified in any species of green algae.ResultsBy using a refined searching strategy, we identified putative nsLTP genes in more than ten species of green algae as one or two genes per haploid genome but not in red and brown algae. The analyses show that the algal nsLTPs carry unique characteristics, including the extended 8CM spacing, larger molecular mass, lower pI value and multiple introns in a gene, which suggests that they could be a novel nsLTP lineage. Moreover, the results of further investigation on the two Chlamydomonas nsLTPs using transcript and protein assays demonstrated their late zygotic stage expression patterns and the canonical nsLTP properties were also verified, such as the fatty acids binding and proteinase resistance activities.ConclusionsIn conclusion, a novel nsLTP lineage is identified in green algae, which carries some unique sequences and molecular features that are distinguishable from those in land plants. Combined with the results of further examinations of the Chlamydomonas nsLTPs in vitro, possible roles of the algal nsLTPs are also suggested. This study not only reveals the existence of the nsLTPs in green algae but also contributes to facilitating future studies on this enigmatic protein family.
Project description:Non-alcoholic-fatty-liver-disease (NAFLD) encompasses conditions associated to fat deposition in the liver, which are generally deteriorated during the aging process. MacroH2A1, a variant of histone H2A, is a key transcriptional regulator involved in tumorigenic processes and cell senescence, and featuring two alternatively splicing isoforms, macroH2A1.1 and macroH2A1.2. MacroH2A1.1 binds with high affinity O-acetyl ADP ribose, a small metabolite produced by the reaction catalysed by NAD+-dependent deacetylase SIRT1, whereas macroH2A1.2 is unable to do so. The functional significance of this binding is unknown. We previously reported that the hepatic levels of macroH2A1.1 and macroH2A1.2 are differentially expressed in mice models of NAFLD. Here we show that over-expression of macroH2A1.1, but not of macroH2A1.2, is able to protect hepatocytes against lipid accumulation. MacroH2A1.1 over-expressing cells display ameliorated glucose metabolism, reduced expression of lipidogenic genes and fatty acids content. SIRT1/macroH2A1.1-dependent epigenetic regulation of lipid metabolism may be relevant to NAFLD development.
Project description:While algal phago-mixotrophs play a major role in aquatic microbial food webs, their diversity remains poorly understood. Recent studies have indicated several species of prasinophytes, early diverging green algae, to be able to consume bacteria for nutrition. To further explore the occurrence of phago-mixotrophy in green algae, we conducted feeding experiments with live fluorescently labeled bacteria stained with CellTracker Green CMFDA, heat-killed bacteria stained with 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (DTAF), and magnetic beads. Feeding was detected via microscopy and/or flow cytometry in five strains of prasinophytes when provided with live bacteria: Pterosperma cristatum NIES626, Pyramimonas parkeae CCMP726, Pyramimonas parkeae NIES254, Nephroselmis pyriformis RCC618, and Dolichomastix tenuilepis CCMP3274. No feeding was detected when heat-killed bacteria or magnetic beads were provided, suggesting a strong preference for live prey in the strains tested. In parallel to experimental assays, green algal bacterivory was investigated using a gene-based prediction model. The predictions agreed with the experimental results and suggested bacterivory potential in additional green algae. Our observations underline the likelihood of widespread occurrence of phago-mixotrophy among green algae, while additionally highlighting potential biases introduced when using prey proxy to evaluate bacterial ingestion by algal cells.
Project description:The aim of the present study was to assess the malodorous spoilages of Spanish-style green table olives through microbial and metabolite composition using current measuring techniques (e.g., high-throughput DNA sequencing, headspace solid-phase microextraction combined with gas chromatography-mass spectrometry). Under different alkaline and washing conditions, the spoilage fermentations were reproduced with Gordal and Manzanilla olive cultivars using a low salt concentration (71 g L-1 NaCl) in the initial brine. The degradation of lactic acid and significant increases in volatile fatty acids and phenols were found in all the spoiled samples in comparison with the unspoiled control samples. According to high-throughput DNA sequencing, Cardiobacteriaceae and Ruminococcus were the dominant bacteria in the spoiled samples. PLS regression and Pearson's correlation coefficient analyses revealed positive and negative correlations among microbial communities, metabolites, and sensory spoilage descriptors. Notably, the "zapatera" descriptor was significantly associated with Propionibacterium, which was positively correlated with acetic acid, propionic acid, succinic acid, and methyl propanoate; while the "butyric" descriptor exhibited a significant positive relationship with the genus Ruminococcus, which gave an almost significant correlation with propionic and butyric acids.
Project description:Trebouxiophyceae are microalgae occupying even extreme environments such as polar regions or deserts, terrestrial or aquatic, and can occur free-living or as lichen photobionts. Yet, it is poorly understood how environmental factors shape their metabolism. Here, we report on responses to light and temperature, and metabolic adjustments to desiccation in Diplosphaera epiphytica, isolated from a lichen, and Edaphochlorella mirabilis, isolated from Tundra soil, assessed via growth and photosynthetic performance parameters. Metabolite profiling was conducted by GC-MS. A meta-analysis together with data from a terrestrial and an aquatic Chlorella vulgaris strain reflected elements of phylogenetic relationship, lifestyle, and relative desiccation tolerance of the four algal strains. For example, compatible solutes associated with desiccation tolerance were up-accumulated in D. epiphytica, but also sugars and sugar alcohols typically produced by lichen photobionts. The aquatic C. vulgaris, the most desiccation-sensitive strain, showed the greatest variation in metabolite accumulation after desiccation and rehydration, whereas the most desiccation-tolerant strain, D. epiphytica, showed the least, suggesting that it has a more efficient constitutive protection from desiccation and/or that desiccation disturbed the metabolic steady-state less than in the other three strains. The authors hope that this study will stimulate more research into desiccation tolerance mechanisms in these under-investigated microorganisms.
Project description:Viruses rely upon host lipid metabolic pathways for successful replication, and there is increasing interest in these pathways as novel therapeutic targets for antiviral drug discovery. Despite this, relatively little is known about the impact of viral infection on cellular lipid metabolism, and the specific lipid metabolites utilized by viruses have not yet been examined. We have applied liquid chromatography-mass spectroscopy (LC-MS) based untargeted metabolite profiling to identify lipid metabolites whose steady-state abundance is significantly altered by replication of hepatitis B virus (HBV), a major human pathogen. Untargeted metabolite profiling indicated that although major lipid classes were unaffected by HBV, an ion of 367 m/z was overabundant in HBV+ cells by 18-fold. As shown by ion fragmentation mass spectrometry and coinjection with standard, the identity of this ion is 7-dehydrocholesterol (7-DHC), an immediate dehydrogenated precursor to cholesterol. While cholesterol has previously been demonstrated to be essential in the replication of many viruses, this is the first to show that viral replication is associated with the selective accumulation of 7-DHC. Most virological studies to date have relied upon methods that deplete all sterols and preclude the observation of any selectivity in sterol utilization by viral pathogens. Our study suggests that HBV may selectively utilize 7-DHC versus other sterols and prompts experiments investigating the functional significance of this enrichment and the elucidation of the mechanism by which it is achieved. The results also highlight the value of untargeted metabolite profiling as a method for identifying critical metabolites for viral infection.