ABSTRACT
Mitochondria play an integral role in cell death, autophagy, immunity, and inflammation. We previously showed that Nur77, an orphan nuclear receptor, induces apoptosis by targeting mitochondria. Here, we report that celastrol, a potent anti-inflammatory pentacyclic triterpene, binds Nur77 to inhibit inflammation and induce autophagy in a Nur77-dependent manner. Celastrol promotes Nur77 translocation from the nucleus to mitochondria, where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase important for inflammatory signaling. The interaction is mediated by an LxxLL motif in TRAF2 and results not only in the inhibition of TRAF2 ubiquitination but also in Lys63-linked Nur77 ubiquitination. Under inflammatory conditions, ubiquitinated Nur77 resides at mitochondria, rendering them sensitive to autophagy, an event involving Nur77 interaction with p62/SQSTM1. Together, our results identify Nur77 as a critical intracellular target for celastrol and unravel a mechanism of Nur77-dependent clearance of inflamed mitochondria to alleviate inflammation.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Autophagy/drug effects , Chemical and Drug Induced Liver Injury/prevention & control , Mitochondria, Liver/drug effects , Mitophagy/drug effects , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , TNF Receptor-Associated Factor 2/metabolism , Triterpenes/pharmacology , Ubiquitination/drug effects , Active Transport, Cell Nucleus , Animals , Anti-Inflammatory Agents/metabolism , Chemical and Drug Induced Liver Injury/genetics , Chemical and Drug Induced Liver Injury/metabolism , Disease Models, Animal , Female , Genotype , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Ligands , Male , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Liver/metabolism , Mitochondria, Liver/pathology , Nuclear Receptor Subfamily 4, Group A, Member 1/deficiency , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Pentacyclic Triterpenes , Phenotype , Protein Binding , Protein Interaction Domains and Motifs , RNA Interference , Sequestosome-1 Protein/genetics , Sequestosome-1 Protein/metabolism , Signal Transduction/drug effects , TNF Receptor-Associated Factor 2/genetics , Transfection , Triterpenes/metabolismABSTRACT
BACKGROUND: Mitochondrial genomic sequences are known to be variable. Comparative analyses of mitochondrial genomes can reveal the nature and extent of their variation. RESULTS: Draft mitochondrial genomes of 16 Tremella fuciformis isolates (TF01-TF16) were assembled from Illumina and PacBio sequencing data. Mitochondrial DNA contigs were extracted and assembled into complete circular molecules, ranging from 35,104 bp to 49,044 bp in size. All mtDNAs contained the same set of 41 conserved genes with identical gene order. Comparative analyses revealed that introns and intergenic regions were variable, whereas genic regions (including coding sequences, tRNA, and rRNA genes) were conserved. Among 24 introns detected, 11 were in protein-coding genes, 3 in tRNA genes, and the other 10 in rRNA genes. In addition, two mobile fragments were found in intergenic regions. Interestingly, six introns containing N-terminal duplication of the host genes were found in five conserved protein-coding gene sequences. Comparison of genes with and without these introns gave rise to the following proposed model: gene fragment exchange with other species can occur via gain or loss of introns with N-terminal duplication of the host genes. CONCLUSIONS: Our findings suggest a novel mechanism of fungal mitochondrial gene evolution: partial foreign gene replacement though intron mobility.
Subject(s)
Basidiomycota/genetics , Mitochondria/genetics , Mitochondrial Proteins/genetics , Sequence Analysis, DNA/methods , Evolution, Molecular , Fungal Proteins/genetics , Gene Order , Genetic Variation , Genome Size , Interspersed Repetitive Sequences , Introns , PhylogenyABSTRACT
Although "histone" methyltransferases and demethylases are well established to regulate transcriptional programs and to use nonhistone proteins as substrates, their possible roles in regulation of heat-shock proteins in the nucleus have not been investigated. Here, we report that a highly conserved arginine residue, R469, in HSP70 (heat-shock protein of 70 kDa) proteins, an evolutionarily conserved protein family of ATP-dependent molecular chaperone, was monomethylated (me1), at least partially, by coactivator-associated arginine methyltransferase 1/protein arginine methyltransferase 4 (CARM1/PRMT4) and demethylated by jumonji-domain-containing 6 (JMJD6), both in vitro and in cultured cells. Functional studies revealed that HSP70 could directly regulate retinoid acid (RA)-induced retinoid acid receptor ß2 (RARß2) gene transcription through its binding to chromatin, with R469me1 being essential in this process. HSP70's function in gene transcriptional regulation appears to be distinct from its protein chaperon activity. R469me1 was shown to mediate the interaction between HSP70 and TFIIH, which involves in RNA polymerase II phosphorylation and thus transcriptional initiation. Our findings expand the repertoire of nonhistone substrates targeted by PRMT4 and JMJD6, and reveal a new function of HSP70 proteins in gene transcription at the chromatin level aside from its classic role in protein folding and quality control.
Subject(s)
Arginine/metabolism , HSP70 Heat-Shock Proteins/metabolism , Receptors, Retinoic Acid/genetics , Tretinoin/pharmacology , Amino Acid Sequence , Chromatin/metabolism , Gene Expression Regulation , HEK293 Cells , HSP70 Heat-Shock Proteins/chemistry , Humans , Methylation , Molecular Sequence Data , Transcription Factor TFIIH/metabolism , Transcription, GeneticABSTRACT
Members of the genus Bacillus and related spore-forming genera are ubiquitous. However, Bacillus-like species isolated from marine sediments have attracted less interest than their terrestrial relatives. Here, we investigated the diversity of Bacillus-like bacterial communities in the sediments of the Bamenwan mangrove wetland in Hainan, China, using culture-dependent and culture-independent methods, and present the first report on this subject. We also discovered some potential novel species from the sediment samples. Four families, Bacillaceae (58%), Paenibacillaceae (22%), Alicyclobacillaceae (15%), and Planococcaceae (5%), and 9 genera, Bacillus (42%), Paenibacillus (16%), Halobacillus (13%), Alicyclobacillus (11%), Rummeliibacillus (5%), Cohnella (5%), Tumebacillus (4%), Pontibacillus (3%), and Aneurinibacillus (2%), were identified by pyrosequencing. In contrast, only 4 genera, Bacillus (57%), Paenibacillus (23%), Halobacillus (14%), and Virgibacillus (6%), were detected by the culture-dependent method. In the 16S rDNA sequencing analysis, the isolates HB12036 and HB12037 were closest to Bacillus okuhidensis Kh10-101T and Paenibacillus xylanilyticus XIL14T with similarities of 94.8% and 95.9%, respectively, indicating that these were novel species. Bacillus sp. HB12035 and HB12040 exhibited antimicrobial activity against Staphylococcus aureus ATCC 25923, and Bacillus sp. HB12033 exhibited antimicrobial activity against Ustilago scitaminea Syd.
Subject(s)
Bacillaceae/isolation & purification , Biodiversity , Geologic Sediments/microbiology , Water Microbiology , Wetlands , Animals , Bacillaceae/classification , Bacillaceae/physiology , China , DNA, Bacterial , DNA, Ribosomal/genetics , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Staphylococcus aureusABSTRACT
As filter-feeding bivalves, oysters can accumulate microorganisms into their gills, causing spoilage and potential safety issues. This study aims to investigate the changes in the gill microbiota of oysters packed under air and modified atmospheres (MAs, 50% CO2: 50% N2, 70% CO2: 30% O2, and 50% CO2: 50% O2) during storage at 4 °C. The diversity of bacterial microbiota in oyster gills was profiled through polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis on the 16S rRNA gene V3 region to describe the variation during the entire storage period. The DGGE profile revealed high bacterial diversity in the air- and MA-packaged oyster gills, and the spoilage bacterial microbiota varied in the MA-packaged oyster gills. Results indicated that CO2:O2 (70%:30%) was suitable for oyster MA packaging and that high bacterial loads in oyster gills need to be considered during storage. In addition, Lactobacillus and Lactococcus species were found to grow dominantly in fresh oyster gills under MA packaging, which supports the potential application of MA packaging for oyster storage.
Subject(s)
Food Packaging , Gills/microbiology , Microbiota , Ostreidae/microbiology , Refrigeration , Seafood/microbiology , Animals , Bacterial Load , Carbon Dioxide , DNA, Bacterial/genetics , Denaturing Gradient Gel Electrophoresis , Food Microbiology , Genetic Variation , Microbiota/genetics , Oxygen , Polymerase Chain Reaction , Shellfish/microbiologyABSTRACT
Seaweed tissue culture (STC) is an important micropropagation tool that has been applied for strain improvement, micropropagation and genetic engineering. Because the mechanisms associated with STC are poorly understood, its application to these organisms lags far behind that of tissue culture propagation of higher plants. Auxin, calcium (Ca2+ ) and hydrogen peroxide (H2 O2 ) fluxes all play key roles during plant growth and development. In this study, we therefore measured indole-3-acetic acid, Ca2+ and H2 O2 fluxes of Gracilaria lichenoides explants during adventitious branches (ABs) formation for the first time using noninvasive micro-test technology. We confirmed that polar auxin transport (PAT) also occurs in the marine red alga G. lichenoides. We additionally found that N-1-naphthylphthalamic acid may suppress auxin efflux via ABCB1 transporters and then inhibit ABs formation from the apical region of G. lichenoides segments. The involvement of Ca2+ and H2 O2 fluxes in PAT-mediated AB formation in G. lichenoides was also investigated. We propose that complex feedback among Ca2+ , H2 O2 and auxin signaling and response systems may occur during ABs polar formation in G. lichenoides explants, similar to that in higher plants. Our results provide innovative insights that should aid future elucidation of mechanisms operative during STC.
Subject(s)
Gracilaria/growth & development , Indoleacetic Acids/metabolism , Calcium/metabolism , Gracilaria/physiology , Hydrogen Peroxide/metabolism , Microtechnology , Tissue Culture TechniquesABSTRACT
Hypomyces aurantius is a mycoparasite that causes cobweb disease, a most serious disease of cultivated mushrooms. Intra-species identification is vital for disease control, however the lack of genomic data makes development of molecular markers challenging. Small size, high copy number, and high mutation rate of fungal mitochondrial genome makes it a good candidate for intra and inter species differentiation. In this study, the mitochondrial genome of H. H.a0001 was determined from genomic DNA using Illumina sequencing. The roughly 72 kb genome shows all major features found in other Hypocreales: 14 common protein genes, large and small subunit rRNAs genes and 27 tRNAs genes. Gene arrangement comparison showed conserved gene orders in Hypocreales mitochondria are relatively conserved, with the exception of Acremonium chrysogenum and Acremonium implicatum. Mitochondrial genome comparison also revealed that intron length primarily contributes to mitogenome size variation. Seventeen introns were detected in six conserved genes: five in cox1, four in rnl, three in cob, two each in atp6 and cox3, and one in cox2. Four introns were found to contain two introns or open reading frames: cox3-i2 is a twintron containing two group IA type introns; cox2-i1 is a group IB intron encoding two homing endonucleases; and cox1-i4 and cox1-i3 both contain two open reading frame (ORFs). Analyses combining secondary intronic structures, insertion sites, and similarities of homing endonuclease genes reveal two group IA introns arranged side by side within cox3-i2. Mitochondrial data for H. aurantius provides the basis for further studies relating to population genetics and species identification.
Subject(s)
Genome, Mitochondrial , Hypocreales/genetics , Introns , Mitochondria/genetics , Mitochondrial Proteins/classification , Mitochondrial Proteins/metabolism , Nucleic Acid Conformation , Open Reading Frames , Phylogeny , RNA, Transfer/chemistry , RNA, Transfer/genetics , RNA, Transfer/metabolismABSTRACT
To evaluate the nutritional and functional value of Sipunculus nudus, a rapid, simple and sensitive analytical method was developed using ultra-high performance liquid chromatography coupled with a triple quadrupole mass detection in multiple-reaction monitoring mode for the simultaneous quantitative determination of 25 free amino acids and 16 nucleosides and nucleobases in S. nudus within 20 min, which was confirmed to be reproducible and accurate. The limits of detection (LODs) and quantification (LOQs) were between 0.003-0.229 µg/mL and 0.008-0.763 µg/mL for the 41 analytes, respectively. The established method was applied to analyze 19 batches of S. nudus samples from four habitats with two different processing methods. The results showed that S. nudus contained a variety of free amino acids, nucleosides and nucleobases in sufficient quantity and reasonable proportion. They also demonstrated that the contents of these compounds in different parts of S. nudus were significantly discriminating, which were in the order: (highest) coelomic fluid > body wall > intestine (lowest). The method is simple and accurate, and could serve as a technical support for establishing quality control of S. nudus and other functional seafoods. Moreover, the research results also laid foundation for further exploitation and development of S. nudus.
Subject(s)
Amino Acids/chemistry , Heterocyclic Compounds/chemistry , Nematoda/chemistry , Nucleosides/chemistry , Amino Acids/isolation & purification , Animals , Chromatography, High Pressure Liquid , Heterocyclic Compounds/isolation & purification , Hydrophobic and Hydrophilic Interactions , Nucleosides/isolation & purification , Tandem Mass SpectrometryABSTRACT
Two Gram-positive actinobacterial strains, designated HA11166(T) and HA12420, were isolated from mangrove sediments in Hainan, China. The bacterial cells grew with 0-9 % (w/v) NaCl, at 15-40 °C and pH 5.0-10.0, with the optimum growth at 1 % NaCl, 30-37 °C and pH 7.0. The organisms had a range of chemical and morphological properties consistent with their classification in the genus Nocardiopsis. Phylogenetic analysis of the 16S rRNA gene sequences indicated that strains HA11166(T) and HA12420 can be affiliated to the genus Nocardiopsis and most closely related to Nocardiopsis trehalosi VKM Ac-942(T) (with the similarity of 97.2 and 97.5 %, respectively). The value of DNA-DNA relatedness between type strain HA11166(T), selected as the representative strain, and N. trehalosi VKM Ac-942(T) was 38.8 %. The DNA G+C content of strain HA11166(T) was 73.7 %. On the basis of these phenotypic and genotypic data, strains HA11166(T) and HA12420 are proposed to represent a novel species of the genus Nocardiopsis, for which the name Nocardiopsis mangrovei sp. nov. is proposed. The type strain is HA11166(T) (=CGMCC 4.7119(T)=DSM 46665(T)).
Subject(s)
Actinobacteria/classification , Actinobacteria/isolation & purification , Geologic Sediments/microbiology , Actinobacteria/genetics , Actinobacteria/physiology , Bacterial Typing Techniques , Base Composition , China , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , Nucleic Acid Hybridization , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sodium Chloride/metabolism , TemperatureABSTRACT
Two aerobic, Gram-stain positive actinobacterial strains with nematicidal activity, designated HA11164(T) and HA12591, were isolated from mangrove sediments in Hainan, China. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strains HA11164(T) and HA12591 belong to the genus Pseudonocardia and are closely related to Pseudonocardia carboxydivorans (with the similarities of 98.30 and 98.24 %, respectively), Pseudonocardia alni (98.23 and 98.16 %, respectively) and Pseudonocardia antimicrobica (98.10 and 98.03 %, respectively). The major polar lipids of the strain HA11164(T), as a representative strain of the two strains, were found to consist of phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, five unidentified glycolipids and four unidentified polar lipids. The predominant menaquinone of strain HA11164(T) was identified as MK-8 (H4), and the major fatty acids were identified as iso-C16:0, C17:1 ω10, C16:0 and C16:1 ω9. The G+C content of strain HA11164(T) was determined to be 74.9 mol%. The DNA-DNA relatedness values between strains HA11164(T) and P. alni, Pseudonocardia tropica, Pseudonocardia antarctica, P. carboxydivorans and Pseudonocardia parietis were 58.3, 56.2, 50.0, 57.1 and 46.0 %, respectively. Based on the results of this polyphasic study, strains HA11164(T) and HA12591 are considered to represent a novel species of the genus Pseudonocardia, for which the name Pseudonocardia nematodicida sp. nov. is proposed. The type strain is HA11164(T) (=CGMCC 4.7118(T) = DSM 45940(T)).
Subject(s)
Actinobacteria/classification , Actinobacteria/isolation & purification , Geologic Sediments/microbiology , Actinobacteria/genetics , Actinobacteria/physiology , Aerobiosis , Bacterial Typing Techniques , Base Composition , China , Cluster Analysis , Cytosol/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Fatty Acids/analysis , Glycolipids/analysis , Molecular Sequence Data , Nucleic Acid Hybridization , Phospholipids/analysis , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Vitamin K 2/analysis , WetlandsABSTRACT
High acetate accumulation was produced during glucose fermentation in high cell density cultures, which is harmful to cell growth. In order to reduce the negative impact of acetate accumulation on the fermentation products, we introduced the Escherichia coli acetyl-CoA synthetase (ACS) gene into the marine microalga Schizochytrium sp. TIO1101, generating genetically modified ACS transformants. The results of PCR and blotting analyses showed that the exogenous ACS gene was incorporated into the genome and successfully expressed. The engineered Schizochytrium increased the pH value and reduced the acetate concentration in the final fermentation medium significantly. Furthermore, the ACS transformants exhibited faster growth and glucose consumption rates than the wild-type strain. The biomass and fatty acid proportion of ACS transformants increased by 29.9 and 11.3 %, respectively. Taken together, the data suggest that ACS overexpression in Schizochytrium might improve the utilization of carbon resource and decrease the production of acetate byproduct. These results demonstrate that application of ACS in metabolic genetic engineering could improve the properties of Schizochytrium significantly.
Subject(s)
Acetate-CoA Ligase/biosynthesis , Biomass , Fatty Acids/metabolism , Gene Expression , Stramenopiles/enzymology , Stramenopiles/metabolism , Acetate-CoA Ligase/genetics , Culture Media/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Glucose/metabolism , Metabolic Engineering , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Stramenopiles/genetics , Stramenopiles/growth & developmentABSTRACT
Malonyl-CoA:acyl-carrier protein transacylase (MCAT), which transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), is a key enzyme in fatty acid biosynthesis. Schizochytrium sp. TIO1101 is a marine protist with high levels of docosahexaenoic acid accumulation. In this study, the putative fabD gene coding MCAT was isolated from Schizochytrium sp. TIO1101. The Schizochytrium MCAT gene (ScTIOfabD) contained an 1176 bp open reading frame encoding a protein of 391 amino acids. The ScTIOfabD gene exhibited high novelty in nucleotide and amino acid sequence. The highest amino acid identity was only 35 % between ScTIOMCAT and the reported MCATs. Further studies demonstrated that ScTIOMCAT could bind malonyl-CoA directly and transfer malonyl group from malonyl-CoA to the ACP domain in vitro. Phylogenetic analysis suggested that ScTIOMCAT was relative close to MCATs of yeast strains. Overexpression of ScTIOMCAT in Saccharomyces cereviseae significantly increased the MCAT activity, without negative effects on the growth rate of the host strain. In addition, ScTIOMCAT generated 16.8 and 62 % increase in biomass and fatty acid accumulation, respectively, and did not alter the profile of fatty acid. Our results indicated that the novel MCAT gene from Schizochytrium sp. TIO1101 was crucial for fatty acid synthesis and had potential applications for genetic modifications of oil-producing species.
Subject(s)
Coenzyme A-Transferases/genetics , Coenzyme A-Transferases/metabolism , Docosahexaenoic Acids/metabolism , Stramenopiles/enzymology , Acyl Carrier Protein/metabolism , Amino Acid Sequence , Cloning, Molecular , Cluster Analysis , Malonyl Coenzyme A/metabolism , Molecular Sequence Data , Open Reading Frames , Phylogeny , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Stramenopiles/genetics , Stramenopiles/metabolismABSTRACT
In this study, we have determined the complete nucleotide sequence of the mitochondrial genome of Octopus minor. It is 15,974 nucleotide pairs and encodes 13 proteins, two ribosomal RNAs and 22 tRNAs of the mitochondrion's own protein synthesizing system. Seven of thirteen proteins are encoded by the H-strand, while the other six proteins, as well as the two ribosomal RNAs are encoded by the L-strand. The nucleotide composition of the proteins showed a nucleotide bias against G encoded by the H-strand, while they showed a nucleotide bias against A and C encoded by the L-strand. Two of the 13 protein coding genes of O. minor began with the unorthodox translation initiation codon ATA and all others use the standard ATG. In addition, six of thirteen mt proteins of O. minor have unambiguous termination codons. There are four cases where tRNA genes appear to overlap. The long noncoding region (LNCR) of O. minor was 930 nucleotides and no repeated sequences were found in this LNCR. The gene arrangements of O. minor showed remarkable similarity to that of O. ocellatus and O. vulgaris. Phylogenetic analysis demonstrated that O. minor appears as sister taxan to the monophyletic group combined by O. ocellatus and O. vulgaris, suggesting a relative distant genetic relationship between O. minor and the other two octopus species.
Subject(s)
DNA, Mitochondrial/genetics , Octopodiformes/genetics , Animals , Base Sequence , Codon/genetics , DNA, Intergenic/genetics , Genes, Mitochondrial/genetics , Genome, Mitochondrial/genetics , Likelihood Functions , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , RNA, Ribosomal/genetics , RNA, Transfer/geneticsABSTRACT
CD133 is extensively used as a surface marker to identify and isolate glioma-initiating cells (GICs) from malignant brain tumors; however, instances of CD133(-) cells exhibiting similar properties have also been reported. To clarify the availability of CD133 as the GIC marker, we first evaluated the ratio of CD133(+) cells and malignancy of glioma spheroids GIC1 and GIC2, respectively. GIC1, which showed a lower percentage of CD133(+) cells, exhibited a highly aggressive behavior in comparison with GIC2. The following experiments demonstrated that tumor suppressor PTEN was lost in GIC1, resulting in the activation of AKT pathway. Overexpression of recombinant PTEN in GIC1 suppressed its proliferation and self-renew without significant effect on CD133 expression level, indicating that the inconsistence between the ratio of CD133(+) cells and proliferation and self-renewal capacity of GIC1 and GIC2 was caused by PTEN deficiency. To further validate our conclusion, a series of GICs were analyzed and the percentages of CD133(+) cells could not reflect the degrees of cell proliferation and self-renewal characteristics in the PTEN deficient GICs, suggesting that the application of CD133 as the GIC maker was restricted by PTEN loss. Furthermore, down-regulation of PTEN in the PTEN-expressing GICs could break the positive correlation between the ratio of CD133(+) cells and proliferation and self-renewal capacity. Our results demonstrated that PTEN status is related to cell proliferation and self-renewal independent of CD133 phenotype in the glioma-initiating cells, resulting in the limitations of CD133 as a biomarker for PTEN deficient GICs.
Subject(s)
Antigens, CD/metabolism , Brain Neoplasms/pathology , Cell Proliferation , Glioma/pathology , Glycoproteins/metabolism , PTEN Phosphohydrolase/genetics , Peptides/metabolism , AC133 Antigen , Brain Neoplasms/genetics , Cell Culture Techniques , Down-Regulation , Glioma/genetics , Humans , Neoplastic Stem Cells/metabolism , Oncogene Protein v-akt/metabolism , PTEN Phosphohydrolase/metabolism , Phosphorylation , Tumor Burden/genetics , Tumor Cells, CulturedABSTRACT
Confronted with inescapable exhaustion of the earth's fossil energy resources, the bio-based process to produce industrial chemicals is receiving significant interest. Biotechnological production of four-carbon 1,4-dicarboxylic acids (C4 diacids) from renewable plant biomass is a promising and attractive alternative to conventional chemistry routes. Although the C4 diacids pathway is well characterized and microorganisms able to convert biomass to these acids have been isolated and described, much still has to be done to make this process economically feasible. Metabolically engineered Escherichia coli has been developed as a biocatalyst to provide new processes for the biosynthesis of many valuable chemicals. However, E. coli does not naturally produce C4 diacids in large quantities. Rational strain development by metabolic engineering based on efficient genetic tools and detailed knowledge of metabolic pathways are crucial to successful production of these compounds. This review summarizes recent efforts and experiences devoted to metabolic engineering of the industrial model bacteria E. coli that led to efficient recombinant biocatalysts for the production of C4 diacids, including succinate, fumarate, malate, oxaloacetate, and aspartate, as well as the key limitations and challenges. Continued advancements in metabolic engineering will help to improve the titers, yields, and productivities of the C4 diacids discussed here.
Subject(s)
Dicarboxylic Acids/metabolism , Escherichia coli/metabolism , Industrial Microbiology , Biomass , Carbon/metabolism , Dicarboxylic Acids/chemistry , Escherichia coli/genetics , Genetic Engineering , Malates/metabolism , Metabolic Networks and Pathways , Succinic Acid/metabolismABSTRACT
In this study, the chloroplast genome of Hariotina reticulata was fully sequenced and compared to other Sphaeropleales chloroplast genomes. It is 210,757 bp larger than most Sphaeropleales cpDNAs. It presents a traditional chloroplast structure, and contains 103 genes, including 68 protein-coding genes, six rRNA genes and 29 tRNA genes. The coding region constitutes of 43% of the whole cpDNA. Eighteen introns are found in 11 genes and six introns are unique for Hariotina. 11 open reading frames are identified among these introns. The synteny between Hariotina and Acutodesmus cpDNAs is in general identical, while within Sphaeropleales order, high variability in cpDNA architecture is indicated by general high DCJ distances. Ankyra judayi exhibits the greatest dissimilarity in gene synteny to the others and share some unique gene clusters with Treubaria triappendiculata. The phylogenomic analyses show that A. judayi is clustered with Treubariaceae species and sister to Chlorophyceae incertae sedis and other Sphaeropleales species. The monophyly of Sphaeropleales is rejected.
Subject(s)
Chloroplasts/genetics , Genome, Chloroplast/genetics , Scenedesmus/genetics , Base Composition/genetics , Base Sequence/genetics , Chlorophyta/genetics , Chromosome Mapping , DNA, Chloroplast/genetics , Genome , Open Reading Frames/genetics , Phylogeny , Whole Genome SequencingABSTRACT
C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A(620)/A(280) ratio) of C-phycocyanin isolated with STREAMLINE column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A(620)/A(650) index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin.
Subject(s)
Chromatography, Liquid/methods , Phycocyanin/isolation & purification , Spirulina/chemistry , Adsorption , Electrophoresis, Polyacrylamide Gel , Metals, Heavy/analysis , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
Adhesion is a key virulence factor of pathogens and can be affected by the environment. Our previously research with RNA-seq indicated that mcp, aer, cheB, and cheV might play roles in the regulation of adhesion in Vibrio alginolyticus (ND-01). In order to determine whether and how environmental factors affect adhesion through these genes, gene silencing was performed followed by quantitative real-time PCR (qRT-PCR), RNAi, transmission electron microscopy, and adhesion, capillary, and motility assays to verify how these genes influence adhesion. Silencing these genes led to deficiencies in adhesion, chemotaxis, flagellar assembly, and motility. The expression levels of cheA, cheW, and cheY, which are important genes closely related to the functions of mcp, aer, cheV, and cheB, were significantly downregulated in all of the RNAi groups. The expression of mcp, aer, cheV, and cheB under different gradients of temperature, pH, and salinity and after starvation for various durations was also detected, which showed that these genes were sensitive to certain environmental stresses, particularly pH and starvation. Our results indicated that mcp, aer, cheB, and cheV: (1) are necessary for ND-01 adhesion; (2) play key roles in the bacterial chemotaxis pathway by controlling the expression of downstream genes; (3) might affect adhesion by impacting motility, though motility is not the only route through which adhesion is affected; and (4) contribute to the regulation of ND-01 adhesion in natural environments with different temperatures, pH levels, and salinities as well as after various starvation periods.
Subject(s)
Bacterial Adhesion , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Chemotactic Factors/metabolism , Gene Expression Regulation, Bacterial , Vibrio alginolyticus/physiology , Bacterial Proteins/genetics , Carrier Proteins/genetics , Chemotactic Factors/genetics , Chemotaxis , Hydrogen-Ion Concentration , Vibrio alginolyticus/geneticsABSTRACT
Chlorella minutissima is considered to be one of the promising feedstocks for biofuels in the future. In this study, the transcriptome from the oil-rich strain UTEX2341 of C. minutissima was generated based on Illumina paired-end sequencing. Through de-novo assembly, a total of 14,905 isogenes were obtained and compacted into 6216 unigenes. A total of 80% of the unigenes were assigned with GO terms and were further subdivided into 55 sub-categories. KEGG analysis demonstrated that 37.2% of the unigenes could be accessed and mapped into 278 pathways. Interestingly, the genes that encoded key enzymes that are involved in the biosynthesis, elongation, and metabolism of fatty acids were identified, including malonyl-CoA-ACP transacylase, 3-ketoacyl-ACP synthase, 3-ketoacyl-ACP reductase, and others. Moreover, the genes that are involved in triacylglycerol (TAG) biosynthesis and metabolism were also observed. Therefore, the transcriptome analysis of C. minutissima UTEX2341 not only supplies comprehensive insight into the molecular pathway that is involved in the biosynthesis of biofuel precursors but also provides substantial valuable genomic resources to accelerate the further development and utilization of biofuels.
Subject(s)
Biofuels , Chlorella/genetics , Gene Expression Regulation, Plant/physiology , Lipids/biosynthesis , Transcriptome , Base Sequence , DNA/genetics , RNA, Plant/genetics , RNA, Plant/metabolismABSTRACT
The complete mitochondrial genome of the green algae Hariotina sp. F30 was obtained in this study using Illumina sequencing data. It is 51 915 bp in length with 36.23% GC content. The genome contains 13 protein-coding genes, 23 tRNA genes and six rRNA genes, all of which are encoded on the heavy strand. AUG is a universal initiation codon among 13 protein-coding genes. UCA is a universal termination codon for most protein-coding gens except UAA in cox1 and cob genes and UGA in nad6 gene. CUU anticodon for tRNA-Lys was detected for the first time in Sphaeropleales.