scholarly journals The Path to Triacylglyceride Obesity in the sta6 Strain of Chlamydomonas reinhardtii

2014 ◽  
Vol 13 (5) ◽  
pp. 591-613 ◽  
Author(s):  
Ursula Goodenough ◽  
Ian Blaby ◽  
David Casero ◽  
Sean D. Gallaher ◽  
Carrie Goodson ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Growth Conditions ◽  
Culture Conditions ◽  
Pentose Phosphate ◽  
Small Subset ◽  
Content Type ◽  
Green Microalga ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Tag Accumulation

ABSTRACT When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then “boosted” after 2 days with additional acetate, the cells become “obese” after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ∼425 genes are upregulated ≥2-fold and ∼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of “sensitive” genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes—encoding a diacylglycerol acyltransferase ( DGTT2 ), a glycerol-3-P dehydrogenase ( GPD3 ), and two candidate lipases (Cre03.g155250 and Cre17.g735600)—are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the “sensitive” genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.

scholarly journals Detailed Identification of Fatty Acid Isomers Sheds Light on the Probable Precursors of Triacylglycerol Accumulation in Photoautotrophically Grown Chlamydomonas reinhardtii

2013 ◽  
Vol 13 (2) ◽  
pp. 256-266 ◽  
Author(s):  
Kenta Sakurai ◽  
Takashi Moriyama ◽  
Naoki Sato
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Chlamydomonas Reinhardtii ◽  
De Novo ◽  
Continuous Production ◽  
Growth Conditions ◽  
High Concentration ◽  
Content Type ◽  
Major Acid ◽  
Tag Accumulation

ABSTRACTChlamydomonas reinhardtiiis a model alga for studying triacylglycerol (TAG) accumulation in the photosynthetic production of biofuel. Previous studies were conducted under photoheterotrophic growth conditions in medium supplemented with acetate and/or ammonium. We wanted to demonstrate TAG accumulation under truly photoautotrophic conditions without reduced elements. We first reidentified all lipid components and fatty acids by mass spectrometry, because the currently used identification knowledge relies on data obtained in the 1980s. Accordingly, various isomers of fatty acids, which are potentially useful in tracing the flow of fatty acids leading to the accumulation of TAG, were detected. In strain CC1010 grown under photoautotrophic conditions, TAG accumulated to about 57.5 mol% of total lipids on a mole fatty acid basis after the transfer to nitrogen-deficient conditions. The content of monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol decreased drastically. The accumulated TAG contained 16:0 as the major acid and 16:4(4,7,10,13), 18:2(9,12), and 18:3(9,12,15), which are typically found in chloroplast lipids. Additionally, 18:1(11) and 18:3(5,9,12), which are specific to extrachloroplast lipids, were also abundant in the accumulated TAG. Photosynthesis and respiration slowed markedly after the shift to nitrogen-deficient conditions. These results suggest that fatty acids for the production of TAG were supplied not only from chloroplast lipids but also from other membranes within the cells, although the possibility ofde novosynthesis cannot be excluded. Under nitrogen-replete conditions, supplementation with a high concentration of CO2promoted TAG production in the cells grown photoautotrophically, opening up the possibility to the continuous production of TAG using CO2produced by industry.

scholarly journals Molecular Epidemiology of NDM-1-Producing Enterobacteriaceae and Acinetobacter baumannii Isolates from Pakistan

2014 ◽  
Vol 58 (9) ◽  
pp. 5589-5593 ◽  
Author(s):  
Anna L. Sartor ◽  
Muhammad W. Raza ◽  
Shahid A. Abbasi ◽  
Kathryn M. Day ◽  
John D. Perry ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Epidemiology ◽  
Genetic Relatedness ◽  
Antibiotic Resistance Genes ◽  
Enterobacter Cloacae ◽  
Content Type ◽  
Plasmid Replicon ◽  
Genes Encoding ◽  
Clonal Spread ◽  
Encoding Genes

ABSTRACTThe molecular epidemiology of 66 NDM-producing isolates from 2 Pakistani hospitals was investigated, with their genetic relatedness determined using repetitive sequence-based PCR (Rep-PCR). PCR-based replicon typing and screening for antibiotic resistance genes encoding carbapenemases, other β-lactamases, and 16S methylases were also performed. Rep-PCR suggested a clonal spread ofEnterobacter cloacaeandEscherichia coli. A number of plasmid replicon types were identified, with the incompatibility A/C group (IncA/C) being the most common (78%). 16S methylase-encoding genes were coharbored in 81% of NDM-producingEnterobacteriaceae.

scholarly journals Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli: TABLE 1

2015 ◽  
Vol 198 (1) ◽  
pp. 7-11 ◽  
Author(s):  
Regine Hengge ◽  
Michael Y. Galperin ◽  
Jean-Marc Ghigo ◽  
Mark Gomelsky ◽  
Jeffrey Green ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
Content Type ◽  
E Coli ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
Systematic Nomenclature ◽  
K 12 ◽  
Encoding Genes

In recent years,Escherichia colihas served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely usedE. coliK-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 “degenerate” enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenicE. colistrains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling inE. coli, we now propose a general and systematicdgcandpdenomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains ofE. coliin future studies.

scholarly journals Proteome Analyses of Strains ATCC 51142 and PCC 7822 of the Diazotrophic Cyanobacterium Cyanothece sp. under Culture Conditions Resulting in Enhanced H2Production

2012 ◽  
Vol 79 (4) ◽  
pp. 1070-1077 ◽  
Author(s):  
Uma K. Aryal ◽  
Stephen J. Callister ◽  
Sujata Mishra ◽  
Xiaohui Zhang ◽  
Janani I. Shutthanandan ◽  
...  
Keyword(s):  
Large Scale ◽  
Genetic Manipulation ◽  
Nitrogenase Activity ◽  
Calvin Cycle ◽  
Proteome Analysis ◽  
Culture Conditions ◽  
Pentose Phosphate ◽  
Diurnal Changes ◽  
Content Type ◽  
Diurnal Cycles

ABSTRACTCultures of the cyanobacterial genusCyanothecehave been shown to produce high levels of biohydrogen. These strains are diazotrophic and undergo pronounced diurnal cycles when grown under N2-fixing conditions in light-dark cycles. We seek to better understand the way in which proteins respond to these diurnal changes, and we performed quantitative proteome analysis ofCyanothecesp. strains ATCC 51142 and PCC 7822 grown under 8 different nutritional conditions. Nitrogenase expression was limited to N2-fixing conditions, and in the absence of glycerol, nitrogenase gene expression was linked to the dark period. However, glycerol induced expression of nitrogenase during part of the light period, together with cytochromecoxidase (Cox), glycogen phosphorylase (Glp), and glycolytic and pentose phosphate pathway (PPP) enzymes. This indicated that nitrogenase expression in the light was facilitated via higher levels of respiration and glycogen breakdown. Key enzymes of the Calvin cycle were inhibited inCyanotheceATCC 51142 in the presence of glycerol under H2-producing conditions, suggesting a competition between these sources of carbon. However, inCyanothecePCC 7822, the Calvin cycle still played a role in cofactor recycling during H2production. Our data comprise the first comprehensive profiling of proteome changes inCyanothecePCC 7822 and allow an in-depth comparative analysis of major physiological and biochemical processes that influence H2production in both strains. Our results revealed many previously uncharacterized proteins that may play a role in nitrogenase activity and in other metabolic pathways and may provide suitable targets for genetic manipulation that would lead to improvement of large-scale H2production.

scholarly journals Physiological roles of small RNA molecules

Microbiology ◽  
2014 ◽  
Vol 160 (6) ◽  
pp. 1007-1019 ◽  
Author(s):  
Charlotte Michaux ◽  
Nicolas Verneuil ◽  
Axel Hartke ◽  
Jean-Christophe Giard
Keyword(s):  
Environmental Changes ◽  
Growth Conditions ◽  
Culture Conditions ◽  
Rna Molecules ◽  
Physiological Processes ◽  
Sensing Systems ◽  
Genes Encoding ◽  
Regulation Mechanisms ◽  
Regulatory Rnas ◽  
Metal Sensing

Unlike proteins, RNA molecules have emerged lately as key players in regulation in bacteria. Most reviews hitherto focused on the experimental and/or in silico methods used to identify genes encoding small RNAs (sRNAs) or on the diverse mechanisms of these RNA regulators to modulate expression of their targets. However, less is known about their biological functions and their implications in various physiological responses. This review aims to compile what is known presently about the diverse roles of sRNA transcripts in the regulation of metabolic processes, in different growth conditions, in adaptation to stress and in microbial pathogenesis. Several recent studies revealed that sRNA molecules are implicated in carbon metabolism and transport, amino acid metabolism or metal sensing. Moreover, regulatory RNAs participate in cellular adaptation to environmental changes, e.g. through quorum sensing systems or development of biofilms, and analyses of several sRNAs under various physiological stresses and culture conditions have already been performed. In addition, recent experiments performed with Gram-positive and Gram-negative pathogens showed that regulatory RNAs play important roles in microbial virulence and during infection. The combined results show the diversity of regulation mechanisms and physiological processes in which sRNA molecules are key actors.

scholarly journals Optimization of Methanotrophic Growth and Production of Poly(3-Hydroxybutyrate) in a High-Throughput Microbioreactor System

2015 ◽  
Vol 81 (14) ◽  
pp. 4767-4773 ◽  
Author(s):  
Eric R. Sundstrom ◽  
Craig S. Criddle
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Medium Optimization ◽  
Nile Red ◽  
Bacterial Biomass ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Culture Conditions ◽  
High Mass ◽  
Content Type

ABSTRACTProduction of poly(3-hydroxybutyrate) (P3HB) from methane has economic and environmental advantages over production by agricultural feedstock. Identification of high-productivity strains and optimal growth conditions is critical to efficient conversion of methane to polymer. Current culture conditions, including serum bottles, shake flasks, and agar plates, are labor-intensive and therefore insufficient for systematic screening and isolation. Gas chromatography, the standard method for analysis of P3HB content in bacterial biomass, is also incompatible with high-throughput screening. Growth in aerated microtiter plates coupled with a 96-well Nile red flow-cytometric assay creates an integrated microbioreactor system for high-throughput growth and analysis of P3HB-producing methanotrophic cultures, eliminating the need for individual manipulation of experimental replicates. This system was tested in practice to conduct medium optimization for P3HB production in pure cultures ofMethylocystis parvusOBBP. Optimization gave insight into unexpected interactions: for example, low calcium concentrations significantly enhanced P3HB production under nitrogen-limited conditions. Optimization of calcium and copper concentrations in the growth medium increased final P3HB content from 18.1% to 49.4% and P3HB concentration from 0.69 g/liter to 3.43 g/liter while reducing doubling time from 10.6 h to 8.6 h. The ability to culture and analyze thousands of replicates with high mass transfer in completely mixed culture promises to streamline medium optimization and allow the detection and isolation of highly productive strains. Applications for this system are numerous, encompassing analysis of biofuels and other lipid inclusions, as well as analysis of heterotrophic and photosynthetic systems.

scholarly journals Prevalence of β-Lactamase-Encoding Genes among Enterobacteriaceae Bacteremia Isolates Collected in 26 U.S. Hospitals: Report from the SENTRY Antimicrobial Surveillance Program (2010)

2013 ◽  
Vol 57 (7) ◽  
pp. 3012-3020 ◽  
Author(s):  
Mariana Castanheira ◽  
Sarah E. Farrell ◽  
Lalitagauri M. Deshpande ◽  
Rodrigo E. Mendes ◽  
Ronald N. Jones
Keyword(s):  
New York ◽  
New York City ◽  
Surveillance Program ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Antimicrobial Surveillance ◽  
Genes Encoding ◽  
Resistant Strains ◽  
User Friendly ◽  
Encoding Genes

ABSTRACTEnterobacteriaceaebacteremia isolates (n= 195; 6.4% overall) collected from 26 U.S. hospitals located in 20 states were screened for various β-lactamase classes. A total of 175 isolates carried one to eight acquired β-lactamase genes of 44 types that were detected in 55 combinations. Eighty-five (43.6%) strains carriedblaCTX-M, andblaCTX-M-15was the most prevalent (33.8%). Genes encoding OXA-1/30 (often associated withblaCTX-M-15), CMY-2, SHV extended-spectrum β-lactamase (ESBLs), and TEM-1 were also prevalent. Among 33 carbapenem-resistant strains, 28 carriedblaKPC-2orblaKPC-3(17 and 11 strains, respectively), and those were recovered mostly in the New York City area (16 strains) and Houston, TX (9 strains). Fourteen new SHV variants were identified amongKlebsiella pneumoniaeisolates carrying one or multiple SHV alleles, three carrying G238S and/or E240K amino acid alterations that confer ESBL activity. Only two of eightK. oxytocaisolates carried acquired β-lactamases, but most had mutations on theblaOXYpromoter region, and three new OXY-encoding genes were characterized. Concordance between a commercial nucleic acid-based microarray (Check-MDR CT101) and reference methods was noted for 105/109 (97.2%) strains. Thirty-two strains having genes that are not targeted by the commercial system were detected (OXA ESBLs, PER, PSE, or intrinsic genes). Overall, a great variety of enzymes were observed, with numerous strains carrying multiple genes. Rates of CTX-M-producing strains appear to be increasing in U.S. hospitals (26.6% in 2007 to 43.8% for 2010) participating in the SENTRY Program. Furthermore, the Check-Points system seems to be a reliable, robust, and user-friendly assay for detection of enzyme-mediated resistance.

scholarly journals Adjustment of Trehalose Metabolism in Wine Saccharomyces cerevisiae Strains To Modify Ethanol Yields

2013 ◽  
Vol 79 (17) ◽  
pp. 5197-5207 ◽  
Author(s):  
D. Rossouw ◽  
E. H. Heyns ◽  
M. E. Setati ◽  
S. Bosch ◽  
F. F. Bauer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Modification ◽  
Target Genes ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Systematic Screening ◽  
Content Type ◽  
Genes Encoding ◽  
Ethanol Yields ◽  
Trehalose Biosynthesis

ABSTRACTThe ability ofSaccharomyces cerevisiaeto efficiently produce high levels of ethanol through glycolysis has been the focus of much scientific and industrial activity. Despite the accumulated knowledge regarding glycolysis, the modification of flux through this pathway to modify ethanol yields has proved difficult. Here, we report on the systematic screening of 66 strains with deletion mutations of genes encoding enzymes involved in central carbohydrate metabolism for altered ethanol yields. Five of these strains showing the most prominent changes in carbon flux were selected for further investigation. The genes were representative of trehalose biosynthesis (TPS1, encoding trehalose-6-phosphate synthase), central glycolysis (TDH3, encoding glyceraldehyde-3-phosphate dehydrogenase), the oxidative pentose phosphate pathway (ZWF1, encoding glucose-6-phosphate dehydrogenase), and the tricarboxylic acid (TCA) cycle (ACO1andACO2, encoding aconitase isoforms 1 and 2). Two strains exhibited lower ethanol yields than the wild type (tps1Δ andtdh3Δ), while the remaining three showed higher ethanol yields. To validate these findings in an industrial yeast strain, theTPS1gene was selected as a good candidate for genetic modification to alter flux to ethanol during alcoholic fermentation in wine. Using low-strength promoters active at different stages of fermentation, the expression of theTPS1gene was slightly upregulated, resulting in a decrease in ethanol production and an increase in trehalose biosynthesis during fermentation. Thus, the mutant screening approach was successful in terms of identifying target genes for genetic modification in commercial yeast strains with the aim of producing lower-ethanol wines.

scholarly journals Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

2015 ◽  
Vol 81 (10) ◽  
pp. 3387-3394 ◽  
Author(s):  
Tian Xia ◽  
Qi Han ◽  
William V. Costanzo ◽  
Yixuan Zhu ◽  
Jeffrey L. Urbauer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sole Carbon Source ◽  
Pentose Phosphate ◽  
Glucose Yield ◽  
Content Type ◽  
E Coli ◽  
Carbon Backbone ◽  
Genes Encoding ◽  
Novel Strategy ◽  
Metabolically Engineered Escherichia Coli

ABSTRACTEscherichia colithat is unable to metabolized-glucose (with knockouts inptsG,manZ, andglk) accumulates a small amount ofd-glucose (yield of about 0.01 g/g) during growth on the pentosesd-xylose orl-arabinose as a sole carbon source. Additional knockouts in thezwfandpfkAgenes, encoding, respectively,d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coliMEC143), increased accumulation to greater than 1 g/literd-glucose and 100 mg/literd-mannose from 5 g/literd-xylose orl-arabinose. Knockouts of other genes associated with interconversions ofd-glucose-phosphates demonstrate thatd-glucose is formed primarily by the dephosphorylation ofd-glucose-6-phosphate. Under controlled batch conditions with 20 g/literd-xylose, MEC143 generated 4.4 g/literd-glucose and 0.6 g/literd-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.

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