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Fatty Acid Activation in Cyanobacteria Mediated by Acyl-Acyl Carrier Protein Synthetase Enables Fatty Acid Recycling
No full textIn cyanobacteria fatty acids destined for lipid synthesis can be synthesized de novo, but also exogenous free fatty acids from the culture medium can be directly incorporated into lipids. Activation of exogenous fatty acids is likely required prior to their utilization. To identify the enzymatic activity responsible for activation we cloned candidate genes from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 and identified the encoded proteins as acyl-acyl carrier protein synthetases (Aas). The enzymes catalyze the ATP-dependent esterification of fatty acids to the thiol of acyl carrier protein. The two protein sequences are only distantly related to known prokaryotic Aas proteins but they display strong similarity to sequences that can be found in almost all organisms that perform oxygenic photosynthesis. To investigate the biological role of Aas activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and S. elongatus PCC 7942. The mutant strains showed two phenotypes characterized by the inability to utilize exogenous fatty acids and by the secretion of endogenous fatty acids into the culture medium. The analyses of extracellular and intracellular fatty acid profiles of aas mutant strains as well as labeling experiments indicated that the detected free fatty acids are released from membrane lipids. The data suggest a considerable turnover of lipid molecules and a role for Aas activity in recycling the released fatty acids. In this model, lipid degradation represents a third supply of fatty acids for lipid synthesis in cyanobacteria
Arabidopsis acyl-acyl carrier protein synthetase AAE15 with medium chain fatty acid specificity is functional in cyanobacteria
No full textCyanobacteria are potential hosts for the biosynthesis of oleochemical compounds. The metabolic precursors for such compounds are fatty acids and their derivatives, which require chemical activation to become substrates in further conversion steps. We characterized the acyl activating enzyme AAE15 of Arabidopsis encoded by At4g14070, which is a homologue of a cyanobacterial acyl-ACP synthetase (AAS). We expressed AAE15 in insect cells and demonstrated its AAS activity with medium chain fatty acid (C10-C14) substrates in vitro. Furthermore, we used AAE15 to complement a Synechocystis aas deletion mutant and showed that the new strain preferentially incorporates supplied medium chain fatty acids into internal lipid molecules. Based on this data we propose that AAE15 can be utilized in metabolic engineering strategies for cyanobacteria that aim to produce compounds based on medium chain fatty acids
Acyl-Acyl Carrier Protein Synthetasen aus Blaualgen und Pflanzen
No full textAllen metabolischen Prozessen von Fettsäuren, geht
deren Aktivierung zu einem Thioesterderivat voraus.
Während die Aktivierung durch Acyl-CoA-Synthetasen in
der Literatur gut etabliert ist, ist für die
alternative Aktivierung mittels Acyl-Carrier-Protein
bislang wenig Information verfügbar. Die Aufgabe dieser
Arbeit war daher die Untersuchung von
Acyl-ACP-Synthetase-Aktivitäten (AAS) in Cyanobakterien
und höheren Pflanzen als Vertretern von
photosynthetischen Organismen.In einer früheren Studie unserer Arbeitsgruppe
konnten Acyl-ACP-Synthetasen aus Cyanobakterien
identifiziert werden und ihre Aktivität nach
heterologer Expression in Escherichia coli
funktional charakterisiert werden. Die biologische
Funktion dieser Aktivitäten konnte jedoch nicht
ermittelt werden. In Arabidopsis wurden mit AAE15 und
AAE16 zwei homologe Sequenzen gefunden. Beide Proteine
wurden zuvor bereits bezüglich ihrer Rolle für die
Elongation von exogenen Fettsäuren in Chloroplasten
untersucht (Koo et al. 2005), jedoch legten die
gewonnenen Daten lediglich für AAE15 eine AAS-Aktivität
nahe.Um die biologische Funktion der AAS-Aktivität in
Cyanobakterien zu untersuchen, wurden
aas-Deletionsmutanten in den Stämmen
Synechocystis sp. PCC 6803 und Synechococcus
elongatus PCC 7942 hergestellt. Die erzeugten
Mutanten zeigten zwei Phänotypen: Die Stämme waren
unfähig exogene Fettsäuren aus dem Medium zu verwerten
und sie sekretierten endogene Fettsäuren in das
Kulturmedium. Durch Komplementation der
aas-Mutante des Stammes Synechocystis sp.
PCC 6803 mit dem aas Gen aus Synechococcus
elongatus PCC 7942 konnte der Wildtyp-Status
widerhergestellt werden. Die Analyse der
extrazellulären und der intrazellulären
Fettsäureprofile von Wildtyp und den
aas-Mutanten Stämmen ergab, dass die gefundenen
Fettsäuren aus Membranlipiden freigesetzt werden. Die
gefundenen Daten deuten auf einen hohen Turnover dieser
Lipide hin. Die AAS-Aktivität scheint für die
Reaktivierung der freigesetzten Fettsäuren erforderlich
zu sein mit dem Ziel diese Fettsäuren dem
Lipidmetabolismus erneut verfügbar zu machen.Das Genom von Arabidopsis kodiert für zwei Sequenzen
mit deutlicher Ähnlichkeit zu den AAS-Proteinen aus
Cyanobakterien. Für diese beiden mit AAE15 und AAE16
benannten Proteine wurde mittels Expression von
EYFP-Fusionsproteinen in Zwiebelzellen die Lokalisation
in Plastiden nachgewiesen. Für eine weitergehende
Charakterisierung wurden beide Proteine in
Insektenzellen exprimiert und ihre Aktivität mittels
in-vitro Enzymtests untersucht. Für AAE15 konnte
AAS-Aktivität mit mittelkettigen Fettsäuren
nachgewiesen werden. Zusätzlich wurde die
aas-Mutante aus Synechocystis sp. PCC
6803 mit AAE15 und AAE16 komplementiert
und die entstandenen Stämme anschließend für
Fütterungsstudien mit Fettsäuren herangezogen. Es
konnte gezeigt werden, dass die Komplementation mit
AAE15 den Wildtyp-Phänotyp wieder herstellte.
Zusätzlich wurde die Spezifität von AAE15 für
mittelkettige Fettsäuren bestätigt. Die Komplementation
mit AAE16 verlief ebenfalls erfolgreich und
deutet eine breite Substratspezifität für dieses
Protein an. In weiteren Studien wurde die Expression
von AAE15 und AAE16 in Abhängigkeit von
Gewebetypen und von Entwicklungsstadien untersucht. Die
histochemischen GUS-Färbungen zeigten für AAE15
und AAE16 hochspezifische Expressionsprofile in
verschiedenen Organen der Blüte und zusätzlich für
AAE16 die Expression in Schließzellen. Diese
Ergebnisse zeigen deutliche Diskrepanzen zu den
Mikroarray-basierten Daten, die durch Genevestigator
verfügbar sind.All metabolic processes involving fatty acids are
preceded by the activation of the fatty acid to form a
thioester derivative. Activation by acyl-CoA synthetase
is well established whereas only little information is
available about the alternative way utilizing acyl
carrier protein (ACP) as acceptor of the acyl group.
The focus of this work was to investigate acyl-ACP
synthetase (AAS) activity in cyanobacteria and plants,
two groups of photosynthetic organisms.In a previous study of our group cyanobacterial AAS
was identified and functionally characterized by
heterologous expression in Escherichia coli.
However, the biological role of this activity within
the cell was not determined. Two homologous sequences,
AAE15 and AAE16 were found in Arabidopsis. Both of them
were investigated by Koo et al. (2005) with
respect to their role in the elongation of exogenous
fatty acids in chloroplasts, but only for AAE15 the AAS
activity was proposed.To elucidate the biological role of AAS activity in
cyanobacteria, aas knockout mutants were
generated in the background of Synechocystis sp.
PCC 6803 and Synechococcus elongatus PCC 7942.
The obtained mutants showed two phenotypes: They were
unable to utilize exogenous fatty acids and they
secreted endogenous fatty acids into the culture
medium. The wild type phenotype was restored by
complementation of the aas knockout mutant of
Synechocystis sp. PCC 6803 with the
corresponding gene from Synechococcus elongatus
PCC 7942. The analysis of extracellular and
intracellular fatty acid profiles of wild type strains
and aas mutant strains showed that fatty acids
are released from membrane lipids indicating a strong
turnover of these lipids. The AAS activity seemed to be
necessary for the reactivation of the released fatty
acid thereby enabling their recycling back into the
lipid metabolism.The genome of Arabidopsis encodes two sequences with
strong amino acid similarity to the AAS of
cyanobacterial origin. For these two proteins termed
AAE15 and AAE16 the localization in plastids was
confirmed by expression of EYFP fusion proteins in
onion cells followed by fluorescent microscopy. To
further characterize these proteins, AAE15 and AAE16
were overexpressed in insect cells and analyzed by
in-vitro assays. It was shown that AAE15
displays AAS activity with substrate specificity
towards medium chain fatty acids. In addition the
aas knockout mutant of Synechocystis sp.
PCC 6803 was complemented individually with
AAE15 and AAE16, and the obtained strains
were fed with labeled fatty acids. It was demonstrated
that complementation with AAE15 restored the
wild type phenotype, and the specificity for medium
chain fatty acids of the enzyme was confirmed. In
addition the complementation experiment gave evidence
for AAS activity of AAE16 and indicated its broad
substrate specificity. In further studies the
expression of AAE15 and AAE16 were
analyzed with respect to tissue specificity and to
dependence on developmental stage. The histochemical
GUS staining indicated highly specific expression
profiles for AAE15 and AAE16 in flower
organs and additionally for AAE16 in guard cells
of different organs. The results revealed some strong
discrepancies to microarray-based data provided by
Genevestigator
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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