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The gamma-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis
No full textPutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-DL-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the gamma-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHP-resistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates
Mutational activation of the RocR activator and of a cryptic rocDEF promoter bypass loss of the initial steps of proline biosynthesis in Bacillus subtilis
No full textThe gamma-glutamyl-phosphate reductase (ProA) interlinks both the anabolic and osmostress adaptive proline biosynthetic routes of Bacillus subtilis. Because no paralogous protein to ProA exists in this microorganism, proA mutants should exhibit a tight proline auxotrophic growth phenotype. Contrary to expectations, proA mutants formed microcolonies on agar plates lacking proline and faster growing Pro(+) suppressor mutants arose. These mutants carried alterations in the rocR-rocDEF region encoding enzymes of the arginine degradation pathway and its transcriptional activator RocR. They were of two types: (i) mutants carrying single amino acid substitutions in RocR resulting in partial inducer-independent variants and (ii) mutants carrying single base-pair changes in the vicinity of the SigL/Sig-54-dependent -12/-24 class rocDEF promoter that activate a cryptic SigA-type promoter. Consequently, enhanced rocDEF transcription should lead to increased cellular amounts of the RocD ornithine aminotransferase, an enzyme that synthesizes the same reaction product as ProA, gamma-glutamic-semialdehyde/delta-1-pyrroline-5-carboxylate. This compound can be enzymatically converted into proline. The Pro(+) suppressors also exhibited a new regulatory pattern by allowing enhanced rocDEF transcription in response to proline availability when ammonium is present. Our work provides an example how flexibly bacteria can genetically develop routes to bypass constraints imposed on their biosynthetic networks and evolve new regulatory mechanisms
Proline as an osmostress protectant and as a nutrient source
No full textIn dem Gram+ Bakterium Bacillus subtilis spielt Prolin eine wichtige Rolle. Als proteinogene Aminosäure ist es essentiell für die Biosynthese von Proteinen, es findet als Kohlen- oder Stickstoffquelle Verwendung und es fungiert außerdem als osmotische Schutzsubstanz.
B. subtilis besitzt zwei Wege um Prolin de novo zu synthetisieren: die anabole und die osmoadaptive Prolinbiosynthese. Beide Wege sind über das gemeinsam genutzte Enzym ProA miteinander verknüpft. Die Deletion von proA führt zu einer Störung in der Prolinbiosynthese. In dieser Arbeit wird gezeigt, dass Suppressor-Mutationen innerhalb der rocR-rocDEF-Region diese Störung durch die Rekrutierung des Argininabbau-Weges für die Prolin Neusynthese ausgleichen können. Zwei Klassen von Mutationen konnten identifiziert werden: (i) einzelner Aminosäureaustausch im Aktivatorprotein RocR führt zu teilweise Induktor unabhängigen RocR*-Proteinen; (ii) Mutationen in der Promotorregion von rocDEF führen zur Aktivierung eines kryptischen SigA-abhängigen Promotors. In beiden Fällen kam es zu einer erhöhten Transkription des rocDEF-Operons, was die Erhöhung der Menge an RocD in der Zelle zur Folge hatte. RocD als Teil des Argininabbau-Weges produziert dabei dasselbe Produkt wie ProA und kann somit dessen Fehlen ersetzen. Außerdem entwickelte sich dabei ein völlig neuer regulatorischer Mechanismus für die Expression des rocDEF-Operons, da dessen Expression nun auch in Anwesenheit von Ammonium durch Prolin induziert werden konnte. Dies demonstriert wie effektiv sich Bakterien an Einschränkungen innerhalb essentieller Stoffwechselwege anpassen können.
In seinem natürlichen Habitat ist B. subtilis ständig wechselnden Umweltbedingungen, wie die Osmolarität, ausgesetzt. Die Anpassung an hochosmolare Bedingungen erfolgt dabei durch die osmotisch kontrollierte de novo Synthese von Prolin, oder durch dessen Aufnahme aus der Umgebung. In dieser Arbeit wird gezeigt, dass prolinhaltige Peptide ebenfalls eine osmoprotektive Wirkung besitzen. Nach dem Import der Peptide durch spezielle Peptidtransporter (App, Dpp, Opp, DtpT) kommt es intrazellulär zu deren Spaltung, was zu einer Freisetzung des Prolins führt. Durch die Akkumulation dieses Prolins kommt es zu einem Schutz der osmotisch gestressten Zelle. Die an der Spaltung der Xaa-Pro bzw. Xaa-Pro-Xaa Peptide beteiligten Peptidasen konnten identifiziert werden: PapA (YqhT) und PapB (YkvY). Dies fügt einen neuen Aspekt in der Nutzung von Prolin als osmotische Schutzsubstanz durch B. subtilis hinzu und demonstriert wie gewinnbringend die im Habitat verfügbaren Ressourcen zum Schutz vor osmotischem Stress genutzt werden können.
B. subtilis besitzt neben den beiden bekannten Prolintransportern (PutP, OpuE) noch ein weiteres, bisher unbekanntes Prolinaufnahmesystem. In dieser Arbeit wurde für dessen Identifizierung ein genetisches Screening-Verfahren entwickelt. In einem Stamm der Prolin nicht mehr aktiv in die Zelle aufnehmen kann, wurden trotz externer Zugabe von Prolin die Gene des Prolinabbau-Weges (putBCP) nicht mehr exprimiert. Dies demonstriert, dass der Import des externen Prolins in die Zelle, die Voraussetzung für die Induktion der Transkription von putBCP ist.
Die im Rahmen dieser Arbeit erlangten Erkenntnisse enthüllen weitere Facetten der physiologischen Rolle von Prolin in B. subtilis und zeigen außerdem sehr eindrucksvoll, wie effektiv sich Bakterien an die unterschiedlichsten Veränderungen anpassen können.Proline is a very important compound and serves several functions for the gram+ bacterium Bacillus subtilis. Prolin is an amino acid and therefore essential for protein synthesis, it can be used by B. subtilis as a single source of carbon and nitrogen and furthermore proline acts as an osmostress protectant.
B. subtilis possess two different ways for the de novo synthesis of proline: the anabolic and the osmoadaptive pathway. Both pathways are connected via the shared ProA protein. The deletion of the proA gene leads to a perturbation in both the anabolic and osmoadaptive proline biosynthesis. Suppressor mutations within the rocR-rocDEF region recruit the arginine degradation pathway for the synthesis of proline. The suppressor mutations were of two types: (i) single amino acid substitutions in the activator protein RocR resulting in partial inducer-independent RocR* variants; (ii) mutants in the promoter region of rocDEF activates a cryptic SigA-type promoter. Both types of mutants enhance the transcription of rocDEF leading to increased amounts of RocD. RocD as part of the arginine degradation pathway synthesizes the same reaction product as the ProA enzyme and thereby bypassing the ProA mediated enzyme reaction. Furthermore, the suppressor mutants also developed a new regulatory mechanism that allows the enhanced rocDEF transcription in response to proline in the presence of ammonium. This demonstrates how effective bacteria can adapt to limitations on their essential biosynthetic pathways.
In his natural habitat B. subtilis is frequently exposed to osmotic fluctuations. B. subtilis can adapt to high osmolarity growth conditions through the osmotically induced de novo synthesis or the uptake of proline. But B. subtilis can also use proline containing peptides as osmostress protectants. Osmoprotection by peptides depends on their import via peptide uptake systems (App, Dpp, Opp, DtpT) and the subsequent hydrolysis to release proline. The liberated proline is then accumulated to protect the cell against the disadvantageous effects of high osmolarity. The peptidases responsible for the hydrolysis of various types of Xaa-Pro and Xaa-Pro-Xaa peptides could be identified: PapA (YqhT) and PapB (YkvY). This adds a new aspect to the use of proline as an osmostress protectant by B. subtilis and demonstrates how gainful available resources can be used by bacteria to adapt to changes in environmental conditions.
In addition to PutP and OpuE, B. subtilis possess a so far uncharacterized proline transport system. To identify the unknown proline transporter a genetic screening approach was developed. In a B. subtilis mutant strain unable to transport proline, the transcription of the proline utilization genes (putBCP) did no longer occur in the presence of proline. This demonstrates the necessity of the proline import to induce putBCP expression.
In conclusion, the knowledge acquired in this dissertation revealed novel facets of the physiological role of proline in B. subtilis and furthermore it illustrates impressively how effective bacteria can adapt on a variety of changing
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Full text link“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
Full text linkWe 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
Full text linkWe 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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