1,720,963 research outputs found
Cyclic Di-AMP Homeostasis in Bacillus subtilis BOTH LACK AND HIGH LEVEL ACCUMULATION OF THE NUCLEOTIDE ARE DETRIMENTAL FOR CELL GROWTH
No full textThe genome of the Gram-positive soil bacterium Bacillus subtilis encodes three potential diadenylate cyclases that may synthesize the signaling nucleotide cyclic di-AMP (c-di-AMP). These enzymes are expressed under different conditions in different cell compartments, and they localize to distinct positions in the cell. Here we demonstrate the diadenylate cyclase activity of the so far uncharacterized enzymes CdaA (previously known as YbbP) and CdaS (YojJ). Our work confirms that c-di-AMP is essential for the growth of B. subtilis and shows that an excess of the molecule is also harmful for the bacteria. Several lines of evidence suggest that the diadenylate cyclase CdaA is part of the conserved essential cda-glm module involved in cell wall metabolism. In contrast, the CdaS enzyme seems to provide c-di-AMP for spores. Accumulation of large amounts of c-di-AMP impairs the growth of B. subtilis and results in the formation of aberrant curly cells. This phenotype can be partially suppressed by elevated concentrations of magnesium. These observations suggest that c-di-AMP interferes with the peptidoglycan synthesis machinery. The activity of the diadenylate cyclases is controlled by distinct molecular mechanisms. CdaA is stimulated by a regulatory interaction with the CdaR (YbbR) protein. In contrast, the activity of CdaS seems to be intrinsically restricted, and a single amino acid substitution is sufficient to drastically increase the activity of the enzyme. Taken together, our results support the idea of an important role for c-di-AMP in B. subtilis and suggest that the levels of the nucleotide have to be tightly controlled.Deutsche Forschungsgemeinschaf
Transcription in Mycoplasma pneumoniae: Analysis of the Promoters of the ackA and ldh genes
No full textThe nucleotide sequences that control transcription initiation and regulation in Mycoplasma pneumoniae are poorly understood. Moreover, only few regulatory events have been reported for M. pneumoniae. We have studied changes in the global protein synthesis pattern in M. pneumoniae in response to the presence of glycerol. The ackA and ldh genes, encoding acetate kinase and lactate dehydrogenase, respectively, were controlled in a carbon source-dependent manner. While the ackA gene was strongly expressed in the presence of glucose, transcription of ldh was induced by glycerol. The promoters of both genes were mapped by primer extension analysis. Molecular analysis of transcription regulatory mechanisms in M. pneumoniae has so far not been possible due to the lack of appropriate reporter systems that can be used to study the activity of promoter fragments and their mutant derivatives in vivo. Recently, a reporter system has been developed which allows cloning of promoter fragments in front of a promoterless lacZ gene and inserting this construct into the genome of M. pneumoniae. To study the requirements of M. pneumoniae RNA polymerase for promoter recognition, a series of fusions of deletion and mutant variants of the ldh promoter was constructed and analyzed in vivo. While mutations affecting the -10 region strongly interfered with gene expression, the -35 region seems to be of minor importance in M. pneumoniae. (c) 2007 Elsevier Ltd. All rights reserved
Determination of the Gene Regulatory Network of a Genome-Reduced Bacterium Highlights Alternative Regulation Independent of Transcription Factors
No full textHere, we determined the relative importance of different transcriptional mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae, by employing an array of experimental techniques under multiple genetic and environmental perturbations. Of the 143 genes tested (21% of the bacterium's annotated proteins), only 55% showed an altered phenotype, highlighting the robustness of biological systems. We identified nine transcription factors (TFs) and their targets, representing 43% of the genome, and 16 regulators that indirectly affect transcription. Only 20% of transcriptional regulation is mediated by canonical TFs when responding to perturbations. Using a Random Forest, we quantified the non-redundant contribution of different mechanisms such as supercoiling, metabolic control, RNA degradation, and chromosome topology to transcriptional changes. Model-predicted gene changes correlate well with experimental data in 95% of the tested perturbations, explaining up to 70% of the total variance when also considering noise. This analysis highlights the importance of considering non-TF-mediated regulation when engineering bacteria.sponsorship: We acknowledge the staff of the Genomics Core facility for their assistance. Also, we thank the CRG/UPF Proteomics Unit, which is part of the Plataforma de Recursos Biomoleculares y Bioinformaticos (Instituto de Salud Carlos III), supported by grant PT13/0001. The project was supported by funds from the Fundacion Marcelino Botin and the Spanish Ministerio de Economia y Competitividad (BIO2007-61762). This project was financed by Instituto de Salud Carlos III and co-financed by Federacion Espanola de Enfermedades Raras under grant agreement PI10/01702 and the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program, under grant agreements no 634942 (MycoSynVac) and 670216 (MYCOCHASSIS). We acknowledge support from the Spanish Ministry of Economy and Competitiveness to the EMBL partnership, Centro de Excelencia Severo Ochoa. We also acknowledge the support of the CERCA Program from the Generalitat de Catalunya, Spain. (Fundacion Marcelino Botin, Spanish Ministerio de Economia y Competitividad|BIO2007-61762, Instituto de Salud Carlos III, Federacion Espanola de Enfermedades Raras|PI10/01702, European Research Council (ERC) under the European Union|634942, European Research Council (ERC) under the European Union|670216, Spanish Ministry of Economy and Competitiveness, CERCA Program from the Generalitat de Catalunya, Spain, grant PT13/0001, European Research Council (ERC)|670216)status: Publishe
Transkription in Mycoplasma pneumoniae
Full text linkDas humanpathogene Bakterium Mycoplasma pneumoniae besitzt lediglich ein kleines Repertoire von Genen, die vermutlich an der Transkriptionsregulation beteiligt sind. Dieser Umstand reflektiert seine Anpassung an ein nährstoffreiches Habitat mit konstanten Umweltbedingungen, dem menschlichen Lungenepithel. Bis heute ist der Hitzeschockregulator HrcA das einzige charakterisierte Protein, welches an der Transkriptionsregulation in diesem Organismus beteiligt ist. Da jedoch zahlreiche andere Transkriptionsregulationen in M. pneumoniae beschrieben worden sind, stellt sich die Frage, welche Mechanismen bzw. welche Regulatoren für diese Ereignisse verantwortlich sind. Das Ziel dieser Arbeit war die Charakterisierung der übrigen Proteine, die vermutlich an Regulationsereignissen beteiligt sind. Die gezielte Suche nach Mutanten, denen diese Proteine fehlen, zeigte dass die Mehrheit dieser Proteine für die Vitalität des Bakteriums essentiell ist. Lediglich Mutanten, denen die weitläufig in Bakterien konservierten Proteine RelA und WhiA fehlen, konnten isoliert werden. Außerdem konnte eine Interaktion zwischen dem Protein Mpn266 und der α-Untereinheit der RNA-Polymerase nachgewiesen werden, ein Hinweis darauf, dass Protein Mpn266 ein Ortholog des Transkriptionsregulators Spx in den Firmicutes ist. Auch dieser mutmaßliche Transkriptionsregulator ist essentiell. Zahlreiche Experimente zur Charakterisierung der Rolle von RelA und des Signalmoleküls (p)ppGpp in M. pneumoniae wurden durchgeführt. Während gezeigt werden konnte, dass die Mechanismen, die zur RelA-abhängigen Synthese von (p)ppGpp in diesem Organismus konserviert sind, konnte, abgesehen von der Unfähigkeit (p)ppGpp herzustellen kein weiterer Phänotyp gefunden werden. Ein Effekt von (p)ppGpp auf die Transkription konnte mittels Mikroarray-Experimenten nicht nachgewiesen werden. Zusätzliche Untersuchungen zur Rolle der HPr-Phosphorylierung konnten zudem zeigen, dass diese keine Rolle bei der Transkriptionsregulation spielen. Statt dessen scheint die HPr-Phosphorylierung die pH-abhängige Zuckeraufnahme in M. pneumoniae zu regulieren. Mikroarrayuntersuchungen einer whiA Mutante zeigen eine konstitutive Hochregulation der Transkription eines großen Operons, welches hauptsächlich aus ribosomalen Genen besteht. Da sowohl der Regulator als auch das Operon in Gram-positiven Bakterien konserviert sind, liegt die Vermutung einer identischen Funktion dieses Regulators in anderen Bakterien nahe. Diese Vermutung wird durch das Vorhandensein eines gemeinsamen DNA Motivs vor diesem Operon gestützt. Zusammengefasst beinhaltet diese Arbeit die ersten Mikroarrayuntersuchungen von M. pneumoniae Mutanten überhaupt und liefert die Grundlage für zukünftige Experimente, die darauf abzielen, Regulationsereignisse in diesen Organismus besser zu verstehen.The human pathogen Mycoplasma pneumoniae has maintained only a small repertoire of genes putatively involved in transcription regulation. This reflects its adaption to a nutrient rich and rather constant habitat, the human lung epithelium. To date, the heat shock response regulator HrcA is the only characterized protein involved in transcription regulation in this organism. However, several transcription regulation events have been described, leading to the question which mechanisms or which regulators are responsible for these events. This work aimed to study the implication of the putative, so far uncharacterized regulators in transcription in M. pneumoniae. Screening of a mutant library showed that the majority of the genes encoding these regulators are essential for viability. Only mutants affected in genes relA and whiA, both widely distributed and highly conserved in bacteria, could be isolated from the library. In addition, interaction of protein Mpn266 with RpoA could be shown, providing evidence that this protein is an orthologue of transcription regulator Spx in the Firmicutes. However, this regulator is essential, too. Several experiments have been performed to characterize the role of RelA and the alarmone (p)ppGpp in M. pneumoniae. Whereas it could be shown that mechanisms leading to (p)ppGpp formation in this organism are conserved, no additional phenotype could be identified in a relA mutant despite an inability to synthesize these alarmones. An effect on transcription regulation could not be shown by microarray analysis. However, evidence was provided that (p)ppGpp formation is not involved in short-term regulation of transcription in M. pneumoniae. Experimental data indicate that the phosphorylation state of HPr is involved in regulating the uptake of glucose and other PTS sugars in a pH dependent manner, rather then in transcription regulation. Microarray analysis of a whiA mutant showed that a large operon consisting mainly of gene s encoding ribosomal proteins is constitutively up-regulated in this strain. Both the regulator and the operon co-occur in Gram-positive bacteria, suggesting a similar function of this regulator in other bacteria. The finding of a conserved DNA sequence upstream of this operon supports this hypothesis. In summary, this work provides the first microarray analyses of M. pneumoniae mutants. Furthermore, the first M. pneumoniae mutant impaired in transcription regulation and a novel putative role of HPr phosphorylation were described. Thus, this work provides a basis for future experiments that could help to understand regulatory mechanisms in this minimal organism
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
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