Structural and functional studies on the transcriptional regulation of flagellar motility and biofilm formation

Part 1: Numerical regulation in the monotrichous bacterium Shewanella putrefaciens Microorganisms have the ability to adapt to changing environmental conditinos. This has enabled them to colonize virtually nearly every niche on the planet Earth. Key to this ability is bacterial motility, which allows bacteria to move away from unfavourable conditions and to move towards favourable conditions. In connection with a sensory system, which detects chemical cues and other stimuli, bacteria can move towards nutrients. Bacterial motility is largely enabled by flagella. The biogenesis of a flagellum is a very costly process, which is for this reason highly regulated. In the monotrichous bacterium Shewanella putrefaciens, FlhF and FlhG are responsible for maintaining number and location of the single polar flagellum. In the course of this work, it could be shown that FlhG limits the number of flagella to one by directly interacting with the master transcriptional regulator of the flagellum, FlrA. Furthermore, FlhG is implicated in assembly of the cytosolic face of the flagellum, the C-Ring. The transcriptional control via FlrA as well as the C-Ring assembly via FliM occur through the same binding site on FlhG. This highlights the central role of FlhG and shows that FlhG integrates the two processes to regulate flagellar number. Taken together, these observations represent an important step towards a complete conceptual description of flagellar biogenesis. Thereby, these results also form the basis for further research. Part 2: Transcriptional regulation of biofilms is mediated by RemA, which interacts with DNA in a histone-like manner Instead of a motile lifestyle, bacteria can also establish a multicellular, sessile lifestyle in the form of biofilms. In biofilms, bacterial cells establish a division of labour and establish an increased resistance against antibiotics and environmental hazardous conditions. This is mediated by the secretion of extracellular proteins and other biological molecules. The protein RemA is central to this process, as it activates the secretion of these extracellular components. Furthermore, RemA is implicated in processes which enable a cellular protection against osmotic pressure, which occurs during biofilm formation. In the context of this work, the structure of RemA from Geobacillus thermodenitrificans could be elucidated. RemA interacts with DNA in a novel and unique way, which is reminiscent of DNA-looping by histone-complexes. By means of biochemical methods, crucial residues of RemA responsible for DNA interaction could be functionally investigated. Furthermore, the structural fate of amino acid mutations, which impair the functionality of RemA, could be investigated. Taken together, this work represents an important step towards the understanding of the transcriptional processes that govern biofilm-formation and osmoprotection in Bacillus subtilis. This work also provides the basis to further investigate the function of RemA in the cellular context. In the future, the structural investigation of RemA-DNA-interaction is facilitated by the insights obtained in the context of this work. Part 3: Membrane protein biogenesis is regulated by a structurally unique, co-translational state of FtsY. Membrane proteins are translated by ribosomes and predominantly inserted into the membrane by the SecYEG-translocon. A factor critical for this process is the SRP-receptor FtsY, which enables co-translational targeting to the translocon in coopration with the SRP-particle FFH and SRP-RNA. In the context of this work it could be shown that a co-translational state of FtsY, the helical domain N2-4, critically mediates membrane targeting of the receptor. By means of crystallographic analyses and studies in solution, it could be shown that the subdomain of N2-4 possesses a different fold when isolated than in the context of the G-domain of FtsY. This observation represents a unique paradigm, which indicates that nascent N2-4 executes a different function during its own translation than when N2-4 is part of the mature FtsY-receptor. These results are an important step towards the conceptual understanding of membrane protein biogenesis and –targeting. Further work could elucidate, whether this concept also applies to homologs of FtsY such as FlhF

A INVENÇÃO DE MARCEL PROUST, SEGUNDO WALTER BENJAMIN

This article proposes a reading act of the essay “The Image of Proust”, 1929, by Walter Benjamin. The way Benjamin translates elements of the proustian Recherche into the essay’s writing reveals a method’s lesson as an effect of the reader's immersion. Taking this into consideration, I analyze two figures of immersion from the essay, within which, in this meantime, we have also submerged. Finally, an image of the reader himself is brought out as a work of art.O presente artigo propõe uma leitura do ensaio “À imagem de Proust”, 1929, de Walter Benjamin. Lê-se de que modo Benjamin traduz elementos da Recherche proustiana à escrita do ensaio, dando a ver uma lição de método, efeito da imersão do leitor na obra. Daí se analisam duas figuras de imersão do ensaio, dentro do qual, nesse meio tempo, também já submergimos. Por fim, resta fazer emergir uma imagem do próprio leitor como obra. REFERÊNCIAS ADORNO, Theodor. Posição do narrador no romance contemporâneo. In: ______. Notas de literatura I. São Paulo: Duas Cidades; 34, 2003, p. 55-64. BENJAMIN, Walter. Gesammelte Schriften: II-1; Aufsätze, Essays, Vorträge. Frankfurt am Main: Suhrkamp Verlag, 1977. ______. Gesammelte Schriften: VI; Fragmente Autobiographische Schriften. Frankfurt am Main: Suhrkamp Verlag, 1991. ______. Obras escolhidas I: Magia e técnica, arte e política: ensaios sobre literatura e história da cultura. Trad. Sergio Paulo Rouanet. Rev. Márcio Seligmann-Silva. São Paulo: Brasiliense, 2012. FLAUBERT, Gustave. Madame Bovary: costumes de província. Tradução Fúlvia M. L. Moretto. São Paulo: Nova Alexandria, 2007. GAGNEBIN, Jeanne Marie. História e narração em Walter Benjamin. São Paulo: Perspectiva, 2011. GENETTE, Gerard. Proust palimpsesto. In: ______.  Figuras. São Paulo: Perspectiva, 1972. p. 41-67. PROUST, Marcel. No caminho de Swann. Tradução Mario Quintana. São Paulo: Globo, 2006. ______. Tempo redescoberto. Tradução Lúcia Miguel Pereira. São Paulo: Globo, 2004

A interpretação encena: ler quem lê

RESUmO: Este trabalho se propõe investigar, a partir da análise das respostas a uma questão de prova aplicada numa turma de 2ª série do Ensino Médio, as operações subjetivas envolvidas na leitura e interpretação de texto, de inferências e suposições até a criação de cenas e enredos. Na questão proposta, sobre a canção “Mil perdões”, de Chico Buarque, os alunos foram convidados a refletir a respeito de um discurso sobre traição e a levantar hipóteses acerca do ato de perdoar tematizado na canção. Organizadas em quatro recorrentes “cenas de linguagem”, conforme propõe Barthes em Fragmentos de um discurso amoroso, as respostas revelam como os alunos adentram o discurso amoroso por vias que reconhecem, remontando determinadas figuras, mais ou menos de acordo com a lei do texto. Trata-se aqui não só de indicar as formas como os alunos preenchem as lacunas de um texto, mas, a partir do lugar de professor, de lê-los em sua errância interpretativa. Por fim, procura-se esboçar uma teoria da leitura em que se destacam as figuras da traição e do ciúme e, ao mesmo tempo, propor uma ética da traição inevitável em todo gesto interpretativo.PALAVRAS-CHAVE: Leitura subjetiva, Formação do leitor, Práticas de leitura, Ensino de literatura, Ciúme

The dedicated chaperone Acl4 escorts ribosomal protein Rpl4 to its nuclear pre-60S assembly site

Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S assembly site in the nucleus

60S ribosome biogenesis requires rotation of the 5S ribonucleoprotein particle

During eukaryotic ribosome biogenesis, nascent ribosomal RNA (rRNA) forms pre-ribosomal particles containing ribosomal proteins and assembly factors. Subsequently, these immature rRNAs are processed and remodelled. Little is known about the premature assembly states of rRNAs and their structural rearrangement during ribosome biogenesis. Using cryo-EM we characterize a pre-60S particle, where the 5S rRNA and its associated ribosomal proteins L18 and L5 (5S ribonucleoprotein (RNP)) are rotated by almost 180 degrees when compared with the mature subunit. Consequently, neighbouring 25S rRNA helices that protrude from the base of the central protuberance are deformed. This altered topology is stabilized by nearby assembly factors (Rsa4 and Nog1),which were identified by fitting their three-dimensional structures into the cryo-EM density. We suggest that the 5S RNP performs a semicircular movement during 60S biogenesis to adopt its final position, fulfilling a chaperone-like function in guiding the flanking 25S rRNA helices of the central protuberance to their final topology

MerA functions as a hypothiocyanous acid reductase and defense mechanism in Staphylococcus aureus

The major pathogen Staphylococcus aureus has to cope with host-derived oxidative stress to cause infections in humans. Here, we report that S. aureus tolerates high concentrations of hypothiocyanous acid (HOSCN), a key antimicrobial oxidant produced in the respiratory tract. We discovered that the flavoprotein disulfide reductase (FDR) MerA protects S. aureus from this oxidant by functioning as a HOSCN reductase, with its deletion sensitizing bacteria to HOSCN. Crystal structures of homodimeric MerA (2.4 Å) with a Cys43–Cys48 intramolecular disulfide, and reduced MerACys43S (1.6 Å) showed the FAD cofactor close to the active site, supporting that MerA functions as a group I FDR. MerA is controlled by the redox-sensitive repressor HypR, which we show to be oxidized to intermolecular disulfides under HOSCN stress, resulting in its inactivation and derepression of merA transcription to promote HOSCN tolerance. Our study highlights the HOSCN tolerance of S. aureus and characterizes the structure and function of MerA as a major HOSCN defense mechanism. Crippling the capacity to respond to HOSCN may be a novel strategy for treating S. aureus infections

Structural Basis for Regulation of the Opposing (p)ppGpp Synthetase and Hydrolase within the Stringent Response Orchestrator Rel

The stringent response enables metabolic adaptation of bacteria under stress conditions and is governed by RelA/SpoT Homolog (RSH)-type enzymes. Long RSH-type enzymes encompass an N-terminal domain (NTD) harboring the second messenger nucleotide (p)ppGpp hydrolase and synthetase activity and a stress-perceiving and regulatory C-terminal domain (CTD). CTD-mediated binding of Rel to stalled ribosomes boosts (p)ppGpp synthesis. However, how the opposing activities of the NTD are controlled in the absence of stress was poorly understood. Here, we demonstrate on the RSH-type protein Rel that the critical regulative elements reside within the TGS (ThrRS, GTPase, and SpoT) subdomain of the CTD, which associates to and represses the synthetase to concomitantly allow for activation of the hydrolase. Furthermore, we show that Rel forms homodimers, which appear to control the interaction with deacylated-tRNA, but not the enzymatic activity of Rel. Collectively, our study provides a detailed molecular view into the mechanism of stringent response repression in the absence of stress