Physiological Consequences of protein translocation stress in Bacillus subtilis

Bacillus subtilis is a soil-inhabiting bacterium, characterized by high-level protein secretion. Since secreted proteins are easy to harvest, B. subtilis is frequently employed in industry for enzyme production. Protein secretion from the cytoplasm, across the cytoplasmic membrane and into the growth medium takes place via the general secretion pathway (Sec) or the so-called twin-arginine (Tat) pathway. Interestingly, B. subtilis cells experience stress when being pushed towards high-level protein secretion. To date, the impact of this secretion stress at the level of the cytoplasmic membrane remained poorly understood, due to the hydrophobic nature and low abundance of membrane proteins. Studies described in this PhD thesis were therefore focused on a definition of protein secretion stress at the membrane level. To this end, a multidisciplinary approach combining biochemical and “omics” technologies was applied. In one series of experiments, the Tat pathway was overexpressed to enhance productivity. This resulted in the identification of a protein that closely interacts with the Tat pathway and modulates its activity. Moreover, high level expression of the Tat pathway resulted in prolonged bacterial growth, altered amino acid metabolism and downregulation of differentiation processes, such as biofilm formation and sporulation. In contrast, absence of the Tat pathway resulted in severe oxidative stress. Lastly, the stressful effects of secreting a staphylococcal antigen via the Sec pathway were analysed by absolute membrane protein quantification, which provided novel insights in the accompanying cellular rearrangements. Altogether, the present studies provide new leads for minimizing secretion stress and enhancing protein production in B. subtilis

¿Fin de la dependencia? repercusiones de la revolución del esquisto sobre las políticas de seguridad de EE.UU.

La política de seguridad de EE.UU. desde la segunda guerra mundial se ha enfocado hacia establecer un suministro seguro de petróleo para su economía, debido a la alta dependencia que tiene de esta fuente energética. Esta dinámica de seguridad puede cambiar ya que gracias a la revolución del esquisto, EE.UU. se acerca al autoabastecimiento y a una posible independencia energética. Este trabajo analiza las repercusiones que tienen estos cambios sobre la política exterior de seguridad de los Estados Unidos.The US security policy since World War II It has been focused on establishing a secure oil supplies for its economy, due to the high dependence of this energy source. This dynamic security can change and thanks to the shale revolution , USA it is about self-sufficiency and possible energy independence. This paper analyzes the impact of these changes on foreign security policy of the United States.InternacionalistaPregrad

La parálisis del proceso y sus consecuencias

Esta investigación abarca el estudio de los efectos e implicaciones que conlleva la falta de impulso procesal como incumplimiento de las partes dentro de un proceso, en razón a la aplicación del desistimiento tácito y la perención en procesos ejecutivos. Se analizan los antecedentes, características y fines de estas figuras, al igual que se comparan con figuras similares en las legislaciones de España, Venezuela y Perú."This investigation includes the study of the effects and implications upon the lack of procedural impulse of the parts within a process, in reason to the application of the ""tacit abandonment"" of the claim and the perención in executive processes. The antecedents, characteristics and aims of these figures are analyzed, as well as they are compared with similar figures in the legislations of Spain, Venezuela and Peru."Abogado (a)Pregrad

Poética de la arquitectura contemporánea

La conceptualización del espacio arquitectónico no ha sido única a través del tiempo, por el contrario, ha sido un proceso de cambios ideológicos que dependen generalmente de la variedad de culturas y de pensamiento. Sin embargo, lo que sí se ha mantenido invariable es que el espacio arquitectónico solo se concreta cuando es experimentado, cuando es percibido y recorrido por el ser humano. En palabras de Tadao Ando, “La arquitectura solo se considera completa con la intervención del ser humano que la experimenta. En otras palabras, el espacio arquitectónico solo cobra vida en correspondencia con la presencia humana que lo percibe” (Jensen & Walker, 1995, p.76). Así mismo Rasmussen (1980) afirma que la arquitectura no solo debe ser vista sino que también debe ser experimentada, apreciada; debe encontrarse cómo y con qué propósito fue diseñada; deben ser habitados cada uno de sus espacios, sentir como nos encierran y conducen hacia otros; se debe analizar cada una de las texturas, descubrir por qué fueron escogidos los colores y cómo esto influencio la orientación del recinto.The conceptualization of the architectural space has not been unique over time, on the contrary, it has been a process of ideological changes that generally depend on the variety of cultures and thought. However, what has remained unchanged is that the architectural space only materializes when it is experienced, when it is perceived and traversed by the human being. In the words of Tadao Ando, ​​"Architecture is considered complete only with the intervention of the human being who experiences it. In other words, the architectural space only comes to life in correspondence with the human presence that perceives it "(Jensen & Walker, 1995, p.76). Likewise, Rasmussen (1980) affirms that architecture must not only be seen, but must also be experienced, appreciated; it must be found how and for what purpose it was designed; Each of its spaces must be inhabited, feel as they lock us up and lead us towards others; each one of the textures must be analyzed, discover why the colors were chosen and how this influenced the orientation of the site

Evaluación del consumo energético a la escala del fragmento urbano

La ciudad ofrece una serie de servicios necesarios para el ser humano. Entre esos servicios, la producción y distribución de energía juegan un rol importante en términos de desarrollo urbano sostenible. La energía interviene sobre tres pilares fundamentales: • El aspecto social: porque garantiza el confort y bienestar humano. • El aspecto económico: todas las industrias y actividades económicas dependen de su aprovisionamiento de energía. • El aspecto ambiental: la producción de energía consume gran parte de los recursos naturales. Debemos interrogarnos sobre el futuro de nuestras sociedades dado que se estima que las reservas petroleras mundiales podrían agotarse en una cincuentena de años (alrededor de 65 años para el gas y 260 para el carbón) (Despretz, 2006).The city offers a series of services necessary for the human being. Among these services, the production and distribution of energy play an important role in terms of sustainable urban development. Energy intervenes on three fundamental pillars: • The social aspect: because it guarantees comfort and human well-being. • The economic aspect: all industries and economic activities depend on their energy supply. • The environmental aspect: the production of energy consumes a large part of natural resources. We must ask ourselves about the future of our societies given that it is estimated that the world oil reserves could be depleted in about fifty years (around 65 years for gas and 260 for coal) (Despretz, 2006)

Membrane Modulation of Super-Secreting “midiBacillus” Expressing the Major Staphylococcus aureus Antigen – A Mass-Spectrometry-Based Absolute Quantification Approach

Bacillus subtilis has been extensively used as a microbial cell factory for industrial enzymes due to its excellent capacities for protein secretion and large-scale fermentation. This bacterium is also an attractive host for biopharmaceutical production. However, the secretion potential of this organism is not fully utilized yet, mostly due to a limited understanding of critical rearrangements in the membrane proteome upon high-level protein secretion. Recently, it was shown that bottlenecks in heterologous protein secretion can be resolved by genome minimization. Here, we present for the first time absolute membrane protein concentrations of a genome-reduced B. subtilis strain (“midiBacillus”) expressing the immunodominant Staphylococcus aureus antigen A (IsaA). We quantitatively characterize the membrane proteome adaptation of midiBacillus during production stress on the level of molecules per cell for more than 400 membrane proteins, including determination of protein concentrations for ∼61% of the predicted transporters. We demonstrate that ∼30% of proteins with unknown functions display a significant increase in abundance, confirming the crucial role of membrane proteins in vital biological processes. In addition, our results show an increase of proteins dedicated to translational processes in response to IsaA induction. For the first time reported, we provide accumulation rates of a heterologous protein, demonstrating that midiBacillus secretes 2.41 molecules of IsaA per minute. Despite the successful secretion of this protein, it was found that there is still some IsaA accumulation occurring in the cytosol and membrane fraction, leading to a severe secretion stress response, and a clear adjustment of the cell’s array of transporters. This quantitative dataset offers unprecedented insights into bioproduction stress responses in a synthetic microbial cell

Double trouble:Bacillus depends on a functional Tat machinery to avoid severe oxidative stress and starvation upon entry into a NaCl-depleted environment

The widely conserved twin-arginine translocases (Tat) allow the transport of fully folded cofactor-containing proteins across biological membranes. In doing so, these translocases serve different biological functions ranging from energy conversion to cell division. In the Gram-positive soil bacterium Bacillus subtilis, the Tat machinery is essential for effective growth in media lacking iron or NaCl. It was previously shown that this phenomenon relates to the Tat-dependent export of the heme-containing peroxidase EfeB, which converts Fe2+ to Fe3+ at the expense of hydrogen peroxide. However, the reasons why the majority of tat mutant bacteria perish upon dilution in NaCl-deprived medium and how, after several hours, a sub-population adapts to this condition was unknown. Here we show that, upon growth in the absence of NaCl, the bacteria face two major problems, namely severe oxidative stress at the membrane and starvation leading to death. The tat mutant cells can overcome these challenges if they are fed with arginine, which implies that severe arginine depletion is a major cause of death and resumed arginine synthesis permits their survival. Altogether, our findings show that the Tat system of B. subtilis is needed to preclude severe oxidative stress and starvation upon sudden drops in the environmental Na+ concentration as caused by flooding or rain

Functional association of the stress-responsive LiaH protein and the minimal TatAyCy protein translocase in Bacillus subtilis

The bacterial twin-arginine (Tat) pathway serves in the exclusive secretion of folded proteins with bound co-factors. While Tat pathways in Gram-negative bacteria and chloroplast thylakoids consist of conserved TatA, TatB and TatC subunits, the Tat pathways of Bacillus species and many other Gram-positive bacteria stand out for their minimalist nature with the core translocase being composed of essential TatA and TatC subunits only. Here we addressed the question whether the minimal TatAyCy translocase of Bacillus subtilis recruits additional cellular components that modulate its activity. To this end, TatAyCy was purified by affinity- and size exclusion chromatography, and interacting co-purified proteins were identified by mass spectrometry. This uncovered the cell envelope stress responsive LiaH protein as an accessory subunit of the TatAyCy complex. Importantly, our functional studies show that Tat expression is tightly trailed by LiaH induction, and that LiaH itself determines the capacity and quality of TatAyCy-dependent protein translocation. In contrast, LiaH has no role in high-level protein secretion via the general secretion (Sec) pathway. Altogether, our observations show that protein translocation by the minimal Tat translocase TatAyCy is tightly intertwined with an adequate bacterial response to cell envelope stress. This is consistent with a critical need to maintain cellular homeostasis, especially when the membrane is widely opened to permit passage of large fully-folded proteins via Tat

Maintaining Integrity Under Stress:Envelope Stress Response Regulation of Pathogenesis in Gram-Negative Bacteria

The Gram-negative bacterial envelope is an essential interface between the intracellular and harsh extracellular environment. Envelope stress responses (ESRs) are crucial to the maintenance of this barrier and function to detect and respond to perturbations in the envelope, caused by environmental stresses. Pathogenic bacteria are exposed to an array of challenging and stressful conditions during their lifecycle and, in particular, during infection of a host. As such, maintenance of envelope homeostasis is essential to their ability to successfully cause infection. This review will discuss our current understanding of the σE- and Cpx-regulated ESRs, with a specific focus on their role in the virulence of a number of model pathogens

Stress-induced adaptive morphogenesis in bacteria

Bacteria thrive in virtually all environments. Like all other living organisms, bacteria may encounter various types of stresses, to which cells need to adapt. In this chapter, we describe how cells cope with stressful conditions and how this may lead to dramatic morphological changes. These changes may not only allow harmless cells to withstand environmental insults but can also benefit pathogenic bacteria by enabling them to escape from the immune system and the activity of antibiotics. A better understanding of stress-induced morphogenesis will help us to develop new approaches to combat such harmful pathogens.Microbial Biotechnolog