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Branched-chain amino acids (BCAAs) act as effector molecules that signal a global transcriptional regulator, CodY, to regulate virulence factors in nutrient depleted environments. Staphylococcus aureus contains three putative BCAA transporters (BrnQ1, BrnQ2, BrnQ3) whose role in BCAA uptake is unknown. We hypothesize that BrnQ transporters are involved in BCAA uptake and contribute to virulence in S. aureus by modulating CodY activity. Results from radioactive uptake assays indicate that BrnQ1 is the predominant BrnQ transporter of isoleucine, valine and leucine. Meanwhile, BrnQ2 is more specific for isoleucine. Furthermore, only the lack of BrnQ1 hinders growth of S. aureus in chemically-defined media with limited concentrations of BCAAs; however, BrnQ2 and BrnQ3 show no such growth phenotype. In vivo, the brnQ1 and brnQ3 mutants are attenuated for virulence in a mouse model of bacteremia, but the brnQ2 mutant was hyper-virulent, suggesting that the gene plays a more important role within the host.

In dieser Arbeit wurde die ABE-Fermentation von Clostridium acetobutylicum mit den Zuckersubstraten der Lignocellulose D-(+)-Glucose, D-(+)-Xylose und L-(-)-Arabinose in synthetischen Medien untersucht. Es konnte erfolgreich ein Chemostatverfahren für die Fermentation mit den Pentosesubstraten entwickelt werden. Es wurde bei dem Substrat D-(+)-Xylose die ABE-Produktivität um das 4,7-fache im Vergleich zum Satzkulturverfahren gesteigert. Ebenfalls zeigte sich D-(+)-Gluconat als ein Substrat von C. acetobutylicum. DNA-Microarray-Studien ergaben Hinweise für mögliche Zuckertransportwege in die Zelle. Durch die Optimierung des Fermentationsmediums konnte der Lösungsmittelertrag über verschiedene Fermentationsmaßstäbe verbessert werden. Die Geninaktivierung von codY in C. acetobutylicum äußerte sich in einer Verbesserung der Butanolproduktion im Satzkultur- und Chemostatverfahren bei unterschiedlichen pH-Werten.

Tez (Yüksek Lisans) -- İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü, 2011, Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2011, Basilisin, Bacillus subtilis tarafından sentezlenen, L-alanin ve L-antikapsin’den oluşan bir dipeptit (125 kDa) antibiyotiktir. Yakın zamanda, B. subtilis’in basilisin biyosentezinde bir polisistronik operon ywfBCDEFG ve bitişiğindeki monosistronik gen ywfH in görev aldığı bulunmuştur. Önceden, basilisin biyosentezinin hücre yoğunluğu sinyali döngüsünün kontrolü altında gerçekleştiği gösterilmiştir. Ek olarak, basilisin üretiminin global düzenleyici Spo0A tarafından direk olarak, pozitif yönde düzenlenirken, geçiş evresi düzenleyicileri CodY ve AbrB tarafından direk olarak, negatif yönde düzenlendiği bulunmuştur. Çok yakın zaman içinde bu global düzenleyicilerin bac operonunun ifadesi üzerindeki etkisi ve bu global düzenleyicilerin bac promoter bölgesi ile olan etkileşimleri keşfedilmiştir. AbrB ve CodY proteinleri bac promotörünü doğrudan baskılayan negatif düzenleyicilerken, ComA ve Spo0A, bac operonun geçiş evresinde direk uyarılmasını sağlayan, pozitif yönde hareket eden düzenleyiciler olduğu gösterilmiştirdir. Bir sonraki çalışma olarak, bu çalışmada, ywfH promotörünün ComA, CodY, AbrB, Spo0A, ve DegU global düzenleyiciler için doğrudan hedef olup olmadığını “Electrophoretic Mobility Shift Assay (EMSA)” yöntemi kullanarak tanımlanması amaçlanmıştır. Bu amaçla, 414bç DNA parçasını içeren ywfH promotör bölgesi, saflaştırılmış C-ucu His6 kuyruklu ComA, Spo0A, AbrB, CodY ve DegU proteinlerinin çeşitli konsantrasyonları ile inkübe edilmiştir. Bunun dışında, AbrB-CodY, Spo0A-ComA, ComA-ComK ve ComA-DegU’nun eş zamanlı mı veya yarışmacı olarak mı PywfH‘ne bağlandığını belirlemek amacıyla iki düzenleyici proteinin varlığında “jel mobility shift” yöntemi tekrarlanmıştır., Bacilysin is a dipeptide antibiotic (125 kDa) composed of L-alanin and L-anticapsin in Bacillus subtilis. Recently, it was found that a polycistronic operon: ywfBCDEFG and adjacent monocistronic gene: ywfH were essential for the production of bacilysin. Previously it was shown that biosynthesis of bacilysin is under the positive control of quorum sensing global regulatory circuit. Additionally, bacilysin production is also positively regulated by global regulatory factor Spo0A and negatively regulated by transition state regulators, CodY and AbrB. Very recently, the effects of global regulatory factors ComA, Spo0A, CodY and AbrB on the expression of bac operon and their interaction with the promoter region of bac operon were investigated. It was shown that ComA and Spo0A act as a positive regulator which participates in the transition state induction of bac operon both directly by interacting with the bac promoter while AbrB and CodY are negative regulators which directly repress the bac promoter. As a further study, the present study aims to identify whether ywfH promoter is direct target for the global regulators; ComA, CodY, AbrB, Spo0A and DegU by using “Electrophoretic Mobility Shift Assay (EMSA)”. For this purpose, the 414 bp DNA fragment harboring the promoter region of ywfH were incubated with the various concentrations of the purified C-terminal His6-tagged-ComA, -Spo0A, -AbrB, -CodY, -DegU proteins. Moreover, the gel mobility shift assays were repeated in the presence of both regulatory proteins to determine whether AbrB-CodY, Spo0A-ComA, ComA-ComK and ComA-DegU could bind to ywfH promoter simultaneously or compete for binding., Yüksek Lisans, M.Sc.

Background Amino acid assimilation is crucial for bacteria and this is particularly true for Lactic Acid Bacteria (LAB) that are generally auxotroph for amino acids. The global response of the LAB model Lactococcus lactis ssp. lactis was characterized during progressive isoleucine starvation in batch culture using a chemically defined medium in which isoleucine concentration was fixed so as to become the sole limiting nutriment. Dynamic analyses were performed using transcriptomic and proteomic approaches and the results were analysed conjointly with fermentation kinetic data. Results The response was first deduced from transcriptomic analysis and corroborated by proteomic results. It occurred progressively and could be divided into three major mechanisms: (i) a global down-regulation of processes linked to bacterial growth and catabolism (transcription, translation, carbon metabolism and transport, pyrimidine and fatty acid metabolism), (ii) a specific positive response related to the limiting nutrient (activation of pathways of carbon or nitrogen metabolism and leading to isoleucine supply) and (iii) an unexpected oxidative stress response (positive regulation of aerobic metabolism, electron transport, thioredoxin metabolism and pyruvate dehydrogenase). The involvement of various regulatory mechanisms during this adaptation was analysed on the basis of transcriptomic data comparisons. The global regulator CodY seemed specifically dedicated to the regulation of isoleucine supply. Other regulations were massively related to growth rate and stringent response. Conclusion This integrative biology approach provided an overview of the metabolic pathways involved during isoleucine starvation and their regulations. It has extended significantly the physiological understanding of the metabolism of L. lactis ssp. lactis. The approach can be generalised to other conditions and will contribute significantly to the identification of the biological processes involved in complex regulatory networks of micro-organisms.

Lactococcus lactis is known to take up extracellular peptides via at least three distinct peptide transporters. The well-described oligopeptide transporter Opp alone is able to ensure the growth of L. lactis in milk, while the di- and tripeptide transporter DtpT is involved in a peptide-dependent signalling mechanism. The oligopeptide Opt transporter displays two peptide-binding proteins, OptA and OptS. We previously demonstrated that OptA-dependent transport is dedicated to nutritional peptides, as an optABCDF mutant (of a strain devoid of Opp) has an impaired capacity to grow in milk. Using isogenic peptide transport mutants, this study shows that biosynthesis of the Opt transporter is much less sensitive to downregulation that is dependent on extracellular peptides taken up by DtpT than is Opp biosynthesis; this peptide-dependent regulation relies on the transcriptional repressor CodY. We demonstrate the dual function of the Opt system; while OptA contributes to the bacterial nutrition during growth in milk, OptS is involved in the transport of signalling peptides derived from milk and controlling opp expression. So, these results shed new light on the peptide-dependent regulation relying on two peptide transporters with different specificities: DtpT and Opt (via OptS).

InBacillus subtilis, motility and chemotaxis require the expression ofhag, which encodes flagellin. This gene is transcribed by theσDform of RNA polymerase and is regulated by a group of proteins called transition state regulators (TSRs). Our studies show thathagtranscription is negatively regulated by the transition state regulator ScoC, by binding to its promoter. Furthermore, ScoC, indirectly, also positively regulateshagby increasing the availability ofσDby downregulating the levels of the anti-σD-factor FlgM. We further show that the positive regulation by ScoC predominates over the negative regulation.

Contains fulltext : 50485.pdf (Publisher’s version ) (Open Access) Several genes involved in nitrogen metabolism are known to contribute to the virulence of pathogenic bacteria. Here, we studied the function of the nitrogen regulatory protein GlnR in the Gram-positive human pathogen Streptococcus pneumoniae. We demonstrate that GlnR mediates transcriptional repression of genes involved in glutamine synthesis and uptake (glnA and glnPQ), glutamate synthesis (gdhA), and the gene encoding the pentose phosphate pathway enzyme Zwf, which forms an operon with glnPQ. Moreover, the expression of gdhA is also repressed by the pleiotropic regulator CodY. The GlnR-dependent regulation occurs through a conserved operator sequence and is responsive to the concentration of glutamate, glutamine, and ammonium in the growth medium. By means of in vitro binding studies and transcriptional analyses, we show that the regulatory function of GlnR is dependent on GlnA. Mutants of glnA and glnP displayed significantly reduced adhesion to Detroit 562 human pharyngeal epithelial cells, suggesting a role for these genes in the colonization of the host by S. pneumoniae. Thus, our results provide a thorough insight into the regulation of glutamine and glutamate metabolism of S. pneumoniae mediated by both GlnR and GlnA.

Genetic competence is a differentiation process initiated by Bacillus subtilis as a result of nutritional deprivation, and is controlled by a complex signal transduction cascade. The promoter of comK, encoding the competence transcription factor, is regulated by at least four different transcription factors: Rok, CodY, DegU and ComK itself. Genetic data have shown that comK expression is influenced by the transition state regulator AbrB as well. In this paper we show that AbrB binds specifically to the comK promoter and covers the RNA polymerase binding site, making it the fifth transcription factor regulating the activity of the comK promoter. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

International audience; Genetic competence is a differentiation process initiated by Bacillus subtilis as a result of nutritional deprivation, and is controlled by a complex signal transduction cascade. The promoter of comK, encoding the competence transcription factor, is regulated by at least four different transcription factors: Rok, CodY, DegU and ComK itself. Genetic data have shown that comK expression is influenced by the transition state regulator AbrB as well. In this paper we show that AbrB binds specifically to the comK promoter and covers the RNA polymerase binding site, making it the fifth transcription factor regulating the activity of the comK promoter.

Lactobacillus casei es una bacteria que desde larga data ha sido empleada por el hombre en procesos fermentativos para la producción de quesos y chacinados y en los últimos años ha adquirido gran relevancia por su uso como probiótico. Durante los procesos mencionados y en el tracto gastrointestinal, Lb casei es sometida a diferentes tipos de estrés, entre ellos el estrés osmótico. El objetivo de este trabajo fue estudiar las modificaciones genéticas, fisiológicas y estructurales de este microorganismo cuando es sometido a un estrés hipersalino, priorizando el estudio de las actividades relacionadas con los procesos de elaboración de alimentos. En esta tesis se demostró que además de los solutos osmocompatibles generales glicín betaína y camitina, di y tripétidos pueden aumentar la osmotolerancia de Lb casei. Además de estimular el crecimiento en un medio hiperosmótico lograron restituir el grado de superenrollamiento del ADN (recuperación del linking number) de modo análogo a la camitina. La construcción y el análisis de una mutante deficiente en el transporte de di-tripéptidos, dtpT, mostró que aquellos péptidos que no son transportados eficientemente no ejercen osmoprotección, mientras que sí lo ejercen aquellos que son transportados de manera eficiente. Así mismo, se describió la pérdida de la represión ejercida por péptidos en las actividades de PrtP y Per , dos de las enzimas principales del sistema proteolítico y principales proveedoras de péptidos en el extracelular e intracelular respectivamente. En el caso de PrtP, se pudo demostrar que el aumento de la actividad se debe a una pérdida de la represión a nivel transcripcional. La demostración de la interacción de CodY (que había sido postulado como el efector de la represión por péptidos de varios componentes del sistema proteolítico en Lactococcus lactis) de Lb casei con la región promotora de prtP apunta a que ésta proteína reguladora mediaría la pérdida de represión en medio hiperosmótico. En un medio carente de péptidos y en alta sal, en cambio, la elevada actividad de PrtP se debe a un aumento de la velocidad máxima de la enzima durante el crecimiento en alta sal y no a un aumento de la actividad transcripcional. Dado que PrtP se encuentra anclada a la pared celular, la modificación de la actividad de la enzima podría ser consecuencia de modificaciones en el entorno de la proteína. Se describió una sensibilidad diferencial a la lisis y a antibióticos que actúan a nivel de envolturas en células provenientes de un medio con alta sal. El aumento de la sensibilidad a la lisis se debe directamente a alteraciones de la pared, ya que el mismo efecto se observó al emplear paredes purificadas. El análisis de las fotografias de microscopía electrónica reveló un aumento en el tamaño de las células crecidas en alta sal y claras diferencias en la pared de la misma. De estos resultados, se desprende la posibilidad de emplear el precrecimiento en NaCl como un método para facilitar la lisis de un microorganismo que normalmente es resistente a la misma. Los resultados obtenidos en esta tesis, contribuyen al conocimiento de la fisiología de bacterias lácticas en presencia de un efector de estrés que normalmente se encuentra durante los procesos en los que estos microorganismos participan. Since long time, Laclobacillus casei has been used for the production of Cheese and sausages. In and the last few years they acquired relevance because of its use as a probiotic. During the fermentation process and in the gastrointestinal tract, Lb casei is subjected to a wide range of stressors and particularly to osmotic stress. The aim of this work was the analysis of the genetic, physiological and structural changes in this microorganism when it grows in a hyperosmotic saline condition, mainly the study of those activities related to the process involved in food production. In this thesis, it was demonstrated that as well as generally osmocompatible solutes, camithine and glycin betaine, di and tripeptides can improve osmotolerance in Lb. casei. Besides their role in stimulation of growth in high osmolarity media, they were able to re-establish the degree of DNA supercoil (recovery of linking number) as camithine did. The construction and analysis of a mutant,dtpT, unable to transport di-tripeptides, enable to show that those inefficiently transported peptides can not exert an osmoprotective role, in distinction from those efficiently transported. The lost of the repression exerted by peptides in PrtP and Per activities, two of the key enzymes of the proteolytic system and main providers of peptides was described. The lost of repression for PrtP took place at the transcriptional level. The interaction of CodY protein of Lb casei with the promoter region of prtP gene was demonstrated, this result suggests that this regulator protein would be involved in the lost of repression by peptides in a hyperosmotic media. In a hypersaline media in the absence of peptides, the high levels of PrtP activity were essentially through an increase of apparent Vmax instead of an increment of transcriptional activity. Since PrtP is anchored to the cell wall, the enzyme activity could be related to changes in the environment of the protein. The differences in the sensitivity to lysis and antibiotics, that exert their effect on the cell envelope in cells grown in the presence of salt, were indicative of these variations. The high level of lysis of the cells grown in NaCl, was also a direct consequence of changes in the cell wall; the same effect was observed when purified cell walls were used instead of whole cells. The analysis of electronic microscopy photographs revealed and increase in the size of cells grown in high salt as well as clear differences in the cell wall. These results raises, that pregrowth in hyperosmotic media could be used as an efficient method to improve lysis of this resistant to lysis bacteria. The results of this study, increase the knowledge about lactic acid bacteria physiology in the presence of NaCl, a stress effector that is usually found in fermentative process. Fil: Piuri, Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

This document records the decision made by the Bureau of Land Management (BLM) for managing approximately 891,600 acres of public land surface and 1,508,200 acres of federal mineral estate administered by the Bureau of Land Management in the Cody Resource Area.

This document addresses alternatives for managing almost 1.1 million acres fo federal land and about 1.5 million acres of federal mineral estate administered by the Cody Resource Area, Worland District, Bureau of Land Management (see map 1). The plan focuses on three resource management issues relating to vegetative resources, special management area designations, and resource accessibility and manageability.