Methioninaufnahme und -export in Corynebacterium glutamicum

Methioninaufnahme und -export in Corynebacterium glutamicum. Die Untersuchung der Methioninaufnahme in C. glutamicum führte zur Charakterisierung von zwei Aufnahmesystemen. Bei dem ersten Transporter handelt es sich um ein hoch-affines System mit einem Km von ca. 0,1 µM und einer Vmax von ca. 0,7 nmol/min (mg TG). Datenbank-Analysen mit dem E. coli Methioninaufnahmesystem MetD als Vorlage führten zur Identifizierung der Gene metI, metN und metQ in C. glutamicum. Die Expression dieses Genclusters, das einen ABC-Transporter für Methionin kodiert, wird durch den Repressor McbR reguliert. Das zweite Aufnahmesystem ist mittel-affin mit einem Km von ca. 88 µM und einer Vmax von ca. 1,65 nmol/min (mg TG). Dieser Methioninimporter, der analog zu E. coli MetP genannt wurde, konnte im metNI-Deletionsstamm charakterisiert werden. Da die Methioninaufnahme über MetP Na+-abhängig ist, handelt es sich um einen sekundären Transporter, der sowohl durch Alanin, Leucin, Isoleucin, Valin als auch Cystein im Überschuss gehemmt wird. Das metP-Gen gehört nicht zum McbR-Regulon. Um den Methioninexport in C. glutamicum zu charakterisieren, wurde ein Beladungssystem mit L-Methionin-haltigen Dipeptiden etabliert. Dabei wurde das Dipeptid von der Zelle aufgenommen und im Cytoplasma hydrolysiert, was zu einem sehr starken Anstieg der internen Methioninkonzentration führte. Anschließend nahm die zellinterne Methioninkonzentration wieder ab. Es wurde gezeigt, dass BrnFE das Haupt-Exportsystem für Methionin ist, wobei die Expression von brnFE von der zellinternen Methioninkonzentration abhängt. Da im brnE-Deletionsstamm noch Methioninexport zu sehen war, wurde ein weiterer Exporter angenommen. Cgl0944, das durch DEUTENBERG (2003) bei einer Genexpressionsanalyse identifiziert wurde, konnte als Methioninexporter ausgeschlossen werden. Eigene Analysen zeigten, dass der zweite Exporter nicht sekundär aktiv ist und seine Aktivität durch die externe Osmolalität beeinflusst wird. Das entsprechende Gen ist zudem nicht expressionsreguliert. Im Rahmen dieser Arbeit konnte außerdem das Vorhandensein von extrazellulären, Membran- oder Zellwand-gebundenen Hydrolasen nachgewiesen werden, die Dipeptide spalten

Proteome turnover in bacteria: current status for Corynebacterium glutamicum and related bacteria

Trötschel C, Albaum S, Poetsch A. Proteome turnover in bacteria: current status for Corynebacterium glutamicum and related bacteria. Microbial biotechnology. 2013;6(6):708-719.With the advent of high-resolution mass spectrometry together with sophisticated data analysis and interpretation algorithms, determination of protein synthesis and degradation rates (i.e. protein turnover) on a proteome-wide scale by employing stable isotope-labelled amino acids has become feasible. These dynamic data provide a deeper understanding of protein homeostasis and stress response mechanisms in microorganisms than well-established 'steady state' proteomics approaches. In this article, we summarize the technological challenges and solutions both on the biochemistry/mass spectrometry and bioinformatics level for turnover proteomics with a focus on chromatographic techniques. Although the number of available case studies for Corynebacterium glutamicum and related actinobacteria is still very limited, our review illustrates the potential of protein turnover studies for an improved understanding of questions in the area of biotechnology and biomedicine. Here, new insights from investigations of growth phase transition and different stress dynamics including iron, acid and heat stress for pathogenic but also for industrial actinobacteria are presented. Finally, we will comment on the advantages of integrated software solutions for biologists and briefly discuss the remaining technical challenges and upcoming possibilities for protein turnover analysis

Characterization of Methionine Export in Corynebacterium glutamicum

Corynebacterium glutamicum is known for its effective excretion of amino acids under particular metabolic conditions. Concomitant activities of uptake and excretion systems would create an energy-wasting futile cycle; amino acid export systems are therefore tightly regulated. We have used a DNA microarray approach to identify genes for membrane proteins which are overexpressed under conditions of elevated cytoplasmic concentrations of methionine. One of these genes was brnF, coding for the larger subunit of BrnFE, a previously identified two-component isoleucine export system. By deletion, complementation, and overexpression of the brnFE genes in a C. glutamicum strain, in which the two uptake systems for methionine were inactivated, we identified BrnFE as being responsible for methionine export. In the presence of both substrates in the cytoplasm, BrnFE was found to transport isoleucine and methionine at similar rates. The expression of the brnFE gene cluster depends on an Lrp-type transcription factor and was shown to be strongly induced by increasing cytoplasmic methionine concentration. Methionine was a better inducer than isoleucine, indicating that methionine rather than isoleucine might be the native substrate of BrnFE. When the synthesis of BrnFE was blocked by chloramphenicol, fast methionine export was still observed, but only at greatly increased cytoplasmic levels of this amino acid. This indicates the presence of at least one other methionine export system, presumably with low affinity but high capacity. Under conditions where cytoplasmic methionine does not exceed a concentration of 50 mM, BrnFE is the dominant export system for this amino acid

The Costs and Benefits of Negotiated Agreements in Social Conflicts

Psychological research on negotiation is shaped by the fundamental belief that social interaction between negotiators is unconditionally beneficial to negotiation outcomes. Over a series of four experiments, we provide the first empirical test of this belief. Drawing on an experimental paradigm established in group performance research, we compare interactive negotiations with non-interactive, nominal negotiation methods, allowing us to identify process gains or losses in different negotiation settings. Our first three experiments show considerable benefits of social interaction in both distributive (Experiments 1 and 2) and integrative (Experiment 3) negotiations, independent of motivational and coordinative challenges. However, our fourth experiment demonstrates that process gains in negotiations are not unconditional. After adding explicit transaction costs to a distributive price negotation, interactive negotiators could no longer surpass their nominal counterparts in terms of negotiation outcomes. On the contrary, by embedding the negotiation in a series of different tasks, we were able to show that longer negotiation durations for interactive negotiators led to a significantly worse overall task performance compared to nominal negotiators

Systems Biology of Acidophile Biofilms for Efficient Metal Extraction

This European Union ERASysApp funded study will investigate one of the major drawbacks of bioleaching of the copper containing mineral chalcopyrite, namely the long lag phase between construction and inoculation of bioleaching heaps and the release of dissolved metals. In practice, this lag phase can be up to three years and the long time period adds to the operating expenses of bioheaps for chalcopyrite dissolution. One of the major time determining factors in bioleaching heaps is suggested to be the speed of mineral colonization by the acidophilic microorganisms present. By applying confocal microscopy, metatranscriptomics, metaproteomics, bioinformatics, and computer modeling the authors aim to investigate the processes leading up to, and influencing the attachment of three moderately thermophilic sulfur-and/or iron-oxidizing model species: Acidithiobacillus caldus, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans. Stirred tank reactors containing chalcopyrite concentrate will be inoculated with these species in various orders and proportions and the effects on the lag phase and rates of metal release will be compared. Meanwhile, confocal microscopy studies of cell attachment to chalcopyrite mineral particles, as well as metatranscriptomics and metaproteomics of the formed biofilms will further increase understanding of the attachment process and help develop a model thereof. By fulfilling our goal to decrease the length of the lag phase of chalcopyrite bioleaching heaps we hope to increase their economic feasibility and therefore, industrial interest in bioleaching as a sustainable technology

Accumulation of Glucosylceramide in the Absence of the Beta-Glucosidase GBA2 Alters Cytoskeletal Dynamics

<div><p>Glycosphingolipids are key elements of cellular membranes, thereby, controlling a variety of cellular functions. Accumulation of the simple glycosphingolipid glucosylceramide results in life-threatening lipid storage-diseases or in male infertility. How glucosylceramide regulates cellular processes is ill defined. Here, we reveal that glucosylceramide accumulation in GBA2 knockout-mice alters cytoskeletal dynamics due to a more ordered lipid organization in the plasma membrane. In dermal fibroblasts, accumulation of glucosylceramide augments actin polymerization and promotes microtubules persistence, resulting in a higher number of filopodia and lamellipodia and longer microtubules. Similar cytoskeletal defects were observed in male germ and Sertoli cells from GBA2 knockout-mice. In particular, the organization of F-actin structures in the ectoplasmic specialization and microtubules in the sperm manchette is affected. Thus, glucosylceramide regulates cytoskeletal dynamics, providing mechanistic insights into how glucosylceramide controls signaling pathways not only during sperm development, but also in other cell types.</p></div

High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3′,5′-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 105 GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces “molecule noise.” Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons

Cytoskeletal defects in GBA2 knockout-testis already occur during the first spermatogenic wave.

<p><b>(A)</b> Immunofluorescent labeling of the cytoskeleton in wild-type (+/+) and GBA2 knockout-testis (-/-) at P7. Microtubules have been labeled using an anti-beta tubulin III antibody (red), F-actin using Alexa Fluor Phalloidin 488 (green), and the DNA using DAPI (blue). Scale bars are indicated. <b>(B)</b> See (A) for P21. <b>(C)</b> See (A) for P23. <b>(D)</b> See (A) for P34. <b>(E)</b> Development of the manchette in spermatids. The manchette was stained with beta-tubulin (red), DNA was labeled with DAPI (blue). Different developmental stages are indicated. <b>(F)</b> Manchette length. The manchette length of spermatids from wild-type (+/+) and GBA2 knockout-mice (-/-) was determined using ImageJ. At least 30 cells have been analyzed per genotype. Data are shown as mean ± S.D.; n numbers and p values calculated using One-Way ANOVA are indicated.</p