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

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

Dermal fibroblasts from GBA2 knockout-mice also display cytoskeletal defects.

<p><b>(A)</b> GBA2 expression in dermal fibroblasts from adult mice. Total protein lysates were probed with a GBA2-specific antibody (2F8) on a Western blot. Heterologously expressed HA-tagged GBA2 was used as a positive control, calnexin (Clnx) as a loading control. <b>(B)</b> Accumulation of GlcCer in GBA2 knockout-fibroblasts. Thin layer chromatography (TLC) analyzing glycosphingolipids from wild-type (+/+) and GBA2 knockout-fibroblasts (-/-). Representative TLC analysis for neutral sphingolipids. GlcCer: glucosylceramide, LacCer: lactosylceramide, Spm: sphingomyelin. GlcCer levels were quantified by densitometry and are presented as mean ± S.D. The fold change in GlcCer levels in GBA2 knockout-fibroblasts was calculated. <b>(C)</b> Fluorescent labeling of the cytoskeleton in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). Cells were transfected with lifeact (green) to visualize F-actin and with EB3-cherry to visualize microtubules. Scale bars are indicated. <b>(D)</b> Fluorescent labeling of F-actin in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). Cells were seeded on CYTOO chips with micropatterns that are coated with fluorescently-labeled fibronectin (purple). F-actin was stained using Alexa Fluor Phalloidin 488 (green) and the DNA was stained with DAPI (blue). Scale bars are indicated. <b>(E)</b> Analysis of cytoskeletal structures. Cells were seeded on the crossbow shape. The number of cells containing filopodia or lamellipodia (left) and the average number of filopodia or lamellipodia per cell (right) were determined. <b>(F)</b> Gene expression-analysis. The mRNA expression level of <i>Cdc42</i>, <i>Rac1</i>, and <i>Rho</i> was analyzed by qRT-PCR. <b>(G)</b> Protein expression-analysis. Total protein lysates were probed with a GBA2- (2F8), a Cdc42-, and a Rac1-specific antibody on a Western blot. Calnexin (Clnx) was used as a loading control. <b>(H)</b> Quantification of protein expression based on (G). <b>(I)</b> Quantification of actin turnover in dermal fibroblasts. Expression levels of G- and F-actin in wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) were determined using Western blot-analysis. Ratio of F-actin/G-actin for wild-type and GBA2 knockout-fibroblasts is expressed relative to the control. <b>(J)</b> See (I) for testis. <b>(K-M)</b> Analysis of microtubule dynamics in dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). <b>(K)</b> Expression of EB3-cherry in dermal fibroblasts. Cells were transfected with EB3-cherry and microtubule dynamics were analyzed. Representative tracks of growing microtubule plus-ends are indicated with white lines. <b>(L)</b> Microtubule growth rate. Wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) were transfected with EB3-cherry and the growth rate of growing plus-ends was analyzed. Per genotype, n = 3 animals with a minimum of 7 cells and 10 tracks per cell were analyzed. Data are presented as mean ± S.D. <b>(M)</b> see (L) for microtubule persistence. For all bar graphs, data are shown as mean ± S.D.; n numbers and p values calculated using One-Way ANOVA are indicated.</p

The absence of GBA2 affects cell migration.

<p><b>(A-B)</b> Wound-healing assay to analyze cell migration of dermal fibroblasts from wild-type (+/+) and GBA2 knockout-mice (-/-). <b>(A)</b> Representative images at different time points after initiating the assay. Scale bars are indicated. <b>(B)</b> Analysis of cell migration. The rate of cell migration has been analyzed. Average data points for wild-type (+/+) and GBA2 knockout-fibroblasts (-/-) for different time points are shown; n numbers and p values using One-Way ANOVA are indicated.</p

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