http://hdl.handle.net/10033/6806 AG Mikrobielle Kommunikation (KOM) Thu, 23 May 2013 18:46:35 GMT 2013-05-23T18:46:35Z http://hdl.handle.net/10033/251212 Title: The Binding Site of the V-ATPase Inhibitor Apicularen Is in the Vicinity of Those for Bafilomycin and Archazolid. Authors: Osteresch, Christin; Bender, Tobias; Grond, Stephanie; von Zezschwitz, Paultheo; Kunze, Brigitte; Jansen, Rolf; Huss, Markus; Wieczorek, Helmut Abstract: The investigation of V-ATPases as potential therapeutic drug targets and hence of their specific inhibitors is a promising approach in osteoporosis and cancer treatment because the occurrence of these diseases is interrelated to the function of the V-ATPase. Apicularen belongs to the novel inhibitor family of the benzolactone enamides, which are highly potent but feature the unique characteristic of not inhibiting V-ATPases from fungal sources. In this study we specify, for the first time, the binding site of apicularen within the membrane spanning V(O) complex. By photoaffinity labeling using derivatives of apicularen and of the plecomacrolides bafilomycin and concanamycin, each coupled to (14)C-labeled 4-(3-trifluoromethyldiazirin-3-yl)benzoic acid, we verified that apicularen binds at the interface of the V(O) subunits a and c. The binding site is in the vicinity to those of the plecomacrolides and of the archazolids, a third family of V-ATPase inhibitors. Expression of subunit c homologues from Homo sapiens and Manduca sexta, both species sensitive to benzolactone enamides, in a Saccharomyces cerevisiae strain lacking the corresponding intrinsic gene did not transfer this sensitivity to yeast. Therefore, the binding site of benzolactone enamides cannot be formed exclusively by subunit c. Apparently, subunit a substantially contributes to the binding of the benzolactone enamides. Fri, 14 Sep 2012 00:00:00 GMT http://hdl.handle.net/10033/251212 2012-09-14T00:00:00Z http://hdl.handle.net/10033/230933 Title: The global impact of the delta subunit RpoE of the RNA polymerase on the proteome of Streptococcus mutans. Authors: Xue, Xiaoli; Li, Jinshan; Wang, Wei; Sztajer, Helena; Wagner-Döbler, Irene Abstract: Transcriptional specificity in low-G+C Gram-positive bacteria is maintained by RpoE, the delta subunit of the RNA polymerase. Here, we studied the effect of RpoE at the proteome level in the human dental pathogen Streptococcus mutans by comparing the ΔrpoE mutant with the wild-type under five conditions: (0) exponential growth, (1) early stationary phase, (2) acid stress, (3) oxidative stress, and (4) combined acid and oxidative stress. A total of 280 cellular protein spots were reproducibly detected, of which 97 differentially expressed protein spots were identified by MALDI-TOF MS. Lack of RpoE caused downregulation of proteins for carbohydrate metabolism and energy production, including phosphoglucomutase (PGM), the phosphopentomutase DeoB and the pyruvate formate-lyase Pfl. The ΔrpoE mutant had extensive changes in the abundance of proteins involved in acid and oxidative tolerance and protein turnover, and of chaperones, at exponential phase in the absence of stress, suggesting a potential internal stress. In addition, the mutant had reduced amounts of proteins for adaptation responses, e.g. the multiple sugar transport and metabolism enzymes required for entering early stationary phase, and the proteins for stress-defence mechanisms and glycolysis under oxidative stress. Comparison of the proteome data with the corresponding transcriptome data suggested that the effects were the result of altered transcriptional and post-transcriptional regulation. The data are consistent with the reduced transcriptional specificity of the RNA polymerase in the ΔrpoE mutant, and suggest a general impact, but not a specific regulatory role, of RpoE in stress adaptation. Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/10033/230933 2012-01-01T00:00:00Z http://hdl.handle.net/10033/228531 Title: Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria. Authors: Brinkhoff, Thorsten; Fischer, Doreen; Vollmers, John; Voget, Sonja; Beardsley, Christine; Thole, Sebastian; Mussmann, Marc; Kunze, Brigitte; Wagner-Döbler, Irene; Daniel, Rolf; Simon, Meinhard Abstract: Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2 m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this cluster was only detected on aggregates up to a few meters above the sediment surface, but never in the fraction of free-living bacteria. A quantitative real-time PCR approach revealed that the MMC constituted up to 13% of total bacterial 16S rRNA genes in surface sediments of the North Sea. In a global survey, including sediments from the Mediterranean Sea, the Atlantic, Pacific and Indian Ocean and various climatic regions, the MMC was detected in most samples and to a water depth of 4300 m. Two fosmids of a library from sediment of the southern North Sea containing 16S rRNA genes affiliated with the MMC were sequenced. Both fosmids have a single unlinked 16S rRNA gene and no complete rRNA operon as found in most bacteria. No synteny to other myxobacterial genomes was found. The highest numbers of orthologues for both fosmids were assigned to Sorangium cellulosum and Haliangium ochraceum. Our results show that the MMC is an important and widely distributed but largely unknown component of marine sediment-associated bacterial communities. Fri, 01 Jun 2012 00:00:00 GMT http://hdl.handle.net/10033/228531 2012-06-01T00:00:00Z http://hdl.handle.net/10033/190851 Title: Subpopulation-specific transcriptome analysis of competence-stimulating-peptide-induced Streptococcus mutans. Authors: Lemme, André; Gröbe, Lothar; Reck, Michael; Tomasch, Jürgen; Wagner-Döbler, Irene Abstract: Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ~3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and noncompetent fraction of CSP-treated S. mutans cells. Sorting was guided by a ComX-green fluorescent protein (ComX-GFP) reporter, and the transcriptome analysis demonstrated the successful combination of both methods, because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, and among them was ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. In contrast, the expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and CipB (mutacin V) confirmed this expression pattern on the single-cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, the uptake of DNA could be shown on the single-cell level. This study demonstrates that all cells in the population respond to CSP through the activation of bacteriocin-related genes. Some of these cells start to activate ComX expression but then segregate into two subpopulations, one becoming competent and another one that lyses, resulting in intrapopulation diversity. Fri, 01 Apr 2011 00:00:00 GMT http://hdl.handle.net/10033/190851 2011-04-01T00:00:00Z