MAPKAP kinase 2 is activated by heat shock and TNF-α: In vivo phosphorylation of small heat shock protein results from stimulation of the MAP kinase cascade

The activation of MAPKAP kinase 2 was investigated under heat-shock conditions in mouse Ehrlich ascites tumor cells and after treatment of human MO7 cells with tumor necrosis factor-α (TNF-α). MAPKAP kinase 2 activity was determined using the small heat-shock proteins (sHsps) Hsp25 and Hsp27 as substrates. In both cell types, about a threefold increase in MAPKAP kinase 2 activity could be detected in a time interval of about 10–15 min after stimulation either by heat shock or TNF-α. Phosphorylation of MAPKAP kinase 2, but not the level of MAPKAP kinase 2 mRNA, was increased after heat shock in EAT cells. It is further shown that activation of MAPKAP kinase 2 in MO7 cells is accompanied by increased MAP kinase activity. These data strongly suggest that increased phosphorylation of the sHsps after heat shock or TNF-α treatment results from phosphorylation by MAPKAP kinase 2, which itself is activated by phosphorylation through MAP kinases. Hence, we demonstrate that MAPKAP kinase 2 is responsible not only for phosphorylation of sHsps in vitro but also in vivo. The findings link sHsp phosphorylation to the MAP kinase cascade, explaining the early phosphorylation of sHsp that is stimulated by a variety of inducers such as mitogens, phorbol esters, thrombin, calcium ionophores, and heat shock.     

Anaerobic degradation of aniline and dihydroxybenzenes by newly isolated sulfate-reducing bacteria and description of Desulfobacterium anilini

A new, rod-shaped, Gram-negative, non-sporing sulfate reducer (strain Ani1) was enriched and isolated from marine sediment with aniline as sole electron donor and carbon source. The strain degraded aniline completely to CO2 and NH3 with stoichiometric reduction of sulfate to sulfide. Strain Ani1 also degraded aminobenzoates and further aromatic and aliphatic compounds. The strain grew in sulfide-reduced mineral medium supplemented only with vitamin B12 and thiamine. Cells contained cytochromes, carbon monoxide dehydrogenase, and sulfite reductase P582, but no desulfoviridin. Strain Ani1 is described as a new species of the genus Desulfobacterium D. anilini. Marine enrichments with the three dihydroxybenzene isomers led to three different strains of sulfate-reducing bacteria; each of them could grow only with the isomer used for enrichment. Two strains isolated with catechol (strain Cat2) or resorcinol (strain Re10) were studied in detail. Both strains oxidized their substrates completely to CO2, and contained cytochromes, carbon monoxide dehydrogenase, and sulfite reductase P 582. Desulfoviridin was not present. Whereas the rod-shaped catechol oxidizer (strain Cat2) was able to grow on 18 aromatic compounds and several aliphatic substrates, the coccoid resorcinol-degrading bacterium (strain Re10) utilized only resorcinol, 2,4-dihydroxybenzoate and 1,3-cyclohexanedion. These strains could not be affiliated with existing species of sulfate-reducing bacteria. A further coccoid sulfate-reducing bacterium (strain Hy5) was isolated with hydroquinone and identified as a subspecies of Desulfococcus multivorans. Most-probable-number enumerations with catechol, phenol, and resorcinol showed relatively large numbers (10(4)-10(6) per ml) of aryl compound-degrading sulfate reducers in marine sediment samples.     

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Highly efficient expression of homologous and heterologous genes in Bacillus megaterium

Summary By developing an efficient transformation system it was possible to reintroduce two different glucose dehydrogenase genes of Bacillus megaterium into this host. These genes were previously cloned, sequenced and expressed in Escherichia coli. Since the expression of one of these genes (gdhA) turned out to be extremely high in B. megaterium, an expression system for genes from closely and distantly related organisms using the controlling region of the gdhA gene was developed.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65th birthday     

Comprehensive glycoproteomics shines new light on the complexity and extent of glycosylation in archaea

Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provide new insights into the roles of N-glycosylation in archaeal cell biology.

A comprehensive glycoproteomic analysis of Haloferax volcanii reveals the extent and complexity of glycosylation in archaea and provides new insights into the roles of this post-translational modification in various cellular processes, including cell shape determination.     

Zonation of spiders (Araneae) and carabid beetles (Coleoptera: Carabidae) in island salt marshes at the North Sea coast

The specific communities of spiders and carabid beetles of island salt marsh habitats of the East Frisian Island chain at the German North Sea coast were investigated. During the vegetation periods of 1997 and 1998 three pitfall trapping transects were installed on the islands of Borkum and Wangerooge. Within the salt marshes, transects extended from 0 m to 175 m. Elevation gradients varied between 10 cm and 232 cm above MHT (mean high tide). On Borkum, 35 traps were exposed in two transects, on Wangerooge 25 traps were placed in one transect. Three to five elevations above MHT were investigated per transect, each one with five traps. Highest species numbers were recorded in the higher elevated salt marshes. In contrast, highest activity values were noticed in the medium elevated salt marshes. Within both groups, spiders and carabids, four communities were distinguished by indirect gradient analysis. Indicator species were assigned to the different communities that were mainly assorted to different elevations of the salt marshes. Thus, the communities of both taxa corresponded well to the vegetational formations. The importance of sea level rise for structuring the communities of salt marsh arthropods is discussed. Overall, still great uncertainties exist on how arthropod communities and salt marshes themselves will develop.     

The low molecular weight proteome of Halobacterium salinarum

Systematic investigation of low molecular weight proteins (LMW, below 20 kDa) in the archaeon Halobacterium salinarum resulted in a 6-fold enhancement of the identification rate, reaching 35% of the theoretical proteome in that size range. This was achieved by optimization of common protocols for protein analysis with general applicability. LMW proteins were rapidly and effectively enriched by filter membrane centrifugation followed by tricine SDS-PAGE. Without staining and with significantly shortened digestion protocols, LMW proteins were identified using an FT-ICR mass spectrometer which allows reliable protein identification by MS3 of a single peptide. In addition to a series of technical challenges, small proteins may show low gene expression levels as suggested by their low average codon adaptation index. Twenty functionally uncharacterized proteins contain a characteristic DNA/RNA binding zinc finger motif which underlines the biological relevance of the small proteome and the necessity of their analysis for systems biology.     

Anticodon nuclease encoding virus-like elements in yeast

The aim of this study was to apply flow cytometric (FCM) analysis to assess the use of sucrose and lecithin vesicles for the protection of probiotic lactic acid bacteria in response to the challenge of gastric acidity and bile salts. FCM analysis in combination with fluorescent probes carboxyfluorescein (cF) and propidium iodide was used to reveal the physiological heterogeneity in the stressed bacteria population. Three subpopulations (intact, stressed, and damaged) were differentiated by FCM in all six examined strains. Significant changes were observed in the presence of the selected protectants. The addition of 20?mM sucrose in the simulated gastric fluid substantially increased the number of intact cells over 20 folds and reduced the damaged subpopulation by half. The presence of 2?% (w/v) lecithin vesicles was shown to protect 50?% more intact cells from the challenge of bile salts. The improved survival as evaluated by FCM analysis was further assessed for the proliferation capacity by sorting a number of cells from each subpopulation on nutrient agar plate. The result confirmed conformity between the proliferation-based cultivability and the probe-indicated viability in the samples of the intact and the damaged subpopulations. However, it also revealed the complexities of the stressed (injured) subpopulation. In conclusion, FCM analysis confirmed that the selected protectants could improve the survival of the probiotic strains in the simulated GI environments. The FCM analysis also proved to be a useful analytical tool for the probiotics research.