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  • 1
    Keywords: Germany ; IN-VIVO ; MODEL ; PATHWAY ; VIVO ; CLONING ; PROTEIN ; PROTEINS ; COMPLEX ; COMPLEXES ; DNA ; BIOLOGY ; RECOGNITION ; ESCHERICHIA-COLI ; CRYSTAL-STRUCTURE ; DEGRADATION ; mutagenesis ; SUBSTRATE ; proteases ; CHAPERONE ; USA ; PROTEOLYSIS ; BACTERIA ; STATE ; DPS ; POLYAMINES
    Abstract: The N-end rule degradation pathway states that the half-life of a protein is determined by the nature of its N-terminal residue. In Escherichia coli the adaptor protein ClpS directly interacts with destabilizing N-terminal residues and transfers them to the ClpA/ClpP proteolytic complex for degradation. The crucial role of ClpS in N-end rule degradation is currently under debate, since ClpA/ClpP was shown to process selected N-terminal degrons harbouring destabilizing residues in the absence of ClpS. Here, we investigated the contribution of ClpS to N-end rule degradation by two approaches. First, we performed a systematic mutagenesis of selected N-degron model substrates, demonstrating that ClpS but not ClpA specifically senses the nature of N-terminal residues. Second, we identified two natural N-end rule substrates of E. coli: Dps and PATase (YgjG). The in vivo degradation of both proteins strictly relied on ClpS, thereby establishing the function of ClpS as the essential discriminator of the E. coli N-end rule pathway
    Type of Publication: Journal article published
    PubMed ID: 19317833
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  • 2
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    Abstract: HtrA proteases and chaperones exhibit important roles in periplasmic protein quality control and stress responses. The genetic inactivation of htrA has been described for many bacterial pathogens. However, in some cases such as the gastric pathogen Helicobacter pylori, HtrA is secreted where it cleaves the tumour-suppressor E-cadherin interfering with gastric disease development, but the generation of htrA mutants is still lacking. Here, we show that the htrA gene locus is highly conserved in worldwide strains. HtrA presence was confirmed in 992 H. pylori isolates in gastric biopsy material from infected patients. Differential RNA-sequencing (dRNA-seq) indicated that htrA is encoded in an operon with two subsequent genes, HP1020 and HP1021. Genetic mutagenesis and complementation studies revealed that HP1020 and HP1021, but not htrA, can be mutated. In addition, we demonstrate that suppression of HtrA proteolytic activity with a newly developed inhibitor is sufficient to effectively kill H. pylori, but not other bacteria. We show that Helicobacter htrA is an essential bifunctional gene with crucial intracellular and extracellular functions. Thus, we describe here the first microbe in which htrA is an indispensable gene, a situation unique in the bacterial kingdom. HtrA can therefore be considered a promising new target for anti-bacterial therapy.
    Type of Publication: Journal article published
    PubMed ID: 26568477
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  • 4
    Abstract: The multicomponent type VI secretion system (T6SS) mediates the transport of effector proteins by puncturing target membranes. T6SSs are suggested to form a contractile nanomachine, functioning similar to the cell-puncturing device of tailed bacteriophages. The T6SS members VipA/VipB form tubular complexes and are predicted to function in analogy to viral tail sheath proteins by providing the energy for secretion via contraction. The ATPase ClpV disassembles VipA/VipB tubules in vitro, but the physiological relevance of tubule disintegration remained unclear. Here, we show that VipA/VipB tubules localize near-perpendicular to the inner membrane of Vibrio cholerae cells and exhibit repetitive cycles of elongation, contraction and disassembly. VipA/VipB tubules are decorated by ClpV in vivo and become static in DeltaclpV cells, indicating that ClpV is required for tubule removal. VipA/VipB tubules mislocalize in DeltaclpV cells and exhibit a reduced frequency of tubule elongation, indicating that ClpV also suppresses the spontaneous formation of contracted, non-productive VipA/VipB tubules. ClpV activity is restricted to the contracted state of VipA/VipB, allowing formation of functional elongated tubules at a T6SS assembly. Targeting of an unrelated ATPase to VipA/VipB is sufficient to replace ClpV function in vivo, suggesting that ClpV activity is autonomously regulated by VipA/VipB conformation.
    Type of Publication: Journal article published
    PubMed ID: 23289512
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  • 5
    Keywords: CELLS ; GROWTH ; KINASE ; PATHWAYS ; GENE ; GENES ; PROTEIN ; SACCHAROMYCES-CEREVISIAE ; ACCUMULATION ; RESPONSES ; mechanisms ; MICROARRAY DATA ; Saccharomyces cerevisiae ; YEAST ; DISPLAY ; GLUCOSE ; REPRESSION ; GENE- EXPRESSION ; GLUCONEOGENIC MESSENGER-RNAS ; HEXOSE TRANSPORTERS ; RAS PROTEINS ; SENSING MECHANISMS
    Abstract: Glucose exerts profound effects upon yeast physiology. In general, the effects of high glucose concentrations (〉1%) upon Saccharomyces cerevisiae have been studied. In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by transcript profiling. We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals. Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of〉 0.1%. We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post- transcriptional mechanisms combine to accelerate the accumulation of ribosomal protein mRNAs. Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose. However, our data indicate that yeast activates ribosome biogenesis only when sufficient glucose is available to make this metabolic investment worthwhile. In contrast, the regulation of metabolic functions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient
    Type of Publication: Journal article published
    PubMed ID: 12694616
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  • 6
    Keywords: CELLS ; CELL ; Germany ; GENE ; GENES ; PROTEIN ; PROTEINS ; SACCHAROMYCES-CEREVISIAE ; transcription ; TIME ; ACID ; ACIDS ; FORM ; DELETION ; MUTANT ; YEAST ; MEMBRANE ; SIGNALING PATHWAY ; WILD-TYPE ; INSTABILITY ; STABILITY ; PLASMA-MEMBRANE ; Jun ; WALL ; AMINO-ACIDS ; MASSES ; ABSENCE ; GENOME-WIDE ANALYSIS ; A-AGGLUTININ ; ALPHA-AGGLUTININ ; BGL2 GENE ; chitin ; DEFECTS ; GROWTH SITES ; KINASE PATHWAY
    Abstract: The c ovalently linked cell wall protein Ccw12p of Saccharomyces cerevisiae is a GPI-anchored protein (V. Mrsa et al., 1999, J Bacteriol 181: 3076-3086). Although only 121 amino acids long, the haemagglutinin-tagged protein released by laminarinase from the cell wall possesses an apparent molecular mass of 〉 300 kDa. A membrane-bound form with an apparent molecular mass of 58 kDa is highly O- and N-glycosylated and contains the GPI anchor. With a half-life of 2 min, the membrane form is transformed to the 〉 300 kDa form. The deletion mutant ccw12Delta grows slower than the wild type, is highly sensitive to Calcofluor white and contains 2.5 times more chitin. Further, compared with wild-type yeast, significantly more proteins are released from intact cells when treated with dithiothreitol. Interestingly, these defects become less pronounced when further GPI-anchored cell wall proteins are deleted. Mutant DeltaGPI (simultaneous deletion of CCW12, CCW13/DAN1, CCW14, TIP1 and CWP1) is similar in many respects to wild-type yeast. To find out how the cell wall is stabilized in mutant DeltaGPI, a genome-wide transcription analysis was performed. Of 159 significantly regulated genes, 14 encode either known or suspected cell wall-associated proteins. Analysis of genes affected in transcription revealed that SED1 and SRL1 in particular are required to reconstruct cell wall stability in the absence of multiple GPI-anchored mannoproteins
    Type of Publication: Journal article published
    PubMed ID: 15165243
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  • 7
    Keywords: ENVIRONMENT ; IN-VITRO ; CELL ; Germany ; human ; IN-VIVO ; VITRO ; VIVO ; ENZYMES ; GENE ; GENOME ; PATIENT ; INFECTION ; SERA ; DONOR ; ANTIGEN ; ANTIGENS ; BIOLOGY ; MOLECULAR-BIOLOGY ; VARIANTS ; SPECTROSCOPY ; ASSAY ; ESCHERICHIA-COLI ; MEMBRANE ; NMR ; BETA ; MONOCLONAL-ANTIBODIES ; INVOLVEMENT ; immune response ; IMMUNE-RESPONSE ; INFECTIONS ; SERUM ; molecular biology ; molecular ; ELISA ; RE ; pathogen ; VARIANT ; CAPACITY ; CERAMIDE ; SUBSTRATE ; ENZYME ; ASSAYS ; function ; in vivo ; microbiology ; SCREENS ; HIGH-AFFINITY ; host ; lipid ; GLYCOLIPIDS ; GLYCOSYLTRANSFERASES ; GUILLAIN-BARRE-SYNDROME ; IMMUNOCHEMICAL ANALYSIS ; MOLECULAR MIMICRY ; SEROLOGICALLY ACTIVE LIPIDS ; T-CELL RECOGNITION
    Abstract: The human pathogen Mycoplasma pneumoniae has a very small genome but with many yet not identified gene functions, e.g. for membrane lipid biosynthesis. Extensive radioactive labelling in vivo and enzyme assays in vitro revealed a substantial capacity for membrane glycolipid biosynthesis, yielding three glycolipids, five phosphoglycolipids, in addition to six phospholipids. Most glycolipids were synthesized in a cell protein/lipid-detergent extract in vitro; galactose was incorporated into all species, whereas glucose only into a few. One (MPN483) of the three predicted glycosyltransferases (GTs; all essential) was both processive and promiscuous, synthesizing most of the identified glycolipids. These enzymes are of a GT-A fold, similar to an established structure, and belong to CAZy GT-family 2. The cloned MPN483 could use both diacylglycerol (DAG) and human ceramide acceptor substrates, and in particular UDP-galactose but also UDP-glucose as donors, making mono-, di- and trihexose variants. MPN483 output and processitivity was strongly influenced by the local lipid environment of anionic lipids. The structure of a major beta 1,6Glc beta GalDAG species was determined by NMR spectroscopy. This, as well as other purified M. pneumoniae glycolipid species, is important antigens in early infections, as revealed from ELISA screens with patient IgM sera, highlighting new aspects of glycolipid function
    Type of Publication: Journal article published
    PubMed ID: 17697098
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  • 8
    Keywords: METABOLISM ; DIFFERENTIATION ; IDENTIFICATION ; STABILITY ; TRANSFORMATION ; GLUCOSE ; BINDING PROTEIN ; PHOSPHOGLYCERATE KINASE ; PROCYCLIC FORMS ; REGULATED MESSENGER-RNAS
    Abstract: When Trypanosoma brucei differentiates from the bloodstream form to the procyclic form, there are decreases in the levels of many mRNAs encoding proteins required for the glycolytic pathway, and the mRNA encoding the RNA recognition motif protein RBP10 decreases in parallel. We show that RBP10 is a cytoplasmic protein that is specific to bloodstream-form trypanosomes, where it is essential. Depletion of RBP10 caused decreases in many bloodstream-form-specific mRNAs, with increases in mRNAs associated with the early stages of differentiation. The changes were similar to, but more extensive than, those caused by glucose deprivation. Conversely, forced RBP10 expression in procyclics induced a switch towards bloodstream-form mRNA expression patterns, with concomitant growth inhibition. Forced expression of RBP10 prevented differentiation of bloodstream forms in response to cis-aconitate, but did not prevent expression of key differentiation markers in response to glucose deprivation. RBP10 was not associated with heavy polysomes, showed no detectable in vivo binding to RNA, and was not stably associated with other proteins. Tethering of RBP10 to a reporter mRNA inhibited translation, and halved the abundance of the bound mRNA. We suggest that RBP10 may prevent the expression of regulatory proteins that are specific to the procyclic form.
    Type of Publication: Journal article published
    PubMed ID: 22296558
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 5 (1991), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Both ATP and an electrochemical potential play roles in translocating proteins across the Inner membrane of Escherichia coli. Recent discoveries have dissected the overall transmembrane movement into separate subreactions with different energy requirements, identified a translocation ATPase, and reconstituted both energy-requiring steps of the reaction from purified components. A more refined understanding of the energetics of this fundamental process is beginning to provide answers about the basic issues of how proteins move across the hydrophobic membrane barrier.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 5 (1991), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Regulation of transcription occurs at different levels, one being in the presence of sequences specifically recognized by different forms of RNA polymerase, i.e. the promoters. Three different kinds of promoter are defined according, among other things, to their dependence on the growth rate of the cell: the house-keeper' promoter of many metabolic genes, the stringent promoter found at several rRNA and ribosomal protein genes, and the ‘gearbox’ at genes whose products are required at higher relative amounts at lower growth rates. The identified gearbox promoters of Escherichia coli share specific homologies in the -10, -35 and upstream regions. Although there may be different types of gearbox promoters, the -10 sequence of one of these promoters has been found to be essential for functioning as a gearbox. This suggests the existence of specific sigma factors for its transcription. RpoS (KatF) is a likely candidate for being one of these sigma factors. Computer simulation allows us to predict that such sigma factors should, in turn, be expressed following a gearbox mode, which would then imply the existence of self-regulated loops contributing to the expression of some genes of bacterial division. Some bacterial gene products need to be synthesized al fixed amounts per cell
    Type of Medium: Electronic Resource
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