Publications

Abstract (Expand)

BioRxiv preprint, 4 April 2018. Abstract: Daily light-dark cycles (LD) drive dynamic regulation of plant and algal transcriptomes via photoreceptor pathways and 24-hour, circadian rhythms. Diel regulation of protein levels and modifications has been less studied. Ostreococcus tauri, the smallest free-living eukaryote, provides a minimal model proteome for the green lineage. Here, we compare transcriptome data under LD to the algal proteome and phosphoproteome, assayed using shotgun mass-spectrometry. Under 10% of 855 quantified proteins were rhythmic but two-thirds of 860 phosphoproteins showed rhythmic modification(s). Most rhythmic proteins peaked in the daytime. Model simulations showed that light-stimulated protein synthesis largely accounts for this distribution of protein peaks. Prompted by apparently dark-stable proteins, we sampled during prolonged dark adaptation, where stable RNAs and very limited change to the proteome suggested a quiescent, cellular “dark state”. In LD, acid-directed and proline-directed protein phosphorylation sites were regulated in antiphase. Strikingly, 39% of rhythmic phospho-sites reached peak levels just before dawn. This anticipatory phosphorylation is distinct from light-responsive translation but consistent with plant phosphoprotein profiles, suggesting that a clock-regulated phospho-dawn prepares green cells for daytime functions.

Authors: Zeenat B. Noordally, Matthew M. Hindle, Sarah F. Martin, Daniel Seaton, Ian Simpson, Thierry Le Bihan, Andrew Millar

Date Published: No date defined

Journal: Not specified

Abstract (Expand)

Genomic aberrations can be used to subtype breast cancer. In this study, we investigated DNA copy number (CN) profiles of 69 cases of male breast cancer (MBC) by array comparative genomic hybridization (aCGH) to detect recurrent gains and losses in comparison with female breast cancers (FBC). Further, we classified these profiles as BRCA1-like, BRCA2-like or non-BRCA-like profiles using previous classifiers derived from FBC, and correlated these profiles with pathological characteristics. We observed large CN gains on chromosome arms 1q, 5p, 8q, 10p, 16p, 17q, and chromosomes 20 and X. Large losses were seen on chromosomes/chromosome arms 1p, 6p, 8p, 9, 11q, 13, 14q, 16q, 17p, and 22. The pattern of gains and losses in estrogen receptor positive (ER+) MBC was largely similar to ER+ FBC, except for gains on chromosome X in MBC, which were uncommon in FBC. Out of 69 MBC patients, 15 patients (22%) had a BRCA2-like profile, of which 2 (3%) were also BRCA1-like. One patient (1%) was only BRCA1-like; the remaining 53 (77%) patients were classified as non-BRCA-like. BRCA2-like cases were more often p53 accumulated than non-BRCA-like cases (P = 0.014). In conclusion, the pattern of gains and losses in ER+ MBC was largely similar to that of its ER+ FBC counterpart, except for gains on chromosome X in MBC, which are uncommon in FBC. A significant proportion of MBC has a BRCA2-like aCGH profile, pointing to a potentially hereditary nature, and indicating that they could benefit from a drug regimen targeting BRCA defects as in FBC.

Authors: H. D. Biesma, P. C. Schouten, M. M. Lacle, J. Sanders, W. Brugman, R. Kerkhoven, I. Mandjes, P. van der Groep, P. J. van Diest, S. C. Linn

Date Published: No date defined

Journal: Genes Chromosomes Cancer

Abstract (Expand)

Atlantic salmon can synthesize polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (20:5n-3), arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) via activities of very long chain fatty acyl elongases (Elovls) and fatty acyl desaturases (Fads), albeit to a limited degree. Understanding molecular mechanisms of PUFA biosynthesis and regulation is a pre-requisite for sustainable use of vegetable oils in aquafeeds as current sources of fish oils are unable to meet increasing demands for omega-3 PUFAs. By generating CRISPR-mediated elovl2 partial knockout (KO), we have shown that elovl2 is crucial for multi-tissue synthesis of 22:6n-3 in vivo and that endogenously synthesized PUFAs are important for transcriptional regulation of lipogenic genes in Atlantic salmon. The elovl2-KOs showed reduced levels of 22:6n-3 and accumulation of 20:5n-3 and docosapentaenoic acid (22:5n-3) in the liver, brain and white muscle, suggesting inhibition of elongation. Additionally, elovl2-KO salmon showed accumulation of 20:4n-6 in brain and white muscle. The impaired synthesis of 22:6n-3 induced hepatic expression of sterol regulatory element binding protein-1 (srebp-1), fatty acid synthase-b, Δ6fad-a, Δ5fad and elovl5. Our study demonstrates key roles of elovl2 at two penultimate steps of PUFA synthesis in vivo and suggests Srebp-1 as a main regulator of endogenous PUFA synthesis in Atlantic salmon.

Authors: Alex K. Datsomor, Nikola Zic, Keshuai Li, Rolf E. Olsen, Yang Jin, Jon Olav Vik, Rolf B. Edvardsen, Fabian Grammes, Anna Wargelius, Per Winge

Date Published: 1st Dec 2019

Journal: Sci Rep

Abstract (Expand)

Chiral 2-substituted 3-hydroxycarboxylic acid derivatives are valuable building blocks for the preparation of naturally occurring and synthetic biologically active molecules. Current methodologies for the preparation of these compounds are still limited for large-scale production due to the high costs, limited microbial strains, low yields, difficult downstream processing, and limited range of structures. We report an effective chemoenzymatic method for the synthesis of enantiomerically pure 2 substituted 3 hydroxycarboxylic esters. The strategy comprises: i) a stereoselective aldol addition of 2 oxoacids to methanal catalyzed by two enantiocomplementary 2 oxoacid aldolases, ii) oxidative decarboxylation, and iii) esterification. Compounds with S-configuration were obtained in 69-80% isolated yields (94-99% ee), and the R enantiomers in 57-88% (88-95% ee), using a substrate concentration range of 0.1-1.0 M. The method developed offers a versatile alternative route to this important class of chiral building blocks, and highlights the exciting opportunities available for using natural enzymes with minimal active site modification.

Authors: Roser Marín-Valls, Karel Hernández, Michael Bolte, Jesús Joglar, Jordi Bujons, Pere Clapés

Date Published: 8th Jul 2019

Journal: ACS Catal.

Abstract (Expand)

The aim of this study was to assess whether fish in Kollevag, a sheltered bay on the western coast of Norway, previously utilized as a waste disposal site, could be affected by environmental contaminants leaking from the waste. Farmed, juvenile Atlantic cod (Gadus morhua) were caged for six weeks at three different locations in Kollevag bay and at one reference location. Sediments and cod samples (bile and liver) were analyzed for polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), brominated flame retardants (BFRs), per-and polyfluoroalkyl substances (PFASs) and polycyclic aromatic hydrocarbon (PAH) metabolites, revealing a contamination gradient at the four stations. Furthermore, hepatosomatic index (HSI) and Fulton's condition factor (CF) were significantly lower in cod caged closest to the disposal site. Levels and activities of biomarker proteins, such as vitellogenin (Vtg), metallothionein (Mt), and biotransformation and oxidative stress enzymes, including cytochrome P450 1a and 3a (Cyp1a, Cyp3a), glutathione s-transferase (Gst) and catalase (Cat), were quantified in blood plasma and liver tissue. Hepatic Cat and Gst activities were significantly reduced in cod caged at the innermost stations in Kollevag, indicating modulation of oxidative stress responses. However, these results contrasted with reduced hepatic lipid peroxidation. Significant increases in transcript levels were observed for genes involved in lipid metabolism (fasn and acly) in cod liver, while transcript levels of ovarian steroidogenic enzyme genes such as p450scc, cyp19, 3beta-hsd and 20beta-hsd showed significant station-dependent increases. Cyp1a and Vtg protein levels were however not significantly altered in cod caged in Kollevag. Plasma levels of estradiol (E2) and testosterone (T) were determined by enzyme immunoassay (EIA) and showed elevated E2 levels, but only at the innermost station. We conclude that the bay of Kollevag did not fullfill adequate environmental condition based on environmental quality standards (EQSs) for chemicals in coastal waters. Following a six weeks caging period, environmental contaminants accumulated in cod tissues and effects were observed on biomarker responses, especially those involved in reproductive processes in cod ovary.

Authors: Karina Dale, M. B. Muller, Zhanna Tairova, Essa Ahsan Khan, K. Hatlen, M. Grung, Fekadu Yadetie, R. Lille-Langoy, Nello Blaser, H. J. Skaug, J. L. Lyche, A. Arukwe, Ketil Hylland, Odd André Karlsen, Anders Goksøyr

Date Published: 26th Feb 2019

Journal: Mar Environ Res

Abstract (Expand)

Caulobacter crescentus is a gram-negative bacterium that can utilize xylose as a substrate using the Weimberg pathway, which converts xylose to α-ketoglutarate in five steps without carbon loss. This is an interesting pathway for heterologous expression in other organisms in order to enable xylose utilization in biorefinery processes. C. crescentus was grown on xylose, arabinose and glucose, and maximum specific growth rates determined for the three substrates were 0.11 h−1, 0.05 h−1, and 0.15 h−1 respectively. Growth was found to be significantly inhibited at sugar concentration of 20 g L−1, shown primarily by an increased lag phase. Enzyme activity assays showed that the Weimberg pathway was active in cells grown, not only on xylose but also on arabinose. No activity was found for growth on glucose. Furthermore, substantial amounts of α-ketoglutarate—up to a yield of 0.4 g g−1—was excreted during growth on xylose, but no other extracellular intermediates in the Weimberg pathway were detected during growth on xylose. Apparently, C. crescentus is not well adapted for efficient growth on high xylose levels, and responds by an extended lag phase and secretion of α-ketoglutarate.

Authors: Henrik Almqvist, Sara Jonsdottir Glaser, Celina Tufvegren, Lisa Wasserstrom, Gunnar Liden

Date Published: 1st Jun 2018

Journal: Fermentation

Abstract (Expand)

Cell volume is an important parameter for modelling cellular processes. Temperature-induced variability of cellular size, volume, intracellular granularity, a fraction of budding cells of yeast Saccharomyces cerevisiae CEN.PK 113–7D (in anaerobic glucose unlimited batch cultures) were measured by flow cytometry and matched with the performance of the biomass growth (maximal specific growth rate (μmax), specific rate of glucose consumption, the rate of maintenance, biomass yield on glucose). The critical diameter of single cells was 7.94 μm and it is invariant at growth temperatures above 18.5°C. Below 18.5°C, it exponentially increases up to 10.2 μm. The size of the bud linearly depends on μmax, and it is between 50% at 5°C and 90% at 31°C of the averaged single cell. The intracellular granularity (side scatter channel (SSC)-index) negatively depends on μmax. There are two temperature regions (5–31°C vs. 33–40°C) where the relationship between SSC-index and various cellular parameters differ significantly. In supraoptimal temperature range (33–40°C), cells are less granulated perhaps due to a higher rate of the maintenance. There is temperature dependent passage through the checkpoints in the cell cycle which influences the μmax. The results point to the existence of two different morphological states of yeasts in these different temperature regions.

Authors: Maksim Zakhartsev, Matthias Reuss

Date Published: 26th Apr 2018

Journal: Not specified

Abstract (Expand)

Pseudomonas is a highly versatile genus containing species that can be harmful to humans and plants while others are widely used for bioengineering and bioremediation. We analysed 432 sequenced Pseudomonas strains by integrating results from a large scale functional comparison using protein domains with data from six metabolic models, nearly a thousand transcriptome measurements and four large scale transposon mutagenesis experiments. Through heterogeneous data integration we linked gene essentiality, persistence and expression variability. The pan-genome of Pseudomonas is closed indicating a limited role of horizontal gene transfer in the evolutionary history of this genus. A large fraction of essential genes are highly persistent, still non essential genes represent a considerable fraction of the core-genome. Our results emphasize the power of integrating large scale comparative functional genomics with heterogeneous data for exploring bacterial diversity and versatility.

Authors: J. J. Koehorst, J. C. van Dam, Ruben Van Heck, E. Saccenti, V. A. Dos Santos, M. Suarez-Diez, P. J. Schaap

Date Published: 7th Dec 2016

Journal: Sci Rep

Abstract (Expand)

The same pathway, such as the mitogen-activated protein kinase (MAPK) pathway, can produce different cellular responses, depending on stimulus or cell type. We examined the phosphorylation dynamics of the MAPK kinase MEK and its targets extracellular signal-regulated kinase 1 and 2 (ERK1/2) in primary hepatocytes and the transformed keratinocyte cell line HaCaT A5 exposed to either hepatocyte growth factor or interleukin-6. By combining quantitative mass spectrometry with dynamic modeling, we elucidated network structures for the reversible threonine and tyrosine phosphorylation of ERK in both cell types. In addition to differences in the phosphorylation and dephosphorylation reactions, the HaCaT network model required two feedback mechanisms, which, as the experimental data suggested, involved the induction of the dual-specificity phosphatase DUSP6 and the scaffold paxillin. We assayed and modeled the accumulation of the double-phosphorylated and active form of ERK1/2, as well as the dynamics of the changes in the monophosphorylated forms of ERK1/2. Modeling the differences in the dynamics of the changes in the distributions of the phosphorylated forms of ERK1/2 suggested that different amounts of MEK activity triggered context-specific responses, with primary hepatocytes favoring the formation of double-phosphorylated ERK1/2 and HaCaT A5 cells that produce both the threonine-phosphorylated and the double-phosphorylated form. These differences in phosphorylation distributions explained the threshold, sensitivity, and saturation of the ERK response. We extended the findings of differential ERK phosphorylation profiles to five additional cultured cell systems and matched liver tumor and normal tissue, which revealed context-specific patterns of the various forms of phosphorylated ERK.

Authors: N. Iwamoto, L. A. D'Alessandro, S. Depner, B. Hahn, B. A. Kramer, P. Lucarelli, A. Vlasov, M. Stepath, M. E. Bohm, D. Deharde, G. Damm, D. Seehofer, W. D. Lehmann, U. Klingmuller, Marcel Schilling

Date Published: 4th Feb 2016

Journal: Sci Signal

Abstract (Expand)

Kinetic data of biochemical reactions are essential for the creation of kinetic models of biochemical networks. One of the main resources of such information is SABIO-RK, a curated database for kinetic data of biochemical reactions and their related information. Despite the importance for computational modelling there has been no simple solution to visualize the kinetic data from SABIO-RK. In this work, I present cy3sabiork, an app for querying and visualization of kinetic data from SABIO-RK in Cytoscape. The kinetic information is accessible via a combination of graph structure and annotations of nodes, with provided information consisting of: (I) reaction details, enzyme and organism; (II) kinetic law, formula, parameters; (III) experimental conditions; (IV) publication; (V) additional annotations. cy3sabiork creates an intuitive visualization of kinetic entries in form of a species-reaction-kinetics graph, which reflects the reaction-centered approach of SABIO-RK. Kinetic entries can be imported in SBML format from either the SABIO-RK web interface or via web service queries. The app allows for easy comparison of kinetic data, visual inspection of the elements involved in the kinetic record and simple access to the annotation information of the kinetic record. I applied cy3sabiork in the computational modelling of galactose metabolism in the human liver.

Author: Matthias König

Date Published: 2016

Journal: F1000Res

Abstract (Expand)

The enzymes in the Embden–Meyerhof–Parnas pathway of Plasmodium falciparum trophozoites were kinetically characterized and their integrated activities analyzed in a mathematical model. For validation of the model, we compared model predictions for steady-state fluxes and metabolite concentrations of the hexose phosphates with experimental values for intact parasites. The model, which is completely based on kinetic parameters that were measured for the individual enzymes, gives an accurate prediction of the steady-state fluxes and intermediate concentrations. This is the first detailed kinetic model for glucose metabolism in P. falciparum, one of the most prolific malaria-causing protozoa, and the high predictive power of the model makes it a strong tool for future drug target identification studies. The modelling workflow is transparent and reproducible, and completely documented in the SEEK platform, where all experimental data and model files are available for download.

Authors: Gerald Penkler, Francois du Toit, Waldo Adams, Marina Rautenbach, Daniel C. Palm, Dawie Van Niekerk, Jacky Snoep

Date Published: 1st Apr 2015

Journal: FEBS J

Abstract (Expand)

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in 'transcription traffic jams' on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.

Authors: Yulia Yuzenkova, Pamela Gamba, M. Herber, L. Attaiech, Sulman Shafeeq, Oscar Kuipers, S. Klumpp, Nikolay Zenkin, Jan-Willem Veening

Date Published: 6th Sep 2014

Journal: Nucleic Acids Res

Abstract (Expand)

Data-based mathematical modeling of biochemical reaction networks, e.g., by nonlinear ordinary differential equation (ODE) models, has been successfully applied. In this context, parameter estimation and uncertainty analysis is a major task in order to assess the quality of the description of the system by the model. Recently, a broadened eigenvalue spectrum of the Hessian matrix of the objective function covering orders of magnitudes was observed and has been termed as sloppiness. In this work, we investigate the origin of sloppiness from structures in the sensitivity matrix arising from the properties of the model topology and the experimental design. Furthermore, we present strategies using optimal experimental design methods in order to circumvent the sloppiness issue and present nonsloppy designs for a benchmark model.

Authors: Christian Tönsing, Jens Timmer, Clemens Kreutz

Date Published: 1st Aug 2014

Journal: Phys. Rev. E

Abstract (Expand)

The Bacillus subtilis catabolite control protein A (CcpA) is a global transcriptional regulator which is controlled by interactions with the phosphoproteins HPrSer46P and CrhP and with low molecular weight effectors depending on the availability of preferred carbon sources like glucose. Distinct point mutations in CcpA abolish regulation of some but not all target genes suggesting additional interactions of CcpA. Therefore, in vivo crosslinking and mass spectrometry were applied to identify CcpA complexes active in repression and activation. To compensate for the excess of promoters only repressed by CcpA, this experiment was accomplished with cells with multiple copies of the activated ackA promoter. Among the identified proteins HPr, RNA polymerase (RNAP) subunits and the global regulator CodY were observed. Bacterial two-hybrid assays combining each RNAP subunit with CcpA localized CcpA binding at the α-subunit (RpoA). In vivo crosslinking combined with immunoblot analyses revealed CcpA-RpoA complexes in cultures with or without glucose whereas CcpA-HPr and CcpA-CodY complexes occurred only or predominantly in cultures with glucose. Surface plasmon resonance (SPR) analyses confirmed binding of CcpA to the N- (αNTD) and C-terminal domains (αCTD) of RpoA as well as to CodY. Furthermore, interactions of CodY with the αNTD and the αCTD were detected by SPR. The K(D) values of complexes of CcpA or CodY with the αNTD or the αCTD are between 5 and 8μM. CcpA and CodY form a loose complex with a K(D) of 60μM. These data were combined to propose a model for a transcription initiation complex at the ackA promoter.

Authors: Andrea Wünsche, Elke Hammer, Maike Bartholomae, Uwe Voelker, Andreas Burkovski, Gerald Seidel, Wolfgang Hillen

Date Published: 20th Apr 2012

Journal: The FEBS journal

Abstract (Expand)

How the human pathogen Streptococcus pneumoniae coordinates cell-wall synthesis during growth and division to achieve its characteristic oval shape is poorly understood. The conserved eukaryotic-type Ser/Thr kinase of S. pneumoniae, StkP, previously was reported to phosphorylate the cell-division protein DivIVA. Consistent with a role in cell division, GFP-StkP and its cognate phosphatase, GFP-PhpP, both localize to the division site. StkP localization depends on its penicillin-binding protein and Ser/Thr-associated domains that likely sense uncross-linked peptidoglycan, because StkP and PhpP delocalize in the presence of antibiotics that target the latest stages of cell-wall biosynthesis and in cells that have stopped dividing. Time-lapse microscopy shows that StkP displays an intermediate timing of recruitment to midcell: StkP arrives shortly after FtsA but before DivIVA. Furthermore, StkP remains at midcell longer than FtsA, until division is complete. Cells mutated for stkP are perturbed in cell-wall synthesis and display elongated morphologies with multiple, often unconstricted, FtsA and DivIVA rings. The data show that StkP plays an important role in regulating cell-wall synthesis and controls correct septum progression and closure. Overall, our results indicate that StkP signals information about the cell-wall status to key cell-division proteins and in this way acts as a regulator of cell division.

Authors: Katrin Beilharz, Linda Nováková, Daniela Fadda, Pavel Branny, Orietta Massidda, Jan-Willem Veening

Date Published: 21st Mar 2012

Journal: Proceedings of the National Academy of Sciences of the United States of America

Abstract (Expand)

Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Authors: Pierre Nicolas, Ulrike Mäder, Etienne Dervyn, Tatiana Rochat, Aurélie Leduc, Nathalie Pigeonneau, Elena Bidnenko, Elodie Marchadier, Mark Hoebeke, Stéphane Aymerich, Dörte Becher, Paola Bisicchia, Eric Botella, Olivier Delumeau, Geoff Doherty, Emma L Denham, Mark J Fogg, Vincent Fromion, Anne Goelzer, Annette Hansen, Elisabeth Härtig, Colin Harwood, Georg Homuth, Hanne Jarmer, Matthieu Jules, Edda Klipp, Ludovic Le Chat, François Lecointe, Rick Lewis, Wolfram Liebermeister, Anika March, Ruben Mars, Priyanka Nannapaneni, David Noone, Susanne Pohl, Bernd Rinn, Frank Rügheimer, Praveen Kumar Sappa, Franck Samson, Marc Schaffer, Benno Schwikowski, Leif Steil, Joerg Stuelke, Thomas Wiegert, Kevin M Devine, Anthony J Wilkinson, Jan Maarten Van Dijl, Michael Hecker, Uwe Voelker, Philippe Bessières, Philippe Noirot

Date Published: 3rd Mar 2012

Journal: Science

Abstract (Expand)

Bacillus subtilis possesses carbon-flux regulating histidine protein (Crh), a paralog of the histidine protein (HPr) of the phosphotransferase system (PTS). Like HPr, Crh becomes (de)phosphorylated in vitro at residue Ser46 by the metabolite-controlled HPr kinase/phosphorylase HPrK/P. Depending on its phosphorylation state, Crh exerts regulatory functions in connection with carbohydrate metabolism. So far, knowledge on phosphorylation of Crh in vivo has been limited and derived from indirect evidence. Here, we studied the dynamics of Crh phosphorylation directly by non-denaturing gel electrophoresis followed by Western analysis. The results confirm that HPrK/P is the single kinase catalyzing phosphorylation of Crh in vivo. Accordingly, phosphorylation of Crh is triggered by the carbon source as observed previously for HPr, but with some differences. Phosphorylation of both proteins occurred during exponential growth and disappeared upon exhaustion of the carbon source. During exponential growth, ~80% of the Crh molecules were phosphorylated when cells utilized a preferred carbon source. The reverse distribution, i.e. around 20% of Crh molecules phosphorylated, was obtained upon utilization of less favorable substrates. This clear-cut classification of the substrates into two groups has not previously been observed for HPr(Ser)~P formation. The likely reason for this difference is the additional PTS-dependent phosphorylation of HPr at His15, which limits accumulation of HPr(Ser)~P.

Authors: Jens J Landmann, Susanne Werner, Wolfgang Hillen, Joerg Stuelke, Boris Görke

Date Published: 28th Nov 2011

Journal: FEMS Microbiol. Lett.

Abstract

Not specified

Authors: Sonja Steinsiek, S. Frixel, Stefan Stagge, Katja Bettenbrock

Date Published: 1st Jun 2011

Journal: Journal of Biotechnology

Abstract (Expand)

The highly processive transcription by multi-subunit RNA polymerases (RNAP) can be interrupted by misincorporation or backtracking events that may stall transcription or lead to erroneous transcripts. Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript. Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre. This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis. Replacement of the TL by Gre factor occurs only in backtracked/misincorporated complexes, and not in correctly elongating complexes. This controlled switching of RNAP activities allows the processivity of elongation to be unaffected by the hydrolytic activity of Gre, while ensuring efficient proofreading of transcription and resolution of backtracked complexes.

Authors: Mohammad Roghanian, Yulia Yuzenkova, Nikolay Zenkin

Date Published: 27th Jan 2011

Journal: Not specified

Abstract

Not specified

Authors: Marco Pittelkow, Erhard Bremer

Date Published: 2011

Journal: Halophiles and Hypersaline Environments

Abstract (Expand)

Genomic tiling array transcriptomics and RNA-seq are two powerful and rapidly developing approaches for unbiased transcriptome analysis. Providing comprehensive identification and quantification of transcripts with an unprecedented resolution, they are leading to major breakthroughs in systems biology. Here we review each step of the analysis from library preparation to the interpretation of the data, with particular attention paid to the possible sources of artifacts. Methodological requirements and statistical frameworks are often similar in both the approaches despite differences in the nature of the data. Tiling array analysis does not require rRNA depletion and benefits from a more mature computational workflow, whereas RNA-Seq has a clear lead in terms of background noise and dynamic range with a considerable potential for evolution with the improvements of sequencing technologies. Being independent of prior sequence knowledge, RNA-seq will boost metatranscriptomics and evolutionary transcriptomics applications.

Authors: Ulrike Mäder, Pierre Nicolas, Hugues Richard, Philippe Bessières, Stéphane Aymerich

Date Published: 10th Nov 2010

Journal: Curr. Opin. Biotechnol.

Abstract (Expand)

Systems biology relies increasingly on collaborations between several groups with different expertise. Therefore, the systems biology community is adopting standards that allow effective communication of concepts, as well as transmission and processing of pathway information. The Systems Biology Graphical Notation (SBGN) is a graphical language for biological pathways that has both a biological as well as a computational meaning. The program CellDesigner allows the codification of biological phenomena in an SBGN compliant form. CellPublisher is a web server that allows the conversion of CellDesigner files to web-based navigatable diagrams based on the user interface of Google maps. Thus, CellPublisher complements CellDesigner by facilitating the understanding of complex diagrams and by providing the possibility to share any CellDesigner diagram online with collaborators and get their feedback. Due to the intuitive interface of the online diagrams, CellPublisher serves as a basis for discovery of novel properties of the modelled networks.

Authors: Lope A. Florez, Christoph R Lammers, Raphael Michna, Joerg Stuelke

Date Published: 14th Oct 2010

Journal: Bioinformatics

Abstract (Expand)

The active center of RNA polymerase can hydrolyze phosphodiester bonds in nascent RNA, a reaction thought to be important for proofreading of transcription. The reaction proceeds via a general two Mg(2+) mechanism and is assisted by the 3' end nucleotide of the transcript. Here, by using Thermus aquaticus RNA polymerase, we show that the reaction also requires the flexible domain of the active center, the trigger loop (TL). We show that the invariant histidine (beta' His1242) of the TL is essential for hydrolysis/proofreading and participates in the reaction in two distinct ways: by positioning the 3' end nucleotide of the transcript that assists catalysis and/or by directly participating in the reaction as a general base. We also show that participation of the beta' His1242 of the TL in phosphodiester bond hydrolysis does not depend on the extent of elongation complex backtracking. We obtained similar results with Escherichia coli RNA polymerase, indicating that the function of the TL in phosphodiester bond hydrolysis is conserved among bacteria.

Authors: Yulia Yuzenkova, Nikolay Zenkin

Date Published: 1st Jun 2010

Journal: Proc. Natl. Acad. Sci. U.S.A.

Abstract (Expand)

Cell surface receptors convert extracellular cues into receptor activation, thereby triggering intracellular signaling networks and controlling cellular decisions. A major unresolved issue is the identification of receptor properties that critically determine processing of ligand-encoded information. We show by mathematical modeling of quantitative data and experimental validation that rapid ligand depletion and replenishment of the cell surface receptor are characteristic features of the erythropoietin (Epo) receptor (EpoR). The amount of Epo-EpoR complexes and EpoR activation integrated over time corresponds linearly to ligand input; this process is carried out over a broad range of ligand concentrations. This relation depends solely on EpoR turnover independent of ligand binding, which suggests an essential role of large intracellular receptor pools. These receptor properties enable the system to cope with basal and acute demand in the hematopoietic system.

Authors: V. Becker, Marcel Schilling, J. Bachmann, U. Baumann, A. Raue, T. Maiwald, J. Timmer, U. Klingmuller

Date Published: 20th May 2010

Journal: Science

Abstract (Expand)

Stability and biological activity of proteins is highly dependent on their physicochemical environment. The development of realistic models of biological systems necessitates quantitative information on the response to changes of external conditions like pH, salinity and concentrations of substrates and allosteric modulators. Changes in just a few variable parameters rapidly lead to large numbers of experimental conditions, which go beyond the experimental capacity of most research groups. We implemented a computer-aided experimenting framework ("robot lab assistant") that allows us to parameterize abstract, human-readable descriptions of micro-plate based experiments with variable parameters and execute them on a conventional 8 channel liquid handling robot fitted with a sensitive plate reader. A set of newly developed R-packages translates the instructions into machine commands, executes them, collects the data and processes it without user-interaction. By combining script-driven experimental planning, execution and data-analysis, our system can react to experimental outcomes autonomously, allowing outcome-based iterative experimental strategies. The framework was applied in a response-surface model based iterative optimization of buffer conditions and investigation of substrate, allosteric effector, pH and salt dependent activity profiles of pyruvate kinase (PYK). A diprotic model of enzyme kinetics was used to model the combined effects of changing pH and substrate concentrations. The 8 parameters of the model could be estimated from a single two-hour experiment using nonlinear least-squares regression. The model with the estimated parameters successfully predicted pH and PEP dependence of initial reaction rates, while the PEP concentration dependent shift of optimal pH could only be reproduced with a set of manually tweaked parameters. Differences between model-predictions and experimental observations at low pH suggest additional protonation-sites at the enzyme or substrates critical for enzymatic activity. The developed framework is a powerful tool to investigate enzyme reaction specifics and explore biological system behaviour in a wide range of experimental conditions.

Authors: Felix Bonowski, Ana Kitanovic, Peter Ruoff, Jinda Holzwarth, Igor Kitanovic, Van Ngoc Bui, Elke Lederer, Stefan Wölfl

Date Published: 23rd Dec 2009

Journal: PLoS ONE

Abstract (Expand)

Bacillus subtilis is the model organism for a large group of Gram-positive bacteria, the Firmicutes. Several online databases have been established over time to manage its genetic and metabolic information, but they differ greatly in their rate of update and their focus on B. subtilis. Therefore, a European systems biology consortium called for an integrated solution that empowers its users to enrich online content. To meet this goal we created SubtiWiki and SubtiPathways, two complementary online tools for gene and pathway information on B. subtilis 168. SubtiWiki (http://subtiwiki.uni-goettingen.de/ ) is a scientific wiki for all genes of B. subtilis and their protein or RNA products. Each gene page contains a summary of the most important information; sections on the gene, its product and expression; sections concerning biological materials and laboratories; and a list of references. SubtiWiki has been seeded with key content and can be extended by any researcher after a simple registration, thus keeping it always up to date. As a complement, SubtiPathways (http://subtipathways.uni-goettingen.de/) is an online tool for navigation of the metabolism of B. subtilis and its regulation. Each SubtiPathways diagram presents a metabolic pathway with its participating enzymes, together with the regulatory mechanisms that act on their expression and activity, in an intuitive interface that is based on Google Maps. Together, SubtiWiki and SubtiPathways provide an integrated view of the processes that make up B. subtilis and its components, making it the most comprehensive web resource for B. subtilis researchers.

Authors: Christoph R Lammers, Lope A. Florez, Arne G Schmeisky, Sebastian F Roppel, Ulrike Mäder, Leendert Hamoen, Joerg Stuelke

Date Published: 3rd Dec 2009

Journal: Microbiology (Reading, Engl.)

Abstract (Expand)

Bacillus subtilis is a well-established cell factory for efficient secretion of many biotechnologically relevant enzymes that are naturally produced by it or related organisms. However, the use of B. subtilis as a host for production of heterologous secretory proteins can be complicated by problems related to inefficient translocation of the foreign proteins across the plasma membrane or to inefficient release of the exported proteins from the cell surface into the surrounding medium. Therefore, there is a clear need for tools that allow more efficient membrane targeting, translocation, and release during the production of these proteins. In the present study, we investigated the contributions of the pre (pre(lip)) and pro (pro(lip)) sequences of a Staphylococcus hyicus lipase to secretion of a heterologous protein, the alkaline phosphatase PhoA of Escherichia coli, by B. subtilis. The results indicate that the presence of the pro(lip)-peptide, in combination with the lipase signal peptide (pre(lip)), contributes significantly to the efficient secretion of PhoA by B. subtilis and that pre(lip) directs PhoA secretion more efficiently than the authentic signal peptide of PhoA. Genome-wide transcriptional analyses of the host cell responses indicate that, under the conditions tested, no known secretion or membrane-cell wall stress responses were provoked by the production of PhoA with any of the pre- and pro-region sequences used. Our data underscore the view that the pre-pro signals of the S. hyicus lipase are very useful tools for secretion of heterologous proteins in B. subtilis.

Authors: Thijs R H M Kouwen, Allan K Nielsen, Emma L Denham, Jean-Yves F Dubois, Ronald Dorenbos, Michael D Rasmussen, Wim J Quax, Roland Freudl, Jan Maarten Van Dijl

Date Published: 30th Nov 2009

Journal: Appl. Environ. Microbiol.

Abstract (Expand)

The molecular mechanisms underlying cell growth, cell division and pathogenesis in Streptococcus pneumoniae are still not fully understood. Single-cell methodologies are potentially of great value to investigate S. pneumoniae cell biology. Here, we report the construction of novel plasmids for single and double cross-over integration of functional fusions to the gene encoding a fast folding variant of the green fluorescent protein (GFP) into the S. pneumoniae chromosome. We have also established a zinc-inducible system for the fine control of gfp-fusion gene expression and for protein depletion experiments in S. pneumoniae. Using this novel single cell toolkit, we have examined the cellular localization of the proteins involved in the essential process of choline decoration of S. pneumoniae teichoic acid. GFP fusions to LicA and LicC, enzymes involved in the activation of choline, showed a cytoplasmic distribution, as predicted from their primary sequences. A GFP fusion to the choline importer protein LicB showed clear membrane localization. GFP fusions to LicD1 and LicD2, enzymes responsible for loading of teichoic acid subunits with choline, are also membrane-associated, even though both proteins lack any obvious membrane spanning domain. These results indicate that the decoration of teichoic acid by the LicD enzymes is a membrane-associated process presumably occurring at lipid-linked teichoic acid precursors.

Authors: Alice Eberhardt, Ling J Wu, Jeff Errington, Waldemar Vollmer, Jan-Willem Veening

Date Published: 8th Sep 2009

Journal: Mol. Microbiol.

Abstract (Expand)

The roles of the two ldh genes of Enterococcus faecalis were studied using knockout mutants. Deletion of ldh-1 causes a metabolic shift from homolactic fermentation to ethanol, formate, and acetoin production, with a high level of formate production even under aerobic conditions. Ldh-2 plays only a minor role in lactate production.

Authors: Maria Jonsson, Zhian Saleihan, Ingolf Nes, Helge Holo

Date Published: 22nd May 2009

Journal: Appl. Environ. Microbiol.

Abstract (Expand)

Antibiotic production is regulated by numerous signals, including the so-called bacterial hormones found in antibiotic producing organisms such as Streptomyces. These signals, the gamma-butyrolactones, are produced in very small quantities, which has hindered their structural elucidation and made it difficult to assess whether they are being produced. In this chapter, we describe a rapid small-scale extraction method from either solid or liquid cultures in scales of one plate or 50 ml of medium. Also described is a bioassay to detect the gamma-butyrolactones by determining either the production of pigmented antibiotic of Streptomyces coelicolor or kanamycin resistant growth on addition of the gamma-butyrolactones. We also describe some insights into the identification of the gamma-butyrolactone receptor and its targets and also the gel retardation conditions with three differently labeled probes.

Authors: Nai-Hua Hsiao, Marco Gottelt, Eriko Takano

Date Published: 21st Apr 2009

Journal: Meth. Enzymol.

Abstract (Expand)

The regulatory proteins AfsR and PhoP control expression of the biosynthesis of actinorhodin and undecylprodigiosin in Streptomyces coelicolor. Electrophoretic mobility shift assays showed that PhoP(DBD) does not bind directly to the actII-ORF4, redD and atrA promoters, but it binds to the afsS promoter, in a region overlapping with the AfsR operator. DNase I footprinting studies revealed a PhoP protected region of 26 nt (PHO box; two direct repeats of 11 nt) that overlaps with the AfsR binding sequence. Binding experiments indicated a competition between AfsR and PhoP; increasing concentrations of PhoP(DBD) resulted in the disappearance of the AfsR-DNA complex. Expression studies using the reporter luxAB gene coupled to afsS promoter showed that PhoP downregulates afsS expression probably by a competition with the AfsR activator. Interestingly, AfsR binds to other PhoP-regulated promoters including those of pstS (a component of the phosphate transport system) and phoRP (encoding the two component system itself). Analysis of the AfsR-protected sequences in each of these promoters allowed us to distinguish the AfsR binding sequence from the overlapping PHO box. The reciprocal regulation of the phoRP promoter by AfsR and of afsS by PhoP suggests a fine interplay of these regulators on the control of secondary metabolism.

Authors: Fernando Santos-Beneit, Antonio Rodríguez-García, Alberto Sola-Landa, Juan-Francisco Martin

Date Published: 11th Feb 2009

Journal: Mol. Microbiol.

Abstract (Expand)

In many bacteria glucose is the preferred carbon source and represses the utilization of secondary substrates. In Bacillus subtilis, this carbon catabolite repression (CCR) is achieved by the global transcription regulator CcpA, whose activity is triggered by the availability of its phosphorylated cofactors, HPr(Ser46-P) and Crh(Ser46-P). Phosphorylation of these proteins is catalyzed by the metabolite-controlled kinase HPrK/P. Recent studies have focused on glucose as a repressing substrate. Here, we show that many carbohydrates cause CCR. The substrates form a hierarchy in their ability to exert repression via the CcpA-mediated CCR pathway. Of the two cofactors, HPr is sufficient for complete CCR. In contrast, Crh cannot substitute for HPr on substrates that cause a strong repression. Determination of the phosphorylation state of HPr in vivo revealed a correlation between the strength of repression and the degree of phosphorylation of HPr at Ser46. Sugars transported by the phosphotransferase system (PTS) cause the strongest repression. However, the phosphorylation state of HPr at its His15 residue and PTS transport activity have no impact on the global CCR mechanism, which is a major difference compared to the mechanism operative in Escherichia coli. Our data suggest that the hierarchy in CCR exerted by the different substrates is exclusively determined by the activity of HPrK/P.

Authors: Kalpana D Singh, Matthias H Schmalisch, Joerg Stuelke, Boris Görke

Date Published: 29th Aug 2008

Journal: J. Bacteriol.

Abstract (Expand)

Most bacteria can selectively use substrates from a mixture of different carbon sources. The presence of preferred carbon sources prevents the expression, and often also the activity, of catabolic systems that enable the use of secondary substrates. This regulation, called carbon catabolite repression (CCR), can be achieved by different regulatory mechanisms, including transcription activation and repression and control of translation by an RNA-binding protein, in different bacteria. Moreover, CCR regulates the expression of virulence factors in many pathogenic bacteria. In this Review, we discuss the most recent findings on the different mechanisms that have evolved to allow bacteria to use carbon sources in a hierarchical manner.

Authors: Boris Görke, Joerg Stuelke

Date Published: 17th Jul 2008

Journal: Nat. Rev. Microbiol.

Abstract (Expand)

The general stress regulon of Bacillus subtilis is controlled by the activity state of sigmaB, a transcription factor that is switched on following exposure to either physical or nutritional stress. ClpP is the proteolytic component of an ATP-dependent protease that is essential for the proper regulation of multiple adaptive responses in B. subtilis. Among the proteins whose abundance increases in ClpP- B. subtilis are several known to depend on sigmaB for their expression. In the current work we examine the relationship of ClpP to the activity of sigmaB. The data reveal that the loss of ClpP in otherwise wild-type B. subtilis results in a small increase in sigmaB activity during growth and a marked enhancement of sigmaB activity following its induction by either physical or nutritional stress. It appears to be the persistence of sigmaB's activity rather than its induction that is principally affected by the loss of ClpP. sigmaB-dependent reporter gene activity rose in parallel in ClpP+ and ClpP- B. subtilis strains but failed to display its normal transience in the ClpP- strain. The putative ClpP targets are likely to be stress generated and novel. Enhanced sigmaB activity in ClpP- B. subtilis was triggered by physical stress but not by the induced synthesis of the physical stress pathway's positive regulator (RsbT). In addition, Western blot analyses failed to detect differences in the levels of the principal known sigmaB regulators in ClpP+ and ClpP- B. subtilis strains. The data suggest a model in which ClpP facilitates the turnover of stress-generated factors, which persist in ClpP's absence to stimulate ongoing sigmaB activity.

Authors: Adam Reeves, Ulf Gerth, Uwe Voelker, W G Haldenwang

Date Published: 22nd Jun 2007

Journal: J. Bacteriol.

Abstract (Expand)

High-quality quantitative data generated under standardized conditions is critical for understanding dynamic cellular processes. We report strategies for error reduction, and algorithms for automated data processing and for establishing the widely used techniques of immunoprecipitation and immunoblotting as highly precise methods for the quantification of protein levels and modifications. To determine the stoichiometry of cellular components and to ensure comparability of experiments, relative signals are converted to absolute values. A major source for errors in blotting techniques are inhomogeneities of the gel and the transfer procedure leading to correlated errors. These correlations are prevented by randomized gel loading, which significantly reduces standard deviations. Further error reduction is achieved by using housekeeping proteins as normalizers or by adding purified proteins in immunoprecipitations as calibrators in combination with criteria-based normalization. Additionally, we developed a computational tool for automated normalization, validation and integration of data derived from multiple immunoblots. In this way, large sets of quantitative data for dynamic pathway modeling can be generated, enabling the identification of systems properties and the prediction of targets for efficient intervention.

Authors: Marcel Schilling, T. Maiwald, S. Bohl, M. Kollmann, C. Kreutz, J. Timmer, U. Klingmuller

Date Published: 13th Dec 2005

Journal: FEBS J

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