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Kinase activity of ArcB from Escherichia coli is subject to regulation by both ubiquinone and demethylmenaquinone

Abstract (Expand)

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is … controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in 'respiratory' electron transfer. This study is an effort to understand how the redox state of the quinone pool(s) is sensed by the cell via the ArcB kinase. We report the relationship between growth, quinone content, ubiquinone redox state, the level of ArcA phosphorylation, and the level of ArcA-dependent gene expression, in a number of mutants of E. coli with specific alterations in their set of quinones, under a range of physiological conditions. Our results provide experimental evidence for a previously formulated hypothesis that not only ubiquinone, but also demethylmenaquinone, can inactivate kinase activity of ArcB. Also, in a mutant strain that only contains demethylmenaquinone, the extent of ArcA phosphorylation can be modulated by the oxygen supply rate, which shows that demethylmenaquinone can also inactivate ArcB in its oxidized form. Furthermore, in batch cultures of a strain that contains ubiquinone as its only quinone species, we observed that the ArcA phosphorylation level closely followed the redox state of the ubiquinone/ubiquinol pool, much more strictly than it does in the wild type strain. Therefore, at low rates of oxygen supply in the wild type strain, the activity of ArcB may be inhibited by demethylmenaquinone, in spite of the fact that the ubiquinones are present in the ubiquinol form.

Authors: P. Sharma, S. Stagge, M. Bekker, K. Bettenbrock, K. J. Hellingwerf

Date Published: 7th Oct 2013

Publication Type: Not specified

[Mesenchymal stroma cells (MSCs) for the treatment of rheumatic disease].

Abstract

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Authors: M. Schmitt, L. P. Muller, G. Keysser, H. M. Lorenz, A. D. Ho, P. Wuchter

Date Published: 6th Sep 2013

Publication Type: Journal

UPARSE: highly accurate OTU sequences from microbial amplicon reads

Abstract (Expand)

Amplified marker-gene sequences can be used to understand microbial community structure, but they suffer from a high level of sequencing and amplification artifacts. The UPARSE pipeline reports operational … taxonomic unit (OTU) sequences with </=1% incorrect bases in artificial microbial community tests, compared with >3% incorrect bases commonly reported by other methods. The improved accuracy results in far fewer OTUs, consistently closer to the expected number of species in a community.

Author: R. C. Edgar

Date Published: 18th Aug 2013

Publication Type: Not specified

Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging

Abstract (Expand)

Green fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localization in vivo, and many different variants are currently available to study bacterial cell … biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two "superfolder" GFPs with codon adaptation specifically for Bacillus subtilis and Streptococcus pneumoniae and have benchmarked them against five other previously available variants of GFP in B. subtilis, S. pneumoniae, and Lactococcus lactis, using promoter-gfp fusions. Surprisingly, the best-performing GFP under our experimental conditions in B. subtilis was the one codon optimized for S. pneumoniae and vice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

Authors: W. Overkamp, K. Beilharz, R. Detert Oude Weme, A. Solopova, H. Karsens, A. Kovacs, J. Kok, ,

Date Published: 16th Aug 2013

Publication Type: Not specified

Organism-Adapted Specificity of the Allosteric Regulation of Pyruvate Kinase in Lactic Acid Bacteria

Abstract

Not specified

Editor:

Date Published: 25th Jul 2013

Publication Type: Not specified

Intermediate instability at high temperature leads to low pathway efficiency for an in vitro reconstituted system of gluconeogenesis in Sulfolobus solfataricus

Abstract (Expand)

Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature … instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/ phosphatase maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1 hour period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the systems uxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3 bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency. This article is protected by copyright. All rights reserved.

Authors: , Dominik Esser, Julia Kort, , ,

Date Published: 20th Jul 2013

Publication Type: Not specified

A model of yeast glycolysis based on a consistent kinetic characterisation of all its enzymes.

Abstract (Expand)

We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an … approximate mathematical model, we employ a "cycle of knowledge" strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom-up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought.

Authors: K. Smallbone, H. L. Messiha, K. M. Carroll, C. L. Winder, N. Malys, W. B. Dunn, E. Murabito, N. Swainston, J. O. Dada, F. Khan, P. Pir, E. Simeonidis, I. Spasic, J. Wishart, D. Weichart, N. W. Hayes, D. Jameson, D. S. Broomhead, S. G. Oliver, S. J. Gaskell, J. E. McCarthy, N. W. Paton, H. V. Westerhoff, D. B. Kell, P. Mendes

Date Published: 9th Jul 2013

Publication Type: Not specified

Dissecting the Catalytic Mechanism of Trypanosoma brucei Trypanothione Synthetase by Kinetic Analysis and Computational Modelling

Abstract (Expand)

In pathogenic trypanosomes, trypanothione synthetase (TryS) catalyzes the synthesis of both glutathionylspermidine (Gsp) and trypanothione [bis(glutathionyl)spermidine, T(SH)2]. Here we present a … thorough kinetic analysis of Trypanosoma brucei TryS in a newly developed phosphate buffer system at pH 7.0 and 37 °C, mimicking the physiological environment of the enzyme in the cytosol of bloodstream parasites. Under these conditions, TryS displays Km-values for GSH, ATP, spermidine and Gsp of 34, 18, 687, and 32 μM, respectively, as well as Ki-values for GSH and T(SH)2 of 1 mM and 360 μM, respectively. As Gsp hydrolysis has a Km-value of 5.6 mM, the in vivo amidase activity is probably negligible. To obtain a deeper insight in the molecular mechanism of TryS, we have formulated alternative kinetic models, with elementary reaction steps represented by linear kinetic equations. The model parameters were fitted to the extensive matrix of steady-state data obtained for different substrate/product combinations under the in vivo-like conditions. The best model describes the full kinetic profile and is able to predict time course data that were not used for fitting. This systems biology approach to enzyme kinetics led us to conclude that (i) TryS follows a ter-reactant mechanism, (ii) the intermediate Gsp dissociates from the enzyme between the two catalytic steps and (iii) T(SH)2 inhibits the enzyme by remaining bound at its product site and, as does the inhibitory GSH, by binding to the activated enzyme complex. The newly detected concerted substrate and product inhibition suggests that TryS activity is tightly regulated.

Editor:

Date Published: 3rd Jul 2013

Publication Type: Not specified

Kinetic characterization of arginine deiminase and carbamate kinase from Streptococcus pyogenes M49

Abstract (Expand)

Streptococcus pyogenes (group A Streptococcus, GAS) is an important human pathogen causing mild superficial infections of skin and mucous membranes, but also life-threatening systemic diseases. S. … pyogenes and other prokaryotic organisms use the arginine deiminase system (ADS) for survival in acidic environments. In this study, the arginine deiminase (AD), and carbamate kinase (CK) from S. pyogenes M49 strain 591 were heterologously expressed in E. coli DH5α, purified, and kinetically characterized. AD and CK from S. pyogenes M49 share high amino acid sequence similarity with the respective enzymes from Lactococcus lactis subsp. lactis IL1403 (45.6% and 53.5% identical amino acids) and Enterococcus faecalis V583 (66.8% and 66.8% identical amino acids). We found that the arginine deiminase of S. pyogenes is not allosterically regulated by the intermediates and products of the arginine degradation (E. g, ATP, citrulline, carbamoyl phosphate). The Km and Vmax values for arginine were 1.13±0.12 mM (mean ± SD) and 1.51±0.07 μmol/min/mg protein. The carbamate kinase is inhibited by ATP but unaffected by arginine and citrulline. The Km and Vmax values for ADP were 0.72±0.08 mM and 1.10±0.10 μmol/min/mg protein and the Km for carbamoyl phosphate was 0.65±0.07 mM. The optimum pH and temperature for both enzymes were 6.5 and 37°C, respectively.

Editor:

Date Published: 1st Jul 2013

Publication Type: Not specified

Regulation of the Activity of Lactate Dehydrogenases from Four Lactic Acid Bacteria

Abstract

Not specified

Authors: , , H. Messiha, , , , , , ,

Date Published: 17th May 2013

Publication Type: Not specified

A shift in the dominant phenotype governs the pH-induced metabolic switch of Clostridium acetobutylicumin phosphate-limited continuous cultures

Abstract (Expand)

In response to changing extracellular pH levels, phosphate-limited continuous cultures of Clostridium acetobutylicum reversibly switches its metabolism from the dominant formation of acids to the … prevalent production of solvents. Previous experimental and theoretical studies have revealed that this pH-induced metabolic switch involves a rearrangement of the intracellular transcriptomic, proteomic and metabolomic composition of the clostridial cells. However, the influence of the population dynamics on the observations reported has so far been neglected. Here, we present a method for linking the pH shift, clostridial growth and the acetone-butanol-ethanol fermentation metabolic network systematically into a model which combines the dynamics of the external pH and optical density with a metabolic model. Furthermore, the recently found antagonistic expression pattern of the aldehyde/alcohol dehydrogenases AdhE1/2 and pH-dependent enzyme activities have been included into this combined model. Our model predictions reveal that the pH-induced metabolic shift under these experimental conditions is governed by a phenotypic switch of predominantly acidogenic subpopulation towards a predominantly solventogenic subpopulation. This model-driven explanation of the pH-induced shift from acidogenesis to solventogenesis by population dynamics casts an entirely new light on the clostridial response to changing pH levels. Moreover, the results presented here underline that pH-dependent growth and pH-dependent specific enzymatic activity play a crucial role in this adaptation. In particular, the behaviour of AdhE1 and AdhE2 seems to be the key factor for the product formation of the two phenotypes, their pH-dependent growth, and thus, the pH-induced metabolic switch in C. acetobutylicum.

Editor:

Date Published: 3rd May 2013

Publication Type: Not specified

Large-scale metabolome analysis and quantitative integration with genomics and proteomics data in Mycoplasma pneumoniae.

Abstract (Expand)

Systems metabolomics, the identification and quantification of cellular metabolites and their integration with genomics and proteomics data, promises valuable functional insights into cellular biology. … However, technical constraints, sample complexity issues and the lack of suitable complementary quantitative data sets prevented accomplishing such studies in the past. Here, we present an integrative metabolomics study of the genome-reduced bacterium Mycoplasma pneumoniae. We experimentally analysed its metabolome using a cross-platform approach. We explain intracellular metabolite homeostasis by quantitatively integrating our results with the cellular inventory of proteins, DNA and other macromolecules, as well as with available building blocks from the growth medium. We calculated in vivo catalytic parameters of glycolytic enzymes, making use of measured reaction velocities, as well as enzyme and metabolite pool sizes. A quantitative, inter-species comparison of absolute and relative metabolite abundances indicated that metabolic pathways are regulated as functional units, thereby simplifying adaptive responses. Our analysis demonstrates the potential for new scientific insight by integrating different types of large-scale experimental data from a single biological source.

Authors: T. Maier, J. Marcos, J. A. Wodke, B. Paetzold, M. Liebeke, R. Gutierrez-Gallego, L. Serrano

Date Published: 20th Apr 2013

Publication Type: Not specified

Dissecting the energy metabolism in Mycoplasma pneumoniae through genome-scale metabolic modeling.

Abstract (Expand)

Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of … understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.

Authors: J. A. Wodke, J. Puchalka, M. Lluch-Senar, J. Marcos, E. Yus, M. Godinho, R. Gutierrez-Gallego, V. A. dos Santos, L. Serrano, E. Klipp, T. Maier

Date Published: 4th Apr 2013

Publication Type: Not specified

Excemplify: a flexible template based solution, parsing and managing data in spreadsheets for experimentalists.

Abstract (Expand)

In systems biology, quantitative experimental data is the basis of building mathematical models. In most of the cases, they are stored in Excel files and hosted locally. To have a public database for … collecting, retrieving and citing experimental raw data as well as experimental conditions is important for both experimentalists and modelers. However, the great effort needed in the data handling procedure and in the data submission procedure becomes the crucial limitation for experimentalists to contribute to a database, thereby impeding the database to deliver its benefit. Moreover, manual copy and paste operations which are commonly used in those procedures increase the chance of making mistakes. Excemplify, a web-based application, proposes a flexible and adaptable template-based solution to solve these problems. Comparing to the normal template based uploading approach, which is supported by some public databases, rather than predefining a format that is potentiall impractical, Excemplify allows users to create their own experiment-specific content templates in different experiment stages and to build corresponding knowledge bases for parsing. Utilizing the embedded knowledge of used templates, Excemplify is able to parse experimental data from the initial setup stage and generate following stages spreadsheets automatically. The proposed solution standardizes the flows of data traveling according to the standard procedures of applying the experiment, cuts down the amount of manual effort and reduces the chance of mistakes caused by manual data handling. In addition, it maintains the context of meta-data from the initial preparation manuscript and improves the data consistency. It interoperates and complements RightField and SEEK as well.

Authors: L. Shi, L. Jong, U. Wittig, P. Lucarelli, M. Stepath, S. Mueller, L. A. D'Alessandro, U. Klingmuller, W. Muller

Date Published: 3rd Apr 2013

Publication Type: Journal

Stealthy annotation of experimental biology by spreadsheets

Abstract (Expand)

The increase in volume and complexity of biological data has led to increased requirements to reuse that data. Consistent and accurate metadata is essential for this task, creating new challenges in … semantic data annotation and in the constriction of terminologies and ontologies used for annotation. The BioSharing community are developing standards and terminologies for annotation, which have been adopted across bioinformatics, but the real challenge is to make these standards accessible to laboratory scientists. Widespread adoption requires the provision of tools to assist scientists whilst reducing the complexities of working with semantics. This paper describes unobtrusive ‘stealthy’ methods for collecting standards compliant, semantically annotated data and for contributing to ontologies used for those annotations. Spreadsheets are ubiquitous in laboratory data management. Our spreadsheet-based RightField tool enables scientists to structure information and select ontology terms for annotation within spreadsheets, producing high quality, consistent data without changing common working practices. Furthermore, our Populous spreadsheet tool proves effective for gathering domain knowledge in the form of Web Ontology Language (OWL) ontologies. Such a corpus of structured and semantically enriched knowledge can be extracted in Resource Description Framework (RDF), providing further means for searching across the content and contributing to Open Linked Data (http://linkeddata.org/)

Authors: , , Matthew Horridge, Simon Jupp, , , , , Robert Stevens,

Date Published: 1st Feb 2013

Publication Type: Journal

Integrative modelling of pH-dependent enzyme activity and transcriptomic regulation of the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum in continuous culture

Abstract (Expand)

In a continuous culture under phosphate limitation the metabolism of Clostridium acetobutylicum depends on the external pH level. By comparing seven steady-state conditions between pH 5.7 and pH 4.5 … we show that the switch from acidogenesis to solventogenesis occurs between pH 5.3 and pH 5.0 with an intermediate state at pH 5.1. Here, an integrative study is presented investigating how a changing external pH level affects the clostridial acetone–butanol–ethanol (ABE) fermentation pathway. This is of particular interest as the biotechnological production of n-butanol as biofuel has recently returned into the focus of industrial applications. One prerequisite is the furthering of the knowledge of the factors determining the solvent production and their integrative regulations. We have mathematically analysed the influence of pH-dependent specific enzyme activities of branch points of the metabolism on the product formation. This kinetic regulation was compared with transcriptomic regulation regarding gene transcription and the proteomic profile. Furthermore, both regulatory mechanisms were combined yielding a detailed projection of their individual and joint effects on the product formation. The resulting model represents an important platform for future developments of industrial butanol production based on C. acetobutylicum.

Editor:

Date Published: 1st Feb 2013

Publication Type: Not specified

Naphthoquinone derivatives exert their antitrypanosomal activity via a multi-target mechanism

Abstract (Expand)

BACKGROUND AND METHODOLOGY: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, … 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. PRINCIPAL FINDINGS: A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC(50) values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. CONCLUSIONS AND SIGNIFICANCE: Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Authors: S. Pieretti, , M. Mazet, R. Perozzo, C. Bergamini, F. Prati, R. Fato, G. Lenaz, G. Capranico, R. Brun, , P. A. Michels, L. Scapozza, M. L. Bolognesi, A. Cavalli

Date Published: 17th Jan 2013

Publication Type: Not specified

Simulations of stressosome activation emphasize allosteric interactions between RsbR and RsbT

Abstract (Expand)

Background The stressosome is a bacterial signalling complex that responds to environmental changes by initiating a protein partner switching cascade, which leads to the release of the alternative sigma … factor, sigmaB. Stress perception increases the phosphorylation of the stressosome sensor protein, RsbR, and the scaffold protein, RsbS, by the protein kinase RsbT. Subsequent dissociation of RsbT from the stressosome activates the sigmaB cascade. However, the sequence of physical events that occur in the stressosome during signal transduction is insufficiently understood. Results Here, we use computational modelling to correlate the structure of the stressosome with the efficiency of the phosphorylation reactions that occur upon activation by stress. In our model, the phosphorylation of any stressosome protein is dependent upon its nearest neighbours and their phosphorylation status. We compare different hypotheses about stressosome activation and find that only the model representing the allosteric activation of the kinase RsbT, by phosphorylated RsbR, qualitatively reproduces the experimental data. Conclusions Our simulations and the associated analysis of published data support the following hypotheses: (i) a simple Boolean model is capable of reproducing stressosome dynamics, (ii) different stressors induce identical stressosome activation patterns, and we also confirm that (i) phosphorylated RsbR activates RsbT, and (ii) the main purpose of RsbX is to dephosphorylate RsbS-P.

Authors: , , Jon Marles-Wright, ,

Date Published: 2013

Publication Type: Not specified

Semantic Data and Models Sharing in Systems Biology: The Just Enough Results Model and the SEEK Platform

Abstract (Expand)

Research in Systems Biology involves integrating data and knowledge about the dynamic processes in biological systems in order to understand and model them. Semantic web technologies should be ideal … for exploring the complex networks of genes, proteins and metabolites that interact, but much of this data is not natively available to the semantic web. Data is typically collected and stored with free-text annotations in spreadsheets, many of which do not conform to existing metadata standards and are often not publically released. Along with initiatives to promote more data sharing, one of the main challenges is therefore to semantically annotate and extract this data so that it is available to the research community. Data annotation and curation are expensive and undervalued tasks that have enormous benefits to the discipline as a whole, but fewer benefits to the individual data producers. By embedding semantic annotation into spreadsheets, however, and automatically extracting this data into RDF at the time of repository submission, the process of producing standards-compliant data, that is available for semantic web querying, can be achieved without adding additional overheads to laboratory data management. This paper describes these strategies in the context of semantic data management in the SEEK. The SEEK is a web-based resource for sharing and exchanging Systems Biology data and models that is underpinned by the JERM ontology (Just Enough Results Model), which describes the relationships between data, models, protocols and experiments. The SEEK was originally developed for SysMO, a large European Systems Biology consortium studying micro-organisms, but it has since had widespread adoption across European Systems Biology.

Editor: David Hutchison and Takeo Kanade and Josef Kittler and Jon M. Kleinberg and Friedemann Mattern and John C. Mitchell and Moni Naor and Oscar Nierstrasz and C. Pandu Rangan and Bernhard Steffen and Madhu Sudan and Demetri Terzopoulos and Doug Tygar and Moshe Y. Vardi and Gerhard Weikum and Camille Salinesi and Moira C. Norrie and Óscar Pastor

Date Published: 2013

Publication Type: Journal

Engineering an anaerobic metabolic regime in Pseudomonas putida KT2440 for the anoxic biodegradation of 1,3-dichloroprop-1-ene

Abstract

Not specified

Authors: Pablo I. Nikel, Víctor de Lorenzo

Date Published: 2013

Publication Type: Not specified

Osmoprotection of Bacillus subtilis through Import and Proteolysis of Proline-Containing Peptides

Abstract

Not specified

Authors: , J. Brill, M. Thuring, G. Wunsche, M. Heun, H. Barzantny, ,

Date Published: 28th Dec 2012

Publication Type: Not specified

Novel twin-arginine translocation pathway-dependent phenotypes of Bacillus subtilis unveiled by quantitative proteomics

Abstract (Expand)

The Twin-arginine Translocation (Tat) pathway is known to translocate fully folded proteins across bacterial, archaeal and organellar membranes. To date, the mechanisms involved in processing, proofreading … and quality control of Tat substrates have remained largely elusive. Bacillus subtilis is an industrially relevant Gram-positive model bacterium. The Tat pathway in B. subtilis differs from that of other well-studied organisms in that it is composed of two complexes operating in parallel. To obtain a better understanding of this pathway in B. subtilis and to identify Tat-associated proteins, the B. subtilis 'Tat proteome' was investigated by quantitative proteomics. Metabolically labeled proteins from cytoplasmic, membrane and extracellular fractions were analyzed by LC-MS/MS. Changes in the amounts of identified peptides allowed for quantitative comparisons of their abundance in tat mutant strains. The observed differences were suggestive of indirect or direct protein-protein relationships. The rich data set generated was then approached in hypothesis-driving and hypothesis-driven manners. The hypothesis-driving approach led to the identification of a novel delayed biofilm phenotype of certain tat mutant strains, whereas the hypothesis-driven approach identified the membrane protein QcrA as a new Tat substrate of B. subtilis. Thus, our quantitative proteomics analyses have unveiled novel Tat pathway-dependent phenotypes in Bacillus.

Authors: Vivianne J Goosens, Andreas Otto, Corinna Glasner, Carmine G Monteferrante, René van der Ploeg, , Dörte Becher,

Date Published: 22nd Dec 2012

Publication Type: Not specified

Know when your numbers are significant

Abstract (Expand)

Experimental biologists, their reviewers and their publishers must grasp basic statistics, urges David L. Vaux, or sloppy science will continue to grow. "And, once in the lab, people generally …y just do what everyone else does, without always understanding why." (D. Vaux)

Author: David L. Vaux

Date Published: 1st Dec 2012

Publication Type: Journal

The DEAD-box RNA helicases in Bacillus subtilis have multiple functions and act independent from each other

Abstract (Expand)

DEAD-box RNA helicases play important roles in remodeling RNA molecules and in facilitating a variety of RNA-protein interactions that are key to many essential cellular processes. In spite of the … importance of RNA, our knowledge about RNA helicases is only limited. In this study we investigated the role of the four DEAD-box RNA helicases in Gram positive model-organism Bacillus subtilis. A strain deleted of all RNA helicases is able to grow at 37°C but not at lower temperatures. Especially the deletion of cshA, cshB or yfmL lead to cold-sensitive phenotypes. Moreover, these mutant strains exhibit unique defects in ribosome biogenesis suggesting distinct functions for the individual enzymes in this process. Based on protein accumulation, severity of the cold-sensitive phenotype and the interaction with components of the RNA degradosome, CshA is the major RNA helicase of B. subtilis. To unravel the functions of CshA in addition to ribosome biogenesis we conducted microarray analysis and identified the ysbAB and frlBONMD mRNAs as targets that are strongly affected by the deletion of the cshA gene. Our findings suggest that the different helicases make distinct contributions to the physiology of B. subtilis. Ribosome biogenesis and RNA degradation are two of their major tasks in B. subtilis.

Authors: Martin Lehnik-Habrink, Leonie Rempeters, Akos T Kovács, Christoph Wrede, Claudia Baierlein, Heike Krebber, ,

Date Published: 24th Nov 2012

Publication Type: Not specified

Osmotic control of opuA expression in Bacillus subtilis and its modulation in response to intracellular glycine betaine and proline pools

Abstract (Expand)

Glycine betaine is an effective osmoprotectant for Bacillus subtilis. Its import into osmotically stressed cells led to the build-up of large pools, whose size was sensitively determined by the degree … of the imposed osmotic stress. The amassing of glycine betaine caused a repression in the formation of an osmostress-adaptive pool of proline, the only osmoprotectant that B. subtilis can synthesize de novo. The ABC transporter OpuA is the main glycine betaine uptake system of B. subtilis. Expression of opuA was up-regulated in response to both sudden and sustained increases in the external osmolarity. Non-ionic osmolytes exerted a stronger inducing effect on transcription than ionic osmolytes, and this was reflected in the development of corresponding OpuA-mediated glycine betaine pools. Primer extension analysis and site-directed mutagenesis pinpointed the osmotically controlled opuA promoter. Deviations from the consensus sequence of SigA-type promoters serve to keep the transcriptional activity of the opuA promoter low in the absence of osmotic stress. Expression of opuA was down regulated in a finely tuned manner in response to increases in the intracellular glycine betaine pool, regardless whether this osmoprotectant was imported or newly synthesized from choline. Such an effect was also exerted by carnitine, an effective osmoprotectant for B. subtilis that is not a substrate for the OpuA transporter. opuA expression was up-regulated in a B. subtilis mutant unable to synthesize proline in response to osmotic stress. Collectively, our data suggest that the intracellular solute pool is a key determinant for the osmotic control of opuA expression.

Authors: , Annette Wensing, Margot Brosius, , ,

Date Published: 24th Nov 2012

Publication Type: Not specified

GraphAlignment: Bayesian pairwise alignment of biological networks

Abstract (Expand)

ABSTRACT: BACKGROUND: With increased experimental availability and accuracy of bio-molecular networks, tools for their comparative and evolutionary analysis are needed. A key component for such studies … is the alignment of networks. RESULTS: We introduce the Bioconductor package GraphAlignment for pairwise alignment of bio-molecular networks. The alignment incorporates information both from network vertices and network edges and is based on an explicit evolutionary model, allowing inference of all scoring parameters directly from empirical data. We compare the performance of our algorithm to an alternative algorithm, Graemlin 2.0.On simulated data, GraphAlignment outperforms Graemlin 2.0 in several benchmarks except for computational complexity. When there is little or no noise in the data, GraphAlignment is slower than Graemlin 2.0. It is faster than Graemlin 2.0 when processing noisy data containing spurious vertex associations. Its typical case complexity grows approximately as O(N^2.6). On empirical bacterial protein-protein interaction networks (PIN) and gene co-expression networks, GraphAlignment outperforms Graemlin 2.0 with respect to coverage and specificity, albeit by a small margin. On large eukaryotic PIN, Graemlin 2.0 outperforms GraphAlignment. CONCLUSIONS: The GraphAlignment algorithm is robust to spurious vertex associations, correctly resolves paralogs, and shows very good performance in identification of homologous vertices defined by high vertex and/or interaction similarity.

Authors: Michal Kolar, Jörn Meier, Ville Mustonen, Michael Lässig,

Date Published: 21st Nov 2012

Publication Type: Not specified

Modelling reveals novel roles of two parallel signalling pathways and homeostatic feedbacks in yeast

Abstract (Expand)

The high osmolarity glycerol (HOG) pathway in yeast serves as a prototype signalling system for eukaryotes. We used an unprecedented amount of data to parameterise 192 models capturing different … hypotheses about molecular mechanisms underlying osmo-adaptation and selected a best approximating model. This model implied novel mechanisms regulating osmo-adaptation in yeast. The model suggested that (i) the main mechanism for osmo-adaptation is a fast and transient non-transcriptional Hog1-mediated activation of glycerol production, (ii) the transcriptional response serves to maintain an increased steady-state glycerol production with low steady-state Hog1 activity, and (iii) fast negative feedbacks of activated Hog1 on upstream signalling branches serves to stabilise adaptation response. The best approximating model also indicated that homoeostatic adaptive systems with two parallel redundant signalling branches show a more robust and faster response than single-branch systems. We corroborated this notion to a large extent by dedicated measurements of volume recovery in single cells. Our study also demonstrates that systematically testing a model ensemble against data has the potential to achieve a better and unbiased understanding of molecular mechanisms.

Authors: J. Schaber, R. Baltanas, A. Bush, E. Klipp, A. Colman-Lerner

Date Published: 15th Nov 2012

Publication Type: Not specified

Reconstruction of Danio rerio metabolic model accounting for subcellular compartmentalisation

Abstract (Expand)

Plant and microbial metabolic engineering is commonly used in the production of functional foods and quality trait improvement. Computational model-based approaches have been used in this important … endeavour. However, to date, fish metabolic models have only been scarcely and partially developed, in marked contrast to their prominent success in metabolic engineering. In this study we present the reconstruction of fully compartmentalised models of the Danio rerio (zebrafish) on a global scale. This reconstruction involves extraction of known biochemical reactions in D. rerio for both primary and secondary metabolism and the implementation of methods for determining subcellular localisation and assignment of enzymes. The reconstructed model (ZebraGEM) is amenable for constraint-based modelling analysis, and accounts for 4,988 genes coding for 2,406 gene-associated reactions and only 418 non-gene-associated reactions. A set of computational validations (i.e., simulations of known metabolic functionalities and experimental data) strongly testifies to the predictive ability of the model. Overall, the reconstructed model is expected to lay down the foundations for computational-based rational design of fish metabolic engineering in aquaculture.

Author: M. Bekaert

Date Published: 14th Nov 2012

Publication Type: Not specified

Malate metabolism in Bacillus subtilis: distinct roles for three classes of malate-oxidizing enzymes

Abstract (Expand)

The Gram-positive soil bacterium Bacillus subtilis uses glucose and malate as the preferred carbon sources. In the presence of either glucose or malate, the expression of genes and operons for the … utilization of secondary carbon sources is subject to carbon catabolite repression. While glucose is a preferred substrate in many organisms from bacteria to man, the factors that contribute to the preference for malate have so far remained elusive. In this work, we have studied the contribution of the different malate-metabolizing enzymes in B. subtilis, and we have elucidated their distinct functions. The malate dehydrogenase and the phosphoenolpyruvate carboxykinase are both essential for malate utilization; they introduce malate into gluconeogenesis. The NADPH-generating malic enzyme YtsJ is important to establish the cellular pools of NADPH for anabolic reactions. Finally, the NADH-generating malic enzymes MaeA, MalS, and MleA are involved in keeping the ATP levels high. Together, this unique array of distinct activities makes malate a preferred carbon source for B. subtilis.

Authors: Frederik M Meyer,

Date Published: 10th Nov 2012

Publication Type: Not specified

Global transcriptome analysis of Atlantic cod (Gadus morhua) liver after in vivo methylmercury exposure suggests effects on energy metabolism pathways

Abstract (Expand)

Methylmercury (MeHg) is a widely distributed contaminant polluting many aquatic environments, with health risks to humans exposed mainly through consumption of seafood. The mechanisms of toxicity of … MeHg are not completely understood. In order to map the range of molecular targets and gain better insights into the mechanisms of toxicity, we prepared Atlantic cod (Gadus morhua) 135k oligonucleotide arrays and performed global analysis of transcriptional changes in the liver of fish treated with MeHg (0.5 and 2 mg/kg of body weight) for 14 days. Inferring from the observed transcriptional changes, the main pathways significantly affected by the treatment were energy metabolism, oxidative stress response, immune response and cytoskeleton remodeling. Consistent with known effects of MeHg, many transcripts for genes in oxidative stress pathways such as glutathione metabolism and Nrf2 regulation of oxidative stress response were differentially regulated. Among the differentially regulated genes, there were disproportionate numbers of genes coding for enzymes involved in metabolism of amino acids, fatty acids and glucose. In particular, many genes coding for enzymes of fatty acid beta-oxidation were up-regulated. The coordinated effects observed on many transcripts coding for enzymes of energy pathways may suggest disruption of nutrient metabolism by MeHg. Many transcripts for genes coding for enzymes in the synthetic pathways of sulphur containing amino acids were also up-regulated, suggesting adaptive responses to MeHg toxicity. By this toxicogenomics approach, we were also able to identify many potential biomarker candidate genes for monitoring environmental MeHg pollution. These results based on changes on transcript levels, however, need to be confirmed by other methods such as proteomics.

Authors: F. Yadetie, O. A. Karlsen, A. Lanzen, K. Berg, P. Olsvik, C. Hogstrand, A. Goksoyr

Date Published: 30th Oct 2012

Publication Type: Not specified

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