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Abstract (Expand)

The mechanism of action of antimicrobial peptides is, to our knowledge, still poorly understood. To probe the biophysical characteristics that confer activity, we present here a molecular-dynamics and biophysical study of a cyclic antimicrobial peptide and its inactive linear analog. In the simulations, the cyclic peptide caused large perturbations in the bilayer and cooperatively opened a disordered toroidal pore, 1-2 nm in diameter. Electrophysiology measurements confirm discrete poration events of comparable size. We also show that lysine residues aligning parallel to each other in the cyclic but not linear peptide are crucial for function. By employing dual-color fluorescence burst analysis, we show that both peptides are able to fuse/aggregate liposomes but only the cyclic peptide is able to porate them. The results provide detailed insight on the molecular basis of activity of cyclic antimicrobial peptides.

Authors: Anna D Cirac, Gemma Moiset, , Armagan Koçer, Pedro Salvador, , Siewert J Marrink, Durba Sengupta

Date Published: 18th May 2011

Publication Type: Not specified

Abstract (Expand)

As a versatile pathogen Staphylococcus aureus can cause various disease patterns, which are influenced by strain specific virulence factor repertoires but also by S. aureus physiological adaptation capacity. Here, we present metabolomic descriptions of S. aureus central metabolic pathways and demonstrate the potential for combined metabolomics- and proteomics-based approaches for the basic research of this important pathogen. This study provides a time-resolved picture of more than 500 proteins and 94 metabolites during the transition from exponential growth to glucose starvation. Under glucose excess, cells exhibited higher levels of proteins involved in glycolysis and protein-synthesis, whereas entry into the stationary phase triggered an increase of enzymes of TCC and gluconeogenesis. These alterations in levels of metabolic enzymes were paralleled by more pronounced changes in the concentrations of associated metabolites, in particular, intermediates of the glycolysis and several amino acids.

Authors: Manuel Liebeke, Kirsten Dörries, Daniela Zühlke, Jörg Bernhardt, Stephan Fuchs, Jan Pané-Farré, Susanne Engelmann, , Rüdiger Bode, Thomas Dandekar, Ulrike Lindequist, ,

Date Published: 1st Apr 2011

Publication Type: Not specified

Abstract (Expand)

The open-reading-frame 3a of SARS coronavirus (SARS-CoV) had been demonstrated previously to form a cation-selective channel that may become expressed in the infected cell and is then involved in virus release. Drugs that inhibit the ion channel formed by the 3a protein can be expected to inhibit virus release, and would be a source for the development of novel therapeutic agents. Here we demonstrate that emodin can inhibit the 3a ion channel of coronavirus SARS-CoV and HCoV-OC43 as well as virus release from HCoV-OC43 with a K1/2 value of about 20 ␮M. We suggest that viral ion channels, in general, may be a good target for the development of antiviral agents.

Authors: Silvia Schwarz, Kai Wang, Wenjing Yu, Bing Sun, Wolfgang Schwarz

Date Published: 1st Apr 2011

Publication Type: Journal

Abstract

Not specified

Authors: Matthew A. Oberhardt, Jacek Puchałka, Vítor A. P. Martins dos Santos, Jason A. Papin

Date Published: 31st Mar 2011

Publication Type: Not specified

Abstract (Expand)

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described "minimal Tat translocase" consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate.

Authors: René van der Ploeg, , Georg Homuth, Marc Schaffer, Emma L Denham, Carmine G Monteferrante, Marcus Miethke, Mohamed A Marahiel, , Theresa Winter, , Haike Antelmann,

Date Published: 30th Mar 2011

Publication Type: Not specified

Abstract (Expand)

Streptomyces coelicolor, the model species of the genus Streptomyces, presents a complex life cycle of successive morphological and biochemical changes involving the formation of substrate and aerial mycelium, sporulation and the production of antibiotics. The switch from primary to secondary metabolism can be triggered by nutrient starvation and is of particular interest as some of the secondary metabolites produced by related Streptomycetes are commercially relevant. To understand these events on a molecular basis, a reliable technical platform encompassing reproducible fermentation as well as generation of coherent transcriptomic data is required. Here, we investigate the technical basis of a previous study as reported by Nieselt et al. (BMC Genomics 11:10, 2010) in more detail, based on the same samples and focusing on the validation of the custom-designed microarray as well as on the reproducibility of the data generated from biological replicates. We show that the protocols developed result in highly coherent transcriptomic measurements. Furthermore, we use the data to predict chromosomal gene clusters, extending previously known clusters as well as predicting interesting new clusters with consistent functional annotations.

Authors: F. Battke, A. Herbig, A. Wentzel, O. M. Jakobsen, M. Bonin, D. A. Hodgson, W. Wohlleben, T. E. Ellingsen, K. Nieselt

Date Published: 25th Mar 2011

Publication Type: Not specified

Abstract (Expand)

Knowledge on absolute protein concentrations is mandatory for the simulation of biological processes in the context of systems biology. A novel approach for the absolute quantification of proteins at a global scale has been developed and its applicability demonstrated using glucose starvation of the Gram-positive model bacterium Bacillus subtilis and the pathogen Staphylococcus aureus as proof-of-principle examples. Absolute intracellular protein concentrations were initially determined for a preselected set of anchor proteins by employing a targeted mass spectrometric method and isotopically labeled internal standard peptides. Known concentrations of these anchor proteins were then used to calibrate two-dimensional (2-D) gels allowing the calculation of absolute abundance of all detectable proteins on the 2-D gels. Using this approach, concentrations of the majority of metabolic enzymes were determined, and thus a quantification of the players of metabolism was achieved. This new strategy is fast, cost-effective, applicable to any cell type, and thus of value for a broad community of laboratories with experience in 2-D gel-based proteomics and interest in quantitative approaches. Particularly, this approach could also be utilized to quantify existing data sets with the aid of a few standard anchor proteins.

Authors: , Susanne Sievers, Daniela Zühlke, Judith Kuzinski, , Jan Muntel, Bernd Hessling, Jörg Bernhardt, Rabea Sietmann, , , Dörte Becher

Date Published: 11th Mar 2011

Publication Type: Not specified

Abstract (Expand)

The cold stress response of Pseudomonas putida KT2440 was investigated by genomewide deep cDNA sequencing and gel-free MS-based protein profiling. Transcriptome and proteome profiles were assessed at 30 degrees C and 2 h after a downshift from 30 to 10 degrees C. Pseudomonas putida adapted to lower ambient temperature by the activation of ribosome-associated functional modules that facilitate translational efficiency. The outer membrane profile was reorganized, anabolic pathways and core as well as energy metabolism were repressed and the alginate regulon and sugar catabolism were activated. At the investigated early time point of cold adaptation, the transcriptome was reprogrammed in almost all functional categories, but the protein profile had still not adapted to the change of living conditions in the cold.

Authors: , F. Schmidt, , C. F. Davenport, M. Gesell Salazar, U. Volker,

Date Published: 1st Mar 2011

Publication Type: Not specified

Abstract (Expand)

We have constructed derivatives of Streptomyces coelicolor M145 as hosts for the heterologous expression of secondary metabolite gene clusters. To remove potentially competitive sinks of carbon and nitrogen, and to provide a host devoid of antibiotic activity, we deleted four endogenous secondary metabolite gene clusters from S. coelicolor M145--those for actinorhodin, prodiginine, CPK and CDA biosynthesis. We then introduced point mutations into rpoB and rpsL to pleiotropically increase the level of secondary metabolite production. Introduction of the native actinorhodin gene cluster and of gene clusters for the heterologous production of chloramphenicol and congocidine revealed dramatic increases in antibiotic production compared with the parental strain. In addition to lacking antibacterial activity, the engineered strains possess relatively simple extracellular metabolite profiles. When combined with liquid chromatography and mass spectrometry, we believe that these genetically engineered strains will markedly facilitate the discovery of new compounds by heterologous expression of cloned gene clusters, particularly the numerous cryptic secondary metabolic gene clusters that are prevalent within actinomycete genome sequences.

Authors: Juan Pablo Gomez-Escribano, Mervyn J. Bibb

Date Published: 1st Mar 2011

Publication Type: Not specified

Abstract (Expand)

Pseudomonas putida KT2440 is a completely sequenced biosafety strain that has retained its capability to survive and function in the environment. The global mRNA expression profiles of the KT2440 strain grown at 10 degrees C and 30 degrees C were determined by deep cDNA sequencing to refine the genome annotation. Transcriptome sequencing identified 36 yet unknown small non-coding RNAs, 143 novel ORFs in 106 intergenic regions, 42 unclassified genes and eight highly expressed leaderless mRNA transcripts. The genome coordinates of eight genes and the organization of 57 operons were corrected. No overrepresented sequence motifs were detected in the 5'-untranslated regions. The 50 most highly expressed genes made up 60% of the total mRNA pool. Comparison of cDNA sequencing, Affymetrix and Progenika microarray data from the same mRNA preparation revealed a higher sensitivity and specificity of cDNA sequencing, a relatively poor correlation between the normalized cDNA reads and microarray signal intensities, and a systematic signal-dependent bias of microarrays in the detection of differentially regulated genes. The study demonstrates the power of next-generation cDNA sequencing for the quantitation of mRNA transcripts and the refinement of bacterial genome annotation.

Authors: , , P. Hagendorf, R. Geffers, U. Schock, T. Pohl, C. F. Davenport,

Date Published: 28th Feb 2011

Publication Type: Not specified

Abstract (Expand)

A constructed lactate dehydrogenase-negative mutant of Enterococcus faecalis V583 grows at the same rate as the wild type, but ferments glucose to ethanol, formate, and acetoin. Microrray analysis showed that LDH deficiency had profound transcriptional effects, 43 genes in the mutant were found to be upregulated and 45 to be downregulated. Most of the upregulated genes encode enzymes of energy metabolism or transport. By 2D gel analysis 45 differentially expressed proteins were identified. A comparison of transcriptomic and proteomic data suggests that for several proteins the level of expression is regulated beyond the level of transcription. Pyruvate catabolic genes, including the truncated ldh, showed highly increased transcription in the mutant. These genes, along with a number of other differentially expressed genes, are preceded by sequences with homology to binding sites for the global redox-sensing repressor, Rex, of Staphylococcus aureus. The data indicate that the genes are transcriptionally regulated by the NADH/NAD ratio and that this ratio plays an important role in the regulatory network controlling energy metabolism in E. faecalis.

Authors: , , Ellen M Fergestad, Geir Mathiesen, ,

Date Published: 8th Feb 2011

Publication Type: Not specified

Abstract (Expand)

To maintain optimal intracellular concentrations of alkali-metal-cations, yeast cells use a series of influx and efflux systems. Nonconventional yeast species have at least three different types of efficient transporters that ensure potassium uptake and accumulation in cells. Most of them have Trk uniporters and Hak K(+) -H(+) symporters and a few yeast species also have the rare K(+) (Na(+) )-uptake ATPase Acu. To eliminate surplus potassium or toxic sodium cations, various yeast species use highly conserved Nha Na(+) (K(+) )/H(+) antiporters and Na(+) (K(+) )-efflux Ena ATPases. The potassium-specific yeast Tok1 channel is also highly conserved among various yeast species and its activity is important for the regulation of plasma membrane potential.

Editor:

Date Published: 1st Feb 2011

Publication Type: Not specified

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, ,

Date Published: 27th Jan 2011

Publication Type: Not specified

Abstract (Expand)

Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Whilst much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here for the first time the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundances of cytochrome bd and bo and outer membrane protein W. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself, or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFinfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis, but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. Measurement of the amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.

Authors: , , , Eleanor W Trotter, H M Shahzad Asif, Guido Sanguinetti, , ,

Date Published: 22nd Jan 2011

Publication Type: Not specified

Abstract (Expand)

Bacteria have developed an impressive ability to survive and propagate in highly diverse and changing environments by evolving phenotypic heterogeneity. Phenotypic heterogeneity ensures that a subpopulation is well prepared for environmental changes. The expression bet hedging is commonly (but often incorrectly) used by molecular biologists to describe any observed phenotypic heterogeneity. In evolutionary biology, however, bet hedging denotes a risk-spreading strategy displayed by isogenic populations that evolved in unpredictably changing environments. Opposed to other survival strategies, bet hedging evolves because the selection environment changes and favours different phenotypes at different times. Consequently, in bet hedging populations all phenotypes perform differently well at any time, depending on the selection pressures present. Moreover, bet hedging is the only strategy in which temporal variance of offspring numbers per individual is minimized. Our paper aims to provide a guide for the correct use of the term bet hedging in molecular biology.

Authors: , Patsy Haccou,

Date Published: 21st Jan 2011

Publication Type: Not specified

Abstract (Expand)

Hepatitis C virus (HCV) is a major causative agent of chronic liver disease in humans. To gain insight into host factor requirements for HCV replication, we performed a siRNA screen of the human kinome and identified 13 different kinases, including phosphatidylinositol-4 kinase III alpha (PI4KIIIalpha), as being required for HCV replication. Consistent with elevated levels of the PI4KIIIalpha product phosphatidylinositol-4-phosphate (PI4P) detected in HCV-infected cultured hepatocytes and liver tissue from chronic hepatitis C patients, the enzymatic activity of PI4KIIIalpha was critical for HCV replication. Viral nonstructural protein 5A (NS5A) was found to interact with PI4KIIIalpha and stimulate its kinase activity. The absence of PI4KIIIalpha activity induced a dramatic change in the ultrastructural morphology of the membranous HCV replication complex. Our analysis suggests that the direct activation of a lipid kinase by HCV NS5A contributes critically to the integrity of the membranous viral replication complex.

Authors: S. Reiss, I. Rebhan, P. Backes, I. Romero-Brey, H. Erfle, P. Matula, L. Kaderali, M. Poenisch, H. Blankenburg, M. S. Hiet, T. Longerich, S. Diehl, F. Ramirez, T. Balla, K. Rohr, A. Kaul, S. Buhler, R. Pepperkok, T. Lengauer, M. Albrecht, R. Eils, P. Schirmacher, V. Lohmann, R. Bartenschlager

Date Published: 18th Jan 2011

Publication Type: Not specified

Abstract (Expand)

Bacillus subtilis possesses interlinked routes for the synthesis of proline. The ProJ-ProA-ProH route is responsible for the production of proline as an osmoprotectant, and the ProB-ProA-ProI route provides proline for protein synthesis. We show here that the transcription of the anabolic proBA and proI genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the proBA and proI genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB-treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the proBA T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of proBA transcription to a control that was responsive to starvation for phenylalanine.

Authors: Jeanette Brill, , Harald Putzer,

Date Published: 13th Jan 2011

Publication Type: Not specified

Abstract (Expand)

Several computational methods exist to suggest rational genetic interventions that improve the productivity of industrial strains. Nonetheless, these methods are less effective to predict possible genetic responses of the strain after the intervention. This problem requires a better understanding of potential alternative metabolic and regulatory pathways able to counteract the targeted intervention.

Authors: , Katrin Gunka, Rafael Polanía, Stefan Tholen,

Date Published: 11th Jan 2011

Publication Type: Not specified

Abstract (Expand)

Clostridium acetobutylicum is able to switch from acidogenic growth to solventogenic growth. We used phosphate-limited continuous cultures that established acidogenic growth at pH 5.8 and solventogenic growth at pH 4.5. These cultures allowed a detailed transcriptomic study of the switch from acidogenesis to solventogenesis that is not superimposed by sporulation and other growth phase-dependent parameters. These experiments led to new insights into the physiological role of several genes involved in solvent formation. The adc gene for acetone decarboxylase is upregulated well before the rest of the sol locus during the switch, and pyruvate decarboxylase is induced exclusively for the period of this switch. The aldehyde-alcohol dehydrogenase gene adhE1 located in the sol operon is regulated antagonistically to the paralog adhE2 that is expressed during acidogenic conditions. A similar antagonistic pattern can be seen with the two paralogs of thiolase genes, thlA and thlB. Interestingly, the genes coding for the putative cellulosome in C. acetobutylicum are exclusively transcribed throughout solventogenic growth. The genes for stress response are only induced during the shift but not in the course of solventogenesis when butanol is present in the culture. Finally, the data clearly indicate that solventogenesis is independent from sporulation.

Authors: Christina Grimmler, , , , , , Wolfgang Liebl,

Date Published: 6th Jan 2011

Publication Type: Not specified

Abstract (Expand)

Fluorescence microscopy is an imaging technique that provides insights into signal transduction pathways through the generation of quantitative data, such as the spatiotemporal distribution of GFP-tagged proteins in signaling pathways. The data acquired are, however, usually a composition of both the GFP-tagged proteins of interest and of an autofluorescent background, which both undergo photobleaching during imaging. We here present a mathematical model based on ordinary differential equations that successfully describes the shuttling of intracellular Mig1-GFP under changing environmental conditions regarding glucose concentration. Our analysis separates the different bleaching rates of Mig1-GFP and background, and the background-to-Mig1-GFP ratio. By applying our model to experimental data, we can thus extract the Mig1-GFP signal from the overall acquired signal and investigate the influence of kinase and phosphatase on Mig1. We found a stronger regulation of Mig1 through its kinase than through its phosphatase when controlled by the glucose concentration, with a constant (de)phosphorylation rate independent of the glucose concentration. By replacing the term for decreasing excited Mig1-GFP concentration with a constant, we were able to reconstruct the dynamics of Mig1-GFP, as it would occur without bleaching and background noise. Our model effectively demonstrates how data, acquired with an optical microscope, can be processed and used for a systems biology analysis of signal transduction pathways.

Authors: Simone Frey, Kristin Sott, Maria Smedh, , Peter Dahl, , Mattias Goksör

Date Published: 2011

Publication Type: Not specified

Abstract (Expand)

Background: Clostridium acetobutylicum is an anaerobic bacterium which is known for its solvent-producing capabilities, namely regarding the bulk chemicals acetone and butanol, the latter being a highly efficient biofuel. For butanol production by C. acetobutylicum to be optimized and exploited on an industrial scale, the effect of pH-induced gene regulation on solvent production by C. acetobutylicum in continuous culture must be understood as fully as possible. Results: We present an ordinary differential equation model combining the metabolic network governing solvent production with regulation at the genetic level of the enzymes required for this process. Parameterizing the model with experimental data from continuous culture, we demonstrate the influence of pH upon fermentation products: at high pH (pH 5.7) acids are the dominant product while at low pH (pH 4.5) this switches to solvents. Through steady-state analyses of the model we focus our investigations on how alteration in gene expression of C. acetobutylicum could be exploited to increase butanol yield in a continuous culture fermentation. Conclusions: Incorporating gene regulation into the mathematical model of solvent production by C. acetobutylicum enables an accurate representation of the pH-induced switch to solvent production to be obtained and theoretical investigations of possible synthetic-biology approaches to be pursued. Steady-state analyses suggest that, to increase butanol yield, alterations in the expression of single solvent-associated genes are insufficient; a more complex approach targeting two or more genes is required.

Editor:

Date Published: 2011

Publication Type: Not specified

Abstract

Not specified

Authors: Marco Pittelkow,

Date Published: 2011

Publication Type: Not specified

Abstract (Expand)

Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations.

Authors: J. Bachmann, A. Raue, M. Schilling, M. E. Bohm, C. Kreutz, D. Kaschek, H. Busch, N. Gretz, W. D. Lehmann, J. Timmer, U. Klingmuller

Date Published: 2011

Publication Type: Not specified

Abstract (Expand)

The topology of nuclear receptor (NR) signaling is captured in a systems biological graphical notation. This enables us to identify a number of ‘design’ aspects of the topology of these networks that might appear unnecessarily complex or even functionally paradoxical. In realistic kinetic models of increasing complexity, calculations show how these features correspond to potentially important design principles, e.g.: (i) cytosolic ‘nuclear’ receptor may shuttle signal molecules to the nucleus, (ii) the active export of NRs may ensure that there is sufficient receptor protein to capture ligand at the cytoplasmic membrane, (iii) a three conveyor belts design dissipating GTP-free energy, greatly aids response, (iv) the active export of importins may prevent sequestration of NRs by importins in the nucleus and (v) the unspecific nature of the nuclear pore may ensure signal-flux robustness. In addition, the models developed are suitable for implementation in specific cases of NR-mediated signaling, to predict individual receptor functions and differential sensitivity toward physiological and pharmacological ligands.

Authors: Alexey N Kolodkin, Frank J Bruggeman, Nick Plant, Martijn J Moné, Barbara M Bakker, Moray J Campbell, Johannes P T M van Leeuwen, Carsten Carlberg, Jacky L Snoep, Hans V Westerhoff

Date Published: 21st Dec 2010

Publication Type: Not specified

Abstract (Expand)

The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be related to the agr-dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.

Authors: Eleni Tsompanidou, Mark J J B Sibbald, Monika A Chlebowicz, Annette Dreisbach, Jaap Willem Back, , Girbe Buist, Emma L Denham

Date Published: 17th Dec 2010

Publication Type: Not specified

Abstract (Expand)

Streptococcus pyogenes (group A Streptococcus [GAS]) is a major human pathogen, causing diseases ranging from mild superficial infections of the skin and pharyngeal mucosal membrane, up to severe systemic and invasive diseases and autoimmune sequelae. The capability of GAS to cause this wide variety of infections is due to the expression of a large set of virulence factors, their concerted transcriptional regulation, and bacterial adaptation mechanisms to various host niches, which we are now beginning to understand on a molecular level. The addition of -omics technologies for GAS pathogenesis investigation, on top of traditional molecular methods, led to fast progress in understanding GAS pathogenesis mechanisms. This article focuses on differential transcriptional analysis performed on the bacterial side as well as on the host cell side. The microarray studies discussed provide new insight into the following five topics: gene-expression patterns under infection-relevant conditions, gene-expression patterns in mutant strains compared with wild-type strains, emergence of exceptionally fit GAS clones, gene-expression patterns of eukaryotic target and immune cells in response to GAS infection, and mechanisms underlying shifts from a pharyngeal to invasive GAS lifestyle.

Authors: , Venelina Sugareva, Nadja Patenge,

Date Published: 8th Dec 2010

Publication Type: Not specified

Abstract (Expand)

In Gram-positive bacteria, carbon catabolite protein A (CcpA) is the master regulator of carbon catabolite control, which ensures optimal energy usage under diverse conditions. Unlike other LacI-GalR proteins, CcpA is activated for DNA binding by first forming a complex with the phosphoprotein HPr-Ser46-P. Bacillus subtilis CcpA functions as both a transcription repressor and activator and binds to more than 50 operators called catabolite response elements (cres). These sites are highly degenerate with the consensus, WTGNNARCGNWWWCAW. How CcpA-(HPr-Ser46-P) binds such diverse sequences is unclear. To gain insight into this question, we solved the structures of the CcpA-(HPr-Ser46-P) complex bound to three different operators, the synthetic (syn) cre, ackA2 cre and gntR-down cre. Strikingly, the structures show that the CcpA-bound operators display different bend angles, ranging from 31° to 56°. These differences are accommodated by a flexible linkage between the CcpA helix-turn-helix-loop-helix motif and hinge helices, which allows independent docking of these DNA-binding modules. This flexibility coupled with an abundance of non-polar residues capable of non-specific nucleobase interactions permits CcpA-(HPr-Ser46-P) to bind diverse operators. Indeed, biochemical data show that CcpA-(HPr-Ser46-P) binds the three cre sites with similar affinities. Thus, the data reveal properties that license this protein to function as a global transcription regulator.

Authors: Maria A Schumacher, Mareen Sprehe, , , Richard G Brennan

Date Published: 26th Nov 2010

Publication Type: Not specified

Abstract (Expand)

Several lactic acid bacteria use homolactic fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium in microaerophilic conditions. However, growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed in glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and dependent on the pH for E. faecalis. In S. pyogenes and L. lactis a change in pH resulted in a clear change in Yatp. The pH that showed the highest Yatp corresponded to the pH of the natural habitat of the organisms.

Authors: , , , , Anja Pritzschke, Nikolai Siemens, , ,

Date Published: 25th Nov 2010

Publication Type: Not specified

Abstract (Expand)

Systems biology is a comprehensive quantitative analysis how the components of a biological system interact over time which requires an interdisciplinary team of investigators. System-theoretic methods are applied to investigate the system's behavior. Using known information about the considered system, a conceptual model is defined. It is transferred in a mathematical model that can be simulated (analytically or numerically) and analyzed using system-theoretic tools. Finally, simulation results are compared with experimental data. However, assumptions, approximations, and requirements to available experimental data are crucial ingredients of this systems biology workflow. Consequently, the modeling of cellular processes creates special demands on the design of experiments: the quality, the amount, and the completeness of data. The relation between models and data is discussed in this chapter. Thereby, we focus on the requirements on experimental data from the perspective of systems biology projects.

Editor:

Date Published: 11th Nov 2010

Publication Type: Not specified

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: , Pierre Nicolas, Hugues Richard, Philippe Bessières, Stéphane Aymerich

Date Published: 10th Nov 2010

Publication Type: Not specified

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