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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

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

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

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

Glucose Transport in Escherichia coli Mutant Strains with Defects in Sugar Transport Systems

Abstract (Expand)

In Escherichia coli several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase … system (glucose-PTS), but also the mannose-PTS, as well as the galactose and maltose transporters can translocate glucose. Mutant strains which lack the EIIBC protein of the glucose-PTS have been previously investigated because their lower rate of acetate formation offers advantages in industrial applications. Nevertheless, a systematic study to analyze the impact of the different glucose uptake systems has not been undertaken. Specifically, how the bacteria cope with the deletion of the major glucose uptake system and which alternative transporters react to compensate for this deficit has not been studied in detail. Therefore, a series of mutant strains were analyzed in aerobic and anaerobic batch cultures, as well as in glucose limited continuous cultivations. Deletion of EIIBC, disturbs glucose transport severely. cAMP-CRP levels rise, induction of the mgl-operon occurs. Nevertheless mgl transcription is not essential, as deletion of this transporter did not affect growth rate; the activities of the remaining transporters seems to be sufficient by induction of the galactose and maltose transporters. Despite the strong up-regulation of mgl under glucose limitations, deletion of this transport-system did not lead to further changes.

Editor:

Date Published: 8th Oct 2012

Publication Type: Not specified

Single cell analysis of gene expression patterns during carbon starvation in Bacillus subtilis reveals large phenotypic variation

Abstract (Expand)

How cells dynamically respond to fluctuating environmental conditions depends on the architecture and noise of the underlying genetic circuits. Most work characterizing stress pathways in the model … bacterium Bacillus subtilis has been performed on bulk cultures using ensemble assays. However, investigating the single cell response to stress is important since noise might generate significant phenotypic heterogeneity. Here, we study the stress response to carbon source starvation and compare both population and single cell data. Using a top-down approach, we investigate the transcriptional dynamics of various stress-related genes of B. subtilis in response to carbon source starvation and to increased cell density. Our data reveal that most of the tested gene-regulatory networks respond highly heterogeneously to starvation and cells show a large degree of variation in gene expression. The level of highly dynamic diversification within B. subtilis populations under changing environments reflects the necessity to study cells at the single cell level.

Editor:

Date Published: 4th Oct 2012

Publication Type: Not specified

‘Domino’ systems biology and the ‘A’ of ATP

Abstract (Expand)

We develop a strategic ‘domino’ approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to … explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in ATP upon glucose addition, (ii) the lack of increase in ADP when ATP is hydrolyzed, and (iii) the rapid disappearance of the ‘A’ (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of AMP explains. Cycling of the ‘A’ moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the ‘A’ component of ATP.

Editor:

Date Published: 1st Sep 2012

Publication Type: Not specified

On the function of the various quinone species in Escherichia coli

Abstract (Expand)

The respiratory chain of Escherichia coli contains three quinones. Menaquinone and demethylmenaquinone have low midpoint potentials and are involved in anaerobic respiration, while ubiquinone, which … has a high midpoint potential, is involved in aerobic and nitrate respiration. Here, we report that demethylmenaquinone plays a role not only in trimethylaminooxide-, dimethylsulfoxide- and fumarate-dependent respiration, but also in aerobic respiration. Furthermore, we demonstrate that demethylmenaquinone serves as an electron acceptor for oxidation of succinate to fumarate, and that all three quinol oxidases of E. coli accept electrons from this naphtoquinone derivative.

Authors: , , Klaas J. Hellingwerf,

Date Published: 1st Sep 2012

Publication Type: Not specified

High- and low-affinity cre boxes for CcpA binding in Bacillus subtilis revealed by genome-wide analysis

Abstract (Expand)

In Bacillus subtilis and its relatives carbon catabolite control, a mechanism enabling to reach maximal efficiency of carbon and energy sources metabolism, is achieved by the global regulator CcpA … (carbon catabolite protein A). CcpA in a complex with HPr-Ser-P (seryl-phosphorylated form of histidine-containing protein, HPr) binds to operator sites called catabolite responsive elements, cre. Depending on the cre box position relative to the promoter, the CcpA/HPr-Ser-P complex can either act as a positive or a negative regulator. The cre boxes are highly degenerate semi-palindromes with a lowly conserved consensus sequence. So far, studies aimed at revealing how CcpA can bind such diverse sites were focused on the analysis of single cre boxes. In this study, a genome-wide analysis of cre sites was performed in order to identify differences in cre sequence and position, which determine their binding affinity.

Authors: , Monika Pabijaniak, Anne de Jong, Robert Dűhring, , ,

Date Published: 17th Aug 2012

Publication Type: Not specified

Systems analysis of transcription factor activities in environments with stable and dynamic oxygen concentrations

Abstract (Expand)

Understanding gene regulation requires knowledge of changes in transcription factor (TF) activities. Simultaneous direct measurement of numerous TF activities is currently impossible. Nevertheless, … statistical approaches to infer TF activities have yielded non-trivial and verifiable predictions for individual TFs. Here, global statistical modelling identifies changes in TF activities from transcript profiles of Escherichia coli growing in stable (fixed oxygen availabilities) and dynamic (changing oxygen availability) environments. A core oxygen-responsive TF network, supplemented by additional TFs acting under specific conditions, was identified. The activities of the cytoplasmic oxygen-responsive TF, FNR, and the membrane-bound terminal oxidases implied that, even on the scale of the bacterial cell, spatial effects significantly influence oxygen-sensing. Several transcripts exhibited asymmetrical patterns of abundance in aerobic to anaerobic and anaerobic to aerobic transitions. One of these transcripts, ndh, encodes a major component of the aerobic respiratory chain and is regulated by oxygen-responsive TFs ArcA and FNR. Kinetic modelling indicated that ArcA and FNR behaviour could not explain the ndh transcript profile, leading to the identification of another TF, PdhR, as the source of the asymmetry. Thus, this approach illustrates how systematic examination of regulatory responses in stable and dynamic environments yields new mechanistic insights into adaptive processes.

Authors: , Andrea Ocone, Melanie R Stapleton, Simon Hall, Eleanor W Trotter, , ,

Date Published: 8th Aug 2012

Publication Type: Not specified

Uncoupling of substrate-level phosphorylation in Escherichia coli during glucose-limited growth

Abstract (Expand)

The respiratory chain of Escherichia coli contains three different cytochrome oxidases. Whereas the cytochrome bo oxidase and the cytochrome bd-I oxidase are well characterized and have been shown to … contribute to proton translocation, physiological data suggested a nonelectrogenic functioning of the cytochrome bd-II oxidase. Recently, however, this view was challenged by an in vitro biochemical analysis that showed that the activity of cytochrome bd-II oxidase does contribute to proton translocation with an H(+)/e(-) stoichiometry of 1. Here, we propose that this apparent discrepancy is due to the activities of two alternative catabolic pathways: the pyruvate oxidase pathway for acetate production and a pathway with methylglyoxal as an intermediate for the production of lactate. The ATP yields of these pathways are lower than those of the pathways that have so far always been assumed to catalyze the main catabolic flux under energy-limited growth conditions (i.e., pyruvate dehydrogenase and lactate dehydrogenase). Inclusion of these alternative pathways in the flux analysis of growing E. coli strains for the calculation of the catabolic ATP synthesis rate indicates an electrogenic function of the cytochrome bd-II oxidase, compatible with an H(+)/e(-) ratio of 1. This analysis shows for the first time the extent of bypassing of substrate-level phosphorylation in E. coli under energy-limited growth conditions.

Authors: , Klaas J Hellingwerf, Maarten J Teixeira de Mattos,

Date Published: 27th Jul 2012

Publication Type: Not specified

Multiple active centers of multi-subunit RNA polymerases

Abstract (Expand)

The active center of multi-subunit RNA polymerase consists of two modules, the Mg(2+) module, holding the catalytic Mg(2+) ion, and a module made of a flexible domain, the Trigger Loop. Uniquely, the … TL module can be substituted by alternative modules, thus changing the catalytic properties of the active center.

Authors: , Mohammad Roghanian,

Date Published: 10th Jul 2012

Publication Type: Not specified

From steady-state to synchronized yeast glycolytic oscillations I: model construction

Abstract (Expand)

An existing detailed kinetic model for the steady-state behavior of yeast glycolysis was tested for its ability to simulate dynamic behavior. Using a small subset of experimental data, the original … model was adapted by adjusting its parameter values in three optimization steps. Only small adaptations to the original model were required for realistic simulation of experimental data for limit-cycle oscillations. The greatest changes were required for parameter values for the phosphofructokinase reaction. The importance of ATP for the oscillatory mechanism and NAD(H) for inter-and intra-cellular communications and synchronization was evident in the optimization steps and simulation experiments. In an accompanying paper [du Preez F et al. (2012) FEBS J doi:10.1111/j.1742-4658.2012.08658.x], we validate the model for a wide variety of experiments on oscillatory yeast cells. The results are important for re-use of detailed kinetic models in modular modeling approaches and for approaches such as that used in the Silicon Cell initiative. Database The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/dupreez/index.html.

Authors: , David D van Niekerk, Bob Kooi, Johann M Rohwer,

Date Published: 21st Jun 2012

Publication Type: Not specified

From steady-state to synchronized yeast glycolytic oscillations II: model validation

Abstract (Expand)

In an accompanying paper [du Preez et al., (2012) FEBS J doi: 10.1111/j.1742-4658.2012.08665.x], we adapt an existing kinetic model for steady-state yeast glycolysis to simulate limit-cycle oscillations. … Here we validate the model by testing its capacity to simulate a wide range of experiments on dynamics of yeast glycolysis. In addition to its description of the oscillations of glycolytic intermediates in intact cells and the rapid synchronization observed when mixing out-of-phase oscillatory cell populations (see accompanying paper), the model was able to predict the Hopf bifurcation diagram with glucose as the bifurcation parameter (and one of the bifurcation points with cyanide as the bifurcation parameter), the glucose- and acetaldehyde-driven forced oscillations, glucose and acetaldehyde quenching, and cell-free extract oscillations (including complex oscillations and mixed-mode oscillations). Thus, the model was compliant, at least qualitatively, with the majority of available experimental data for glycolytic oscillations in yeast. To our knowledge, this is the first time that a model for yeast glycolysis has been tested against such a wide variety of independent data sets. Database The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/dupreez/index.html.

Authors: , David D van Niekerk,

Date Published: 13th Jun 2012

Publication Type: Not specified

Optimized submerged batch fermentation strategy for systems scale studies of metabolic switching in Streptomyces coelicolor A3(2)

Abstract (Expand)

BACKGROUND: Systems biology approaches to study metabolic switching in Streptomyces coelicolor A3(2) depend on cultivation conditions ensuring high reproducibility and distinct phases of culture growth … and secondary metabolite production. In addition, biomass concentrations must be sufficiently high to allow for extensive time-series sampling before occurrence of a given nutrient depletion for transition triggering. The present study describes for the first time the development of a dedicated optimized submerged batch fermentation strategy as the basis for highly time-resolved systems biology studies of metabolic switching in S. coelicolor A3(2). RESULTS: By a step-wise approach, cultivation conditions and two fully defined cultivation media were developed and evaluated using strain M145 of S. coelicolor A3(2), providing a high degree of cultivation reproducibility and enabling reliable studies of the effect of phosphate depletion and L-glutamate depletion on the metabolic transition to antibiotic production phase. Interestingly, both of the two carbon sources provided, D-glucose and L-glutamate, were found to be necessary in order to maintain high growth rates and prevent secondary metabolite production before nutrient depletion. Comparative analysis of batch cultivations with (i) both L-glutamate and D-glucose in excess, (ii) L-glutamate depletion and D-glucose in excess, (iii) L-glutamate as the sole source of carbon and (iv) D-glucose as the sole source of carbon, reveal a complex interplay of the two carbon sources in the bacterium's central carbon metabolism. CONCLUSIONS: The present study presents for the first time a dedicated cultivation strategy fulfilling the requirements for systems biology studies of metabolic switching in S. coelicolor A3(2). Key results from labelling and cultivation experiments on either or both of the two carbon sources provided indicate that in the presence of D-glucose, L-glutamate was the preferred carbon source, while D-glucose alone appeared incapable of maintaining culture growth, likely due to a metabolic bottleneck at the oxidation of pyruvate to acetyl-CoA.

Authors: A. Wentzel, P. Bruheim, A. Overby, O. M. Jakobsen, H. Sletta, W. A. Omara, D. A. Hodgson, T. E. Ellingsen

Date Published: 9th Jun 2012

Publication Type: Not specified

Synthesis, release, and recapture of compatible solute proline by osmotically stressed Bacillus subtilis cells

Abstract (Expand)

Bacillus subtilis synthesizes large amounts of the compatible solute proline as a cellular defense against high osmolarity to ensure a physiologically appropriate level of hydration of the cytoplasm … and turgor. It also imports proline for this purpose via the osmotically inducible OpuE transport system. Unexpectedly, an opuE mutant was at a strong growth disadvantage in high-salinity minimal media lacking proline. Appreciable amounts of proline were detected in the culture supernatant of the opuE mutant strain, and they rose concomitantly with increases in the external salinity. We found that the intracellular proline pool of severely salinity-stressed cells of the opuE mutant was considerably lower than that of its opuE(+) parent strain. This loss of proline into the medium and the resulting decrease in the intracellular proline content provide a rational explanation for the observed salt-sensitive growth phenotype of cells lacking OpuE. None of the known MscL- and MscS-type mechanosensitive channels of B. subtilis participated in the release of proline under permanently imposed high-salinity growth conditions. The data reported here show that the OpuE transporter not only possesses the previously reported role for the scavenging of exogenously provided proline as an osmoprotectant but also functions as a physiologically highly important recapturing device for proline that is synthesized de novo and subsequently released by salt-stressed B. subtilis cells. The wider implications of our findings for the retention of compatible solutes by osmotically challenged microorganisms and the roles of uptake systems for compatible solutes are considered.

Authors: , Carsten von Blohn, Agnieszka Stanek, Susanne Moses, Helena Barzantny,

Date Published: 8th Jun 2012

Publication Type: Not specified

My data are your data

Abstract (Expand)

Encouraging more broad and inclusive data sharing in today's world will involve concerted community efforts to overcome technical barriers and human foibles. Vivien Marx investigates. (includes …s comments from Carole Goble, and mentions SysMO, SEEK and RightField).

Author: Vivien Marx

Date Published: 7th Jun 2012

Publication Type: Not specified

Sustained glycolytic oscillations in individual isolated yeast cells

Abstract (Expand)

Yeast glycolytic oscillations have been studied since the 1950s in cell-free extracts and intact cells. For intact cells, sustained oscillations have so far only been observed at the population level, … i.e. for synchronized cultures at high biomass concentrations. Using optical tweezers to position yeast cells in a microfluidic chamber, we were able to observe sustained oscillations in individual isolated cells. Using a detailed kinetic model for the cellular reactions, we simulated the heterogeneity in the response of the individual cells, assuming small differences in a single internal parameter. This is the first time that sustained limit-cycle oscillations have been demonstrated in isolated yeast cells. Database The mathematical model described here has been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/database/gustavsson/index.html free of charge.

Authors: Anna-Karin Gustavsson, David D van Niekerk, Caroline B Adiels, , Mattias Goksör,

Date Published: 23rd May 2012

Publication Type: Not specified

RNA degradation in Bacillus subtilis: an interplay of essential endo- and exoribonucleases

Abstract (Expand)

RNA processing and degradation are key processes in the control of transcript accumulation and thus in the control of gene expression. In Escherichia coli, the underlying mechanisms and components of … RNA decay are well characterized. By contrast, Gram-positive bacteria do not possess several important players of E. coli RNA degradation, most notably the essential enzyme RNase E. Recent research on the model Gram-positive organism, Bacillus subtilis, has identified the essential RNases J1 and Y as crucial enzymes in RNA degradation. While RNase J1 is the first bacterial exoribonuclease with 5'-to-3' processivity, RNase Y is the founding member of a novel class of endoribonucleases. Both RNase J1 and RNase Y have a broad impact on the stability of B. subtilis mRNAs; a depletion of either enzyme affects more than 25% of all mRNAs. RNases J1 and Y as well as RNase J2, the polynucleotide phosphorylase PNPase, the RNA helicase CshA and the glycolytic enzymes enolase and phosphofructokinase have been proposed to form a complex, the RNA degradosome of B. subtilis. This review presents a model, based on recent published data, of RNA degradation in B. subtilis. Degradation is initiated by RNase Y-dependent endonucleolytic cleavage, followed by processive exoribonucleolysis of the generated fragments both in 3'-to-5' and in 5'-to-3' directions. The implications of these findings for pathogenic Gram-positive bacteria are also discussed.

Authors: Martin Lehnik-Habrink, , ,

Date Published: 8th May 2012

Publication Type: Not specified

Proliferating bloodstream-form Trypanosoma brucei use a negligible part of consumed glucose for anabolic processes

Abstract (Expand)

Our quantitative knowledge of carbon fluxes in the long slender bloodstream form (BSF) Trypanosoma brucei is mainly based on non-proliferating parasites, isolated from laboratory animals and kept in … buffers. In this paper we present a carbon balance for exponentially growing bloodstream form trypanosomes. The cells grew with a doubling time of 5.3h, contained 46 mu mol of carbon (10(8) cells)(-1) and had a glucose consumption flux of 160 nmol min(-1) (10(8) cells)(-1). The molar ratio of pyruvate excreted versus glucose consumed was 2.1. Furthermore, analysis of the (13)C label distribution in pyruvate in (13)C-glucose incubations of exponentially growing trypanosomes showed that glucose was the sole substrate for pyruvate production. We conclude that the glucose metabolised in glycolysis was hardly, if at all, used for biosynthetic processes. Carbon flux through glycolysis in exponentially growing trypanosomes was 10 times higher than the incorporation of carbon into biomass. This biosynthetic carbon is derived from other precursors present in the nutrient rich growth medium. Furthermore, we found that the glycolytic flux was unaltered when the culture went into stationary phase, suggesting that most of the ATP produced in glycolysis is used for processes other than growth.

Authors: , A. van Tuijl, J. van Dam, W. van Winden, A. G. Tielens, J. J. van Hellemond,

Date Published: 8th May 2012

Publication Type: Not specified

Hypothesis: emergence of translation as a result of RNA helicase evolution

Abstract (Expand)

The origin of translation and the genetic code is one of the major mysteries of evolution. The advantage of templated protein synthesis could have been achieved only when the translation apparatus had … already become very complex. This means that the translation machinery, as we know it today, must have evolved towards some different essential function that subsequently sub-functionalised into templated protein synthesis. The hypothesis presented here proposes that translation originated as the result of evolution of a primordial RNA helicase, which has been essential for preventing dying out of the RNA organism in sterile double-stranded form. This hypothesis emerges because modern ribosome possesses RNA helicase activity that likely dates back to the RNA world. I hypothesise that codon-anticodon interactions of tRNAs with mRNA evolved as a mechanism used by RNA helicase, the predecessor of ribosomes, to melt RNA duplexes. In this scenario, peptide bond formation emerged to drive unidirectional movement of the helicase via a molecular ratchet mechanism powered by Brownian motion. I propose that protein synthesis appeared as a side product of helicase activity. The first templates for protein synthesis were functional RNAs (ribozymes) that were unwound by the helicase, and the first synthesised proteins were of random or non-sense sequence. I further suggest that genetic code emerged to avoid this randomness. The initial genetic code thus emerged as an assignment of amino acids to codons according to the sequences of the pre-existing RNAs to take advantage of the side products of RNA helicase function.

Editor:

Date Published: 28th Apr 2012

Publication Type: Not specified

CcpA forms complexes with CodY and RpoA in Bacillus subtilis

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, , , Andreas Burkovski, ,

Date Published: 20th Apr 2012

Publication Type: Not specified

Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

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,

Date Published: 21st Mar 2012

Publication Type: Not specified

The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: highlights from a multi-level omics approach

Abstract (Expand)

BACKGROUND: Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made … biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures. RESULTS: We found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g.(g.h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system. CONCLUSION: This is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.

Authors: , I. F. Escapa, C. Jager, J. Puchalka, , , ,

Date Published: 20th Mar 2012

Publication Type: Not specified

Extracting regulator activity profiles by integration of de novo motifs and expression data: characterizing key regulators of nutrient depletion responses in Streptomyces coelicolor

Abstract (Expand)

Determining transcriptional regulator activities is a major focus of systems biology, providing key insight into regulatory mechanisms and co-regulators. For organisms such as Escherichia coli, … transcriptional regulator binding site data can be integrated with expression data to infer transcriptional regulator activities. However, for most organisms there is only sparse data on their transcriptional regulators, while their associated binding motifs are largely unknown. Here, we address the challenge of inferring activities of unknown regulators by generating de novo (binding) motifs and integrating with expression data. We identify a number of key regulators active in the metabolic switch, including PhoP with its associated directed repeat PHO box, candidate motifs for two SARPs, a CRP family regulator, an iron response regulator and that for LexA. Experimental validation for some of our predictions was obtained using gel-shift assays. Our analysis is applicable to any organism for which there is a reasonable amount of complementary expression data and for which motifs (either over represented or evolutionary conserved) can be identified in the genome.

Authors: M. Iqbal, Y. Mast, R. Amin, D. A. Hodgson, W. Wohlleben, N. J. Burroughs

Date Published: 13th Mar 2012

Publication Type: Not specified

Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis

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, , 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, , Georg Homuth, Hanne Jarmer, Matthieu Jules, Edda Klipp, Ludovic Le Chat, François Lecointe, , Wolfram Liebermeister, Anika March, , , David Noone, Susanne Pohl, Bernd Rinn, Frank Rügheimer, , Franck Samson, Marc Schaffer, Benno Schwikowski, , , Thomas Wiegert, Kevin M Devine, Anthony J Wilkinson, , , , Philippe Bessières, Philippe Noirot

Date Published: 3rd Mar 2012

Publication Type: Not specified

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