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1.  A Model for Sigma Factor Competition in Bacterial Cells 
PLoS Computational Biology  2014;10(10):e1003845.
Sigma factors control global switches of the genetic expression program in bacteria. Different sigma factors compete for binding to a limited pool of RNA polymerase (RNAP) core enzymes, providing a mechanism for cross-talk between genes or gene classes via the sharing of expression machinery. To analyze the contribution of sigma factor competition to global changes in gene expression, we develop a theoretical model that describes binding between sigma factors and core RNAP, transcription, non-specific binding to DNA and the modulation of the availability of the molecular components. The model is validated by comparison with in vitro competition experiments, with which excellent agreement is found. Transcription is affected via the modulation of the concentrations of the different types of holoenzymes, so saturated promoters are only weakly affected by sigma factor competition. However, in case of overlapping promoters or promoters recognized by two types of sigma factors, we find that even saturated promoters are strongly affected. Active transcription effectively lowers the affinity between the sigma factor driving it and the core RNAP, resulting in complex cross-talk effects. Sigma factor competition is not strongly affected by non-specific binding of core RNAPs, sigma factors and holoenzymes to DNA. Finally, we analyze the role of increased core RNAP availability upon the shut-down of ribosomal RNA transcription during the stringent response. We find that passive up-regulation of alternative sigma-dependent transcription is not only possible, but also displays hypersensitivity based on the sigma factor competition. Our theoretical analysis thus provides support for a significant role of passive control during that global switch of the gene expression program.
Author Summary
Bacteria respond to changing environmental conditions by switching the global pattern of expressed genes. A key mechanism for global switches of the transcriptional program depends on alternative sigma factors that bind the RNA polymerase core enzyme and direct it towards the appropriate stress response genes. Competition of different sigma factors for a limited amount of RNA polymerase is believed to play a central role in this global switch. Here, a theoretical approach is used towards a quantitative understanding of sigma factor competition and its effects on gene expression. The model is used to quantitatively describe in vitro competition assays and to address the question of indirect or passive control in the stringent response upon amino acids starvation. We show that sigma factor competition provides a mechanism for a passive up-regulation of the stress specific sigma-driven genes due to the increased availability of RNA polymerase in the stringent response. Moreover, we find that active separation of sigma factor from the RNA polymerase during early transcript elongation weakens the sigma factor-RNA polymerase equilibrium constant, raising the question of how their in vitro measure is relevant in the cell.
PMCID: PMC4191881  PMID: 25299042
2.  Control of transcription elongation by GreA determines rate of gene expression in Streptococcus pneumoniae 
Nucleic Acids Research  2014;42(17):10987-10999.
Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in ‘transcription traffic jams’ on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.
PMCID: PMC4176173  PMID: 25190458
3.  Emergence of robust growth laws from optimal regulation of ribosome synthesis 
Molecular Systems Biology  2014;10(8):747.
Bacteria must constantly adapt their growth to changes in nutrient availability; yet despite large-scale changes in protein expression associated with sensing, adaptation, and processing different environmental nutrients, simple growth laws connect the ribosome abundance and the growth rate. Here, we investigate the origin of these growth laws by analyzing the features of ribosomal regulation that coordinate proteome-wide expression changes with cell growth in a variety of nutrient conditions in the model organism Escherichia coli. We identify supply-driven feedforward activation of ribosomal protein synthesis as the key regulatory motif maximizing amino acid flux, and autonomously guiding a cell to achieve optimal growth in different environments. The growth laws emerge naturally from the robust regulatory strategy underlying growth rate control, irrespective of the details of the molecular implementation. The study highlights the interplay between phenomenological modeling and molecular mechanisms in uncovering fundamental operating constraints, with implications for endogenous and synthetic design of microorganisms.
PMCID: PMC4299513  PMID: 25149558
growth control; metabolic control; phenomenological model; resource allocation; synthetic biology
4.  Probing the Mechanical Properties of Magnetosome Chains in Living Magnetotactic Bacteria 
Nano Letters  2014;14(8):4653-4659.
The mechanical properties of cytoskeletal networks are intimately involved in determining how forces and cellular processes are generated, directed, and transmitted in living cells. However, determining the mechanical properties of subcellular molecular complexes in vivo has proven to be difficult. Here, we combine in vivo measurements by optical microscopy, X-ray diffraction, and transmission electron microscopy with theoretical modeling to decipher the mechanical properties of the magnetosome chain system encountered in magnetotactic bacteria. We exploit the magnetic properties of the endogenous intracellular nanoparticles to apply a force on the filament-connector pair involved in the backbone formation and stabilization. We show that the magnetosome chain can be broken by the application of external field strength higher than 30 mT and suggest that this originates from the rupture of the magnetosome connector MamJ. In addition, we calculate that the biological determinants can withstand in vivo a force of 25 pN. This quantitative understanding provides insights for the design of functional materials such as actuators and sensors using cellular components.
PMCID: PMC4133184  PMID: 25003507
Biomineralization; cytoskeleton; mechanical properties; magnetotactic bacteria; magnetosomes; X-ray diffraction
5.  Influence of Magnetic Fields on Magneto-Aerotaxis 
PLoS ONE  2014;9(7):e101150.
The response of cells to changes in their physico-chemical micro-environment is essential to their survival. For example, bacterial magnetotaxis uses the Earth's magnetic field together with chemical sensing to help microorganisms move towards favoured habitats. The studies of such complex responses are lacking a method that permits the simultaneous mapping of the chemical environment and the response of the organisms, and the ability to generate a controlled physiological magnetic field. We have thus developed a multi-modal microscopy platform that fulfils these requirements. Using simultaneous fluorescence and high-speed imaging in conjunction with diffusion and aerotactic models, we characterized the magneto- aerotaxis of Magnetospirillum gryphiswaldense. We assessed the influence of the magnetic field (orientation; strength) on the formation and the dynamic of a micro-aerotactic band (size, dynamic, position). As previously described by models of magnetotaxis, the application of a magnetic field pointing towards the anoxic zone of an oxygen gradient results in an enhanced aerotaxis even down to Earth's magnetic field strength. We found that neither a ten-fold increase of the field strength nor a tilt of 45° resulted in a significant change of the aerotactic efficiency. However, when the field strength is zeroed or when the field angle is tilted to 90°, the magneto-aerotaxis efficiency is drastically reduced. The classical model of magneto-aerotaxis assumes a response proportional to the cosine of the angle difference between the directions of the oxygen gradient and that of the magnetic field. Our experimental evidence however shows that this behaviour is more complex than assumed in this model, thus opening up new avenues for research.
PMCID: PMC4077765  PMID: 24983865
6.  Selecting for Function: Solution Synthesis of Magnetic Nanopropellers 
Nano Letters  2013;13(11):5373-5378.
We show that we can select magnetically steerable nanopropellers from a set of carbon coated aggregates of magnetic nanoparticles using weak homogeneous rotating magnetic fields. The carbon coating can be functionalized, enabling a wide range of applications. Despite their arbitrary shape, all nanostructures propel parallel to the vector of rotation of the magnetic field. We use a simple theoretical model to find experimental conditions to select nanopropellers which are predominantly smaller than previously published ones.
PMCID: PMC3885197  PMID: 24127909
Nanopropeller; self-assembly; magnetic nanoparticles; hydrodynamics; hydrothermal carbonization
7.  Differences between cotranscriptional and free riboswitch folding 
Nucleic Acids Research  2013;42(4):2687-2696.
Riboswitches are part of noncoding regions of messenger RNA (mRNA) that act as RNA sensors regulating gene expression of the downstream gene. Typically, one out of two distinct conformations is formed depending on ligand binding when the transcript leaves RNA polymerase (RNAP). Elongation of the RNA chain by RNAP, folding and binding all occurs simultaneously and interdependently on the seconds’ timescale. To investigate the effect of transcript elongation velocity on folding for the S-adenosylmethionine (SAM)-I and adenine riboswitches we employ two complementary coarse-grained in silico techniques. Native structure-based molecular dynamics simulations provide a 3D, atomically resolved model of folding with homogenous energetics. Energetically more detailed kinetic Monte Carlo simulations give access to longer timescale by describing folding on the secondary structure level and feature the incorporation of competing aptamer conformations and a ligand-binding model. Depending on the extrusion scenarios, we observe and quantify different pathways in structure formation with robust agreements between the two techniques. In these scenarios, free-folding riboswitches exhibit different folding characteristics compared with transcription-rate limited folding. The critical transcription rate distinguishing these cases is higher than physiologically relevant rates. This result suggests that in vivo folding of the analyzed SAM-I and adenine riboswitches is transcription-rate limited.
PMCID: PMC3936736  PMID: 24275497
8.  Dilution and the theoretical description of growth-rate dependent gene expression 
Expression of a gene is not only tuned by direct regulation, but also affected by the global physiological state of the (host) cell. This global dependence complicates the quantitative understanding of gene regulation and the design of synthetic gene circuits. In bacteria these global effects can often be described as a dependence on the growth rate. Here we discuss how growth-rate dependence can be incorporated in mathematical models of gene expression by comparing data for unregulated genes with the predictions of different theoretical descriptions of growth-rate dependence. We argue that a realistic description of growth effects requires a growth-rate dependent protein synthesis rate in addition to dilution by growth.
PMCID: PMC3847955  PMID: 24041253
Genetic circuits; Modeling; Bacterial growth; Dilution; Growth-rate dependence
9.  Population Dynamics of Bacterial Persistence 
PLoS ONE  2013;8(5):e62814.
Persistence is a prime example of phenotypic heterogeneity, where a microbial population splits into two distinct subpopulations with different growth and survival properties as a result of reversible phenotype switching. Specifically, persister cells grow more slowly than normal cells under unstressed growth conditions, but survive longer under stress conditions such as the treatment with bactericidal antibiotics. We analyze the population dynamics of such a population for several typical experimental scenarios, namely a constant environment, shifts between growth and stress conditions, and periodically switching environments. We use an approximation scheme that allows us to map the dynamics to a logistic equation for the subpopulation ratio and derive explicit analytical expressions for observable quantities that can be used to extract underlying dynamic parameters from experimental data. Our results provide a theoretical underpinning for the study of phenotypic switching, in particular for organisms where detailed mechanistic knowledge is scarce.
PMCID: PMC3652822  PMID: 23675428
10.  On Ribosome Load, Codon Bias and Protein Abundance 
PLoS ONE  2012;7(11):e48542.
Different codons encoding the same amino acid are not used equally in protein-coding sequences. In bacteria, there is a bias towards codons with high translation rates. This bias is most pronounced in highly expressed proteins, but a recent study of synthetic GFP-coding sequences did not find a correlation between codon usage and GFP expression, suggesting that such correlation in natural sequences is not a simple property of translational mechanisms. Here, we investigate the effect of evolutionary forces on codon usage. The relation between codon bias and protein abundance is quantitatively analyzed based on the hypothesis that codon bias evolved to ensure the efficient usage of ribosomes, a precious commodity for fast growing cells. An explicit fitness landscape is formulated based on bacterial growth laws to relate protein abundance and ribosomal load. The model leads to a quantitative relation between codon bias and protein abundance, which accounts for a substantial part of the observed bias for E. coli. Moreover, by providing an evolutionary link, the ribosome load model resolves the apparent conflict between the observed relation of protein abundance and codon bias in natural sequences and the lack of such dependence in a synthetic gfp library. Finally, we show that the relation between codon usage and protein abundance can be used to predict protein abundance from genomic sequence data alone without adjustable parameters.
PMCID: PMC3492488  PMID: 23144899
11.  Interplay of Magnetic Interactions and Active Movements in the Formation of Magnetosome Chains 
PLoS ONE  2012;7(3):e33562.
Magnetotactic bacteria assemble chains of magnetosomes, organelles that contain magnetic nano-crystals. A number of genetic factors involved in the controlled biomineralization of these crystals and the assembly of magnetosome chains have been identified in recent years, but how the specific biological regulation is coordinated with general physical processes such as diffusion and magnetic interactions remains unresolved. Here, these questions are addressed by simulations of different scenarios for magnetosome chain formation, in which various physical processes and interactions are either switched on or off. The simulation results indicate that purely physical processes of magnetosome diffusion, guided by their magnetic interactions, are not sufficient for the robust chain formation observed experimentally and suggest that biologically encoded active movements of magnetosomes may be required. Not surprisingly, the chain pattern is most resembling experimental results when both magnetic interactions and active movement are coordinated. We estimate that the force such active transport has to generate is compatible with forces generated by the polymerization or depolymerization of cytoskeletal filaments. The simulations suggest that the pleiotropic phenotypes of mamK deletion strains may be due to a defect in active motility of magnetosomes and that crystal formation in magneteosome vesicles is coupled to the activation of their active motility in M. gryphiswaldense, but not in M. magneticum.
PMCID: PMC3307741  PMID: 22442698
12.  Growth-Rate Dependence Reveals Design Principles of Plasmid Copy Number Control 
PLoS ONE  2011;6(5):e20403.
Genetic circuits in bacteria are intimately coupled to the cellular growth rate as many parameters of gene expression are growth-rate dependent. Growth-rate dependence can be particularly pronounced for genes on plasmids; therefore the native regulatory systems of a plasmid such as its replication control system are characterized by growth-rate dependent parameters and regulator concentrations. This natural growth-rate dependent variation of regulator concentrations can be used for a quantitative analysis of the design of such regulatory systems. Here we analyze the growth-rate dependence of parameters of the copy number control system of ColE1-type plasmids in E. coli. This analysis allows us to infer the form of the control function and suggests that the Rom protein increases the sensitivity of control.
PMCID: PMC3103578  PMID: 21647376
13.  Growth-rate dependent global effects on gene expression in bacteria 
Cell  2009;139(7):1366.
Bacterial gene expression depends not only on specific regulations but also directly on bacterial growth, because important global parameters such as the abundance of RNA polymerases and ribosomes are all growth-rate dependent. Understanding these global effects is necessary for a quantitative understanding of gene regulation and for the robust design of synthetic genetic circuits. The observed growth-rate dependence of constitutive gene expression can be explained by a simple model using the measured growth-rate dependence of the relevant cellular parameters. More complex growth dependences for genetic circuits involving activators, repressors and feedback control were analyzed, and salient features were verified experimentally using synthetic circuits. The results suggest a novel feedback mechanism mediated by general growth-dependent effects and not requiring explicit gene regulation, if the expressed protein affects cell growth. This mechanism can lead to growth bistability and promote the acquisition of important physiological functions such as antibiotic resistance and tolerance (persistence).
PMCID: PMC2818994  PMID: 20064380
growth rate; constitutive gene expression; gene regulation; genetic circuits; bistability; growth feedback; antibiotics; persister cells
14.  Traffic patrol in the transcription of ribosomal RNA 
RNA biology  2009;6(4):392-394.
Synthesis of ribosomal RNA (rRNA) is essential for fast cell growth and rRNA transcription is typically characterized by dense traffic of RNA polymerases along the rRNA genes. However, dense traffic is susceptible to traffic jams which may arise inevitably due to stochastic pausing of the polymerases. Based on recent theoretical and experimental results, we suggest that the “traffic viewpoint” provides a unique perspective towards understanding the control of ribosome synthesis in both bacterial and eukaryotic cells.
PMCID: PMC2859892  PMID: 19502817
rRNA; transcription; RNA polymerase; transcript elongation; antitermination; growth rate
15.  Transport of Beads by Several Kinesin Motors 
Biophysical Journal  2007;94(2):532-541.
The movements of beads pulled by several kinesin-1 (conventional kinesin) motors are studied both theoretically and experimentally. While the velocity is approximately independent of the number of motors pulling the beads, the walking distance or run-length is strongly increased when more motors are involved. Run-length distributions are measured for a wide range of motor concentrations and matched to theoretically calculated distributions using only two global fit parameters. In this way, the maximal number of motors pulling the beads is estimated to vary between two and seven motors for total kinesin concentrations between 0.1 and 2.5 μg/ml or between 0.27 and 6.7 nM. In the same concentration regime, the average number of pulling motors is found to lie between 1.1 and 3.2 motors.
PMCID: PMC2157241  PMID: 17872957

Results 1-15 (15)