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BMC Bioinformatics Jul 2011The lac operon genetic switch is considered as a paradigm of genetic regulation. This system has a positive feedback loop due to the LacY permease boosting its own...
BACKGROUND
The lac operon genetic switch is considered as a paradigm of genetic regulation. This system has a positive feedback loop due to the LacY permease boosting its own production by the facilitated transport of inducer into the cell and the subsequent de-repression of the lac operon genes. Previously, we have investigated the effect of stochasticity in an artificial lac operon network at the single cell level by comparing corresponding deterministic and stochastic kinetic models.
RESULTS
This work focuses on the dynamics of cell populations by incorporating the above kinetic scheme into two Monte Carlo (MC) simulation frameworks. The first MC framework assumes stochastic reaction occurrence, accounts for stochastic DNA duplication, division and partitioning and tracks all daughter cells to obtain the statistics of the entire cell population. In order to better understand how stochastic effects shape cell population distributions, we develop a second framework that assumes deterministic reaction dynamics. By comparing the predictions of the two frameworks, we conclude that stochasticity can create or destroy bimodality, and may enhance phenotypic heterogeneity.
CONCLUSIONS
Our results show how various sources of stochasticity act in synergy with the positive feedback architecture, thereby shaping the behavior at the cell population level. Further, the insights obtained from the present study allow us to construct simpler and less computationally intensive models that can closely approximate the dynamics of heterogeneous cell populations.
Topics: Computer Simulation; Escherichia coli; Feedback; Gene Expression Regulation; Gene Regulatory Networks; Humans; Lac Operon; Monte Carlo Method; Stochastic Processes
PubMed: 21791088
DOI: 10.1186/1471-2105-12-301 -
The Journal of Cell Biology May 2003We use the lac operon in Escherichia coli as a prototype system to illustrate the current state, applicability, and limitations of modeling the dynamics of cellular... (Review)
Review
We use the lac operon in Escherichia coli as a prototype system to illustrate the current state, applicability, and limitations of modeling the dynamics of cellular networks. We integrate three different levels of description (molecular, cellular, and that of cell population) into a single model, which seems to capture many experimental aspects of the system.
Topics: Animals; Computational Biology; Escherichia coli; Humans; Lac Operon; Lactose; Models, Biological; Nonlinear Dynamics
PubMed: 12743100
DOI: 10.1083/jcb.200301125 -
Plasmid 2016Few Escherichia coli cloning vectors are available that can both be stably maintained and efficiently cured. One such vector is pAM34, a pBR332 derivative constructed by...
Few Escherichia coli cloning vectors are available that can both be stably maintained and efficiently cured. One such vector is pAM34, a pBR332 derivative constructed by Gil and Bouché (1991). Replication of this plasmid is driven by the lacZYA promoter under control of a gratuitous inducer. However, lac operator-repressor interactions are also used to regulate many expression systems which limit the utility of pAM34. In this report pAM34 has been modified by replacement of the lac regulatory elements with those of the transposon Tn10 tetracycline resistance module. This resulted in medium copy number plasmids that are dependent on the presence of tetracycline (or less satisfactorily, anhydrotetracycline) for replication. The tetracycline-dependent plasmids are rapidly lost in the absence of tetracycline and plasmid loss is markedly accelerated when the host strain expresses a tetracycline efflux pump.
Topics: Cloning, Molecular; DNA Replication; DNA Transposable Elements; Escherichia coli; Genetic Vectors; Lac Operon; Plasmids; Promoter Regions, Genetic; Tetracycline; Tetracycline Resistance
PubMed: 26876942
DOI: 10.1016/j.plasmid.2016.02.004 -
Protein Engineering, Design & Selection... Nov 2009Transcriptional regulation is an essential component of all metabolic pathways. At the most basic level, a protein binds to a particular DNA sequence (operator) on the...
Transcriptional regulation is an essential component of all metabolic pathways. At the most basic level, a protein binds to a particular DNA sequence (operator) on the genome and either positively or negatively alters the level of transcription. Together, the protein and its operator form an epigenetic switch that regulates gene expression. In an effort to produce a 'better' switch, we have discovered novel facets of the lac operon that are responsible for optimal functionality. We have uncovered a relationship between operator binding affinity and inducibility and demonstrated that the operator DNA is not a passive component of a genetic switch; it is responsible for establishing binding affinity, specificity as well as translational efficiency. In addition, an operator's directionality can indirectly affect gene expression. Unraveling the basic properties of this classical epigenetic switch demonstrates that multiple factors must be optimized in designing a better switch.
Topics: Base Sequence; DNA; Green Fluorescent Proteins; Lac Operon; Lac Repressors; Models, Molecular; Nucleic Acid Conformation; Protein Biosynthesis; Protein Conformation; Protein Engineering; RNA, Messenger; Transcription, Genetic
PubMed: 19729374
DOI: 10.1093/protein/gzp051 -
Journal of Theoretical Biology May 2013The quasi-equilibrium approximation is acceptable when molecular interactions are fast enough compared to circuit dynamics, but is no longer allowed when cellular...
The quasi-equilibrium approximation is acceptable when molecular interactions are fast enough compared to circuit dynamics, but is no longer allowed when cellular activities are governed by rare events. A typical example is the lactose operon (lac), one of the most famous paradigms of transcription regulation, for which several theories still coexist to describe its behaviors. The lac system is generally analyzed by using equilibrium constants, contradicting single-event hypotheses long suggested by Novick and Weiner (1957). Enzyme induction as an all-or-none phenomenon. Proc. Natl. Acad. Sci. USA 43, 553-566) and recently refined in the study of (Choi et al., 2008. A stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science 322, 442-446). In the present report, a lac repressor (LacI)-mediated DNA immunoprecipitation experiment reveals that the natural LacI-lac DNA complex built in vivo is extremely tight and long-lived compared to the time scale of lac expression dynamics, which could functionally disconnect the abortive expression bursts and forbid using the standard modes of lac bistability. As alternatives, purely kinetic mechanisms are examined for their capacity to restrict induction through: (i) widely scattered derepression related to the arrival time variance of a predominantly backward asymmetric random walk and (ii) an induction threshold arising in a single window of derepression without recourse to nonlinear multimeric binding and Hill functions. Considering the complete disengagement of the lac repressor from the lac promoter as the probabilistic consequence of a transient stepwise mechanism, is sufficient to explain the sigmoidal lac responses as functions of time and of inducer concentration. This sigmoidal shape can be misleadingly interpreted as a phenomenon of equilibrium cooperativity classically used to explain bistability, but which has been reported to be weak in this system.
Topics: Animals; Lac Operon; Lac Repressors; Models, Genetic; Stochastic Processes; beta-Galactosidase
PubMed: 23454080
DOI: 10.1016/j.jtbi.2013.02.005 -
Infection and Immunity Sep 1984Previous studies have demonstrated that expression of virulence in Shigella spp. is controlled by growth temperature. To study the regulation of virulence (vir) genes,...
Previous studies have demonstrated that expression of virulence in Shigella spp. is controlled by growth temperature. To study the regulation of virulence (vir) genes, we set out to develop a rapid, easily-assayed phenotype with which to measure expression of virulence. This report described a procedure for isolating vir-lac operon fusions in S. flexneri 2a by using the specialized transducing bacteriophage Mu d1(Apr lac) of Casadaban and Cohen (M. Casadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1976). Mu d1(Apr lac) lysogens were isolated and screened for loss of virulence and for temperature-dependent expression of the lactose genes on Mu d1(Apr lac). A recombinant plasmid carrying the Mu immunity gene was also introduced into lysogens of interest to stabilize the Mu d1(Apr lac) insertion and prevent possible thermal induction at 37 degrees C. The mutant which we isolated failed to penetrate tissue culture cells in the assay for virulence and produced almost 15-fold more beta-galactosidase when grown at 37 degrees C than when grown at 30 degrees C. The site of insertion of Mu d1(Apr lac) in this strain was shown to be in the 140-megadalton plasmid pSf2a140, which is known to be associated with virulence. P1L4-mediated transduction of the insertion into a virulent recipient demonstrated genetic linkage of Mu d1(Apr lac) with loss of virulence and temperature-dependent expression of beta-galactosidase. All of these features fulfill the phenotype expected for a Mu d1(Apr lac)-induced vir-lac operon fusion. This mutant provides us with a means of measuring expression of a gene function required for virulence by assaying for beta-galactosidase. The insertion will also serve as a starting point for mapping of genes on pSf2a140 which are necessary for expression of virulence.
Topics: Bacteriophage mu; Gene Expression Regulation; Genes, Bacterial; Genetic Linkage; Lac Operon; Operon; Plasmids; Shigella flexneri; Transcription, Genetic
PubMed: 6236150
DOI: 10.1128/iai.45.3.642-648.1984 -
Molecular Biology and Evolution Jun 2021Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that...
Populations of Escherichia coli selected in constant and fluctuating environments containing lactose often adapt by substituting mutations in the lacI repressor that cause constitutive expression of the lac operon. These mutations occur at a high rate and provide a significant benefit. Despite this, eight of 24 populations evolved for 8,000 generations in environments containing lactose contained no detectable repressor mutations. We report here on the basis of this observation. We find that, given relevant mutation rates, repressor mutations are expected to have fixed in all evolved populations if they had maintained the same fitness effect they confer when introduced to the ancestor. In fact, reconstruction experiments demonstrate that repressor mutations have become neutral or deleterious in those populations in which they were not detectable. Populations not fixing repressor mutations nevertheless reached the same fitness as those that did fix them, indicating that they followed an alternative evolutionary path that made redundant the potential benefit of the repressor mutation, but involved unique mutations of equivalent benefit. We identify a mutation occurring in the promoter region of the uspB gene as a candidate for influencing the selective choice between these paths. Our results detail an example of historical contingency leading to divergent evolutionary outcomes.
Topics: Adaptation, Biological; Biological Evolution; Escherichia coli; Escherichia coli Proteins; Gene Expression; Gene Expression Regulation, Bacterial; Genetic Fitness; Lac Operon; Lac Repressors; Membrane Proteins; Mutation
PubMed: 33744956
DOI: 10.1093/molbev/msab077 -
Biophysical Journal Feb 2009The lac operon has been a paradigm for genetic regulation with positive feedback, and several modeling studies have described its dynamics at various levels of detail.... (Comparative Study)
Comparative Study
The lac operon has been a paradigm for genetic regulation with positive feedback, and several modeling studies have described its dynamics at various levels of detail. However, it has not yet been analyzed how stochasticity can enrich the system's behavior, creating effects that are not observed in the deterministic case. To address this problem we use a comparative approach. We develop a reaction network for the dynamics of the lac operon genetic switch and derive corresponding deterministic and stochastic models that incorporate biological details. We then analyze the effects of key biomolecular mechanisms, such as promoter strength and binding affinities, on the behavior of the models. No assumptions or approximations are made when building the models other than those utilized in the reaction network. Thus, we are able to carry out a meaningful comparison between the predictions of the two models to demonstrate genuine effects of stochasticity. Such a comparison reveals that in the presence of stochasticity, certain biomolecular mechanisms can profoundly influence the region where the system exhibits bistability, a key characteristic of the lac operon dynamics. For these cases, the temporal asymptotic behavior of the deterministic model remains unchanged, indicating a role of stochasticity in modulating the behavior of the system.
Topics: Extracellular Space; Gene Expression Regulation, Bacterial; Gene Regulatory Networks; Lac Operon; Models, Genetic; Sensitivity and Specificity; Stochastic Processes; Thiogalactosides; Time Factors
PubMed: 19186128
DOI: 10.1016/j.bpj.2008.10.028 -
PLoS Genetics Jul 2011Genes are regulated because their expression involves a fitness cost to the organism. The production of proteins by transcription and translation is a well-known cost...
Genes are regulated because their expression involves a fitness cost to the organism. The production of proteins by transcription and translation is a well-known cost factor, but the enzymatic activity of the proteins produced can also reduce fitness, depending on the internal state and the environment of the cell. Here, we map the fitness costs of a key metabolic network, the lactose utilization pathway in Escherichia coli. We measure the growth of several regulatory lac operon mutants in different environments inducing expression of the lac genes. We find a strikingly nonlinear fitness landscape, which depends on the production rate and on the activity rate of the lac proteins. A simple fitness model of the lac pathway, based on elementary biophysical processes, predicts the growth rate of all observed strains. The nonlinearity of fitness is explained by a feedback loop: production and activity of the lac proteins reduce growth, but growth also affects the density of these molecules. This nonlinearity has important consequences for molecular function and evolution. It generates a cliff in the fitness landscape, beyond which populations cannot maintain growth. In viable populations, there is an expression barrier of the lac genes, which cannot be exceeded in any stationary growth process. Furthermore, the nonlinearity determines how the fitness of operon mutants depends on the inducer environment. We argue that fitness nonlinearities, expression barriers, and gene-environment interactions are generic features of fitness landscapes for metabolic pathways, and we discuss their implications for the evolution of regulation.
Topics: Biological Evolution; Environment; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genetic Fitness; Genotype; Lac Operon; Metabolic Networks and Pathways; Phenotype
PubMed: 21814515
DOI: 10.1371/journal.pgen.1002160 -
Research in Microbiology Jun 2004The neo-Darwinists suggested that evolution is constant and gradual, and thus that genetic changes that drive evolution should be too. However, more recent understanding... (Review)
Review
The neo-Darwinists suggested that evolution is constant and gradual, and thus that genetic changes that drive evolution should be too. However, more recent understanding of phenomena called adaptive mutation in microbes indicates that mutation rates can be elevated in response to stress, producing beneficial and other mutations. We review evidence that, in Escherichia coli, two separate mechanisms of stress-induced genetic change occur that revert a lac frameshift allele allowing growth on lactose medium. First, compensatory frameshift ("point") mutations occur by a mechanism that includes DNA double-strand breaks and (we have suggested) their error-prone repair. Point mutation requires induction of the RpoS-dependent general stress response, and the SOS DNA damage response leading to upregulation of the error-prone DNA polymerase DinB (Pol IV), and occurs during a transient limitation of post-replicative mismatch repair activity. A second mechanism, adaptive amplification, entails amplification of the leaky lac allele to 20-50 tandem repeats. These provide sufficient beta-galactosidase activity for growth, thereby apparently deflecting cells from the point mutation pathway. Unlike point mutation, amplification neither occurs in hypermutating cells nor requires SOS or DinB, but like point mutation, amplification requires the RpoS-dependent stress response. Similar processes are being found in other bacterial systems and yeast. Stress-induced genetic changes may underlie much of microbial evolution, pathogenesis and antibiotic resistance, and also cancer formation, progression and drug resistance.
Topics: Adaptation, Biological; DNA Repair; Escherichia coli; Escherichia coli Proteins; Evolution, Molecular; F Factor; Gene Amplification; Lac Operon; Models, Genetic; Mutagenesis; Mutation; Plasmids; Recombination, Genetic; SOS Response, Genetics
PubMed: 15207867
DOI: 10.1016/j.resmic.2004.01.020