Bacteria evolved genes whose single-cell distributions of expression levels are broad, or even bimodal. Evidence suggests that they might enhance phenotypic diversity for coping with fluctuating environments. We identified seven genes in E. coli with bimodal (low and high) single-cell expression levels under standard growth conditions and studied how their dynamics are modified by environmental and antibiotic stresses known to target gene expression. We found that all genes lose bimodality under some, but not under all, stresses. Also, bimodality can reemerge upon cells returning to standard conditions, which suggests that the genes can switch often between high and low expression rates. As such, these genes could become valuable components of future multi-stable synthetic circuits. Next, we proposed models of bimodal transcription dynamics with realistic parameter values, able to mimic the outcome of the perturbations studied. We explored several models' tunability and boundaries of parameter values, beyond which it shifts to unimodal dynamics. From the model results, we predict that bimodality is robust, and yet tunable, not only by RNA and protein degradation rates, but also by the fraction of time that promoters remain unavailable for new transcription events. Finally, we show evidence that, although the empirical expression levels are influenced by many factors, the bimodality emerges during transcription initiation, at the promoter regions and, thus, may be evolvable and adaptable.

Closely spaced promoters in tandem formation are abundant in bacteria. We investigated the evolutionary conservation, biological functions, and the RNA and single-cell protein expression of genes regulated by tandem promoters in E. coli. We also studied the sequence (distance between transcription start sites 'dTSS', pause sequences, and distances from oriC) and potential influence of the input transcription factors of these promoters. From this, we propose an analytical model of gene expression based on measured expression dynamics, where RNAP-promoter occupancy times and dTSS are the key regulators of transcription interference due to TSS occlusion by RNAP at one of the promoters (when dTSS ≤ 35 bp) and RNAP occupancy of the downstream promoter (when dTSS > 35 bp). Occlusion and downstream promoter occupancy are modeled as linear functions of occupancy time, while the influence of dTSS is implemented by a continuous step function, fit to in vivo data on mean single-cell protein numbers of 30 natural genes controlled by tandem promoters. The best-fitting step is at 35 bp, matching the length of DNA occupied by RNAP in the open complex formation. This model accurately predicts the squared coefficient of variation and skewness of the natural single-cell protein numbers as a function of dTSS. Additional predictions suggest that promoters in tandem formation can cover a wide range of transcription dynamics within realistic intervals of parameter values. By accurately capturing the dynamics of these promoters, this model can be helpful to predict the dynamics of new promoters and contribute to the expansion of the repertoire of expression dynamics available to synthetic genetic constructs.

Summary: Each cell is a phenotypically unique individual that is influenced by internal and external processes, operating in parallel. To characterize the dynamics of cellular processes one needs to observe many individual cells from multiple points of view and over time, so as to identify commonalities and variability. With this aim, we engineered a software, 'SCIP', to analyze multi-modal, multi-process, time-lapse microscopy morphological and functional images. SCIP is capable of automatic and/or manually corrected segmentation of cells and lineages, automatic alignment of different microscopy channels, as well as detect, count and characterize fluorescent spots (such as RNA tagged by MS2-GFP), nucleoids, Z rings, Min system, inclusion bodies, undefined structures, etc. The results can be exported into *mat files and all results can be jointly analyzed, to allow studying not only each feature and process individually, but also find potential relationships. While we exemplify its use on Escherichia coli, many of its functionalities are expected to be of use in analyzing other prokaryotes and eukaryotic cells as well. We expect SCIP to facilitate the finding of relationships between cellular processes, from small-scale (e.g. gene expression) to large-scale (e.g. cell division), in single cells and cell lineages. Availability and implementation: http://www.ca3-uninova.org/project_scip. Supplementary information: Supplementary data are available at Bioinformatics online.

BACKGROUND: sgnesR (Stochastic Gene Network Expression Simulator in R) is an R package that provides an interface to simulate gene expression data from a given gene network using the stochastic simulation algorithm (SSA). The package allows various options for delay parameters and can easily included in reactions for promoter delay, RNA delay and Protein delay. A user can tune these parameters to model various types of reactions within a cell. As examples, we present two network models to generate expression profiles. We also demonstrated the inference of networks and the evaluation of association measure of edge and non-edge components from the generated expression profiles. RESULTS: The purpose of sgnesR is to enable an easy to use and a quick implementation for generating realistic gene expression data from biologically relevant networks that can be user selected. CONCLUSIONS: sgnesR is freely available for academic use. The R package has been tested for R 3.2.0 under Linux, Windows and Mac OS X.

Several major advances in Cell and Molecular Biology have been made possible by recent advances in live-cell microscopy imaging. To support these efforts, automated image analysis methods such as cell segmentation and tracking during a time-series analysis are needed. To this aim, one important step is the validation of such image processing methods. Ideally, the “ground truth” should be known, which is possible only by manually labelling images or in artificially produced images. To simulate artificial images, we have developed a platform for simulating biologically inspired objects, which generates bodies with various morphologies and kinetics and, that can aggregate to form clusters. Using this platform, we tested and compared four tracking algorithms: Simple Nearest-Neighbour (NN), NN with Morphology and two DBSCAN-based methods. We show that Simple NN works well for small object velocities, while the others perform better on higher velocities and when clustering occurs. Our new platform for generating new benchmark images to test image analysis algorithms is openly available at (http://griduni.uninova.pt/Clustergen/ClusterGen_vl.0.zip).

MOTIVATION: Single-molecule measurements of live Escherichia coli transcription dynamics suggest that this process ranges from sub- to super-Poissonian, depending on the conditions and on the promoter. For its accurate quantification, we propose a model that accommodates all these settings, and statistical methods to estimate the model parameters and to select the relevant components. RESULTS: The new methodology has improved accuracy and avoids overestimating the transcription rate due to finite measurement time, by exploiting unobserved data and by accounting for the effects of discrete sampling. First, we use Monte Carlo simulations of models based on measurements to show that the methods are reliable and offer substantial improvements over previous methods. Next, we apply the methods on measurements of transcription intervals of different promoters in live E. coli, and show that they produce significantly different results, both in low- and high-noise settings, and that, in the latter case, they even lead to qualitatively different results. Finally, we demonstrate that the methods can be generalized for other similar purposes, such as for estimating gene activation kinetics. In this case, the new methods allow quantifying the inducer uptake dynamics as opposed to just comparing them between cases, which was not previously possible. We expect this new methodology to be a valuable tool for functional analysis of cellular processes using single-molecule or single-event microscopy measurements in live cells. AVAILABILITY AND IMPLEMENTATION: Source code is available under Mozilla Public License at http://www.cs.tut.fi/%7Ehakkin22/censored/ CONTACT: [email protected] or [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcription kinetics is limited by its initiation steps, which differ between promoters and with intra- and extracellular conditions. Regulation of these steps allows tuning both the rate and stochasticity of RNA production. We used time-lapse, single-RNA microscopy measurements in live Escherichia coli to study how the rate-limiting steps in initiation of the Plac/ara-1 promoter change with temperature and induction scheme. For this, we compared detailed stochastic models fit to the empirical data in maximum likelihood sense using statistical methods. Using this analysis, we found that temperature affects the rate limiting steps unequally, as nonlinear changes in the closed complex formation suffice to explain the differences in transcription dynamics between conditions. Meanwhile, a similar analysis of the PtetA promoter revealed that it has a different rate limiting step configuration, with temperature regulating different steps. Finally, we used the derived models to explore a possible cause for why the identified steps are preferred as the main cause for behavior modifications with temperature: we find that transcription dynamics is either insensitive or responds reciprocally to changes in the other steps. Our results suggests that different promoters employ different rate limiting step patterns that control not only their rate and variability, but also their sensitivity to environmental changes.

MOTIVATION: MS2-GFP-tagging of RNA is currently the only method to measure intervals between consecutive transcription events in live cells. For this, new transcripts must be accurately detected from intensity time traces. RESULTS: We present a novel method for automatically estimating RNA numbers and production intervals from temporal data of cell fluorescence intensities that reduces uncertainty by exploiting temporal information. We also derive a robust variant, more resistant to outliers caused e.g. by RNAs moving out of focus. Using Monte Carlo simulations, we show that the quantification of RNA numbers and production intervals is generally improved compared with previous methods. Finally, we analyze data from live Escherichia coli and show statistically significant differences to previous methods. The new methods can be used to quantify numbers and production intervals of any fluorescent probes, which are present in low copy numbers, are brighter than the cell background and degrade slowly. AVAILABILITY: Source code is available under Mozilla Public License at http://www.cs.tut.fi/%7ehakkin22/jumpdet/.

MOTIVATION: Present research on gene expression using live cell imaging and fluorescent proteins or tagged RNA requires accurate automated methods of quantification of these molecules from the images. Here, we propose a novel automated method for classifying pixel intensities of fluorescent spots to RNA numbers. RESULTS: The method relies on a new model of intensity distributions of tagged RNAs, for which we estimated parameter values in maximum likelihood sense from measurement data, and constructed a maximum a posteriori classifier to estimate RNA numbers in fluorescent RNA spots. We applied the method to estimate the number of tagged RNAs in individual live Escherichia coli cells containing a gene coding for an RNA with MS2-GFP binding sites. We tested the method using two constructs, coding for either 96 or 48 binding sites, and obtained similar distributions of RNA numbers, showing that the method is adaptive. We further show that the results agree with a method that uses time series data and with quantitative polymerase chain reaction measurements. Lastly, using simulated data, we show that the method is accurate in realistic parameter ranges. This method should, in general, be applicable to live single-cell measurements of low-copy number fluorescence-tagged molecules. AVAILABILITY AND IMPLEMENTATION: MATLAB extensions written in C for parameter estimation and finding decision boundaries are available under Mozilla public license at http://www.cs.tut.fi/%7ehakkin22/estrna/ CONTACT: [email protected].

MOTIVATION: Cell division in Escherichia coli is morphologically symmetric. However, as unwanted protein aggregates are segregated to the cell poles and, after divisions, accumulate at older poles, generate asymmetries in sister cells' vitality. Novel single-molecule detection techniques allow observing aging-related processes in vivo, over multiple generations, informing on the underlying mechanisms. RESULTS: CellAging is a tool to automatically extract information on polar segregation and partitioning in division of aggregates in E.coli, and on cellular vitality. From time-lapse, parallel brightfield and fluorescence microscopy images, it performs cell segmentation, alignment of brightfield and fluorescence images, lineage construction and pole age determination, and it computes aging-related features. We exemplify its use by analyzing spatial distributions of fluorescent protein aggregates from images of cells across generations. AVAILABILITY: CellAging, instructions and an example are available at http://www.cs.tut.fi/%7esanchesr/cellaging/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: Zebrafish embryos have recently been established as a xenotransplantation model of the metastatic behaviour of primary human tumours. Current tools for automated data extraction from the microscope images are restrictive concerning the developmental stage of the embryos, usually require laborious manual image preprocessing, and, in general, cannot characterize the metastasis as a function of the internal organs. METHODS: We present a tool, ZebIAT, that allows both automatic or semi-automatic registration of the outer contour and inner organs of zebrafish embryos. ZebIAT provides a registration at different stages of development and an automatic analysis of cancer metastasis per organ, thus allowing to study cancer progression. The semi-automation relies on a graphical user interface. RESULTS: We quantified the performance of the registration method, and found it to be accurate, except in some of the smallest organs. Our results show that the accuracy of registering small organs can be improved by introducing few manual corrections. We also demonstrate the applicability of the tool to studies of cancer progression. CONCLUSIONS: ZebIAT offers major improvement relative to previous tools by allowing for an analysis on a per-organ or region basis. It should be of use in high-throughput studies of cancer metastasis in zebrafish embryos.

BACKGROUND: Cell imaging is becoming an indispensable tool for cell and molecular biology research. However, most processes studied are stochastic in nature, and require the observation of many cells and events. Ideally, extraction of information from these images ought to rely on automatic methods. Here, we propose a novel segmentation method, MAMLE, for detecting cells within dense clusters. METHODS: MAMLE executes cell segmentation in two stages. The first relies on state of the art filtering technique, edge detection in multi-resolution with morphological operator and threshold decomposition for adaptive thresholding. From this result, a correction procedure is applied that exploits maximum likelihood estimate as an objective function. Also, it acquires morphological features from the initial segmentation for constructing the likelihood parameter, after which the final segmentation is obtained. CONCLUSIONS: We performed an empirical evaluation that includes sample images from different imaging modalities and diverse cell types. The new method attained very high (above 90%) cell segmentation accuracy in all cases. Finally, its accuracy was compared to several existing methods, and in all tests, MAMLE outperformed them in segmentation accuracy.

SUMMARY: We present Mytoe, a tool for analyzing mitochondrial morphology and dynamics from fluorescence microscope images. The tool provides automated quantitative analysis of mitochondrial motion by optical flow estimation and of morphology by segmentation of individual branches of the network-like structure of the organelles. Mytoe quantifies several features of individual branches, such as length, tortuosity and speed, and of the macroscopic structure, such as mitochondrial area and degree of clustering. We validate the methods and apply them to the analysis of sequences of images of U2OS human cells with fluorescently labeled mitochondria. AVAILABILITY: Source code, Windows software and Manual available at http://www.cs.tut.fi/%7Esanchesr/mito SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: [email protected]; [email protected].

MOTIVATION: Cell growth and division affect the kinetics of internal cellular processes and the phenotype diversity of cell populations. Since the effects are complex, e.g. different cellular components are partitioned differently in cell division, to account for them in silico, one needs to simulate these processes in great detail. RESULTS: We present SGNS2, a simulator of chemical reaction systems according to the Stochastic Simulation Algorithm with multi-delayed reactions within hierarchical, interlinked compartments which can be created, destroyed and divided at runtime. In division, molecules are randomly segregated into the daughter cells following a specified distribution corresponding to one of several partitioning schemes, applicable on a per-molecule-type basis. We exemplify its use with six models including a stochastic model of the disposal mechanism of unwanted protein aggregates in Escherichia coli, a model of phenotypic diversity in populations with different levels of synchrony, a model of a bacteriophage's infection of a cell population and a model of prokaryotic gene expression at the nucleotide and codon levels. AVAILABILITY: SGNS2, instructions and examples available at www.cs.tut.fi/~lloydpri/sgns2/ (open source under New BSD license). CONTACT: [email protected]. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: Evidence suggests that in prokaryotes sequence-dependent transcriptional pauses affect the dynamics of transcription and translation, as well as of small genetic circuits. So far, a few pause-prone sequences have been identified from in vitro measurements of transcription elongation kinetics. RESULTS: Using a stochastic model of gene expression at the nucleotide and codon levels with realistic parameter values, we investigate three different but related questions and present statistical methods for their analysis. First, we show that information from in vivo RNA and protein temporal numbers is sufficient to discriminate between models with and without a pause site in their coding sequence. Second, we demonstrate that it is possible to separate a large variety of models from each other with pauses of various durations and locations in the template by means of a hierarchical clustering and a random forest classifier. Third, we introduce an approximate likelihood function that allows to estimate the location of a pause site. CONCLUSIONS: This method can aid in detecting unknown pause-prone sequences from temporal measurements of RNA and protein numbers at a genome-wide scale and thus elucidate possible roles that these sequences play in the dynamics of genetic networks and phenotype.

MOTIVATION: Production and degradation of RNA and proteins are stochastic processes, difficulting the distinction between spurious fluctuations in their numbers and changes in the dynamics of a genetic circuit. An accurate method of change detection is key to analyze plasticity and robustness of stochastic genetic circuits. RESULTS: We use automatic change point detection methods to detect non-spurious changes in the dynamics of delayed stochastic models of gene networks at run time. We test the methods in detecting changes in mean and noise of protein numbers, and in the switching frequency of a genetic switch. We also detect changes, following genes' silencing, in the dynamics of a model of the core gene regulatory network of Saccharomyces cerevisiae with 328 genes. Finally, from images, we determine when RNA molecules tagged with fluorescent proteins are first produced in Escherichia coli. Provided prior knowledge on the time scale of the changes, the methods detect them accurately and are robust to fluctuations in protein and RNA levels. AVAILABILITY: Simulator: www.cs.tut.fi/~sanchesr/SGN/SGNSim.html CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BACKGROUND: In prokaryotes, transcription and translation are dynamically coupled, as the latter starts before the former is complete. Also, from one transcript, several translation events occur in parallel. To study how events in transcription elongation affect translation elongation and fluctuations in protein levels, we propose a delayed stochastic model of prokaryotic transcription and translation at the nucleotide and codon level that includes the promoter open complex formation and alternative pathways to elongation, namely pausing, arrests, editing, pyrophosphorolysis, RNA polymerase traffic, and premature termination. Stepwise translation can start after the ribosome binding site is formed and accounts for variable codon translation rates, ribosome traffic, back-translocation, drop-off, and trans-translation. RESULTS: First, we show that the model accurately matches measurements of sequence-dependent translation elongation dynamics. Next, we characterize the degree of coupling between fluctuations in RNA and protein levels, and its dependence on the rates of transcription and translation initiation. Finally, modeling sequence-specific transcriptional pauses, we find that these affect protein noise levels. CONCLUSIONS: For parameter values within realistic intervals, transcription and translation are found to be tightly coupled in Escherichia coli, as the noise in protein levels is mostly determined by the underlying noise in RNA levels. Sequence-dependent events in transcription elongation, e.g. pauses, are found to cause tangible effects in the degree of fluctuations in protein levels.

Stochasticity in gene expression affects many cellular processes and is a source of phenotypic diversity between genetically identical individuals. Events in elongation, particularly RNA polymerase pausing, are a source of this noise. Since the rate and duration of pausing are sequence-dependent, this regulatory mechanism of transcriptional dynamics is evolvable. The dependency of pause propensity on regulatory molecules makes pausing a response mechanism to external stress. Using a delayed stochastic model of bacterial transcription at the single nucleotide level that includes the promoter open complex formation, pausing, arrest, misincorporation and editing, pyrophosphorolysis, and premature termination, we investigate how RNA polymerase pausing affects a gene's transcriptional dynamics and gene networks. We show that pauses' duration and rate of occurrence affect the bursting in RNA production, transcriptional and translational noise, and the transient to reach mean RNA and protein levels. In a genetic repressilator, increasing the pausing rate and the duration of pausing events increases the period length but does not affect the robustness of the periodicity. We conclude that RNA polymerase pausing might be an important evolvable feature of genetic networks.

MOTIVATION: Gene regulatory networks (GRNs) are stochastic, thus, do not have attractors, but can remain in confined regions of the state space, i.e. the 'noisy attractors', which define the cell type and phenotype. RESULTS: We propose a gamma-Bernoulli mixture model clustering algorithm (GammaBMM), tailored for quantizing states from gamma and Bernoulli distributed data, to determine the noisy attractors of stochastic GRN. GammaBMM uses multiple data sources, naturally selects the number of states and can be extended to other parametric distributions according to the number and type of data sources available. We apply it to protein and RNA levels, and promoter occupancy state of a toggle switch and show that it can be bistable, tristable or monostable depending on its internal noise level. We show that these results are in agreement with the patterns of differentiation of model cells whose pathway choice is driven by the switch. We further apply GammaBMM to a model of the MeKS module of Bacillus subtilis, and the results match experimental data, demonstrating the usability of GammaBMM. AVAILABILITY: Implementation software is available upon request.

UNLABELLED: We present CellLine, a simulator of the dynamics of gene regulatory networks (GRN) in the cells of a lineage. From user-defined reactions and initial substance quantities, it generates cell lineages, i.e. genealogic pedigrees of cells related through mitotic division. Each cell's dynamics is driven by a delayed stochastic simulation algorithm (delayed SSA), allowing multiple time delayed reactions. The cells of the lineage can be individually subject to 'perturbations', such as gene deletion, duplication and mutation. External interventions, such as adding or removing a substance at a given moment, can be specified. Cell differentiation lineages, where differentiation is stochastically driven or externally induced, can be modeled as well. Finally, CellLine can generate and simulate the dynamics of multiple copies of any given cell of the lineage. As examples of CellLine use, we simulate the following systems: cell lineages containing a model of the P53-Mdm2 feedback loop, a differentiation lineage where each cell contains a 4 gene repressilator (a bistable circuit), a model of the differentiation of the cells of the retinal mosaic required for color vision in Drosophila melanogaster, where the differentiation pathway depends on one substance's concentration that is controlled by a stochastic process, and a 9 gene GRN to illustrate the advantage of using CellLine rather than simulating multiple independent cells, in cases where the cells of the lineage are dynamically correlated. AVAILABILITY: The CellLine program, instructions and examples are available at www.ucalgary.ca/~aribeiro/CellLine/CellLine.html

UNLABELLED: We present SGNSim, 'Stochastic Gene Networks Simulator', a tool to model gene regulatory networks (GRN) where transcription and translation are modeled as multiple time delayed events and its dynamics is driven by a stochastic simulation algorithm (SSA) able to deal with multiple time delayed events. The delays can be drawn from several distributions and the reaction rates from complex functions or from physical parameters. SGNSim can generate ensembles of GRNs, within a set of user-defined parameters, such as topology. It can also be used to model specific GRNs and systems of chemical reactions. Perturbations, e.g. gene deletion, over-expression, copy and mutation, can be modeled as well. As examples, we present a model of a toggle switch without cooperative binding subject to perturbations, a system of reactions within a compartmentalized environment where membrane crossing is controlled by a negative feedback mechanism and a simulation based on the yeast transcriptional network. AVAILABILITY: SGNSim program, instructions and examples available at www.ucalgary.ca/~aribeiro/SGNtheSim/SGNtheSim.html.