70 research outputs found
Studies of Bacterial Branching Growth using Reaction-Diffusion Models for Colonial Development
Various bacterial strains exhibit colonial branching patterns during growth
on poor substrates. These patterns reflect bacterial cooperative
self-organization and cybernetic processes of communication, regulation and
control employed during colonial development. One method of modeling is the
continuous, or coupled reaction-diffusion approach, in which continuous time
evolution equations describe the bacterial density and the concentration of the
relevant chemical fields. In the context of branching growth, this idea has
been pursued by a number of groups. We present an additional model which
includes a lubrication fluid excreted by the bacteria. We also add fields of
chemotactic agents to the other models. We then present a critique of this
whole enterprise with focus on the models' potential for revealing new
biological features.Comment: 1 latex file, 40 gif/jpeg files (compressed into tar-gzip). Physica
A, in pres
Modeling branching and chiral colonial patterning of lubricating bacteria
In nature, microorganisms must often cope with hostile environmental
conditions. To do so they have developed sophisticated cooperative behavior and
intricate communication capabilities, such as: direct cell-cell physical
interactions via extra-membrane polymers, collective production of
extracellular "wetting" fluid for movement on hard surfaces, long range
chemical signaling such as quorum sensing and chemotactic (bias of movement
according to gradient of chemical agent) signaling, collective activation and
deactivation of genes and even exchange of genetic material. Utilizing these
capabilities, the colonies develop complex spatio-temporal patterns in response
to adverse growth conditions. We present a wealth of branching and chiral
patterns formed during colonial development of lubricating bacteria (bacteria
which produce a wetting layer of fluid for their movement). Invoking ideas from
pattern formation in non-living systems and using ``generic'' modeling we are
able to reveal novel survival strategies which account for the salient features
of the evolved patterns. Using the models, we demonstrate how communication
leads to self-organization via cooperative behavior of the cells. In this
regard, pattern formation in microorganisms can be viewed as the result of the
exchange of information between the micro-level (the individual cells) and the
macro-level (the colony). We mainly review known results, but include a new
model of chiral growth, which enables us to study the effect of chemotactic
signaling on the chiral growth. We also introduce a measure for weak chirality
and use this measure to compare the results of model simulations with
experimental observations.Comment: 50 pages, 24 images in 44 GIF/JPEG files, Proceedings of IMA
workshop: Pattern Formation and Morphogenesis (1998
Eukaryotic Transcription: What Does It Mean for a Gene to Be ‘on’?
Until recently, transcription could only be observed by measuring mRNA production of cell populations, thus obscuring the kinetics at the level of individual transcription events. A new study now shows that eukaryotic transcription, visualised in individual living cells, occurs in bursts — much as it does in prokaryotes
Following Cell-fate in E. coli After Infection by Phage Lambda
The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired
Protein synthesis molecule by molecule
Since the earliest days of molecular biology it has been known that even a seemingly uniform culture of bacteria is made up of cells very different from each other in terms of their levels of a given protein. This individuality has now finally been quantified at single-molecule resolution, as reported in two recent papers
Stochastic simulation and statistical inference platform for visualization and estimation of transcriptional kinetics
Recent advances in single-molecule fluorescent imaging have enabled quantitative measurements of transcription at a single gene copy, yet an accurate understanding of transcriptional kinetics is still lacking due to the difficulty of solving detailed biophysical models. Here we introduce a stochastic simulation and statistical inference platform for modeling detailed transcriptional kinetics in prokaryotic systems, which has not been solved analytically. The model includes stochastic two-state gene activation, mRNA synthesis initiation and stepwise elongation, release to the cytoplasm, and stepwise co-transcriptional degradation. Using the Gillespie algorithm, the platform simulates nascent and mature mRNA kinetics of a single gene copy and predicts fluorescent signals measurable by time-lapse single-cell mRNA imaging, for different experimental conditions. To approach the inverse problem of estimating the kinetic parameters of the model from experimental data, we develop a heuristic optimization method based on the genetic algorithm and the empirical distribution of mRNA generated by simulation. As a demonstration, we show that the optimization algorithm can successfully recover the transcriptional kinetics of simulated and experimental gene expression data. The platform is available as a MATLAB software package at https://data.caltech.edu/records/1287
Lambda-prophage induction modeled as a cooperative failure mode of lytic repression
We analyze a system-level model for lytic repression of lambda-phage in E.
coli using reliability theory, showing that the repressor circuit comprises 4
redundant components whose failure mode is prophage induction. Our model
reflects the specific biochemical mechanisms involved in regulation, including
long-range cooperative binding, and its detailed predictions for prophage
induction in E. coli under ultra-violet radiation are in good agreement with
experimental data.Comment: added referenc
Rapid Two-Photon Imaging with Nanometer Accuracy of Individual Quantum Dots in a Biological Environment
Quantitative transcription factor binding kinetics at the single-molecule level
We have investigated the binding interaction between the bacteriophage lambda
repressor CI and its target DNA using total internal reflection fluorescence
microscopy. Large, step-wise changes in the intensity of the red fluorescent
protein fused to CI were observed as it associated and dissociated from
individually labeled single molecule DNA targets. The stochastic association
and dissociation were characterized by Poisson statistics. Dark and bright
intervals were measured for thousands of individual events. The exponential
distribution of the intervals allowed direct determination of the association
and dissociation rate constants, ka and kd respectively. We resolved in detail
how ka and kd varied as a function of 3 control parameters, the DNA length L,
the CI dimer concentration, and the binding affinity. Our results show that
although interaction with non-operator DNA sequences are observable, CI binding
to the operator site is not dependent on the length of flanking non-operator
DNA.Comment: 34 pages, 10 figures, accepted by Biophysical Journa
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