Gene expression is an intrinsically stochastic process. Fluctuations in transcription and translation lead to cell-to-cell variations in mRNA and protein levels affecting cellular function and cell fate. Here, using fluorescence time-lapse microscopy, we quantify noise dynamics in an artificial operon in Escherichia coli, which is based on the native operon of ColicinE2, a toxin. In the natural system, toxin expression is controlled by a complex regulatory network; upon induction of the bacterial SOS response, ColicinE2 is produced (cea gene) and released (cel gene) by cell lysis. Using this ColicinE2-based operon, we demonstrate that upon induction of the SOS response noise of cells expressing the operon is significantly lower for the (mainly) transcriptionally regulated gene cea compared to the additionally post-transcriptionally regulated gene cel. Likewise, we find that mutations affecting the transcriptional regulation by the repressor LexA do not significantly alter the population noise, whereas specific mutations to post-transcriptionally regulating units, strongly influence noise levels of both genes. Furthermore, our data indicate that global factors, such as the plasmid copy number of the operon encoding plasmid, affect gene expression noise of the entire operon. Taken together, our results provide insights on how noise in a native toxin-producing operon is controlled and underline the importance of post-transcriptional regulation for noise control in this system.

The bacterial SOS response is a cellular reaction to DNA damage, that, among other actions, triggers the expression of colicin - toxic bacteriocins in Escherichia coli that are released to kill close relatives competing for resources. However, it is largely unknown, how the complex network regulating toxin expression controls the time-point of toxin release to prevent premature release of inefficient protein concentrations. Here, we study how different regulatory mechanisms affect production and release of the bacteriocin ColicinE2 in Escherichia coli. Combining experimental and theoretical approaches, we demonstrate that the global carbon storage regulator CsrA controls the duration of the delay between toxin production and release and emphasize the importance of CsrA sequestering elements for the timing of ColicinE2 release. In particular, we show that ssDNA originating from rolling-circle replication of the toxin-producing plasmid represents a yet unknown additional CsrA sequestering element, which is essential in the ColicinE2-producing strain to enable toxin release by reducing the amount of free CsrA molecules in the bacterial cell. Taken together, our findings show that CsrA times ColicinE2 release and reveal a dual function for CsrA as an ssDNA and mRNA-binding protein, introducing ssDNA as an important post-transcriptional gene regulatory element.

The production of bacteriocins in response to worsening environmental conditions is one means of bacteria to outcompete other microorganisms. Colicins, one class of bacteriocins in Escherichia coli, are effective against closely related Enterobacteriaceae. Current research focuses on production, release and uptake of these toxins by bacteria. However, little is known about the quantitative aspects of these dynamic processes. Here, we quantitatively study expression dynamics of the Colicin E2 operon in E. coli on a single cell level using fluorescence time-lapse microscopy. DNA damage, triggering SOS response leads to the heterogeneous expression of this operon including the cea gene encoding the toxin, Colicin E2, and the cel gene coding for the induction of cell lysis and subsequent colicin release. Advancing previous whole population investigations, our time-lapse experiments reveal that at low exogenous stress levels all cells eventually respond after a given time (heterogeneous timing). This heterogeneous timing is lost at high stress levels, at which a synchronized stress response of all cells 60 min after induction via stress can be observed. We further demonstrate, that the amount of colicin released is dependent on cel (lysis) gene expression, independent of the applied exogenous stress level. A heterogeneous response in combination with heterogeneous timing can be biologically significant. It might enable a bacterial population to endure low stress levels, while at high stress levels an immediate and synchronized population wide response can give single surviving cells of the own species the chance to take over the bacterial community after the stress has ceased.

We report on a highly sensitive approach to measure and quantify the time dependent changes of the flow generated by the flagella bundle rotation of single bacterial cells. This is achieved by observing the interactions between a silica particle and a bacterium, which are both trapped next to each other in a dual beam optical tweezer. In this configuration, the particle serves as a sensitive detector where the fast-Fourier analysis of the particle trajectory renders, it possible to access information about changes of bacterial activity. (C) 2014 AIP Publishing LLC.

Background Two-component signal transduction systems are one means of bacteria to respond to external stimuli. The LiaFSR two-component system of Bacillus subtilis consists of a regular two-component system LiaRS comprising the core Histidine Kinase (HK) LiaS and the Response Regulator (RR) LiaR and additionally the accessory protein LiaF, which acts as a negative regulator of LiaRS-dependent signal transduction. The complete LiaFSR system was shown to respond to various peptide antibiotics interfering with cell wall biosynthesis, including bacitracin. Methodology and Principal Findings Here we study the response of the LiaFSR system to various concentrations of the peptide antibiotic bacitracin. Using quantitative fluorescence microscopy, we performed a whole population study analyzed on the single cell level. We investigated switching from the non-induced ‘OFF’ state into the bacitracin-induced ‘ON’ state by monitoring gene expression of a fluorescent reporter from the RR-regulated liaI promoter. We found that switching into the ‘ON’ state occurred within less than 20 min in a well-defined switching window, independent of the bacitracin concentration. The switching rate and the basal expression rate decreased at low bacitracin concentrations, establishing clear heterogeneity 60 min after bacitracin induction. Finally, we performed time-lapse microscopy of single cells confirming the quantitative response as obtained in the whole population analysis for high bacitracin concentrations. Conclusion The LiaFSR system exhibits an immediate, heterogeneous and graded response to the inducer bacitracin in the exponential growth phase.

Summary form only given. Analyzing bacteria activity is the key to gain insight on the viability and response of prokaryotic cells to changes in their environment. Classical methods to measure bacteria viability, however, are often time consuming or in the need of high bacterial densities for successful analysis. Here, we demonstrate how the movement of a single cell can be quantified by observing the interactions between an optically trapped bacterium and a silica bead, which are aligned next to each other in a dual beam optical tweezers configuration (Fig.1).The motion of a microparticle held in an optical trap is subject to thermal fluctuations as long as there is no external disturbance [1]. Non-equilibrium noise, however, leads to a slight, frequency dependent change of the particle position in the harmonic potential [2]. We utilize this basic principle to perform measurements on bacteria cells. Any vibrations generated as the bacterium tries to escape from the first optical trap in a dual beam setup are picked-up by the detector bead that is held in the second. The position trajectory of the trapped microparticle therefore renders information on the frequency and intensity of the activity of the whole bacteria cell. We used Bacillus Subtilis, a bacterium strain that holds peritrichous flagellation, as a model system to demonstrate the feasibility of this approach. The flagella of B. Subtilis are bundled together and rotate in clock-wise or counter-clock-wise direction in order to push the bacterium forward in liquid media [3]. We demonstrate that small, time-dependent changes of the flagellar rotation can be resolved to distinguish different states of bacteria activity without the requirement of staining, fluorescence labelling, or any further treatment of the bacteria cell.

Carbohydrate-based sensors, that specifically detect sugar binding molecules or cells, are increasingly important in medical diagnostic and drug screening. Here we demonstrate that cantilever arrays functionalized with different mannosides allow the real-time detection of several Escherichia coli strains in solution. Cantilever deflection is thereby dependent on the bacterial strain studied and the glycan used as the sensing molecule. The cantilevers exhibit specific and reproducible deflection with a sensitivity range over four orders of magnitude.

Advances in carbohydrate sequencing technologies have revealed the tremendous complexity of the glycome. This complexity reflects the structural and chemical diversity of carbohydrates and is greater than that of proteins and oligonucleotides. The next step in understanding the biological function of carbohydrates requires the identification and quantification of carbohydrate interactions with other biomolecules, in particular, with proteins. To this end, we have developed a cantilever array biosensor with a self-assembling carbohydrate-based sensing layer that selectively and sensitively detects carbohydrate protein binding interactions. Specifically, we examined binding of mannosides and the protein cyanovirin-N, which binds and blocks the human immunodeficiency virus (HIV). Cyanovirin-N binding to immobilized oligomannosides on the cantilever resulted In mechanical surface stress that Is transduced into a mechanical force and cantilever bending. The degree and duration of cantilever deflection correlates with the interaction's strength, and comparative binding experiments reveal molecular binding preferences. This study establishes that carbohydrate-based cantilever biosensors are a robust, label-free, and scalable means to analyze carbohydrate-protein interactions and to detect picomolar concentrations of carbohydrate-binding proteins.