A synthetic Escherichia coli predator–prey ecosystem

We have constructed a synthetic ecosystem consisting of two Escherichia coli populations, which communicate bi-directionally through quorum sensing and regulate each other's gene expression and survival via engineered gene circuits. Our synthetic ecosystem resembles canonical predator–prey systems in terms of logic and dynamics. The predator cells kill the prey by inducing expression of a killer protein in the prey, while the prey rescue the predators by eliciting expression of an antidote protein in the predator. Extinction, coexistence and oscillatory dynamics of the predator and prey populations are possible depending on the operating conditions as experimentally validated by long-term culturing of the system in microchemostats. A simple mathematical model is developed to capture these system dynamics. Coherent interplay between experiments and mathematical analysis enables exploration of the dynamics of interacting populations in a predictable manner

Long-Term Monitoring of Bacteria Undergoing Programmed Population Control in a Microchemostat

Using an active approach to preventing biofilm formation, we implemented a microfluidic bioreactor that enables long-term culture and monitoring of extremely small populations of bacteria with single-cell resolution. We used this device to observe the dynamics of Escherichia coli carrying a synthetic “population control” circuit that regulates cell density through a feedback mechanism based on quorum sensing. The microfluidic bioreactor enabled long-term monitoring of unnatural behavior programmed by the synthetic circuit, which included sustained oscillations in cell density and associated morphological changes, over hundreds of hours

Confinement-Induced Drug-Tolerance in Mycobacteria Mediated by an Efflux Mechanism

<div><p>Tuberculosis (TB) is the world’s deadliest curable disease, responsible for an estimated 1.5 million deaths annually. A considerable challenge in controlling this disease is the prolonged multidrug chemotherapy (6 to 9 months) required to overcome drug-tolerant mycobacteria that persist in human tissues, although the same drugs can sterilize genetically identical mycobacteria growing in axenic culture within days. An essential component of TB infection involves intracellular <i>Mycobacterium tuberculosis</i> bacteria that multiply within macrophages and are significantly more tolerant to antibiotics compared to extracellular mycobacteria. To investigate this aspect of human TB, we created a physical cell culture system that mimics confinement of replicating mycobacteria, such as in a macrophage during infection. Using this system, we uncovered an epigenetic drug-tolerance phenotype that appears when mycobacteria are cultured in space-confined bioreactors and disappears in larger volume growth contexts. Efflux mechanisms that are induced in space-confined growth environments contribute to this drug-tolerance phenotype. Therefore, macrophage-induced drug tolerance by mycobacteria may be an effect of confined growth among other macrophage-specific mechanisms.</p></div

Loss of MmpL11 function reduced resistance to rifampicin in the 200pL cultures.

<p>Effect of rifampicin (350 μg/ml) on the growth of <i>M</i>. <i>smegmatis mmpL11</i> mutants in 200pL cultures. The growth curves in the right-most column represent the average cell density of the individual reactors shown on the left. All 12 (100%) mutant drug-free cultures grew (black growth curves). However, only 11 of 16 (69%) mutant cultures grew in rifampicin medium and growth in these cultures was generally slower. All cultures were cultivated simultaneously on the same chip at 37°C.</p

The microdialyser system.

<p><b>(A)</b> Schematic diagram of a microdialyser unit with elements such as the growth and conditioning chambers, separated by a link valve labelled. <b>(B)</b> Once the link valve is open, passive diffusive exchange occurs between the growth and conditioning chambers: nutrient molecules (represented by red dots) diffuse into the growth chamber while mycobacterial metabolic waste products (blue crosses) diffuse into the conditioning chamber. <b>(C)</b> Functional illustration of diffusive exchange in the microdialyser showing the dye concentration in the growth chamber (orange diamonds) and the conditioning chamber (light blue circles). The fluid in the conditioning chamber is cycled between DYE (cycles 2–20 and 30–45) and WATER (cycles 1; 21–29). The dye concentration in the growth chamber depends on the concentration in the conditioning chamber. This result was reproduced over 160 times in 9 different chips.</p

(A) Growth of <i>M</i>. <i>smegmatis ΔFtsEX</i> cells with rifampicin [350 μg/ml] with the time interval between consecutive microdialysis steps doubled to 2 hours—across the various microdialyser growth chambers sizes—1700pL (red curves), 1200pL (green), 500pL (blue) and 200pL (black). The growth curves on the right represent the average cell density of the individual reactors shown on the left. With the slower dilution rate, the cell populations in the larger reactors become more tolerant to rifampicin in accordance to the reactor volume: the fraction of reactors with growth decreases as the volume increases. All cultures were cultivated simultaneously on the same chip at 37°C. (B) The percentage of microdialyser cultures with positive growth at different dilution rates with rifampicin (350μg/ml) or in drug-free medium.

<p>At a dilution (microdialysis) interval of one hour, all drug-free cultures (black boxes) reactors registered positive growth independent of culture volume. Five of six 200pL cultures and among the larger cultures, only one of the eight 500pL cultures tolerated rifampicin (red circles). With the dilution interval doubled to two hours, the yield of the drug-containing cultures (blue crosses) increased albeit in a volume-dependent fashion, whereby the fraction of cultures with positive growth decreased as the culture volume increased. The cultures were cultivated at 37°C.</p

(A) Use of the microdialyser system to resolve the bacteriostatic/bactericidal credentials of antimicrobial drugs. The graphs in the left column represent a typical growth curves for each growth chamber size and those in the right column represent a set of growth curves for the same growth chamber volume. The cultures represented by the red curves were first cultured in drug-free medium for 15 hours. Using the microdialysis process, at 15 hours, rifampicin (350 μg/ml) was introduced to these cultures and then withdrawn at 150 hours. Although microbial growth in the bigger volume reactors—seven 500, eight 1200 and eight 1700pL—was effectively suppressed when rifampicin was applied, cell growth resumed when the drug was withdrawn, suggesting that rifampicin was bacteriostatic under these conditions. The cells in one of the eight 500pL maintained slow growth in the presence of the drug. Microbial growth in five of six small (200pL) growth chambers was only slightly decreased by the drug a

<p>The X-axis represents the cell density reached by ΔFtsEX cells in the reactors shown above (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136231#pone.0136231.g008" target="_blank">Fig 8A</a>) after culturing for 150 hours in medium with rifampicin (350 μg/ml) during the first phase of culture. The Y-axis represents the new cell density attained in the same reactors after 150 additional hours of culturing in medium without rifampicin. Accordingly, data points in quadrants 1, 2 and 3 represent reactors in which the cells are dead, static or resistant respectively. In general <i>M</i>. <i>smegmatis</i> Δ<i>ftsEX</i> cells were resistant to rifampicin in the 200pL reactors and static in the rest.</p

Growth dynamics of <i>M</i>. <i>smegmatis ΔftsEX</i> cells with or without rifampicin (350 μg/ml) across the various microdialyser growth chambers sizes: 1700pL (green boxes), 1200pL (yellow boxes), 500pL (blue boxes) and 200pL (brown boxes).

<p>The growth curves in the right-most column represent the average cell density of the individual reactors shown on the left. In drug-free medium, the cells exhibited positive growth across all the growth chamber volumes as indicated (black growth curves). With rifampicin, growth in five of six of the 200pL reactors was only slightly affected with the exception of one of the growth chambers in which the cells did not grow. Growth in the larger reactors (eight 1700pL, eight 1200pL and seven 500pL) was inhibited (red curves), except in one of the 500pL growth chambers. The cultures under each drug condition were cultivated simultaneously on the same chip at 37°C.</p

Efflux inhibition reduces drug tolerance.

<p>Effect of verapamil (100μg/ml) on 200pL ΔFtsEX cultures with or without rifampicin (350 μg/ml). The right-most growth curves represent the average cell density of the individual reactors shown in each row on the left. The control (cyan growth curves), rifampicin only (blue) and verapamil only (black) cultures registered positive growth. Rifampicin tolerance was dramatically reduced in cultures that also contained verapamil (red). All cultures were cultivated simultaneously on the same chip at 37°C.</p