Programmed population control by cell-cell communication and regulated killing Lingchong You, Robert Sidney Cox III, Ron Weiss & Frances H. Arnold Programmed population control by cell-cell communication and regulated killing. You, Lingchong et al. Letters to Nature 428 (2004)1 Victoria Hsiao

What They Did They built and characterized a “population control circuit” which can automatically regulate the density of an E.coli population. Quorum sensing- when bacteria regulate gene expression based on population density (which they sense based on the density of signaling molecules). Negative feedback loop: Bacteria produce signaling molecule  as # of bac increases, so does the density of the signal  at a certain threshold, the quorum sensing kicks in, which leads to cell death. Programmed population control by cell-cell communication and regulated killing. You, Lingchong et al. Letters to Nature 428 (2004)2

The Plasmids pLuxR12 contains the genes for the LuxR/LuxI system from the marine bacterium V. fischeri. LuxI  The LuxI protein, which makes acyl-homoserine lactone (AHL) – a small diffusible signaling molecule. LuxR  LuxR transcriptional regulator  when activated with AHL, induces the expression of the “killer gene” pluxCcdB3 contains the “killer gene” lacZ α -ccdB, which is a fusion protein (referred to as E in the next slide). The lacZ α part allows fusion protein levels to be measured with a LacZ assay The ccdB part kills susceptible cells by poisoning the DNA gyrase complex Programmed population control by cell-cell communication and regulated killing. You, Lingchong et al. Letters to Nature 428 (2004)3

The Circuit LuxI protein produces AHL, which accumulates in the medium and inside the cells. Once the AHL reaches a high enough concentration, it will bind and activate the LuxR transcriptional regulator, which binds to the luxI promoter. E, the “killer protein,” is then expressed and causes cells death at high enough levels. Programmed population control by cell-cell communication and regulated killing. You, Lingchong et al. Letters to Nature 428 (2004)4

Mathematical Model N (mL -1 ) = viable cell density k (h -1 ) =growth rate N m (mL -1 ) = carrying capacity E (nM) = concentration of killer protein d (nM -1 h -1 ) =death rate constant A= concentration of AHL k E & d E = growth and degradation rate constants of E vA & dA = same for AHL Programmed population control by cell-cell communication and regulated killing. You, Lingchong et al. Letters to Nature 428 (2004)5 Eq. 1) Cell Growth and Death Eq. 2) Production and Degradation of the Killer Protein Eq. 3) Production and Degradation of AHL

Mathematical Model Using the mathematical model, Arnold et al. predicted that the system would reach a stable cell density for all realistic parameter values, though it may or may not have damped oscillations while going to steady state. Predicts that steady-state density increases proportionally with the AHL degradation rate constant. Programmed population control by cell-cell communication and regulated killing. You, Lingchong et al. Letters to Nature 428 (2004)6

Experimental Results Culture with circuit OFF Culture with circuit ON CFU = colony-forming units (per mL) LacZ activity shows how much killer protein is being expressed Insets show the ON data on a linear scale. Programmed population control by cell-cell communication and regulated killing. You, Lingchong et al. Letters to Nature 428 (2004)7

Controlling steady-state density with AHL To confirm that the killer protein production rate was limited by AHL production in the ON circuit, 200mL of exogenous AHL were added to the media. As expected, it did not affect bacteria without the circuit or with the OFF, but prevented growth completely in the bacteria with the ON circuit. They were able to change the steady-state density of the E.coli population by using AHL degradation rate as a “dial.” The AHL degradation rate was controlled by changing the pH of the medium ( ↑pH= ↑N s ) Programmed population control by cell-cell communication and regulated killing. You, Lingchong et al. Letters to Nature 428 (2004)8

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The big ideas Using cell-cell communication to coordinate behavior across the population. Population-control circuits that have cell-cell communication actually require phenotypic variation to work. This way, cells have different tolerances for the killer protein. Otherwise, if all the bacteria had the same phenotype, then once the killer protein reached a critical density, all the cells would die. Programmed population control by cell-cell communication and regulated killing. You, Lingchong et al. Letters to Nature 428 (2004)10

Discussion Liked: ◦ Self-regulating system based on a single negative feedback loop. I liked the idea that once you added the plasmids, you could sort of stand back and see what happens. ◦ Worked the best with phenotypic variation. I just thought it was cool that the system accounted for, and actually depended on, genetic variation in the bacteria. ◦ The final steady-state density can be tuned by changing the pH of the medium, which seems like a simple and easy way to set the final state of the system. Disliked: ◦ Bacteria already have this sort of feedback loop in response to crowding/nutrient depletion, so while it was cool that they could change the environmental cue that triggered cell death, it also seemed sort of basic. ◦ But this is a foundational paper, which is supposed to lead to building synthetic ecosystems with programmed interactions between bacterial communities. Programmed population control by cell-cell communication and regulated killing. You, Lingchong et al. Letters to Nature 428 (2004)11