A stabilized microbial ecosystem of self-limiting bacteria using synthetic quorum-regulated lysis

Abstract

Microbial ecologists are increasingly turning to small, synthesized ecosystems15 as a reductionist tool to probe the complexity of native microbiomes6,7. Concurrently, synthetic biologists have gone from single-cell gene circuits811 to controlling whole populations using intercellular signalling1216. The intersection of these fields is giving rise to new approaches in waste recycling17, industrial fermentation18, bioremediation19 and human health16,20. These applications share a common challenge7 well-known in classical ecology21,22—stability of an ecosystem cannot arise without mechanisms that prohibit the faster-growing species from eliminating the slower. Here, we combine orthogonal quorum-sensing systems and a population control circuit with diverse self-limiting growth dynamics to engineer two ‘ortholysis’ circuits capable of maintaining a stable co-culture of metabolically competitive Salmonella typhimurium strains in microfluidic devices. Although no successful co-cultures are observed in a two-strain ecology without synthetic population control, the ‘ortholysis’ design dramatically increases the co-culture rate from 0% to approximately 80%. Agent-based and deterministic modelling reveal that our system can be adjusted to yield different dynamics, including phase-shifted, antiphase or synchronized oscillations, as well as stable steady-state population densities. The ‘ortholysis’ approach establishes a paradigm for constructing synthetic ecologies by developing stable communities of competitive microorganisms without the need for engineered co-dependency.

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Figure 1: Experimental investigation into the space of the population dynamics of a self-communicating synchronized lysis circuit.
Figure 2: Experimental demonstration of long-term co-culture of competitive species with unequal growth rates using signal orthogonal self-lysis.
Figure 3: Agent-based model elucidating experimental dynamics.
Figure 4: Prediction of synchronized lysis circuit dynamics in a dual-strain population using various communication motifs.

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Acknowledgements

This material is based on work supported by NIH/NIGMS grant no. RO1-GM069811 and by the San Diego Center for Systems Biology under NIH/NIGMS grant no. P50-GM085764. S.R.S. was partially funded by the National Science Foundation Graduate Research Fellowship under grant no. DGE-1144086. P.B. acknowledges support from HFSP fellowship LT000840/2014-C. L.X. and L.S.T. were partially supported by ONR grant no. N00014-16-1-2093. Any opinion, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the funding agencies.

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All authors contributed extensively to the work presented in this paper.

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Correspondence to Jeff Hasty.

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Supplementary information

Supplementary information

Supplementary Figures 1–5; Supplementary Table 1 (PDF 1447 kb)

41564_2017_BFnmicrobiol201783_MOESM2_ESM.avi

This video shows timelapse fluorescence microscopy of the SLC in strain 2 (ssrA tag on LuxI) at 20x magnification. Images were taken every 2 min of a 100 × 100 μm chamber. Timestamp is in minutes. (AVI 22816 kb)

Supplementary Video 1

This video shows timelapse fluorescence microscopy of the SLC in strain 2 (ssrA tag on LuxI) at 20x magnification. Images were taken every 2 min of a 100 × 100 μm chamber. Timestamp is in minutes. (AVI 22816 kb)

41564_2017_BFnmicrobiol201783_MOESM3_ESM.avi

This video shows timelapse fluorescence microscopy of the SLC in strain 1 (no ssrA tag on LuxI) at 20x magnification. Images were taken every 2 min of a 100 × 100 μm chamber. Timestamp is in minutes. (AVI 22966 kb)

Supplementary Video 2

This video shows timelapse fluorescence microscopy of the SLC in strain 1 (no ssrA tag on LuxI) at 20x magnification. Images were taken every 2 min of a 100 × 100 μm chamber. Timestamp is in minutes. (AVI 22966 kb)

41564_2017_BFnmicrobiol201783_MOESM4_ESM.avi

Video of non-lysis co-culture on a microfluidic device at 20x magnification. Strain 4 (non-lysis Lux-CFP) and strain 6 (non-lysis Rpa-GFP). Timelapse fluorescence microscopy images were taken every 3 min. Timestamp is in minutes. (AVI 28306 kb)

Supplementary Video 3

Video of non-lysis co-culture on a microfluidic device at 20x magnification. Strain 4 (non-lysis Lux-CFP) and strain 6 (non-lysis Rpa-GFP). Timelapse fluorescence microscopy images were taken every 3 min. Timestamp is in minutes. (AVI 28306 kb)

41564_2017_BFnmicrobiol201783_MOESM5_ESM.avi

Video of dual lysis co-culture on a microfluidic device at 20x magnification. Strain 5 (lysis Lux-CFP) and Strain 7 (lysis Rpa-GFP). Timelapse fluorescence microscopy images were taken every 3 min. Timestamp is in minutes. (AVI 27373 kb)

Supplementary Video 4

Video of dual lysis co-culture on a microfluidic device at 20x magnification. Strain 5 (lysis Lux-CFP) and Strain 7 (lysis Rpa-GFP). Timelapse fluorescence microscopy images were taken every 3 min. Timestamp is in minutes. (AVI 27373 kb)

41564_2017_BFnmicrobiol201783_MOESM6_ESM.avi

Timelapse agent-based simulation of two lysis strains both in the oscillatory regime. (AVI 4083 kb)

Supplementary Video 5

Timelapse agent-based simulation of two lysis strains both in the oscillatory regime. (AVI 4083 kb)

41564_2017_BFnmicrobiol201783_MOESM7_ESM.avi

Timelapse agent-based simulation of two lysis strains: one in the oscillatory regime and the other in the constant lysis regime. (AVI 5447 kb)

Supplementary Video 6

Timelapse agent-based simulation of two lysis strains: one in the oscillatory regime and the other in the constant lysis regime. (AVI 5447 kb)

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Scott, S., Din, M., Bittihn, P. et al. A stabilized microbial ecosystem of self-limiting bacteria using synthetic quorum-regulated lysis. Nat Microbiol 2, 17083 (2017). https://doi.org/10.1038/nmicrobiol.2017.83

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