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Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria

Nature Microbiology volume 3, pages 1043–1053 (2018)Cite this article

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Abstract

Engineering microorganisms to promote human or plant health will require manipulation of robust bacteria that are capable of surviving in harsh, competitive environments. Genetic engineering of undomesticated bacteria can be limited by an inability to transfer DNA into the cell. Here we developed an approach based on the integrative and conjugative element from Bacillus subtilis (ICEBs1) to overcome this problem. A donor strain (XPORT) was built to transfer miniaturized integrative and conjugative elements (mini-ICEBs1) to undomesticated bacteria. The strain was engineered to enable inducible control over conjugation, to integrate delivered DNA into the chromosome of the recipient, to restrict spread of heterologous DNA through separation of the type IV secretion system from the transferred DNA, and to enable simple isolation of engineered bacteria through a d-alanine auxotrophy. Efficient DNA transfer (10–1 to 10–7 conjugation events per donor) is demonstrated using 35 Gram-positive strains isolated from humans (skin and gut) and soil. Mini-ICEBs1 was used to rapidly characterize the performance of an isopropyl-β-d-thiogalactoside (IPTG)-inducible reporter across dozens of strains and to transfer nitrogen fixation to four Bacillus species. Finally, XPORT was introduced to soil to demonstrate DNA transfer under non-ideal conditions.

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Fig. 1: Development of XPORT based on B. subtilis ICE.
Fig. 2: Engineering diverse bacterial species with XPORT.
Fig. 3: Engineering undomesticated soil bacteria.
Fig. 4: Introducing new DNA to mini-ICE.

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Acknowledgements

We thank E. Alm (Massachusetts Institute of Technology) and R. Xavier (Broad Institute) for the bacterial isolates obtained from human gut and skin. This work was supported by the US Defense Advanced Research Projects Agency’s Biological Robustness in Complex Settings program award (HR0011-15-2-0033), the US Office of the Secretary of Defense Laboratory University Collaborative Initiative fellowship, the Office of the Secretary of Defense Applied Research for the Advancement of S&T Priorities program on Synthetic Biology for Military Environments, and the National Institute of General Medical Sciences (GM041934).

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Authors and Affiliations

  1. Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jennifer A. N. Brophy, Alexander J. Triassi & Christopher A. Voigt

  2. US Army Research Laboratory, Adelphi, MD, USA

    Bryn L. Adams & Dimitra N. Stratis-Cullum

  3. General Technical Services, Adelphi, MD, USA

    Rebecca L. Renberg

  4. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Alan D. Grossman

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Contributions

J.A.N.B., C.A.V. and A.D.G. conceived the study and designed the ICEBs1 engineering and characterization experiments. J.A.N.B. and A.J.T. performed ICEBs1 engineering and characterization experiments and analysed the data. B.L.A. and D.N.S.-C. conceived and designed soil sensor experiments. B.L.A. and R.L.R. performed the soil sensor experiments and analysed the data. J.A.N.B., C.A.V., B.L.A. and R.L.R. wrote the manuscript.

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Correspondence to Christopher A. Voigt.

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Supplementary information

Supplementary Information

Supplementary Figures 1–15, Supplementary Tables 1–5.

Reporting Summary

Supplementary File 1

Sequence file for B. subtilis JAB1000 mini-ICEBs1_M1.

Supplementary File 2

Sequence file for B. subtilis JAB447 mini-ICEBs1_M2.

Supplementary File 3

Sequence file for B. subtilis JAB854 mini-ICEBs1_M3.

Supplementary File 4

Sequence file for B. subtilis JAB927 mini-ICEBs1_M4.

Supplementary File 5

Sequence file for B. subtilis JAB932 mini-ICEBs1_M1.

Supplementary File 6

Sequence file for B. subtilis JAB943 mini-ICEBs1_M7.

Supplementary File 7

Sequence file for B. subtilis JAB944 mini-ICEBs1_M6.

Supplementary File 8

Sequence file for B. subtilis JAB951 mini-ICEBs1_M5.

Supplementary File 9

Sequence file for B. subtilis JAB981 mini-ICEBs1_M1.

Supplementary File 10

Sequence file for B. subtilis JH642 ICEBs1.

Supplementary File 11

Sequence file for plasmid pJAB205.

Supplementary File 12

Sequence file for plasmid pJAB273.

Supplementary File 13

Sequence file for plasmid pJAB309.

Supplementary File 14

Sequence file for plasmid pJAB423.

Supplementary File 15

Sequence file for plasmid pJAB463.

Supplementary File 16

Sequence file for plasmid pJAB716.

Supplementary File 17

Sequence file for plasmid pJAB775.

Supplementary File 18

Sequence file for plasmid pJAB778.

Supplementary File 19

Sequence file for plasmid pJAB980.

Supplementary File 20

Sequence file for plasmid pJAB988.

Supplementary File 21

Sequence file for plasmid pJAB-001.

Supplementary File 22

Sequence file for plasmid pJAB-002.

Supplementary File 23

Sequence file for plasmid pJAB-003.

Supplementary File 24

Sequence file for plasmid pJAB-005.

Supplementary File 25

Sequence file for plasmid pJAB- 009.

Supplementary File 26

Sequence file for plasmid pJAB- 017.

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Brophy, J.A.N., Triassi, A.J., Adams, B.L. et al. Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria. Nat Microbiol 3, 1043–1053 (2018). https://doi.org/10.1038/s41564-018-0216-5

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