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Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi

Nature Microbiology volume 4, pages 1105–1113 (2019)Cite this article

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Abstract

The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time1,2 and metabolic prowess3,4. However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.

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Fig. 1: Profiling of V. natriegens growth and genome.
Fig. 2: CRISPRi screen in rich and minimal media.
Fig. 3: CRISPRi in rich and minimal media.
Fig. 4: Analysis of V. natriegens core genes.

Data availability

Genome sequences are available from NCBI (GenBank: CP009977, CP009978; RefSeq: NZ_CP009977, NZ_CP009978). Sequencing data for gRNA counts are available at NCBI Sequence Read Archive under BioProject PRJNA511728 (SRR8369136, SRR8369137, SRR8369138, SRR8369139) and transcriptome data is available at the Gene Expression Omnibus under accession number GSE126544 (GSM3603279, GSM3603280, GSM3603281, GSM3603282, GSM3603283, GSM3603284). All other data are available in the Supplementary Information or upon request from the corresponding authors.

Code availability

Custom code is available at https://github.com/citizenlee/vnat_glib or will be made available upon request.

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Acknowledgements

We acknowledge J. F. Juárez, J. Teramoto, M. Mee, A. Camilli and J. Aach for comments and discussions; C. Mancuso and M. Joung; Lyubov Golubeva for the pRSF plasmid; B. Davis and M. Waldor for V. cholerae strains O395 and BAH-2, and the pCTX-Km and pCTX-Ap plasmids; V. de Lorenzo for the pBAM1 plasmid; D. E. Cameron and J. Mekalanos for the pTnFGL3 plasmid and B. Wanner for the BW29427 strain. This work was supported by Department of Energy Grant DE-FG02-02ER63445 (to G.M.C.), AWS Cloud Credits for Research programme (to H.H.L.) and a National Science Foundation CAREER Award MCB-1350949 (to A.S.K.).

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Author notes

  1. These authors contributed equally: Henry H. Lee, Nili Ostrov.

Authors and Affiliations

  1. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Henry H. Lee, Nili Ostrov, Michaela A. Gold & George M. Church

  2. Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA, USA

    Brandon G. Wong & Ahmad S. Khalil

  3. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

    Ahmad S. Khalil & George M. Church

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  1. Henry H. Lee
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Contributions

H.H.L. and N.O. designed and performed experiments, analysed data and wrote the paper. B.G.W. and A.S.K. designed and performed single-cell microfluidics experiments and provided input on the paper. M.A.G. contributed to the electroporation experiments and formulated recovery media. G.M.C. supervised the project.

Corresponding authors

Correspondence to Henry H. Lee or George M. Church.

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Competing interests

H.H.L., N.O. and G.M.C. have filed patents related to this work.

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Supplementary information

Supplementary Information

Supplementary Discussion, Supplementary Figures 1–17, Supplementary Tables 1–4 and Supplementary References.

Reporting Summary

Supplementary Table 5

CRISPRi library.

Supplementary Table 6

Core genes.

Supplementary Table 7

Transcriptome analysis.

Supplementary Table 8

Transposon coverage.

Supplementary Video 1

Single cell growth of V. natriegens (LB3) and E. coli (LB) at 37 °C.

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Lee, H.H., Ostrov, N., Wong, B.G. et al. Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi. Nat Microbiol 4, 1105–1113 (2019). https://doi.org/10.1038/s41564-019-0423-8

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