Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    Eagon, R. G. Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. J. Bacteriol. 83, 736–737 (1962).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Weinstock, M. T., Hesek, E. D., Wilson, C. M. & Gibson, D. G. Vibrio natriegens as a fast-growing host for molecular biology. Nat. Methods 13, 849–851 (2016).

    CAS  Article  Google Scholar 

  3. 3.

    Long, C. P., Gonzalez, J. E., Cipolla, R. M. & Antoniewicz, M. R. Metabolism of the fast-growing bacterium Vibrio natriegens elucidated by 13C metabolic flux analysis. Metab. Eng. 44, 191–197 (2017).

    CAS  Article  Google Scholar 

  4. 4.

    Hoffart, E. et al. High substrate uptake rates empower Vibrio natriegens as production host for industrial biotechnology. Appl. Environ. Microbiol. 83, e01614-17 (2017).

    Article  Google Scholar 

  5. 5.

    Aiyar, S. E., Gaal, T. & Gourse, R. L. rRNA promoter activity in the fast-growing bacterium Vibrio natriegens. J. Bacteriol. 184, 1349–1358 (2002).

    CAS  Article  Google Scholar 

  6. 6.

    Overbeek, R. et al. The SEED and the rapid annotation of microbial genomes using subsystems technology (RAST). Nucleic Acids Res. 42, D206–D214 (2013).

    Article  Google Scholar 

  7. 7.

    Maida, I. et al. Draft genome sequence of the fast-growing bacterium Vibrio natriegens strain DSMZ 759. Genome Announc. 1, e00648–13 (2013).

    Article  Google Scholar 

  8. 8.

    Thompson, J. R. & Polz, M. F. in The Biology of Vibrios(eds Thompson, F. L. et al.) 190–203 (American Society of Microbiology, 2006).

  9. 9.

    Heidelberg, J. F. et al. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406, 477–483 (2000).

    CAS  Article  Google Scholar 

  10. 10.

    Chan, P. P. & Lowe, T. M. GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res. 37, D93–D97 (2009).

    CAS  Article  Google Scholar 

  11. 11.

    Brown, C. T., Olm, M. R., Thomas, B. C. & Banfield, J. F. Measurement of bacterial replication rates in microbial communities. Nat. Biotechnol. 34, 1256–1263 (2016).

    CAS  Article  Google Scholar 

  12. 12.

    van Opijnen, T., Lazinski, D. W. & Camilli, A. Genome-wide fitness and genetic interactions determined by Tn-seq, a high-throughput massively parallel sequencing method for microorganisms. Curr. Protoc. Microbiol. 106, 7.16.1–7.16.24 (2010).

    Google Scholar 

  13. 13.

    Rock, J. M. et al. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat. Microbiol. 2, 16274 (2017).

    CAS  Article  Google Scholar 

  14. 14.

    Peters, J. M. et al. A comprehensive, CRISPR-based functional analysis of essential genes in bacteria. Cell 165, 1493–1506 (2016).

    CAS  Article  Google Scholar 

  15. 15.

    Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    CAS  Article  Google Scholar 

  16. 16.

    Liu, X. et al. High‐throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Mol. Syst. Biol. 13, 931 (2017).

    Article  Google Scholar 

  17. 17.

    Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).

    Article  Google Scholar 

  18. 18.

    Tatusova, T. et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 44, 6614–6624 (2016).

    CAS  Article  Google Scholar 

  19. 19.

    Chao, M. C. et al. High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model-based analyses of transposon-insertion sequencing data. Nucleic Acids Res. 41, 9033–9048 (2013).

    CAS  Article  Google Scholar 

  20. 20.

    Okada, K., Iida, T., Kita-Tsukamoto, K. & Honda, T. Vibrios commonly possess two chromosomes. J. Bacteriol. 187, 752–757 (2005).

    CAS  Article  Google Scholar 

  21. 21.

    Yamazaki, Y., Niki, H. & Kato, J.-I. Profiling of Escherichia coli chromosome database. Methods Mol. Biol. 416, 385–389 (2008).

    CAS  Article  Google Scholar 

  22. 22.

    Julio, S. M. et al. DNA adenine methylase is essential for viability and plays a role in the pathogenesis of Yersinia pseudotuberculosis and Vibrio cholerae. Infect. Immun. 69, 7610–7615 (2001).

    CAS  Article  Google Scholar 

  23. 23.

    Egan, E. S., Fogel, M. A. & Waldor, M. K. Divided genomes: negotiating the cell cycle in prokaryotes with multiple chromosomes. Mol. Microbiol. 56, 1129–1138 (2005).

    CAS  Article  Google Scholar 

  24. 24.

    Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).

    CAS  Article  Google Scholar 

  25. 25.

    Schleicher, L. et al. Vibrio natriegens as host for expression of multisubunit membrane protein complexes. Front. Microbiol. 9, 2537 (2018).

    Article  Google Scholar 

  26. 26.

    Lenz, D. H. et al. The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae. Cell 118, 69–82 (2004).

    CAS  Article  Google Scholar 

  27. 27.

    Val, M.-E. et al. A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae. Sci. Adv. 2, e1501914 (2016).

    Article  Google Scholar 

  28. 28.

    Mee, M. T., Collins, J. J., Church, G. M. & Wang, H. H. Syntrophic exchange in synthetic microbial communities. Proc. Natl Acad. Sci. USA 111, E2149–E2156 (2014).

    CAS  Article  Google Scholar 

  29. 29.

    Cui, L. et al. A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nat. Commun. 9, 1912 (2018).

    Article  Google Scholar 

  30. 30.

    Zhang, S. & Voigt, C. A. Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design. Nucleic Acids Res. 46, 11115–11125 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Vega, N. M., Allison, K. R., Khalil, A. S. & Collins, J. J. Signaling-mediated bacterial persister formation. Nat. Chem. Biol. 8, 431–433 (2012).

    CAS  Article  Google Scholar 

  32. 32.

    Martínez-García, E., Calles, B., Arévalo-Rodríguez, M. & de Lorenzo, V. pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. BMC Microbiol. 11, 38 (2011).

    Article  Google Scholar 

  33. 33.

    Cameron, D. E., Urbach, J. M. & Mekalanos, J. J. A defined transposon mutant library and its use in identifying motility genes in Vibrio cholerae. Proc. Natl Acad. Sci. USA 105, 8736–8741 (2008).

    CAS  Article  Google Scholar 

  34. 34.

    Huerta-Cepas, J. et al. Fast genome-wide functional annotation through orthology assignment by eggNOG-mapper. Mol. Biol. Evol. 34, 2115–2122 (2017).

    CAS  Article  Google Scholar 

  35. 35.

    Dalia, T. N. et al. Multiplex genome editing by natural transformation (MuGENT) for synthetic biology in Vibrio natriegens. ACS Synth. Biol. 6, 1650–1655 (2017).

    CAS  Article  Google Scholar 

Download references

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.).

Author information

Affiliations

Authors

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.

Ethics declarations

Competing interests

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

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Search

Quick links

Sign up for the Nature Briefing newsletter for a daily update on COVID-19 science.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing