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.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Custom code is available at https://github.com/citizenlee/vnat_glib or will be made available upon request.
Eagon, R. G. Pseudomonas natriegens, a marine bacterium with a generation time of less than 10 minutes. J. Bacteriol. 83, 736–737 (1962).
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).
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).
Hoffart, E. et al. High substrate uptake rates empower Vibrio natriegens as production host for industrial biotechnology. Appl. Environ. Microbiol. 83, e01614-17 (2017).
Aiyar, S. E., Gaal, T. & Gourse, R. L. rRNA promoter activity in the fast-growing bacterium Vibrio natriegens. J. Bacteriol. 184, 1349–1358 (2002).
Overbeek, R. et al. The SEED and the rapid annotation of microbial genomes using subsystems technology (RAST). Nucleic Acids Res. 42, D206–D214 (2013).
Maida, I. et al. Draft genome sequence of the fast-growing bacterium Vibrio natriegens strain DSMZ 759. Genome Announc. 1, e00648–13 (2013).
Thompson, J. R. & Polz, M. F. in The Biology of Vibrios(eds Thompson, F. L. et al.) 190–203 (American Society of Microbiology, 2006).
Heidelberg, J. F. et al. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 406, 477–483 (2000).
Chan, P. P. & Lowe, T. M. GtRNAdb: a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Res. 37, D93–D97 (2009).
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).
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).
Rock, J. M. et al. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat. Microbiol. 2, 16274 (2017).
Peters, J. M. et al. A comprehensive, CRISPR-based functional analysis of essential genes in bacteria. Cell 165, 1493–1506 (2016).
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Liu, X. et al. High‐throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Mol. Syst. Biol. 13, 931 (2017).
Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).
Tatusova, T. et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 44, 6614–6624 (2016).
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).
Okada, K., Iida, T., Kita-Tsukamoto, K. & Honda, T. Vibrios commonly possess two chromosomes. J. Bacteriol. 187, 752–757 (2005).
Yamazaki, Y., Niki, H. & Kato, J.-I. Profiling of Escherichia coli chromosome database. Methods Mol. Biol. 416, 385–389 (2008).
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).
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).
Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).
Schleicher, L. et al. Vibrio natriegens as host for expression of multisubunit membrane protein complexes. Front. Microbiol. 9, 2537 (2018).
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).
Val, M.-E. et al. A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae. Sci. Adv. 2, e1501914 (2016).
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).
Cui, L. et al. A CRISPRi screen in E. coli reveals sequence-specific toxicity of dCas9. Nat. Commun. 9, 1912 (2018).
Zhang, S. & Voigt, C. A. Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design. Nucleic Acids Res. 46, 11115–11125 (2018).
Vega, N. M., Allison, K. R., Khalil, A. S. & Collins, J. J. Signaling-mediated bacterial persister formation. Nat. Chem. Biol. 8, 431–433 (2012).
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).
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).
Huerta-Cepas, J. et al. Fast genome-wide functional annotation through orthology assignment by eggNOG-mapper. Mol. Biol. Evol. 34, 2115–2122 (2017).
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).
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.).
H.H.L., N.O. and G.M.C. have filed patents related to this work.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Discussion, Supplementary Figures 1–17, Supplementary Tables 1–4 and Supplementary References.
Single cell growth of V. natriegens (LB3) and E. coli (LB) at 37 °C.
About this article
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
ACS Synthetic Biology (2020)
Environmental Microbiology (2020)
Current Opinion in Biotechnology (2020)
Establishment of CRISPR interference in Methylorubrum extorquens and application of rapidly mining a new phytoene desaturase involved in carotenoid biosynthesis
Applied Microbiology and Biotechnology (2020)
Trends in Biotechnology (2020)