Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Measuring transcription at a single gene copy reveals hidden drivers of bacterial individuality

Nature Microbiology volume 4, pages 2118–2127 (2019)Cite this article

Subjects

Abstract

Single-cell measurements of mRNA copy numbers inform our understanding of stochastic gene expression1,2,3, but these measurements coarse-grain over the individual copies of the gene, where transcription and its regulation take place stochastically4,5. Here, we combine single-molecule quantification of mRNA and gene loci to measure the transcriptional activity of an endogenous gene in individual Escherichia coli bacteria. When interpreted using a theoretical model for mRNA dynamics, the single-cell data allow us to obtain the probabilistic rates of promoter switching, transcription initiation and elongation, mRNA release and degradation. Unexpectedly, we find that gene activity can be strongly coupled to the transcriptional state of another copy of the same gene present in the cell, and to the event of gene replication during the bacterial cell cycle. These gene-copy and cell-cycle correlations demonstrate the limits of mapping whole-cell mRNA numbers to the underlying stochastic gene activity and highlight the contribution of previously hidden variables to the observed population heterogeneity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Detecting active transcription at a single gene copy.
Fig. 2: Analysing nascent mRNA reveals the stochastic kinetics of transcript initiation, elongation, release and degradation.
Fig. 3: Promoter activity is coupled to the activity of additional gene copies in the cell.
Fig. 4: Promoter activity is coupled to the cell-cycle event of gene replication.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding author on request.

Code availability

The custom MATLAB routines used for processing and analysing the fluorescence microscopy data are freely available from the corresponding author on request.

References

  1. Sanchez, A. & Golding, I. Genetic determinants and cellular constraints in noisy gene expression. Science 342, 1188–1193 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Golding, I. et al. Real-time kinetics of gene activity in individual bacteria. Cell 123, 1025–1036 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Taniguchi, Y. et al. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329, 533–538 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Jones, D. L., Brewster, R. C. & Phillips, R. Promoter architecture dictates cell-to-cell variability in gene expression. Science 346, 1533–1536 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Sepúlveda, L. A. et al. Measurement of gene regulation in individual cells reveals rapid switching between promoter states. Science 351, 1218–1222 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Raj, A. et al. Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 4, e309 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. So, L. H. et al. General properties of transcriptional time series in Escherichia coli. Nat. Genet. 43, 554–560 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Peterson, J. R. et al. Effects of DNA replication on mRNA noise. Proc. Natl Acad. Sci. USA 112, 15886–15891 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zenklusen, D., Larson, D. R. & Singer, R. H. Single-RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15, 1263–1271 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Joshi, M. C. et al. Escherichia coli sister chromosome separation includes an abrupt global transition with concomitant release of late-splitting intersister snaps. Proc. Natl Acad. Sci. USA 108, 2765–2770 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Skinner, S. O. et al. Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization. Nat. Protoc. 8, 1100–1113 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. French, S. L. & Miller, O. L. Jr. Transcription mapping of the Escherichia coli chromosome by electron microscopy. J. Bacteriol. 171, 4207–4216 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Golding, I. & Cox, E. C. RNA dynamics in live Escherichia coli cells. Proc. Natl Acad. Sci. USA 101, 11310–11315 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Elf, J., Li, G. W. & Xie, X. S. Probing transcription factor dynamics at the single-molecule level in a living cell. Science 316, 1191–1194 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Choubey, S., Kondev, J. & Sanchez, A. Deciphering transcriptional dynamics in vivo by counting nascent RNA molecules. PLoS Comput. Biol. 11, e1004345 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Xu, H. et al. Stochastic kinetics of nascent RNA. Phys. Rev. Lett. 117, 128101 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Chen, H. et al. Genome-wide study of mRNA degradation and transcript elongation in Escherichia coli. Mol. Syst. Biol. 11, 781 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Kuhlman, T. et al. Combinatorial transcriptional control of the lactose operon of Escherichia coli. Proc. Natl Acad. Sci. USA 104, 6043–6048 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Moffitt, J. R. et al. Spatial organization shapes the turnover of a bacterial transcriptome. eLife 5, e13065 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Proshkin, S. et al. Cooperation between translating ribosomes and RNA polymerase in transcription elongation. Science 328, 504–508 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Epshtein, V. & Nudler, E. Cooperation between RNA polymerase molecules in transcription elongation. Science 300, 801–805 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Senecal, A. et al. Transcription factors modulate c-Fos transcriptional bursts. Cell Rep. 8, 75–83 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Skinner, S. O. et al. Single-cell analysis of transcription kinetics across the cell cycle. eLife 5, e12175 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Neidhardt, F. C., Ingraham, J. L. & Schaechter, M. Physiology of the Bacterial Cell: a Molecular Approach (Sinauer Associates, 1990).

  25. Wallden, M. et al. The synchronization of replication and division cycles in individual E. coli cells. Cell 166, 729–739 (2016).

    Article  CAS  PubMed  Google Scholar 

  26. Elowitz, M. B. et al. Stochastic gene expression in a single cell. Science 297, 1183–1186 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Guptasarma, P. Does replication-induced transcription regulate synthesis of the myriad low copy number proteins of Escherichia coli? BioEssays 17, 987–997 (1995).

    Article  CAS  PubMed  Google Scholar 

  28. Hammar, P. et al. Direct measurement of transcription factor dissociation excludes a simple operator occupancy model for gene regulation. Nat. Genet. 46, 405–408 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Golding, I. Infection by bacteriophage lambda: an evolving paradigm for cellular individuality. Curr. Opin. Microbiol. 43, 9–13 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Symmons, O. & Raj, A. What’s luck got to do with it: single cells, multiple fates, and biological nondeterminism. Mol. Cell 62, 788–802 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lau, I. F. et al. Spatial and temporal organization of replicating Escherichia coli chromosomes. Mol. Microbiol. 49, 731–743 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. Baba, T. et al. Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Ellermeier, C. D., Janakiraman, A. & Slauch, J. M. Construction of targeted single copy lac fusions using λ Red and FLP-mediated site-specific recombination in bacteria. Gene 290, 153–161 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Thomason, L. et al. Recombineering: genetic engineering in bacteria using homologous recombination. Curr. Protoc. Mol. Biol. 78, 1.16.1–1.16.24 (2007).

    Article  Google Scholar 

  35. Cherepanov, P. P. & Wackernagel, W. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158, 9–14 (1995).

    Article  CAS  PubMed  Google Scholar 

  36. Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Schmidl, S. R. et al. Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS Synth. Biol. 3, 820–831 (2014).

    Article  CAS  PubMed  Google Scholar 

  38. Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step, precision cloning method with high throughput capability. PloS ONE 3, e3647 (2008).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Bernstein, J. A. et al. Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays. Proc. Natl Acad. Sci. USA 99, 9697–9702 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Llopis, J. et al. Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc. Natl Acad. Sci. USA 95, 6803–6808 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Young, J. W. et al. Measuring single-cell gene expression dynamics in bacteria using fluorescence time-lapse microscopy. Nat. Protoc. 7, 80–88 (2012).

    Article  CAS  Google Scholar 

  42. Olivo-Marin, J.-C. Extraction of spots in biological images using multiscale products. Pattern Recognit. 35, 1989–1996 (2002).

    Article  Google Scholar 

  43. Bates, D. & Kleckner, N. Chromosome and replisome dynamics in E. coli: loss of sister cohesion triggers global chromosome movement and mediates chromosome segregation. Cell 121, 899–911 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Nielsen, H. J. et al. Progressive segregation of the Escherichia coli chromosome. Mol. Microbiol. 61, 383–393 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Nielsen, H. J. et al. Dynamics of Escherichia coli chromosome segregation during multifork replication. J. Bacteriol. 189, 8660–8666 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Xu, H. et al. Combining protein and mRNA quantification to decipher transcriptional regulation. Nat. Methods 12, 739–742 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We are grateful to the following people for their generous advice and for providing reagents: D. Bates, J. Elf, H. Garcia, M. Girard, J. Halliday, C. Herman, M. Joshi, D. Magnan, J. Moffitt, E. Nudler, R. Phillips, A. Sarrion-Perdigones, S. Sebastian, L. Sepúlveda, A. Singh, P. Sivaramakrishnan, S. Skinner, A. Sokac, J. Tabor, K. Venken and all the members of the Golding lab. Work in the Golding lab is supported by grants from the National Institutes of Health (grant no. R01 GM082837), the National Science Foundation (grant nos. PHY 1147498, PHY 1430124 and PHY 1427654), the Welch Foundation (grant no. Q-1759) and the John S. Dunn Foundation (Collaborative Research Award). H.X. was supported by the Burroughs Wellcome Fund Career Award at the Scientific Interface (grant no. 1013907), the Thousand Talents Plan of China (Programme for Young Professionals), the National Natural Science Foundation of China (grant no. 11774225), the National Key Research and Development Programme of China (grant no. 2018YFC0310800) and the National Science Foundation of Shanghai (grant no. 18ZR1419800). We gratefully acknowledge the computing resources provided by the CIBR Center of the Baylor College of Medicine.

Author information

Author notes

  1. These authors contributed equally: Mengyu Wang, Jing Zhang.

Authors and Affiliations

  1. Department of Physics, University of Illinois at Urbana−Champaign, Urbana, IL, USA

    Mengyu Wang & Ido Golding

  2. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA

    Mengyu Wang, Jing Zhang & Ido Golding

  3. Graduate Program in Quantitative and Computational Biosciences, Baylor College of Medicine, Houston, TX, USA

    Mengyu Wang

  4. School of Physics and Astronomy, Shanghai Jiao Tong University, Shanghai, China

    Heng Xu

  5. Institute of Natural Sciences, Shanghai Jiao Tong University, Shanghai, China

    Heng Xu

  6. Department of Microbiology, University of Illinois at Urbana−Champaign, Urbana, IL, USA

    Ido Golding

Authors
  1. Mengyu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Heng Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ido Golding
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.W., J.Z., H.X. and I.G. designed the experiments. M.W. and J.Z. performed the experiments. M.W., J.Z. and H.X. analysed the data. M.W., J.Z. and I.G. wrote this Letter.

Corresponding author

Correspondence to Ido Golding.

Ethics declarations

Competing interests

The authors declare no competing interests.

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 Note, Supplementary Figs. 1−37, Supplementary Tables 1−5 and Supplementary References.

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, M., Zhang, J., Xu, H. et al. Measuring transcription at a single gene copy reveals hidden drivers of bacterial individuality. Nat Microbiol 4, 2118–2127 (2019). https://doi.org/10.1038/s41564-019-0553-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41564-019-0553-z

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing