Please wait a minute...

Quantitative Biology

Quant. Biol.    2017, Vol. 5 Issue (1) : 67-75     DOI: 10.1007/s40484-017-0099-0
RESEARCH ARTICLE |
Visualization of phage DNA degradation by a type I CRISPR-Cas system at the single-cell level
Jingwen Guan1,2,3,Xu Shi1,2,Roberto Burgos1,Lanying Zeng1,2,3()
1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA
2. Center for Phage Technology, Texas A&M University, College Station, TX 77843, USA
3. Molecular and Environmental Plant Sciences, Texas A&M University, College Station, TX 77843, USA
Download: PDF(1177 KB)   HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
Abstract

Background: The CRISPR-Cas system is a widespread prokaryotic defense system which targets and cleaves invasive nucleic acids, such as plasmids or viruses. So far, a great number of studies have focused on the components and mechanisms of this system, however, a direct visualization of CRISPR-Cas degrading invading DNA in real-time has not yet been studied at the single-cell level.

Methods: In this study, we fluorescently label phage lambda DNA in vivo, and track the labeled DNA over time to characterize DNA degradation at the single-cell level.

Results: At the bulk level, the lysogenization frequency of cells harboring CRISPR plasmids decreases significantly compared to cells with a non-CRISPR control. At the single-cell level, host cells with CRISPR activity are unperturbed by phage infection, maintaining normal growth like uninfected cells, where the efficiency of our anti-lambda CRISPR system is around 26%. During the course of time-lapse movies, the average fluorescence of invasive phage DNA in cells with CRISPR activity, decays more rapidly compared to cells without, and phage DNA is fully degraded by around 44 minutes on average. Moreover, the degradation appears to be independent of cell size or the phage DNA ejection site suggesting that Cas proteins are dispersed in sufficient quantities throughout the cell.

Conclusions: With the CRISPR-Cas visualization system we developed, we are able to examine and characterize how a CRISPR system degrades invading phage DNA at the single-cell level. This work provides direct evidence and improves the current understanding on how CRISPR breaks down invading DNA.

Author Summary  The CRISPR-Cas system is a widespread evolutionary adaptation in prokaryotes including archaea and bacteria, defending against invasive nucleic acids, such as plasmids or viruses. We aim to visualize and characterize how a CRISPR system acts within E. coli cells to destroy a phage invader at the single-cell level. By fluorescently labeling and tracking phage lambda DNA after infection using microscopy, we find that CRISPR rapidly degrades phage DNA to allow the cell to live on, and discover some parameters accounting for the cell-to-cell variability of the CRISPR functions, providing insights on how CRISPR systems protect bacteria.
Keywords bacteriophage lambda      CRISPR-Cas      fluorescence microscopy      single-cell analysis      type I CRISPR     
PACS:     
Fund: 
Corresponding Authors: Lanying Zeng   
Issue Date: 22 March 2017
 Cite this article:   
Jingwen Guan,Xu Shi,Roberto Burgos, et al. Visualization of phage DNA degradation by a type I CRISPR-Cas system at the single-cell level[J]. Quant. Biol., 2017, 5(1): 67-75.
 URL:  
http://journal.hep.com.cn/qb/EN/10.1007/s40484-017-0099-0
http://journal.hep.com.cn/qb/EN/Y2017/V5/I1/67
Service
E-mail this article
E-mail Alert
RSS
Articles by authors
Jingwen Guan
Xu Shi
Roberto Burgos
Lanying Zeng
Fig.1  CRISPR system reduces lysogenization efficiency.

(A) The lysogenization frequencies of CRISPR versus control system for our phage DNA reporter strain at a range of APIs (low API of 1 – 6 and high API of 15 – 65). The control spacer: blue cross marker with blue lines as the mean for low and high APIs; the CRISPR spacer: red circle with red lines as the mean of the low and high APIs. (B) The lysogenization efficiency of the CRISPR system, defined as the ratio of average lysogenization frequency of the CRISPR system relative to that of the control system for low and high APIs. Error bar represents S.D.

Fig.2  CRISPR system apparently degrades labeled phage DNA in single cells.

The DNA of the fluorescently labeled infecting phage (red spot, pointed by purple arrows) is ejected into the host E. coli cell forming a fluorescent spot (green spot, pointed by yellow arrows) at 0 min. The phage DNA (green spot) intensity decreases over time and finally disappears. (A) A lytic cell in a control movie. The green or yellow spot (yellow color is a result of overlay by green and red fluorescence) indicating the phage DNA disappears around 110 min. At 120 min, the cell lyses. (B) A lytic cell in a CRISPR movie. The green or yellow spot indicating the phage DNA disappears around 120 min or persists until cell lysis. At 120 min, the cell lyses. (C) A CRISPR cell (top) and uninfected cell (bottom) in a CRISPR movie. The green spot indicating the phage DNA in the CRISPR cell disappears around 45 min, which is much earlier than that in the lytic cell in (A) and (B). At 70 min, the cell divides, similar to that of the uninfected cell.

Fig.3  Phage DNA intensity decreases faster in CRISPR-active cells.

(A) In the CRISPR movies, the phage DNA intensities of the CRISPR cells (red line, N= 167) decrease much faster than those of the lytic cells (blue line, N= 423) indicating CRISPR is actively functioning to degrade the invading phage DNA. The averages are shown as the thick lines. (B) The histogram of phage DNA spot disappearance time corresponding to the degradation time for CRISPR cells is well fitted to a Gaussian distribution (red line). The time to totally degrade phage DNA is around 43.9±1.5 minutes. (C) The histogram of phage DNA spot disappearance time accounting for the photobleaching and/or phage DNA packaging into the phage head. Around 50% of the lytic cells still have the phage DNA spot at the end of the movies (120 min). (D) The phage DNA spot disappearance time is correlated with cell lysis time with a correlation coefficient of 0.91, p-value of 0.01. Error bar represents S.E.M.

Fig.4  Phage DNA degradation correlates with spot intensity, not with cell size or initial DNA location.

(A) The complete phage DNA degradation or spot disappearance time positively correlates with the maximum intensity of the spot at the beginning of the movie with a correlation coefficient of 0.97, p-value of 0.03. The binned data were obtained with a bin interval of 1.5±105 A.U. (B) The spot disappearance time does not change with the initial cell size with a correlation coefficient of-0.55, p-value of 0.45. The binned data were obtained with a bin interval of 1 mm. (C) The efficiency of CRISPR is very similar for the initial invading phage DNA at polar/mid-cell (0.27±0.04) or non-polar (0.25±0.02) positions with a p-value of 0.02. The diagram of the cell is shown on the top right. (D) The spot disappearance time does not seem to correlate with the initial phage DNA location showing similar disappearance time for polar/mid-cell (42.1±2.4 min) or non-polar (44.7±1.8 min) cell location with a p-value of 0.02. Error bar represents S.E.M.

Bacterial strains, plasmids, and phages
Strain name Relevant genotype Source/Ref.
Bacterial strains
BA16 MG1655, dam-, seqA-yfp, CmR [22]
LZ1436 BA16[pWUR397A, pWUR400, pWUR477], AmpR, StrR, CmR This work
LZ1437 BA16[pWUR397A, pWUR400, pWUR478], AmpR, StrR, CmR This work
Phage strains
lLZ760 Fully methylated, gpD-mosaic, lD-mTurquoise2cI857 bor::KanR [28]
Plasmids
pWUR397A cas3 in pRSF-1b [21]
pWUR400 casA-casB-casC-casD-casE in pCDF-1b [20]
pWUR477 Non-targeting CRISPR/ spacers from E. coli K12 with no homology to phage lambda in pACYCDuet-1 [20]
pWUR478 Template CRISPR/ template strand of lambda genes J, O, R and E in pACYCDuet-1 [20]
Tab.1  The bacteria, plasmids and phages used in this study.
1 Mohanraju, P., Makarova, K. S., Zetsche, B., Zhang, F., Koonin, E. V. and van der Oost, J. (2016) Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science, 353, aad5147
doi: 10.1126/science.aad5147.
2 Deveau, H., Garneau, J. E. and Moineau, S. (2010) CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol., 64, 475–493
doi: 10.1146/annurev.micro.112408.134123.
3 van Erp, P. B., Jackson, R. N., Carter, J., Golden, S. M., Bailey, S. and Wiedenheft, B. (2015) Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli. Nucleic Acids Res., 43, 8381–8391
doi: 10.1093/nar/gkv793.
4 Bhaya, D., Davison, M. and Barrangou, R. (2011) CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu. Rev. Genet., 45, 273–297
doi: 10.1146/annurev-genet-110410-132430.
5 Sternberg, S. H., Richter, H., Charpentier, E. and Qimron, U. (2016) Adaptation in CRISPR-Cas systems. Mol. Cell, 61, 797–808
doi: 10.1016/j.molcel.2016.01.030.
6 van der Oost, J., Westra, E. R., Jackson, R. N. and Wiedenheft, B. (2014) Unravelling the structural and mechanistic basis of CRISPR-Cas systems. Nat. Rev. Microbiol., 12, 479–492
doi: 10.1038/nrmicro3279.
7 Huo, Y., Nam, K. H., Ding, F., Lee, H., Wu, L., Xiao, Y., Farchione, M. D. Jr, Zhou, S., Rajashankar, K., Kurinov, I., (2014) Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation. Nat. Struct. Mol. Biol., 21, 771– 777
doi: 10.1038/nsmb.2875.
8 Hatoum-Aslan, A., Maniv, I. and Marraffini, L. A. (2011) Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured by a ruler mechanism anchored at the precursor processing site. Proc. Natl. Acad. Sci. USA, 108, 21218–21222
doi: 10.1073/pnas.1112832108.
9 Sinkunas, T., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath, P. and Siksnys, V. (2011) Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system. EMBO J., 30, 1335–1342
doi: 10.1038/emboj.2011.41.
10 Barrangou, R. (2015) Diversity of CRISPR-Cas immune systems and molecular machines. Genome Biol., 16, 247
doi: 10.1186/s13059-015-0816-9.
11 Mulepati, S. and Bailey, S. (2013) In vitro reconstitution of an Escherichia coli RNA-guided immune system reveals unidirectional, ATP-dependent degradation of DNA target. J. Biol. Chem., 288, 22184–22192
doi: 10.1074/jbc.M113.472233.
12 Amitai, G. and Sorek, R. (2016) CRISPR-Cas adaptation: insights into the mechanism of action. Nat. Rev. Microbiol., 14, 67–76
doi: 10.1038/nrmicro.2015.14.
13 Künne, T., Kieper, S. N., Bannenberg, J. W., Vogel, A. I., Miellet, W. R., Klein, M., Depken, M., Suarez-Diez, M. and Brouns, S. J. (2016) Cas3-derived target DNA degradation fragments fuel primed CRISPR adaptation. Mol. Cell, 63, 852–864
doi: 10.1016/j.molcel.2016.07.011.
14 McGinn, J. and Marraffini, L. A. (2016) CRISPR-Cas systems optimize their immune response by specifying the site of spacer integration. Mol. Cell, 64, 616–623
doi: 10.1016/j.molcel.2016.08.038.
15 Jackson, R. N., Golden, S. M., van Erp, P. B., Carter, J., Westra, E. R., Brouns, S. J., van der Oost, J., Terwilliger, T. C., Read, R. J. and Wiedenheft, B. (2014) Crystal structure of the CRISPR RNA-guided surveillance complex from Escherichia coli. Science, 345, 1473–1479
doi: 10.1126/science.1256328.
16 Hochstrasser, M. L., Taylor, D. W., Bhat, P., Guegler, C. K., Sternberg, S. H., Nogales, E.and Doudna, J. A. (2014) CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference. Proc. Natl. Acad. Sci. USA, 111, 6618–6623
doi: 10.1073/pnas.1405079111.
17 Westra, E. R., van Erp, P. B., Kunne, T., Wong, S. P., Staals, R. H., Seegers, C. L., Bollen, S., Jore, M. M., Semenova, E., Severinov, K., (2012) CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol. Cell, 46, 595–605
doi: 10.1016/j.molcel.2012.03.018.
18 Mulepati, S., Heroux, A. and Bailey, S. (2014) Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target. Science, 345, 1479–1484
doi: 10.1126/science.1256996.
19 Redding, S., Sternberg, S. H., Marshall, M., Gibb, B., Bhat, P., Guegler, C. K., Wiedenheft, B., Doudna, J. A. and Greene, E. C. (2015) Surveillance and processing of foreign DNA by the Escherichia coli CRISPR-Cas system. Cell, 163, 854–865
doi: 10.1016/j.cell.2015.10.003.
20 Brouns, S. J., Jore, M. M., Lundgren, M., Westra, E. R., Slijkhuis, R. J., Snijders, A. P., Dickman, M. J., Makarova, K. S., Koonin, E. V. and van der Oost, J. (2008) Small CRISPR RNAs guide antiviral defense in prokaryotes. Science, 321, 960–964
doi: 10.1126/science.1159689.
21 Edgar, R. and Qimron, U. (2010) The Escherichia coli CRISPR system protects from lambda lysogenization, lysogens, and prophage induction. J. Bacteriol., 192, 6291–6294
doi: 10.1128/JB.00644-10.
22 Babic, A., Lindner, A. B., Vulic, M., Stewart, E. J. and Radman, M. (2008) Direct visualization of horizontal gene transfer. Science, 319, 1533–1536
doi: 10.1126/science.1153498.
23 Shao, Q., Hawkins, A. and Zeng, L. (2015) Phage DNA dynamics in cells with different fates. Biophys. J., 108, 2048–2060
doi: 10.1016/j.bpj.2015.03.027.
24 Pul, U., Wurm, R., Arslan, Z., Geissen, R., Hofmann, N. and Wagner, R. (2010) Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS. Mol. Microbiol., 75, 1495–1512
doi: 10.1111/j.1365-2958.2010.07073.x.
25 Lu, M., Campbell, J. L., Boye, E. and Kleckner, N. (1994) SeqA: a negative modulator of replication initiation in E. coli. Cell, 77, 413–426
doi: 10.1016/0092-8674(94)90156-2.
26 Slater, S., Wold, S., Lu, M., Boye, E., Skarstad, K. and Kleckner, N. (1995) E. coli SeqA protein binds oriC in two different methyl-modulated reactions appropriate to its roles in DNA replication initiation and origin sequestration. Cell, 82, 927–936
doi: 10.1016/0092-8674(95)90272-4.
27 Pougach, K., Semenova, E., Bogdanova, E., Datsenko, K. A., Djordjevic, M., Wanner, B. L. and Severinov, K. (2010) Transcription, processing and function of CRISPR cassettes in Escherichia coli. Mol. Microbiol., 77, 1367–1379
doi: 10.1111/j.1365-2958.2010.07265.x.
28 Shao, Q., Trinh, J. T., McIntosh, C. S., Christenson, B., Balazsi, G. and Zeng, L. (2017) Lysis-lysogeny coexistence: prophage integration during lytic development. MicrobiologyOpen 6
doi: 10.1002/mbo3.395.
29 Van Valen, D., Wu, D., Chen, Y. J., Tuson, H., Wiggins, P. and Phillips, R. (2012) A single-molecule Hershey-Chase experiment. Curr. Biol., 22, 1339–1343
doi: 10.1016/j.cub.2012.05.023.
30 Edgar, R., Rokney, A., Feeney, M., Semsey, S., Kessel, M., Goldberg, M. B., Adhya, S. and Oppenheim, A. B. (2008) Bacteriophage infection is targeted to cellular poles. Mol. Microbiol., 68, 1107–1116
doi: 10.1111/j.1365-2958.2008.06205.x.
31 Rothenberg, E., Sepulveda, L. A., Skinner, S. O., Zeng, L., Selvin, P. R. and Golding, I. (2011) Single-virus tracking reveals a spatial receptor-dependent search mechanism. Biophys. J., 100, 2875–2882
doi: 10.1016/j.bpj.2011.05.014.
32 Zeng, L., Skinner, S. O., Zong, C., Sippy, J., Feiss, M. and Golding, I. (2010) Decision making at a subcellular level determines the outcome of bacteriophage infection. Cell, 141, 682–691
doi: 10.1016/j.cell.2010.03.034.
33 Zeng, L. and Golding, I. (2011) Following cell-fate in E. coli after infection by phage lambda. J. Vis. Exp., 56, e3363,
doi: 10.3791/3363.
34 Sliusarenko, O., Heinritz, J., Emonet, T. and Jacobs-Wagner, C. (2011) High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics. Mol. Microbiol., 80, 612–627
doi: 10.1111/j.1365-2958.2011.07579.x.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed