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Real-time observations of single bacteriophage λ DNA ejections in vitro

Grayson, Paul and Han, Lin and Winther, Tabita and Phillips, Rob (2007) Real-time observations of single bacteriophage λ DNA ejections in vitro. Proceedings of the National Academy of Sciences of the United States of America, 104 (37). pp. 14652-14657. ISSN 0027-8424. PMCID PMC1976217. https://resolver.caltech.edu/CaltechAUTHORS:GRApnas07

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[img] Video (MPEG) (Movie 1 (.mpg). Phage lambda cI60 (48.5 kbp genome) in Mg buffer.) - Supplemental Material
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[img] Video (MPEG) (Movie 2 (.mpg). Phage lambda cI60 (48.5 kbp genome) in Na buffer.) - Supplemental Material
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[img] Video (MPEG) (Movie 3 (.mpg) Phage lambda cI26 b221 (38 kbp) in Mg buffer.) - Supplemental Material
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[img] Video (MPEG) (Movie 4 (.mpg) Phage lambda cI26 b221 (38 kbp) in Na buffer.) - Supplemental Material
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PDF (Supporting Information Figure 6. Portions of single DNA ejection events judged as "bad" and not selected for further analysis.)
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PDF (Supporting Information Figure 7. Three steps in analyzing images of ejected DNA fragments.)
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PDF (Supporting Information Figure 8. Calibration for measuring ejected DNA lengths. The plot shows the observed length of the DNA in pixels as a function of its length in kbp.)
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PDF (Supporting Information Figure 9. Graphs of the single ejection trajectories, showing the effect of the flow on the DNA.)
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PDF (Supporting Information Figure 10. Comparison of the velocity of DNA translocation at two different flow rates: 10 and 40 mL/min, corresponding to shear flows of 14 and 57 s-1, respectively.)
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PDF (Supporting Information Figure 11. Single trajectories from cI60 ejections in buffer A. The plot shows the amount of DNA ejected as a function of time, with trajectories aligned for visual comparison. Ejections take »4 s to reach completion in this buffer)
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Abstract

The physical, chemical, and structural features of bacteriophage genome release have been the subject of much recent attention. Many theoretical and experimental studies have centered on the internal forces driving the ejection process. Recently, Mangenot et al. [Mangenot S, Hochrein M, Rädler J, Letellier L (2005) Curr Biol 15:430–435.] reported fluorescence microscopy of phage T5 ejections, which proceeded stepwise between DNA nicks, reaching a translocation speed of 75 kbp/s or higher. It is still unknown how high the speed actually is. This paper reports real-time measurements of ejection from phage {lambda}, revealing how the speed depends on key physical parameters such as genome length and ionic state of the buffer. Except for a pause before DNA is finally released, the entire 48.5-kbp genome is translocated in {approx}1.5 s without interruption, reaching a speed of 60 kbp/s. The process gives insights particularly into the effects of two parameters: a shorter genome length results in lower speed but a shorter total time, and the presence of divalent magnesium ions (replacing sodium) reduces the pressure, increasing ejection time to 8–11 s. Pressure caused by DNA–DNA interactions within the head affects the initiation of ejection, but the close packing is also the dominant source of friction: more tightly packed phages initiate ejection earlier, but with a lower initial speed. The details of ejection revealed in this study are probably generic features of DNA translocation in bacteriophages and have implications for the dynamics of DNA in other biological systems.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1976217/PubMed CentralArticle
https://doi.org/10.1073/pnas.0703274104DOIUNSPECIFIED
https://doi.org/10.1073/pnas.0703274104DOIUNSPECIFIED
ORCID:
AuthorORCID
Phillips, Rob0000-0003-3082-2809
Additional Information:© 2007 by The National Academy of Sciences of the USA. Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved July 6, 2007 (received for review April 11, 2007). Published online on September 5, 2007, 10.1073/pnas.0703274104 We thank A. Graff (University of Basel, Basel, Switzerland) and E. Berkane (University of Würzburg, Würzburg, Germany) for providing protocols for the purification of LamB and the pop154 E. coli strain. Michael Feiss (University of Iowa, Ames, IA) kindly sent us samples of the {lambda}b221cI26 and {lambda}cI60 phages used here. M. Inamdar (ITT, Bombay, India) kindly provided data from his calculations on the pressure within {lambda}. We thank Douglas Rees, S. Fraser, and G. Jensen for laboratory space, and I. Molineux, J. Widom, J. Kondev, W. Gelbart, C. Knobler, A. Grosberg, M. Rubinstein, and others for very helpful conversations; and S. Quake as well as the anonymous reviewers for critical reading of the manuscript. This work was supported by a grant from the Keck Foundation (to R.P.), a National Institutes of Health Director's Pioneer Award (to R.P.), and National Science Foundation Grant CMS-0301657 (to R.P.). P.G. was partially supported by a National Science Foundation graduate research fellowship. Author contributions: P.G. and R.P. designed research; P.G. and T.W. performed research; P.G. and L.H. contributed new reagents/analytic tools; P.G. analyzed data; and P.G. and R.P. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/cgi/content/full/0703274104/DC1. Movies 1-4. Real time recordings of bacteriophage lambda DNA ejection events. Recordings lasting 1,000 frames (250 s) were performed as described in the text, then single ejection events were selected from the movies and analyzed to reveal the DNA ejection process. Samples of four experimental conditions are shown. For each movie, a key presents the size scale, the elapsed time, and the frame count.
Subject Keywords:fluorescence; microscopy; virus; genome
Issue or Number:37
PubMed Central ID:PMC1976217
Record Number:CaltechAUTHORS:GRApnas07
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:GRApnas07
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9785
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:16 Mar 2008
Last Modified:08 Oct 2019 22:38

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